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==== Front Neurol Sci Neurol Sci Neurological Sciences 1590-1874 1590-3478 Springer International Publishing Cham 36454442 6531 10.1007/s10072-022-06531-9 Abstracts Abstracts of the 52nd Annual Conference of the Italian Society of Neurology 1 12 2022 2022 43 Suppl 1 1530 © Fondazione Società Italiana di Neurologia 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. issue-copyright-statement© Fondazione Società Italiana di Neurologia 2022 ==== Body pmcPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	36454442	PMC9713129	NO-CC CODE	2022-12-06 23:15:26	no	Neurol Sci. 2022 Dec 1; 43(Suppl 1):1-530	utf-8	Neurol Sci	2,022	10.1007/s10072-022-06531-9	oa_other
==== Front Ir J Med Sci Ir J Med Sci Irish Journal of Medical Science 0021-1265 1863-4362 Springer International Publishing Cham 3241 10.1007/s11845-022-03241-1 Letter to the Editor Real life experience of molnupiravir as a treatment of SARS-CoV-2 infection in vaccinated and unvaccinated patients: a letter on its effectiveness at preventing hospitalization Scioscia Giulia 12 De Pace Cosimo Carlo [email protected] 12 Giganti Giulio 2 Tondo Pasquale 12 Foschino Barbaro Maria Pia 12 Lacedonia Donato 12 1 grid.10796.39 0000000121049995 Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy 2 Respiratory Medicine Unit, “Policlinico Foggia” University Hospital, Foggia, Italy 1 12 2022 13 21 11 2022 24 11 2022 © The Author(s), under exclusive licence to Royal Academy of Medicine in Ireland 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background The SARS-CoV-2 pandemic has prompted clinicians to develop an early and effective treatment of viral infections. To date, vaccines, monoclonal antibodies, and antivirals are the cornerstone of therapy for SARS-CoV-2. AIFA approved the prescription of molnupiravir on 30/12/2021. Molnupiravir is a prodrug that causes the accumulation of errors in the viral genome. Methods We prescribed molnupiravir to a total of 74 patients in a range between 26 and 96 years old and followed-up them for 30 days. 10 patients affected by idiopathic pulmonary fibrosis (IPF) were treated. Results The follow-up showed that all of the treated patients presented a regression of symptoms. No patients were hospitalized and/or showed sequelae after the infection by SARS-CoV-2, even though the examined population was older and with more co-morbidities than other patients treated with different antivirals. Conclusion Molnupiravir is safe and well-tolerated by patients with high-risk of progression to severe COVID. No patients were hospitalized or showed sequelae, including all patients affected by IPF. Keywords COVID-19 IPF Molnupiravir Real life ==== Body pmcIntroduction The SARS-CoV-2 pandemic has prompted clinicians to develop an early and effective treatment of viral infections. To date, vaccines, monoclonal antibodies, and antivirals are the cornerstone of therapy for SARS-CoV-2. AIFA, the Italian Medicines Agency, approved the prescription of molnupiravir [1, 2] on 30/12/2021. Molnupiravir is a prodrug metabolized to the ribonucleoside analog N-hydroxycytidine (NHC). NHC spreads into cells, where it is phosphorylated to form the pharmacologically active ribonucleoside triphosphate (N-hydroxycytidine triphosphate, NHC-TP). Incorporating NHC-TP into viral RNA by the use of viral RNA polymerase causes an accumulation of errors in the viral genome, which inhibits its replication. The prescribed dosage is 4 tablets (200 mg each) every 12 h for 5 days. The treatment has to be started within 5 days after the onset of symptoms. AIFA laid out indications when prescribing the antiviral. Molnupiravir may be prescribed to patients with mild to moderate symptoms who do not need oxygen supplementation nor an increase in oxygen demand in the event of at-home baseline oxygen therapy due to chronic conditions. Patients cannot be already hospitalized for COVID-19. Patients also must have at least one of the following risk factors associated with the evolution into severe disease:Severe pulmonary and/or cardiovascular disease Oncological or onco-hematological disease in active phase Primary or acquired immunodeficiency Obesity (BMI ≥ 30) Chronic renal failure Uncontrolled diabetes mellitus No dose adjustments are required in case of renal or hepatic impairment. Molnupiravir should not be used in dialysis patients or in patients with eGFR < 30 ml/min/1.73 m2. Molnupiravir should not be prescribed during pregnancy. Women of childbearing potential must use contraceptive methods for the duration of treatment and for at least four additional days after the end of treatment; men who are partners of women of childbearing potential must use contraceptive methods for the entire duration of treatment and for at least three months after the end of treatment with molnupiravir. Possible side effects [3] are dizziness, headache, diarrhea, nausea, vomiting, rash, and urticaria. To our knowledge, there are few real-life studies about molnupiravir [4] and no reports about at-home treatment in European population. Our experience We prescribed molnupiravir to a total of 74 patients (39 male, 35 female) with an average age of 72.3 (ds 14.5) in a range between 26 and 96 and followed-up for 30 days. The most frequent symptoms in these patients were fever, coughing, and pharyngodynia. 70 patients were vaccinated in the six months before positivity to SARS-CoV-2. Among vaccinated patients, only 65 had underwent a complete vaccination course of three doses. 5 treated patients were under 55; four of them had indication to antiviral therapy for immunodeficiency, and one had indication to oxygen therapy and home ventilation for severe chronic respiratory failure. Only one patient had an indication for an oncological condition. He had been vaccinated with three doses in the previous six months. 3 patients were treated with the indication for chronic kidney disease associated with cardiovascular disease. 17 patients were eligible for being treated with molnupiravir due to severe pulmonary disease (10 of these patients were diagnosed with idiopathic pulmonary fibrosis (IPF)), 10 patients due to severe pulmonary disease associated with cardiovascular disease (of which one was diagnosed with IPF), and one patient due to severe pulmonary disease associated with cardiovascular disease and obesity. 9 patients were treated with the indication for immunodeficiency; one of them underwent lung transplantation 5 years prior to being treated with molnupiravir. Obesity was the risk factor to determine eligibility in 7 patients. In two cases, obesity was associated with cardiovascular diseases, in one more case with cardiovascular severe pulmonary diseases, and in a further one with diabetes mellitus. 8 patients were treated with the indication for uncontrolled diabetes mellitus (of which one was associated with obesity, 3 with cardiovascular diseases, and one with both cardiovascular disease and chronic renal failure). 39 patients were treated due to risks associated with severe cardiovascular disease (22 of them had only this indication with no other co-morbidities) (Table 1).Table 1 Characteristics of population Number of patients 74 Gender 39 male, 35 female Age AV 72,3 (SD 14,5) Vaccinated 70 Oncological disease 1 Chronic renal disease 3 Severe pulmonary disease 17 (10 affected by IPF) Immunodeficiency 9 Obesity 7 Uncontrolled diabetes mellitus 8 Severe cardiovascular disease 39 We observed an average negative result of nasopharyngeal control swab on day 12. This is due to the fact that regional follow-up protocol allows control tests to be carried at least 7 days after the first positive result, with more flexible further calendar, i.e., the test can be performed on resolution of symptoms. The most remarkable result of this study is that all of the treated patients presented a regression of symptoms during follow-up. No patients were hospitalized and/or showed sequelae after the infection by SARS-CoV-2, even though the examined population was older and presented more co-morbidities than other patients who underwent treatment with other available antivirals. No patients complained about side effects related to the assumption of Molnupiravir. Furthermore, experience shows that molnupiravir is easier to access when compared to other approved antiviral drug, due to lack of significant restrictions (e.g., drug interactions and dose adjustment). Finally, to the best of our knowledge, this is the first report about the use of molnupiravir in patients affected by IPF, with extremely positive results. In conclusion, molnupiravir is safe and well-tolerated by patients with high-risk of progression to severe COVID. According to these results, molnupiravir can lead to a potential risk reduction of 100%, especially when associated with vaccination. Larger studies may shed brighter light on the actual effectiveness of early treatment at home. Acknowledgements We would like to thank Cosimo Rosario Di Martino for his language review. Funding The authors declare that they have not received funding for this work. Data Availability All data are available contacting the corresponding author. Declarations Conflict of Interests The authors declare no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Kabinger F, Stiller C, Schmitzová J et al (2021) Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat Struct Mol Biol 28(9):740–746. 10.1038/s41594-021-00651-0. Epub 2021 Aug 11. PMID: 34381216; PMCID: PMC8437801 2. Singh AK, Singh A, Singh R, Misra A (2021) Molnupiravir in COVID-19: a systematic review of literature. Diabetes Metab Syndr 15(6):102329. 10.1016/j.dsx.2021.102329. Epub 2021 Oct 30. PMID: 34742052; PMCID: PMC8556684 3. Painter WP, Holman W, Bush JA et al (2021) Human safety, tolerability, and pharmacokinetics of molnupiravir, a novel broad-spectrum oral antiviral agent with activity against SARS-CoV-2. Antimicrob Agents Chemother 65(5):e02428–20. 10.1128/AAC.02428-20. Epub ahead of print. PMID: 33649113; PMCID: PMC8092915 4. Flisiak R, Zarębska-Michaluk D, Rogalska M et al (2022) Real-world experience with molnupiravir during the period of SARS-CoV-2 Omicron variant dominance. Pharmacological reports : PR, 1–7. Advance online publication. 10.1007/s43440-022-00408-6	36454535	PMC9713130	NO-CC CODE	2022-12-02 23:22:08	no	Ir J Med Sci. 2022 Dec 1;:1-3	utf-8	Ir J Med Sci	2,022	10.1007/s11845-022-03241-1	oa_other
==== Front Nature Nature Nature 0028-0836 1476-4687 Nature Publishing Group UK London 36265511 5433 10.1038/s41586-022-05433-2 Article Additive manufacturing of micro-architected metals via hydrogel infusion http://orcid.org/0000-0003-3846-2908 Saccone Max A. [email protected] 1 http://orcid.org/0000-0001-6516-2180 Gallivan Rebecca A. 2 http://orcid.org/0000-0002-3867-8234 Narita Kai 2 http://orcid.org/0000-0002-4114-6167 Yee Daryl W. [email protected] 2 http://orcid.org/0000-0002-9675-1508 Greer Julia R. [email protected] 2 1 grid.20861.3d 0000000107068890 Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA USA 2 grid.20861.3d 0000000107068890 Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA USA 20 10 2022 16 23 11 2021 7 10 2022 © The Author(s), under exclusive licence to Springer Nature Limited 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Metal additive manufacturing (AM) enables the production of high value and high performance components1 with applications from aerospace2 to biomedical3 fields. Layer-by-layer fabrication circumvents the geometric limitations of traditional metalworking techniques, allowing topologically optimized parts to be made rapidly and efficiently4,5. Existing AM techniques rely on thermally initiated melting or sintering for part shaping, a costly and material-limited process6–8. We report an AM technique that produces metals and alloys with microscale resolution via vat photopolymerization (VP). Three-dimensional-architected hydrogels are infused with metal precursors, then calcined and reduced to convert the hydrogel scaffolds into miniaturized metal replicas. This approach represents a paradigm shift in VP; the material is selected only after the structure is fabricated. Unlike existing VP strategies, which incorporate target materials or precursors into the photoresin during printing9–11, our method does not require reoptimization of resins and curing parameters for different materials, enabling quick iteration, compositional tuning and the ability to fabricate multimaterials. We demonstrate AM of metals with critical dimensions of approximately 40 µm that are challenging to fabricate by using conventional processes. Such hydrogel-derived metals have highly twinned microstructures and unusually high hardness, providing a pathway to create advanced metallic micromaterials. An additive manufacturing technique that infuses 3D printed hydrogels with metallic precursors leads to metallic micromaterials, providing new opportunities for the fabrication of energy materials, micro-electromechanical systems and biomedical devices. Subject terms Metals and alloys Materials chemistry Design, synthesis and processing ==== Body pmcMain Metal AM is mostly achieved via powder bed fusion12 and directed energy deposition13 processes. Layer-by-layer processes enable fabrication of metal multimaterials14 and functionally graded composites15, but such laser-based processes struggle to produce materials such as copper; high thermal conductivity and low laser absorptivity cause difficulties in thermal initiation and localization of melting or sintering16. Vat photopolymerization (VP) is a promising alternative that uses light-initiated free radical polymerization to shape parts. Digital light processing (DLP) printing accomplishes this by projecting two-dimensional images of ultraviolet light into a photoresin bath to cure an entire layer of the three-dimensional (3D) structure simultaneously. DLP is capable of high print speeds17, has been demonstrated with submicrometre resolution,18 and has diverse commercial applications from the direct manufacturing of shoe soles19 to COVID-19 test swabs20. VP was developed predominantly for use with polymers21–23 and has also been demonstrated for glasses9 and ceramics10. However, inorganic material selection remains limited owing to challenges with incorporating appropriate precursors into photoresins as solutions24, slurries25 or inorganic–organic mixtures26. Consequently, the fabrication of metals via VP remains a challenge. Oran et al. demonstrated AM of nanoscale silver by using hydrogels as ‘nanomanufacturing reactors’27,28 in which two-photon activation guides the infiltration of precursors to volumetrically deposit 3D materials. Vyatskikh et al. demonstrated AM of nanoscale nickel by using two-photon lithography to pattern inorganic–organic resins containing nickel acrylates, followed by pyrolysis and H2 reduction26. However, these pioneering works are limited in material scope, requiring complex resin design and optimization for each new material. Other less commonly used metal AM techniques such as direct ink writing and material jetting use extrusion from a nozzle and controlled deposition of a binder, respectively, to define part shape. These methods circumvent the challenges of using heat to define part shape; copper materials have been fabricated via direct ink writing29 and material jetting30, but neither technique has produced copper parts with feature sizes under 100 µm. Hydrogel infusion fabrication We have developed a VP-based AM technique, coined hydrogel infusion additive manufacturing (HIAM), which enables fabrication of a wide range of micro-architected metals and alloys from a single photoresin composition. We use 3D architected hydrogel scaffolds as platforms for subsequent in situ material synthesis reactions, shown schematically in Fig. 1a. To fabricate metal microlattices, we use DLP (schematic in Supplementary Fig. 1) to print N,N-dimethylformamide (DMF)/polyethylene glycol diacrylate (PEGda)-based architected organogels (see Supplementary Information Discussion 1 for resin design). The DLP printing step defines the shape of the final part; the designed octet lattice shape used throughout this work can be found in Supplementary Fig. 2. Details of the resin composition and DLP printing and swelling parameters can be found in Supplementary Tables 1 and 2. After printing, a solvent exchange replaces DMF with water, converting the organogels into hydrogels. The hydrogel structures are then soaked in a metal salt precursor solution to allow metal ions to swell the hydrogel scaffold. Calcination in air converts the metal-salt-swollen hydrogels to metal oxides, and subsequent reduction in forming gas (95% N2, 5% H2) yields metal or alloy replicas of the designed architecture. Throughout the process, the part shape, defined during DLP printing, is maintained, with each dimension undergoing approximately 60–70% linear shrinkage, with a concomitant 65–90% approximate mass loss during calcination (see Supplementary Table 3 for shrinkage and mass loss for several materials).Fig. 1 HIAM process and materials. a, Schematic of HIAM process. A DMF/PEGda-based 3D printed organogel structure is converted to an infused hydrogel replica after leaching out photoactive compounds, solvent exchange, and infusing an appropriate aqueous precursor. Subsequent calcination in air forms metal oxide structures, which are reduced to metals in forming gas. b–e, Optical images of the HIAM process for Cu metal, showing: b, printed organogel; c, infused hydrogel; d, calcined metal oxide; and e, reduced metal. f, Additional metals fabricated via HIAM including Ag and Ni, binary alloy CuNi, high-entropy alloy CuNiCoFe and refractory alloy W–Ni. g, An octet lattice infused with Cu(NO3)2 from one end and Co(NO3)2 from the other. h, After calcination and reduction, the Cu/Co gel is transformed to a Cu/Co multimaterial. i, Parallel calcination of several different infused gels. Scale bars: b,c, 5 mm; d–f, 1 mm; g, 1 cm; h, 2 mm; i, 2 cm. To demonstrate the versatility of HIAM compared with previous VP AM techniques24,27, we used HIAM to fabricate octet lattice structures of copper (process steps shown in Fig. 1b–e), nickel, silver and alloys thereof (Supplementary Fig. 3), as well as more complex materials such as the high-entropy alloy CuNiCoFe and the refractory alloy W–Ni (Fig. 1f). Additional development was required for these materials; fabrication and characterization of CuNiCoFe and W–Ni are described in Supplementary Figs. 4 and 5, and Supplementary Information Discussions 2 and 3, respectively. We also fabricated multimaterials such as Cu/Co (Fig. 1g,h). HIAM is distinguished by its ability to be parallelized; several organogels can be printed simultaneously, swelled in separate solutions and then calcined/reduced together. Figure 1i shows eight hydrogel lattices (precursors of Cu, CuNi, CuNiCoFe and CuNiCoFeCr) being simultaneously calcined to form oxides. This parallelization is impossible with existing VP methods and is a direct consequence of the temporal separation of part shaping and material selection. Compared with existing techniques that include precursors in the resin or introduce precursors through chemically directed swelling, HIAM enables exploration of a much larger compositional space, including multimaterials. From here, we focus on characterization of one pure metal and one alloy: Cu and CuNi. Structural characterization The external and internal morphologies of metal microlattices were investigated by using a combination of scanning electron microscopy (SEM) and Ga+ source focused ion beam (FIB) milling. SEM imaging revealed that Cu and CuNi samples maintained their octet lattice shape during thermal treatment (Fig. 2a,e), with beam diameters of approximately 40 µm (Fig. 2b,f). We FIB-milled representative cross-sections at nodes and observed dense and relatively defect-free structures. The Cu showed some less than 5 µm diameter pores and a lamellar crack (Fig. 2c), whereas the CuNi alloy (Fig. 2g) exhibited a similarly dense structure with micrometre-sized spherical pores, but no observed lamellar cracks. Energy dispersive X-ray spectroscopy (EDS) mapping showed homogenous distribution of Cu in the Cu lattice (Fig. 2d), and homogenous distribution of Cu and Ni in the CuNi lattice (Fig. 2h). See Supplementary Fig. 6 for structural characterization of additional materials including Ni, Ag and CuAg alloy.Fig. 2 Morphology of Cu and CuNi microlattices. a–c,e–g, SEM images of Cu (a–c) and CuNi (e–g) octet lattices, showing multiple unit cells from the top (a,e), a single node (b,f) and a FIB-milled cross-section showing the internal structure of a node from 52° tilt (c,g). d,h, EDS elemental mapping, showing uniform distribution of Cu (d) and uniform distribution of Cu and Ni (h). Scale bars: a,e, 100 µm; b,f, 50 µm; c,g, 20 µm; d,h, 50 µm. Chemical characterization To understand the chemical and microstructural evolution of these materials during calcination and reduction, we investigated the chemical composition of the metal microlattices by using X-ray diffraction (XRD), EDS, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). EDS analyses (Supplementary Fig. 7) of Cu and CuNi microlattices show that these materials contain, by weight, 93% and 86% of the target materials, respectively. The balance is made up of carbon, which is difficult to accurately quantify and probably includes some adventitious carbon (Supplementary Information Discussion 4), and aluminosilicate contamination from the furnace tubes. EDS analysis shows that the atomic ratio of Cu:Ni in our CuNi material is 1.21:1, or stoichiometrically Cu55Ni45. This deviation of alloy composition from swelling solution composition is probably due to different affinities of PEGda with the metal ions31. However, by adjusting the swelling solutions to account for preferential incorporation of certain ions, target compositions can be achieved with precision. For example, to target a Cu50Ni50 alloy, we swelled a hydrogel precursor in a 1:1.21 molar ratio of Cu(NO3)2:Ni(NO3)2. After calcination and reduction, EDS analysis showed that the stoichiometry of this cupronickel alloy was within 1% of the target composition, at Cu50.5Ni49.5 (Supplementary Fig. 8). Calcination of metal-nitrate-infused gels in air at 700 °C with gas flow rate of 50 standard cubic centimetres per minute (sccm) produces metal oxide replicas of the architectures. The XRD patterns in Fig. 3a show that the calcined Cu precursor gel, which contained Cu(NO3)2, and the CuNi precursor gel, which contained Cu(NO3)2/Ni(NO3)2, were fully converted to CuO and CuO/NiO, respectively (see Supplementary Fig. 9 for additional materials). Notably, the CuO/NiO XRD pattern shows the presence of the individual CuO and NiO phases in the calcined material. Reduction of these metal oxides in forming gas (900 °C, 150 sccm) converts the CuO and CuO/NiO lattices to Cu and a homogenous CuNi alloy, respectively (Fig. 3b). As both CuNi and Cu have face-centred cubic (FCC) crystal structures, the single set of FCC reflections in the CuNi pattern shift to higher diffraction angles, which is a result of decreased lattice spacing due to the incorporation of the smaller Ni atom into the structure.Fig. 3 Chemical characterization of hydrogel infusion produced metals and alloys. a, XRD patterns of calcined gels: Cu(NO3)2 gel is converted to CuO and Cu(NO3)2/Ni(NO3)2 gel is converted to CuO/NiO. b, XRD patterns of oxides reduced to parent metals: CuO is converted to Cu and CuO/NiO is converted to a homogenous CuNi alloy, as evidenced by the single set of FCC reflections. c, TGA profiles of metal-ion-infused gels heated to 700 °C in air at 1 °C min−1 reveal rapid mass loss events reaching maxima at 353 °C for Cu and 331 °C for CuNi. d, DSC profiles of metal-ion-infused gels heated to 400 °C in air at 1 °C min−1 reveal exothermic (Exo) events with maximum heat flow at 308 °C for Cu and at 304 °C for CuNi. AU, arbitrary units. Ref. ICSD #, reference pattern from Inorganic Crystal Structure Database (see Methods). Figure 3c contains TGA measurements of Cu and CuNi gels heated in air at 1 °C min−1. During calcination the Cu and CuNi precursor gels retain 12.7% and 15.8% of the original mass, respectively, reaching mass stabilization indicative of full conversion between 370 °C and 380 °C. The derivative of sample weight with respect to temperature, dW/dT, shows the regions of highest mass loss rate (approximately 1 wt% °C−1) occur at 353 °C for Cu and at 331 °C for CuNi. Guides to the eye are placed at 110 °C, where initial dW/dT peaks occur for both Cu and CuNi, and 265 °C, where a subsequent dW/dT peak occurs for only Cu. Fig. 3d contains DSC profiles of Cu and CuNi precursor gels heated in air at 1 °C min−1 (see Supplementary Fig. 10 for additional materials). Both gels exhibit similar normalized heat flow profiles; exothermic peaks begin at approximately 235 °C for both and reach a maximum heat flow of −1.5 W g−1 at 308 °C for Cu and −2.6 W g−1 at 304 °C for CuNi. Morphology and microstructure characterization Our HIAM-fabricated metals are microcrystalline, with randomly oriented micrograins that are densely populated by annealing twins. The presence of micrometre-scale twinned regions in Cu is seen clearly in Ga+ ion-channelling images (Fig. 4a, yellow arrows point to twins) and electron backscatter diffraction (EBSD) maps (Fig. 4b).Fig. 4 Microstructure and mechanical properties of hydrogel infusion fabricated metals and alloys. a,b, Ga+ ion-channelling image (a) and EBSD map of Cu (b) show annealing twins. Cu has a complex micrograined structure and multiple twinned regions denoted by yellow arrows. c, TEM images of Cu show well-formed grain boundaries and aluminosilicate inclusions. FCC copper is observed from the diffraction pattern in the inset of c. d, TEM image of a twin boundary and aluminosilicate inclusion. e, Nanoindentation hardness of HIAM-fabricated Cu and CuNi samples is higher than predicted based on Hall–Petch scaling (dashed lines show calculated twin-induced hardening). Error bars show standard deviations of grain size and nanoindentation hardness. Insets: area-weighted grain size distribution for Cu and CuNi. Sample sizes: Cu hardness, n = 22; CuNi hardness, n = 44; Cu grain size, n = 246; CuNi grain size, n = 309. Scale bars: a, 50 µm; b, 20 µm; c, 2 µm, inset 10 nm−1; d, 500 nm. Both Cu and CuNi have high crystallographic twin densities32, defined as the twin boundary length per cross-sectional area, of 1.7 × 106 m−1 for Cu and 1.3 × 106 m−1 for CuNi. For Cu and CuNi each grain contains on average 4.8 and 3.8 twin boundaries, respectively, with 88% and 75% of all grains containing at least one twin boundary. Additional twinning statistics measured by EBSD are reported in Supplementary Table 4. Transmission electron microscopy (TEM) analysis of HIAM-fabricated Cu (Fig. 4c) shows more microstructural detail. We observe that grain boundaries and twin boundaries are well formed, and do not observe voids at triple junctions or secondary phases (for example, unreduced CuO or amorphous carbon). However, there exist aluminosilicate inclusions (see Supplementary Fig. 11 for TEM EDS) that result from contamination from the furnace tube (Fig. 4d). Image analysis of SEM micrographs (see Supplementary Information Discussion 5 and Supplementary Figs. 12 and 13) show that Cu has an area-weighted average grain size of 13.74 ± 8.43 µm, and CuNi has an area-weighted average grain size of 9.81 ± 4.79 µm (Fig. 4e, insets). Mechanical characterization Nanoindentation experiments performed on HIAM-fabricated Cu and CuNi revealed the hardness of Cu to be 1.81 ± 0.37 GPa and that of CuNi to be 2.15 ± 0.22 GPa. To contextualize these results, Fig. 4e contains a plot of nanoindentation hardness versus grain size for HIAM-fabricated Cu and CuNi compared with literature data for the same metals produced via traditional processing techniques (see Supplementary Table 5 for tabulated data). The plot also contains the expected hardness based on the Hall–Petch relation33 H = H0 + kd−1/2 that relates nanoindentation hardness H to grain size d for ductile metals, in which H0 is an intrinsic hardness for a single crystalline material and k is a scaling factor related to grain-boundary-induced hardening. Twin boundaries disrupt dislocation motion during deformation32,34, increasing measured hardness. To account for this effect, we introduce a modified Hall–Petch relation35. To calculate an upper bound on twin-induced hardening, we assume that twin boundaries and grain boundaries equally impede dislocation motion; the grain size d effectively shrinks and is replaced by d/(1+N), where N is the number of twin boundaries per grain (see Supplementary Information Discussion 6 for derivation of this expression). Discussion Chemical composition and processing effects Thermogravimetric and DSC analysis of Cu and CuNi calcination reveals that the water initially bound to the hydrogel polymer network evaporates below approximately 100 °C, indicated by the endothermic heat flow of approximately 0.25 W g−1 present in the DSC profiles36. Multiple thermally induced processes occur simultaneously upon further heating. Between 100 °C and 400 °C, the Cu sample undergoes (1) dehydration of Cu(NO3)2⋅xH2O to anhydrous Cu(NO3)2, (2) thermal decomposition of anhydrous Cu(NO3)2 and (3) an exothermic combustion reaction37 in which the nitrate salt acts as an oxidizer of the PEGda (C26H46O13) polymer through the reaction Cu(NO3)2 + η C26H46O13 + (31η − 2.5) O2 → CuO + 23η H2O + 26η CO2 + N2, for which η is the molar ratio of PEGda to nitrate salt and (4) thermal decomposition of PEGda. TGA and DSC profiles of the CuNi samples suggest a similar process, with the copper and nickel nitrate salts simultaneously decomposing and acting as oxidizers for polymer combustion. Both Cu and CuNi precursor gels exhibit exothermic events around 300 °C that are attributed to the combustion of the PEGda polymer scaffold and oxidation of the metal ions. The rates and temperatures of maximum heat flow are −1.5 W g−1 at 308 °C for Cu, and −2.6 W g−1 at 304 °C for CuNi (Fig. 3d) and indicate that during calcination the CuNi gel releases heat more rapidly. This finding is consistent with our observation that the rate of maximum heat flow in the Ni gel is even greater, at −3.97 W g−1 at 333 °C (Supplementary Information Discussion 7 and Supplementary Table 6). The onset of this exothermic event is similar for Cu and CuNi, (approximately 235 °C), whereas the onset of the corresponding event in the Ni gel occurs at a substantially higher temperature of approximately 295 °C. This trend suggests that the heat released from the exothermic combustion of the copper nitrate salt in the CuNi gel is sufficient to quickly increase the local temperature in the gel to the point where the nickel nitrate salt also contributes to the oxidation reaction at an apparently lower temperature, as is common in combustion synthesis38. The Cu and CuNi samples contain fewer defects and pores compared with pure Ni and Ag (Supplementary Fig. 6). The presence of defects and pores is correlated with higher maximum dW/dT. During calcination, rapid thermal decomposition inhibits global, isotropic part shrinkage and drives a kinetic competition with mass loss, resulting in internal voids and pore formation. Slowing the rate of mass loss during calcination increased HIAM part density; we achieved this through a slow ramp rate of 0.25 °C min−1 and low pressure of approximately 6 Torr. Mechanical characterization and twinning The high annealing twin densities we observed, formed without the ordinarily requisite melting and recrystallization39, highlight the complex interplay of kinetic processes (for example, solid-state diffusion and grain nucleation) and thermodynamic grain coalescence during calcination and reduction. However, this high boundary density does not fully explain the 47% and 15% increase in hardness for Cu and CuNi, respectively. The observed aluminosilicate nanoinclusions are heterogeneously distributed and contribute to local variations rather than an increase in average hardness. With no evidence for secondary phases or grain boundary complexions (Fig. 4c,d), the high hardness of HIAM metals probably stems from atomic-scale features (for example, dissolved carbon) formed during the HIAM process (Supplementary Information Discussion 8). Material selection after part shaping In nearly every AM process so far, the material is decided before part shaping, that is, the AM material feedstock is the desired material or contains precursors that are subsequently converted into the desired material. Thus, the feedstock is inseparable from the final material composition. In addition, fabricating any new material necessarily requires a different feedstock. For ceramic and metal VP with precursors incorporated into the photoresin, the fabrication of new inorganic materials requires non-trivial photoresin design and print-parameter optimization. HIAM is fundamentally different from modern AM processes in that the material is selected after part shaping. A blank organogel structure fabricated from a single resin formulation can be transformed into a vast number of different compositions. Because the inorganic precursors are infused after part shaping, HIAM is unlike the traditional slurry or inorganic–organic hybrid resin approaches in that only a single photoresin composition needs to be designed and optimized for VP, simplifying the material development process. We have shown that this capability also allows HIAM to fabricate complex materials such as refractory metals and high-entropy alloys as well as multimaterial metal structures, which were previously impossible tasks for VP. Finally, HIAM is generalizable to other gel-producing AM processes such as direct ink writing and two-photon lithography. Conclusion The HIAM process enables the creation of micro-architected metal 3D structures by using a versatile VP approach. The conversion of metal salts within polymer scaffolds to metal oxides and their subsequent reduction to metals and alloys requires only that the target material has water-soluble precursors and that the intermediate oxide formed after calcination can be reduced by hydrogen gas. The ability to fabricate metals by using this accessible and high-resolution process provides new opportunities for fabrication of energy materials, micro-electromechanical systems and biomedical devices. Because the material is selected only after the part is shaped, directed infusion enables fabrication of metallic multimaterials. Unprecedented compositional flexibility enables the fabrication of multicomponent alloys such as high-entropy alloys and refractory alloys, known to have intermetallic phases that lead to superior high-temperature behaviour and enhanced yield strength40. HIAM has direct implications for industrial use, as it provides a practical and powerful capability to integrate into the burgeoning VP printing ecosystem. Methods Fabrication of metal microlattices 3D printing resin preparation Here 28 ml of DMF (Sigma-Aldrich, >99.9%) was mixed with 35 ml PEGda Mn = 575 (Sigma-Aldrich). Separately, 347 mg 2-dimethylamino-2-(4-methyl-benzyl)-1-(4-morpholin-4-yl-phenyl)-butan-1-one (Irgacure 379; iGM Resins), 229 mg bis[4-(dimethylamino)phenyl]methanone (Michler’s ketone; Sigma-Aldrich) and 10.3 mg 1-(phenyldiazenyl)naphthalen-2-ol (Sudan I; Sigma-Aldrich) was stirred into 7 ml of DMF. This solution was then added to the DMF/PEGda mixture and swirled until completely homogenous. DLP 3D printing The resin was formed into 3D organogel structures by using a commercial 405 nm wavelength DLP 3D printer (Autodesk Ember). Lattice structures were designed to consist of octet lattices with 200 µm beam diameter and 1.5 mm unit cell size. Post-processing washes After printing, the organogel lattices were yellow in colour due to the presence of the ultraviolet blocker Sudan I. To remove unreacted photoresin components, each 3D printed organogel structure was soaked in DMF for 1 h on a hot plate at 70 °C. After the first DMF rinse, the DMF was decanted, and the organogel was soaked again in fresh DMF for 1 h at 70 °C. After this process, the lattice appeared clear. Subsequently, each organogel structure was soaked in deionized water for 1 h at 70 °C, followed by a second soak in fresh deionized water for 1 h at 70 °C to convert the structures from organogel lattices to hydrogel lattices. The solvent-exchange step is needed to remove residual DMF in the structure, which can lead to formation of porosity upon calcination and reduction (Supplementary Fig. 14). The presence of DMF can also cause precipitation of the metal salt during the swelling process, leading to an inhomogeneous distribution of the metal precursors. Swelling in metal salt solutions 2 M solutions of copper nitrate, nickel nitrate, iron nitrate, cobalt nitrate, silver nitrate, chromium nitrate and ammonium metatungstate were prepared with deionized water. Hydrogel structures were immersed in the appropriate metal salt solution for 24 h at 70 °C or 2 weeks at 70 °C (only for W-Ni). Calcination and reduction Metal-ion-swelled hydrogel structures were calcined in a vacuum tube furnace (MTI, OTF-1500X). The samples were placed in and covered by alumina boats within an alumina tube (MTI, 80 cm length, 51 mm inner diameter) that itself sat inside a larger fused quartz tube (MTI, 1 m length, 92 mm inner diameter). The furnace was set up in this fashion to prevent copper vapours resulting from the heating process from interacting with the quartz tube, which has been shown to lead to devitrification of the amorphous quartz tube and potential deposition of SiO2(ref. 41). The addition of the inner alumina tube substantially reduced the amount of SiO2 particles observed after thermal treatment. During calcination, a 0.25 °C min−1 ramp rate was used up to a maximum temperature of 700 °C, followed by a 3 h isothermal hold and cooling at 2 °C min−1, under a compressed air flow of 50 sccm at a pressure of approximately 7 Torr. The slow ramp rate of 0.25 °C min−1 during calcination was found to be critical for controlling the highly exothermic decomposition of the nitrate salts and minimizing porosity in the fabricated samples. After calcination of metal-ion-swelled gels, metal oxide structures were produced, except for gels infused with silver nitrate salts, which produced elemental silver or silver composite lattices. All calcined structures except pure Ag and W-Ni were then reduced in forming gas at a flow rate of 150 sccm at a pressure of approximately 22 Torr by heating at 3 °C min−1 to 900 °C or 700 °C (only for CuAg) followed by a 6 h isothermal hold, resulting in copper, nickel, homogenous cupronickel alloy and heterogeneous copper–silver alloy lattices. The W-Ni structures were calcined and reduced under different conditions; W-Ni samples underwent calcination at 0.25 °C min−1 to 500 °C, then 1 °C min−1 to 700 °C, and a 3-hour isothermal hold. The W-Ni samples were reduced following a thermal profile of 3 °C min−1 to 1,200 °C and a 1-hour isothermal hold, under forming gas at a flow rate of 500 sccm at atmospheric pressure (through a gas bubbler). Characterization and measurement SEM and FIB milling Samples were imaged via SEM (FEI Versa 3D DualBeam) at an accelerating voltage of 10–20 kV. Elemental analysis was performed in the same instrument by using EDS (Bruker Quantax 200, XFlash 6\|60 detector), with an applied voltage of 20 kV or 5 kV. The applied voltage was selected to ensure the greatest accuracy when quantifying metal elements (Supplementary Information Discussion 4 and Supplementary Fig. 15). Gallium FIB milling was performed in the same instrument to mill lattice cross-sections by using an accelerating voltage of 30 kV and a current of 50 nA. FIB cleaning of the cross-sections was performed by using an accelerating voltage of 16 kV and a current of 25 nA. Electron backscatter diffraction With the z direction and build direction aligned, metal lattice samples were loaded into an Oxford EBSD System in a Zeiss 11550VP SEM and were imaged by using a 120 µm aperture at 20 kV. Data analysis for the Kikuchi maps was done in AZtecHKL software. All maps display the inverse pole figure in the z direction. TEM Lamellae with thicknesses of less than 100 nm were prepared for TEM by using a liftout procedure in an SEM (FEI Versa 3D DualBeam). The top surface of the liftout region was protected with an approximately 100 nm thick layer of Pt deposited via a gas injection system, followed by an approximately 400 nm thick layer of Ga+ FIB-deposited Pt in the same chamber. Next, a Ga+ ion beam was used to carve out trenches into the node of the lattice, forming a U-cut to free the approximately 1 µm thick metal lamella base from the rest of the lattice. A tungsten needle (EZlift program) was attached to the lamella with FIB-deposited Pt before being cut free of the sample and transferred to a copper halfmoon grid. The lamella was attached to the Cu grid with FIB-deposited Pt and the tungsten needle was cut away to free the sample. After detaching the tungsten needle, a series of FIB cuts with a decreasing Ga+ voltage/current (30 kV/100 pA; 30 kV/10 pA; 16 kV/23 pA) were used to progressively thin the cross-section of the lamella structure to less than 100 nm. TEM imaging was performed in a Jeol JEM-2800 transmission electron microscope with a 200 kV beam. XRD Powder XRD (PANalytical X’Pert Pro) patterns were collected by using a Cu Kα1 source at 45 kV and 40 mA. Samples were either ground into powders or flattened and attached to an amorphous zero-background sample holder by using clay before XRD analysis. Experimental patterns were compared to references from the Inorganic Crystal Structure Database42. TGA TGA (TA Instruments TGA 550A) was performed by heating samples to 700 °C at a rate of 1 °C min−1 in an air flow of 25 ml min−1 while the mass of the sample was continuously measured. DSC DSC (TA Instruments DSC 25) was performed by heating samples to 700 °C at a rate of 1 °C min−1 in an air flow of 25 ml min−1 while heat flow to the sample was continuously measured. Nanoindentation Samples were prepared for nanoindentation by mounting them in acrylic (Beuhler SamplKwik) and curing for 12 h in silicone moulds. The samples were polished first with 300 grit until the metal structure was exposed. The samples then were polished with 600 grit, followed by 1200 grit and subsequently a 0.25 µm grit suspension (Beuhler MetaDi Polycrystalline Diamond Slurry). Indentation was performed by using an Agilent G200 Nano Indenter with XP module by using a Berkovich tip with an area function given by A = 24.5h2 + 688h, where A is the tip contact area and h is the contact depth. Samples were indented at 10−3 strain rate to a maximum depth of 1 µm, followed by a 2 s hold and subsequent unloading. Grain sizes were determined via SEM image analysis that consisted of tracing grains on the surface of the sample and by using ImageJ to analyse the resulting shapes. These values were corroborated by EBSD mapping analysis of grain size. Experimental data was compared to a Hall–Petch fit of reference data from the literature43–47. Online content Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-05433-2. Supplementary information Supplementary Information Supplementary Figs. 1–15, Tables 1–6 and Discussions 1–8. Supplementary information The online version contains supplementary material available at 10.1038/s41586-022-05433-2. Acknowledgements This work was supported by the US Department of Energy, Office of Science, Basic Energy Sciences under award no. DE-SC0016945. We thank C. Ma for support and assistance with instruments in the Geological and Planetary Sciences Division Analytical Facility at Caltech, as well as M. Xu and X. Pan for assistance with TEM experiments at UC Irvine Materials Research Institute. M.A.S. acknowledges a graduate fellowship from the Resnick Sustainability Institute at Caltech. R.A.G. acknowledges the AI4SCIENCE graduate fellowship at Caltech. K.N. acknowledges a fellowship from the Masason Foundation. Author contributions M.A.S, D.W.Y, and J.R.G. conceived of and designed the experiments. M.A.S. and D.W.Y. designed the photoresin, printing parameters and swelling protocol and fabricated samples. M.A.S., D.W.Y., and K.N. performed the thermal treatments and FIB/SEM/EDS experiments. M.A.S. performed the DSC, TGA and XRD experiments. R.A.G. performed the nanoindentation and EBSD experiments and prepared TEM samples. All authors analysed data and discussed the findings. M.A.S., R.A.G., D.W.Y. and J.R.G. wrote the manuscript. All authors edited and approved the manuscript. Peer review Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Data availability The data generated and/or analysed during the current study are available from the corresponding authors on reasonable request. Code availability The code generated during the current study is available from the corresponding authors on reasonable request. Competing interests K.N. founded the company 3D Architech, Inc., which has an option and right to acquire an exclusive license to US Patent 11318435B2. 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==== Front J Racial Ethn Health Disparities J Racial Ethn Health Disparities Journal of Racial and Ethnic Health Disparities 2197-3792 2196-8837 Springer International Publishing Cham 36449129 1468 10.1007/s40615-022-01468-3 Article Willingness to Receive the COVID-19 Vaccine in California: Disparities by Race and Citizenship Status http://orcid.org/0000-0003-0157-4754 Bacong Adrian Matias [email protected] 12 Haro-Ramos Alein Y. 3 1 grid.168010.e 0000000419368956 Stanford University Center for Asian Health Research and Education, Stanford, CA USA 2 grid.168010.e 0000000419368956 Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA USA 3 grid.47840.3f 0000 0001 2181 7878 School of Public Health, University of California Berkeley, Berkeley, CA USA 30 11 2022 110 17 7 2022 18 11 2022 21 11 2022 © W. Montague Cobb-NMA Health Institute 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Although it is widely acknowledged that racialized minorities may report lower COVID-19 vaccine willingness compared to non-Hispanic white individuals, what is less known, however, is whether the willingness to receive the COVID-19 vaccine also differs by citizenship. Understanding disparities in vaccine willingness by citizenship is particularly important given the misleading rhetoric of some political leaders regarding vaccine eligibility by citizenship status. This study used the 2020 California Health Interview Survey (n = 21,949) to examine disparities in vaccine willingness by race/ethnicity and citizenship among Asian, Latinx, and non-Hispanic white individuals. Overall, 77.7% of Californians indicated that they were willing to receive the COVID-19 vaccine if it was made available. However, there were distinct differences by race/ethnicity and citizenship. Asian people, regardless of citizenship, had the highest predicted probability of vaccine willingness, accounting for demographic, socioeconomic, and health factors. Non-citizen Latinx and non-citizen non-Hispanic white people had higher predicted probabilities of vaccine willingness compared to their US-born counterparts, accounting for demographic, socioeconomic, and health factors. Our results reveal that although vaccine willingness may be high among non-citizen individuals, it may not necessarily translate into actual vaccine uptake. Furthermore, while individual-level factors may account for some of the differences in vaccine willingness by race/ethnicity and citizenship, other institutional and structural barriers prevent vaccine uptake. Keywords COVID-19 Vaccination Race/ethnicity Citizenship Health equity http://dx.doi.org/10.13039/100006545 National Institute on Minority Health and Health Disparities F31MD015931 Bacong Adrian Matias ==== Body pmcIntroduction COVID-19 vaccine disparities in CA are evident by race/ethnicity, with Black and Latinx individuals experiencing lower vaccination rates than their White and Asian counterparts [1]. However, while racial disparities are well known, a paucity of research examines the degree to which racial subgroups of varying citizenship statuses fare in COVID-19 vaccine willingness. Immigrants and racialized minorities have an elevated risk of COVID-19 infection, morbidity, and mortality [2] due to social and occupational factors that lead to differential exposure, such as being employed in public-facing jobs in essential industries and low access to health care [3]. Given the multiple intertwining COVID-19 risk factors that vulnerable groups experience, it is crucial to understand whether beliefs and willingness regarding the COVID-19 vaccines among racialized minorities vary across immigration statuses. Previous studies have found that even before the COVID-19 pandemic, immigrants experienced disparities in vaccination compared to those born in the USA [4]. For example, vaccination coverage was lower among foreign-born compared to US-born individuals in pneumococcal, HPV, and Tdap, even after accounting for confounding factors [4]. While vaccination rates were generally lower among foreign-born people, differences were starker among noncitizens, with Hispanic/Latinx noncitizens having the lowest coverage for several vaccines. Other work has also documented racial-ethnic disparities in vaccination rates for diseases like the flu, with the most pervasive disparities found between Black and Latinxs adults compared to Whites [5, 6]. Given the variation in vaccination rates, it is imperative to consider the attitudes toward the vaccine within citizenship status and by race/ethnicity. Preexisting deterrents to preventive health care services, such as language barriers, financial limitations, and low health insurance rates [7, 8], are now compounded by a turbulent US political environment [9], mistrust in health care in marginalized communities [10], and the novelty of the COVID-19 vaccine [11]. In particular, the implementation of the new public charge rule, which went into effect just before the pandemic began in February 2020, led to a decline in enrollment in safety-net programs (i.e., Medicaid, WIC, and SNAP) among US-born children, particularly in regions with a higher share of noncitizens. Specifically, 260,000 fewer people were covered in children’s Medicaid coverage occurred after the public charge announcement by then-President Donald Trump [12]. This specific public charge rule broadened the criteria by which noncitizen immigrants could become ineligible for permanent resident status. In this case, any nonpermanent resident immigrant could be denied permanent residency if they received public benefits such as food assistance, housing, or Medicaid [12]. Given that the public charge rule was implemented a month before the national state of emergency went into effect, it is plausible that immigrants without legal status may have feared accessing health care and preventive services, including immunization against SARS-CoV2. Along with the political environment, organizational barriers to getting vaccinated have been cited at vaccination sites nationwide. While the COVID-19 vaccine is free and accessible to everyone regardless of health insurance or legal status [13], various businesses administering the vaccine require patients to provide a Social Security number or health insurance information [14]. The resulting obstacles are more consequential for vulnerable subgroups, such as undocumented immigrants, who are less likely to advocate for themselves. Overall, there is a need for a more comprehensive understanding of the willingness and attitudes toward the COVID-19 vaccine among racial minorities of distinct citizenship statuses. These data can inform the tailoring of vaccination programs and communication strategies to improve vaccination uptake among marginalized communities at the intersection of race/ethnicity and citizenship status. To address these gaps, this study examines differences in the willingness to receive the COVID-19 vaccine by citizenship status and race/ethnicity among a representative sample of Californians. Methods We used data from the public use 2020 California Health Interview Survey (CHIS) (n = 21,949). The CHIS is an annual survey intended to provide state-wide estimates of the health, social, and economic profiles of all Californians. The 2020 CHIS is a particularly novel dataset to study the effects of the COVID-19 pandemic because it was immediately redesigned during CA’s “work from home” orders to include questions about the effects of the pandemic [15]. The redesign of the questionnaire at the onset of the pandemic allowed the CHIS to provide real-time data on the social and health effects of the pandemic. We restrict our data to the 2020 iteration as it is the most currently available data of the CHIS. Of the 21,949 people who completed the 2020 CHIS, we restrict our analysis to 20,536 individuals who had complete data on all of the variables of interest outlined below. Since these data are de-identified, public use data, they are not human subjects research and do not require Institutional Review Board approval. Outcome Vaccine willingness was our outcome variable of interest and was asked as follows: “If a vaccine becomes available for COVID-19, would you get it?” Two response categories were available and were coded as 0 = no and 1 = yes. Independent Variable Participants’ race/ethnicity and citizenship status were the independent variable of interest. The CHIS provides separate variables for participants’ self-identified race/ethnicity and citizenship status. We combined both variables to create the following nine categories: “0 = US-born White,” “1 = naturalized White,” “2 = non-citizen White,” “3 = US-born Latinx,” “4 = naturalized Latinx,” “5 = noncitizen Latinx,” “6 = US-born Asian,” “7 = naturalized Asian,” and “8 = noncitizen Asian.” In our preliminary analyses, we attempted to examine vaccine willingness among Black and African American people, American Indian and Alaska Native people, Native Hawaiians, and Pacific Islander people, in addition to people who identified as “other races” or as “multiracial.” However, sample sizes for certain groups (e.g., noncitizen Black) were small, thereby leading to unstable estimates. We acknowledge that this is a limitation of this study and that more work is needed to include those who do not identify as non-Hispanic White, Latino, or Asian. Covariates We account for four sets of covariates that could explain the association between vaccine uptake, race/ethnicity, and citizenship. Our demographic factors included age category (18–34 years old, 35–49 years old, 50–64 years old, and 65 + years old) and gender (female or male). Given privacy concerns, the public-use version of the CHIS did not provide additional categories for other gender identities. Social and socioeconomic factors included family type (single with no kids, married with no kids, married with kids, and single with kids), urbanicity (urban or rural), educational attainment (less than high school, high school graduate, some college, associate’s degree, bachelor’s degree, or more), employment status (currently employed versus not), and whether participants had a usual source of care other than the emergency room. We also examined the federal poverty level (FPL) but only presented its distribution in the univariate and bivariate analyses (> 100% FPL vs. not) due to issues of collinearity with other socioeconomic predictors. We examined three types of COVID-19-related factors: whether participants experienced racial discrimination due to the COVID-19 pandemic, whether participants work in “essential work,” and whether participants worked from home due to the COVID-19 pandemic. Finally, we examined physical health factors that could influence vaccine uptake. These factors included whether participants were overweight or obese, were diagnosed with diabetes, were diagnosed with heart disease, or were diagnosed as pre-hypertensive/hypertensive according to their doctor. Analysis Plan We began our analysis by first examining the weighted univariate distribution of vaccine uptake, demographic, social, socioeconomic, COVID-19, and health factors. Next, we examined the characteristics of the sample by race/ethnicity and citizenship status. Differences in each factor by race/ethnicity and citizenship were determined using a chi-square test. For our multivariable analyses, we examined a series of five nested binary logistic regressions. Model 1 examined the bivariate relationship between vaccine willingness and race/ethnicity and citizenship status. Model 2 introduced demographic factors as possible confounders in the association between vaccine willingness and race/ethnicity and citizenship status. Model 3 included social and socioeconomic factors as alternative explanatory variables for differences in vaccine willingness. Model 4 examined the additional associations of COVID-19-related factors as explanatory factors for vaccine willingness. Finally, model 5 included health factors as additional confounding factors. To provide ease of interpretation in disparities in vaccine willingness by race/ethnicity and citizenship status, we calculated predicted probabilities using the “margins” command in Stata. We also conduct pairwise comparisons of predicted probabilities by race/ethnicity and citizenship status using the “pwcompare” command in Stata and adjust for multiple comparisons using the Sidak method [16]. All data cleaning, recoding, and analysis were done using Stata Version 17.0 [17]. All analyses were weighted to be representative of the Californian population using the methods recommended by CHIS [18]. To summarize, these weights account for both the telephone and web data collection methods used by the CHIS in addition to the oversampling of certain minoritized groups to provide estimates for all counties, large and small, in CA. Furthermore, the weighting procedure accounts for the differential probability of selection of households, nonresponse, and sample differences among less-represented groups. Weights are based on 2010 Census stratum counts projections. Weighted analyses with the CHIS use Jackknife variance estimation calculation with replicate weights in order to produce reliable estimates that are representative of the Californian population [18]. Results Table 1 presents the weighted sample characteristics of the 2020 CHIS. Overall, 77.7% of participants indicated that they would receive the COVID-19 vaccine if it was available. However, there were distinct differences in vaccine willingness by race and citizenship status (p < 0.001). In general, vaccine willingness was highest among US-born people within each race/ethnic group, except for Hispanic/Latinx people, where vaccine willingness was highest among noncitizen Hispanic/Latinx people. When examining differences in vaccine willingness by race/ethnicity, Asian people had the highest vaccine willingness, followed by non-Hispanic White and Hispanic/Latinx respondents. Interestingly, vaccine willingness was lowest among naturalized Hispanic/Latinx people.Table 1 Weighted sample characteristics by race and citizenship status, 2020 California Health Interview Survey (CHIS), n = 20,536 Asian Hispanic/Latinx Non-Hispanic White Total (n = 20,536) US-born (n = 870) Naturalized (n = 1438) Non-citizen (n = 437) US-born (n = 2769) Naturalized (n = 1049) Non-citizen (n = 499) US-born (n = 12,452) Naturalized (n = 793) Non-citizen (n = 229) Variables % % % % % % % % % % P-value Would get COVID-19 vaccine if available 77.7 89.6 86.1 83.8 70.4 69.2 71.6 82.1 81.1 87.8 < 0.001 Demographic factors Age category < 0.001 18–34 years 30.6 56.6 11.1 47.0 53.6 15.0 26.1 22.2 18.5 27.8 35–49 years 25.2 23.3 27.2 29.6 24.6 26.4 42.4 20.6 19.2 41.1 50–64 years 23.3 10.6 33.9 18.8 12.5 36.5 25.9 25.4 27.0 12.9 65 + years 20.9 9.6 27.8 4.6 9.3 22.1 5.6 31.8 35.3 18.2 Female gender 50.8 56.0 54.4 45.5 49.8 51.0 51.2 51.1 44.8 49.6 0.033 Social factors Family type < 0.001 Single, no kids 39.6 59.5 27.8 36.3 46.5 27.0 24.8 43.4 34.7 30.4 Married, no kids 30.5 18.9 43.4 23.2 16.4 38.3 22.9 37.1 45.7 35.2 Married with kids 20.4 15.3 25.5 32.5 18.5 25.0 34.7 15.3 17.7 30.2 Single with kids 9.5 6.2 3.3 8.0 18.6 9.7 17.7 4.2 2.0 4.3 Living in rural area 11.7 3.2 5.3 3.5 9.3 8.6 11.5 17.6 6.8 3.7 < 0.001 Socioeconomic factors Educational attainment < 0.001 Less than high school 16.0 2.6 18.0 16.5 10.8 43.9 55.0 3.7 15.5 9.0 High school graduate 21.6 7.1 12.8 14.5 27.5 22.1 22.5 22.3 14.3 12.4 Some college 15.9 15.5 8.8 8.5 21.2 12.4 11.3 17.0 13.7 12.7 Associates degree 5.5 4.5 4.3 2.2 8.4 3.0 1.0 6.2 4.4 2.4 Bachelor’s degree or more 41.0 70.3 56.2 58.2 32.1 18.6 10.2 50.9 52.2 63.6 Currently employed 55.0 63.7 54.9 55.1 57.0 55.0 58.6 52.2 51.0 59.4 0.003 FPL > = 100% 86.5 91.3 90.2 79.4 81.8 79.9 70.4 93.4 93.1 96.9 < 0.001 Has usual source of care 76.0 81.8 91.3 76.0 81.0 83.9 71.7 89.3 90.2 81.9 < 0.001 COVID-19-related factors Experience discrimination based on race and COVID-19 1.6 7.2 2.7 3.8 1.8 0.8 3.0 0.6 0.3 0.6 < 0.001 Works as an “essential worker” 17.3 17.1 12.8 12.1 22.5 18.7 19.0 15.4 10.0 9.6 < 0.001 Works from home 22.2 41.1 24.4 27.3 23.2 14.2 7.1 24.6 18.8 28.2 < 0.001 Physical health factors Overweight or obese 61.3 42.9 39.5 43.7 68.3 73.2 78.3 57.9 58.4 50.2 < 0.001 Diabetes diagnosed by doctor 10.7 4.6 15.6 8.2 9.9 19.5 12.7 8.6 7.8 5.9 < 0.001 Heart disease diagnosed by doctor 6.5 3.2 5.8 1.3 3.6 5.1 2.9 10.2 9.0 8.6 < 0.001 Prehypertension or hypertension diagnosed by doctor 32.1 26.0 39.7 18.9 25.8 35.4 23.8 36.8 40.7 29.3 < 0.001 FPL, federal poverty level The majority of the sample was between 18 and 34 years old (30.6%), female (50.8%), and single without kids (39.6%). For socioeconomic factors, most participants had at least a bachelor’s degree (41.0%), were currently employed (55.0%), and had a usual source of healthcare other than the emergency room (76.0%). Approximately 1.6% of the sample indicated that they had experienced discrimination based on their race within the context of the COVID-19 pandemic, yet US-born Asian respondents were 3.5 times more likely to report experiences of racial discrimination during the pandemic (7.2%). Moreover, while 17.3% of all participants indicated that they were “essential workers,” US-born Latinxs were more likely (22.5%) to report being “essential workers.” Approximately 22.2% of participants indicated that they worked from home, yet noncitizen Latinxs were less likely to report being able to work from home (7.1%). Table 2 presents the weighted binary logistic regression results of the association of vaccine willingness and citizenship status and race/ethnicity. In the crude bivariate model (model 1), all Hispanic/Latinx groups had lower odds of COVID-19 vaccine willingness compared to US-born non-Hispanic White people. In comparison, noncitizen White (OR = 1.57, 95% CI = 1.10, 2.25), US-born (OR = 1.87, 95% CI = 1.45, 2.43) and naturalized Asian (OR = 1.35, 95% CI = 1.06, 1.72) people had higher odds of COVID-19 vaccine willingness compared to US-born White people.Table 2 Weighted multivariable binary logistic regression of COVID-19 vaccine willingness on immigration status and race, 2020 California Health Interview Survey (n = 20,536) Model 1 Model 2 Model 3 Model 4 Model 5 Variables OR 95% CI OR 95% CI OR 95% CI OR 95% CI OR 95% CI Citizenship status and race US-born White Ref Ref Ref Ref Ref Ref Ref Ref Ref Ref Naturalized White 0.94 0.69–1.28 0.91 0.67–1.24 0.94 0.69–1.29 0.97 0.70–1.33 0.98 0.71–1.35 Noncitizen White 1.57* 1.10–2.25 1.66** 1.15–2.39 1.62* 1.11–2.35 1.65** 1.14–2.38 1.62* 1.12–2.35 US-born Latinx 0.52* 0.46–0.59 0.56* 0.49–0.63 0.66* 0.58–0.76 0.67* 0.58–0.77 0.67* 0.58–0.77 Naturalized Latinx 0.49* 0.42–0.58 0.51* 0.43–0.59 0.76 0.64–0.92 0.77 0.64 –0.93 0.76 0.64–0.92 Noncitizen Latinx 0.55* 0.44–0.68 0.60* 0.48–0.76 1.09 0.85–1.40 1.14 0.89–1.47 1.15 0.90–1.47 US-born Asian 1.87* 1.45–2.43 2.05* 1.55–2.70 1.80* 1.37–2.37 1.78* 1.34–2.34 1.74*** 1.32–2.29 Naturalized Asian 1.35* 1.06–1.72 1.38 1.09–1.75 1.44 1.14–1.83 1.46 1.15–1.85 1.41 1.11–1.79 Noncitizen Asian 1.13 0.79–1.60 1.22 0.86–1.74 1.37 + 0.95–1.97 1.39 + 0.97–2.00 1.36 + 0.95–1.95 Demographic factors Age category 18–34 years old Ref Ref Ref Ref Ref Ref Ref Ref 35–49 years old 0.96 0.82–1.12 1.02 0.87–1.19 1.02 0.88–1.20 1.02 0.87–1.18 50–64 years old 1.00 0.85–1.19 1.10 0.92–1.31 1.13 0.94–1.34 1.09 0.91–1.30 65 + years old 1.42* 1.20–1.68 1.62* 1.37–1.92 1.72* 1.45–2.04 1.59* 1.33–1.90 Gender Male Ref Ref Ref Ref Ref Ref Ref Ref Female 0.80* 0.71–0.89 0.78* 0.70–0.87 0.77* 0.69–0.86 0.77* 0.69–0.86 Social and socioeconomic factors Family type Single, no kids Ref Ref Ref Ref Ref Ref Married, no kids 0.95 0.81–1.11 0.95 0.81–1.11 0.95 0.82–1.11 Married with kids 0.76 0.62–0.92 0.75 0.62–0.91 0.77 0.63–0.93 Single with kids 1.08 0.88–1.32 1.08 0.89–1.32 1.10 0.90–1.33 Urbanicity Urban Ref Ref Ref Ref Ref Ref Living in rural area 0.81 0.72–0.92 0.83 0.73–0.94 0.84 0.74–0.95 Educational attainment Less than high school Ref Ref Ref Ref Ref Ref High school graduate 1.49 1.18–1.89 1.50 1.18–1.91 1.51* 1.19–1.92 Some college 1.54* 1.26–1.89 1.51* 1.24–1.85 1.53* 1.25–1.87 Associates degree 1.78* 1.39–2.28 1.74* 1.35–2.23 1.75* 1.36–2.26 Bachelor’s degree or more 3.17* 2.57–3.91 2.88* 2.33–3.56 2.91* 2.34–3.61 Employment status Currently unemployed Ref Ref Ref Ref Ref Ref Currently employed 0.96 0.86–1.08 0.91 0.80–1.03 0.92 0.80–1.04 Has usual source of care Does not have usual source of care Ref Ref Ref Ref Ref Ref Has usual source of care 1.29 1.08–1.54 1.27 1.07–1.52 1.26* 1.05–1.51 COVID-related factors Experience of discrimination based on race and COVID-19 Did not experience discrimination based on race and COVID-19 Ref Ref Ref Ref Experienced discrimination based on race and COVID-19 0.95 0.61–1.48 0.96 0.61–1.49 Essential worker status Not an “essential worker” Ref Ref Ref Ref Works as an “essential worker” 1.03 0.88–1.21 1.03 0.87–1.20 Work from home status due to COVID-19 Does not work at home Ref Ref Ref Ref Works at home due 1.52* 1.31–1.77 1.52* 1.31–1.77 Health-related factors Weight status Not overweight or obese Ref Ref Overweight or obese 0.89 + 0.78–1.01 Diabetes status No diabetes diagnosed by doctor Ref Ref Diabetes diagnosis by doctor 1.36 1.11–1.66 Heart disease status No heart disease diagnosed by doctor Ref Ref Heart disease diagnosed by doctor 1.08 0.84–1.39 Hypertension status No pre-hypertension/hypertension diagnosed by doctor Ref Ref Pre-hypertension/hypertension diagnosed by doctor 1.04 0.92–1.17 Constant 4.59* 4.28–4.92 4.71* 4.04–5.49 1.89* 1.42–2.52 1.84* 1.38–2.46 1.93* 1.42–2.62 + , p < 0.10, , p < 0.05, , p < 0.01, and **, p < 0.001. Ref., reference category Model 2, which included age category and gender as demographic confounders, showed similar results to model 1. Hispanic/Latinx groups, regardless of citizenship, continued to have lower odds of vaccine willingness compared to US-born White individuals. US-born and naturalized Asian people continued to have higher odds of vaccine willingness compared to US-born White people. Results remained similar when accounting for social and socioeconomic factors (model 3), with a couple of exceptions. Non-citizen Latinx people now had similar odds of vaccine willingness when compared to US-born White people. Following US-born and naturalized Asian people, Non-citizen Asian people had marginally higher odds of vaccine willingness when compared to US-born White people (OR = 1.37, 95% CI = 0.95, 1.97). These trends seen for all groups remained robust when accounting for COVID-19-related factors (model 4) and health factors (model 5). Figure 1 presents a visualization of the predicted probabilities of vaccine hesitancy by race and citizenship status (based on Table 2, model 5, fully adjusted for demographic, social, socioeconomic, COVID-19, and health factors). In addition, we conducted pairwise comparisons to evaluate if differences in the predicted probability of vaccine willingness by race and citizenship status group were statistically significant using the Sidak method. In general, we see that vaccine willingness is similar among all Asian people regardless of citizenship status (US-born vs. naturalized Asian: p = 1.000; US-born vs. noncitizen Asian: p = 1.000; naturalized vs. non-citizen Asian: p = 1.000) In contrast, there are distinct differences between US-born and non-citizen groups for non-Hispanic White and Latinx groups. Starting with Latinx people first, we see that vaccine willingness is significantly higher among noncitizen Latinx people than among US-born Latinx (p = 0.010). A similar trend is seen between noncitizens and US-born non-Hispanic White. Noncitizen White individuals had higher vaccine willingness than US-born White individuals, albeit not statistically significant when accounting for multiple comparisons (p = 0.328).Fig. 1 Predicted probability of willingness to obtain COVID-19 vaccine by citizenship status and race. Note: Based on model 5 of Table 2, fully adjusted for demographic, social, socioeconomic, COVID-19, and health factors When comparing race/ethnic and citizenship groups, US-born Asian people had significantly higher vaccine willingness compared to US-born White (p = 0.006), US-born Latinx people (p < 0.001), and naturalized Latinx (p < 0.001). In addition, naturalized Asian (p < 0.001) and noncitizen Asian (p = 0.019) people had significantly higher vaccine willingness compared to US-born Latinx. Finally, noncitizen White people had higher vaccine willingness compared to US-born Latinx (p = 0.001) and naturalized Latinx (p = 0.014). Overall, vaccine willingness was lowest among US-born Latinx people, while vaccine willingness was highest among US-born Asian and non-citizen White people. Discussion Our results reveal the varied disparities in COVID-19 vaccine willingness by both race/ethnicity and citizenship status. Overall, we found that at least 70% of Californians were willing to receive the COVID-19 vaccine when it became available. However, there were distinct differences by race and citizenship status. Asian people had the highest predicted probability of willingness to receive the COVID-19 vaccine, regardless of citizenship status, followed by non-Hispanic white and Latinx individuals. Furthermore, noncitizen Latinx and White individuals had a greater predicted probability of willingness to receive the COVID-19 vaccine relative to their naturalized and US-born counterparts. Although the high willingness to become vaccinated among all Californians, regardless of citizenship, is encouraging, this willingness may not translate into actual vaccine uptake. As of July 2022, 71.5% of all Californians have been fully vaccinated for COVID-19 (i.e., 2 doses of Moderna or Pfizer or 1 dose of Johnson & Johnson) [1]. However, only 58.1% of Californians have received their first booster dose. Thus, it is possible that some of the disparities in vaccine uptake may be related to barriers that noncitizen or racially marginalized individuals may face, such as the threat of being labeled a public charge or the inability to get a vaccine because of demanding work schedules. The higher vaccine willingness among Asian people is particularly interesting. The conflation of COVID-19 to Asian people [19–21] would lead some Asian people to be more willing to receive the vaccine to combat these stereotypes. Furthermore, the higher rates of vaccine willingness may be related to the presence of Asian people in essential work and healthcare. For example, although Asian people comprise about 7% of all healthcare workers [22] there has been disproportionate COVID-19 infection and mortality among certain Asian ethnic groups, like Filipinos, who disproportionately comprise a large share of the nursing workforce [23, 24]. The lower vaccine willingness among Latinx individuals as a whole is also concerning, especially given that Latinx immigrants comprise a large share of the essential workforce. These lower rates of willingness to get vaccinated may be related to institutional and structural barriers for workers that may not allow Latinx immigrants to take paid time off to receive the vaccine. For example, Latinx immigrants are more likely to work in precarious industries where employer abuse is pervasive [25] and taking time off from work is difficult [26]. Another explanation may be Latinx Californians’ fear of immigration enforcement and becoming a public charge. For instance, a recent study found that exposures to immigration enforcement, such as avoidance of health and social services due to immigration fears or experiences of detention or deportation, were associated with a lower likelihood of accepting the COVID-19 vaccine among a sample of undocumented young adults in CA [27]. Alternatively, some Latinx people may have a general mistrust due to discrimination by medical providers or histories of racial discrimination [22, 28, 29]. Thus, although vaccine willingness may be slightly higher among non-citizen Latinx compared to US-born and naturalized Latinx people, structural barriers may ultimately affect the actual uptake of the COVID-19 vaccine. Finally, while the willingness to receive the COVID-19 vaccine was generally high among non-Hispanic White individuals, it was interesting to see that rates were lowest among US-born Whites, despite accounting for educational attainment. The lower willingness rates among US-born individuals, in general, could be related to potential political views on vaccination. In the USA, race/ethnicity has become a key predictor of voting behavior and political affiliation, whereby White voters are more likely to lean Republican [30]. Given the politicization of the COVID-19 pandemic, political views are a significant factor in vaccine resistance. For instance, political ideology is associated with vaccine uptake, whereby regions with a higher share of Republicans have a lower share of individuals who have received the COVID-19 vaccine [30, 31]. Unfortunately, the 2020 CHIS did not include questions related to political preferences. These results are balanced by a number of additional limitations. First, as previously mentioned in our methods, we were unable to examine vaccine willingness disparities among Black, Indigenous, Native Hawaiian, Pacific Islander, and multi-racial populations. Previous work has noted the disheartening toll that COVID-19 has had among these communities [32, 33], which may encourage these communities to be more willing to receive the COVID-19 vaccine. However, future work should examine how issues of citizenship and immigration affect the Black community, especially. Second, given our use of public data, we are unable to examine how vaccine willingness may differ within ethnic subgroups (e.g., Mexican and Filipino). Although we provide our reports in the aggregate, it is possible that vaccine willingness may vastly differ between certain subgroups depending on the impact COVID-19 has had on them. Finally, there may be some other unmeasured factors that we were unable to account for in our analysis. As previously mentioned, we were unable to examine the role that political preferences or policies such as “public charge” may have in explaining differences in vaccine willingness by race and citizenship. Furthermore, while CHIS is intended to be representative of all of CA, we are unable to examine how larger area-level factors could affect vaccine willingness. However, this study provides two key contributions to the literature on COVID-19 vaccine health inequities by race, ethnicity, and citizenship. First, although previous studies have examined COVID-19 inequities by race and ethnicity [10, 11, 28] or have speculated how there may be disparities by citizenship [8, 9, 12, 27, 34], our study provides a detailed look at how willingness to vaccinate may be different by race/ethnicity and citizenship. Examining the intersections of race, ethnicity, and citizenship allows us to examine how potential vulnerability compounds to produce health inequities [35]. Second, our results provided the first look into the general willingness of individuals to become vaccinated against COVID-19 prior to vaccination is available. This is important as the USA continues to deal with upsurges in COVID-19 and future outbreaks. Conclusion Overall, our study found that COVID-19 vaccine willingness at the beginning of the pandemic among Asian, Latinx, and non-Hispanic White individuals was high. However, there were distinct differences by citizenship status. Although the high rates of willingness to receive the COVID-19 vaccine are encouraging, they may not translate into actual uptake. As booster doses and COVID-19 antiviral treatment (i.e., Paxlovid) become available, it is important to consider some of the large institutional and structural barriers that may prevent vaccine willingness from becoming vaccine uptake. Finally, it is important to remove these barriers to ensure that vaccine uptake is equitable for all. Author Contribution AMB conceptualized the study, led the data analysis and interpretation, wrote the methods, results, discussion, and conclusion, and prepared the manuscript for submission. AYHR supported AMB in the data analysis and interpretation, wrote the introduction, and edited the manuscript for clarity. Funding Adrian M. Bacong was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under award number F31MD015931. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Declarations Ethics Approval Because this study is an observational study using de-identified public use data, it does not fit the definition of “human subjects research.” Thus, institutional review board approval was not required. Competing Interests The authors declare no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. California Department of Public Health. Statewide vaccination data [Web]. 2022 [updated July 7, 2022; cited 2022 July 13, 2022]. Available from: https://covid19.ca.gov/vaccination-progress-data/. 2. Hayward SE Deal A Cheng C Crawshaw A Orcutt M Vandrevala TF Clinical outcomes and risk factors for COVID-19 among migrant populations in high-income countries: a systematic review J Migr Health 2021 3 100041 10.1016/j.jmh.2021.100041 33903857 3. Ojeda RH, Robinson S, Dominguez-Villegas R, Telles E, Valenzuela A, Aguilar J. 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==== Front J Gen Intern Med J Gen Intern Med Journal of General Internal Medicine 0884-8734 1525-1497 Springer International Publishing Cham 36451013 7914 10.1007/s11606-022-07914-6 Original Research: Qualitative Research Harmful by Design—a Qualitative Study of the Health Impacts of Immigration Detention http://orcid.org/0000-0003-2201-086X Diaz Chanelle MD MPH [email protected] 1 Ortiz Veronica MD MHS 2 Sanchez Lesly BS 3 Fernandez Jose BS 3 Andrade Elí A. MPH 1 Akiyama Matthew J. MD MSc 4 Ross Jonathan MD MSc 1 1 grid.240283.f 0000 0001 2152 0791 Division of General Internal Medicine, Department of Medicine, Montefiore Medical Center/Albert Einstein College of Medicine, 3300 Kossuth Avenue, Bronx, NY 10467 USA 2 grid.5386.8 000000041936877X Department of Pediatrics, New York Presbyterian Hospital, Weill Cornell Medicine, New York, NY USA 3 grid.251993.5 0000000121791997 Albert Einstein College of Medicine, Bronx, New York, USA 4 grid.240283.f 0000 0001 2152 0791 Divisions of General Internal Medicine and Infectious Disease, Department of Medicine, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, New York, USA 30 11 2022 18 23 6 2022 28 10 2022 © The Author(s), under exclusive licence to Society of General Internal Medicine 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background The USA has the largest immigration detention system in the world with over 20,000 individuals imprisoned by Immigration and Customs Enforcement (ICE) daily. Numerous reports have documented human rights abuses in immigration detention, yet little is known about its health impacts. Objective To characterize how the US immigration detention system impacts health from the perspective of people who were recently detained by ICE. Design Qualitative study using anonymous, semi-structured phone interviews in English or Spanish conducted between July 2020 and February 2021. Participants Adults who had been detained by ICE for at least 30 days in the New York City metropolitan area within the previous 2 years, and that were fluent in English and/or Spanish. Approach We explored participants’ health histories and experiences trying to meet physical and mental health needs while in detention and after release. We conducted a reflective thematic analysis using an inductive approach. Key Results Of 16 participants, 13 identified as male; five as lesbian, gay, bisexual, or queer; and four as Black; they were from nine countries. Participants had spent a median of 20 years living in the USA and spent a median of 11 months in immigration detention. Four themes emerged from our analysis: (1) poor conditions and inhumane treatment, (2) a pervasive sense of injustice, (3) structural barriers limiting access to care, and (4) negative health impacts of immigration detention. Conclusions The narratives illustrate how structural features of immigration detention erode health while creating barriers to accessing needed medical care. Clinicians caring for immigrant communities must be cognizant of these health impacts. Community-based alternatives to immigration detention should be prioritized to mitigate health harms. Supplementary Information The online version contains supplementary material available at 10.1007/s11606-022-07914-6. KEY WORDS immigration detention structural vulnerability COVID-19 ==== Body pmcINTRODUCTION The USA has the largest immigration detention system in the world, with nearly half a million individuals detained annually by Immigration and Customs Enforcement (ICE).1–3 ICE uses databases, information-sharing systems, and raids to target people for deportation. Individuals arrested at the border or interior by local or federal law enforcement can be transferred to ICE custody. The US immigration detention system is intricately connected to the mass incarceration system. Detained immigrants are typically held in local jails or for-profit detention centers contracted by ICE that effectively operate as prisons.3 Most immigrants in ICE custody have no criminal record,4 yet as civil detainees, they have fewer legal protections than criminal defendants with no guaranteed right to appointed counsel.2 Immigrants are at risk of poor health outcomes due to their structural vulnerability,5, 6 a confluence of social, economic, and political structures (e.g., language barriers, health insurance access, and anti-immigrant sentiment) that interact to negatively impact this population.7–9 This structural vulnerability is substantially amplified for those in immigration detention where the conditions of incarceration and embedded inequities in access to medical care can exacerbate health-related harms.10–13 During the COVID-19 pandemic, their vulnerability was evident as cases and death rates in US immigration detention far outpaced national averages.14 Despite the increased use of immigration detention, ICE does not routinely collect or report data on health risks or outcomes of those in its custody. A recent study found that adults in immigration detention reported a high burden of serious health conditions including cancer, HIV, diabetes, and mental illnesses, and frequently experienced disruptions in care.15 Reports from international settings demonstrate that immigration detention is deleterious to the physical and mental health of those who experience it.16–21 However, research to understand the specific health impacts of detention in the US context is needed to inform national policy and interventions to mitigate harms. We sought to characterize how the US immigration detention system impacts health from the perspective of people who were recently detained. We used qualitative interviews to gain a detailed understanding of the complex ways one’s health can be affected during detention and post-release. METHODS Setting We conducted this study in the New York City (NYC) metropolitan area. We partnered with several community-based and legal organizations in NYC that work directly with persons impacted by immigration detention for recruitment. Participants and Recruitment Participants were recruited through convenience sampling. They were referred from partner organizations that provided a first name or pseudonym and phone number to study staff, who then contacted the individuals and screened for inclusion criteria. Eligible individuals were offered participation in the study without further selection. Inclusion criteria were as follows: (1) release from immigration detention within 2 years of study enrollment, (2) detained for ≥ 30 days, (3) age ≥ 18 years, (4) fluent in English or Spanish, (5) currently residing in the NYC metro area, and (6) able to provide informed consent. A total of 25 individuals were referred for the study: five declined participation, four were unreachable, and 16 completed interviews. Research Team The research team included two physician investigators with experience conducting medico-legal evaluations of individuals in immigration detention (CD, JR), a physician investigator whose research focuses on infectious disease in carceral settings (MJA), three bilingual (English/Spanish) medical students with qualitative research training (LA, JF, VO), and a bilingual (English/Spanish) doctoral student in epidemiology (EA). The interviews were conducted by LA and JF; data analysis and interpretation were done by VO, EA, MJA, CD, and JR. Interviews We conducted in-depth interviews by phone in the participants’ preferred language (English or Spanish). Interviews were audio recorded using an encrypted application and professionally transcribed for analysis; interviews conducted in Spanish were professionally translated and transcribed simultaneously. To ensure confidentiality, verbal consent was obtained, no personal identifiers were recorded, transcripts were de-identified for analysis, and recordings were destroyed after the transcription was reviewed for accuracy. Interviews lasted approximately 1 hour. A 17-question interview guide (Supplementary File 1) was developed to elicit participants’ health-related experiences during and after detention. We explored the development of physical and mental health conditions during detention, barriers to and facilitators of receiving medical care during and after detention, perceptions of health care quality, and experiences related to the COVID-19 pandemic. The interview guide was piloted with 3 study participants and iteratively revised for clarity and as new topics emerged during interviews. The research team met regularly to debrief, discuss issues encountered during the interviews, and consider revisions to the interview guide. Data Analysis Preliminary analysis of interviews was carried out concurrently with data collection. Reflective thematic analysis was used to identify repeated patterns across interviews.22 The process of analysis was inductive: we reviewed the initial transcripts, wrote memos reflecting on emerging themes, and then met to draft a codebook. We used Dedoose Version 9.0.17 (2021) to manage the data coding. Each transcript was coded independently by at least two analysts. We met regularly to discuss provisional themes and further refine the codebook until the salient patterns repeated across and within transcripts were identified and agreed upon. Discrepancies in coding were discussed and resolved by consensus. Throughout the analysis, we were reflexive about how our assumptions and experiences may affect our interpretation of the data. Saturation was reached when the three coding authors agreed that no new themes emerged from analysis of the final transcripts. The quotes presented have been edited slightly with some punctuation added to facilitate reading. This qualitative study followed the COnsolidated criteria for REporting Qualitative research (COREQ).23 Ethical Considerations Ethical approval for the research was granted by the Albert Einstein College of Medicine Institutional Review Board. To ensure anonymity, quotes have not been attributed to specific participants. RESULTS Of 16 participants, 13 identified as male; five as lesbian, gay, bisexual, or queer; and four as Black; they were from nine countries (Table 1). They had spent a median of 20 years living in the USA (IQR 8.5, 26) and had spent a median of 11 months in immigration detention (IQR 6.5, 16.5). Fourteen experienced prolonged detention (≥ 6 months). Nine experienced solitary confinement while detained, and twelve were detained during the COVID-19 pandemic. Table 1 Study Participants’ Characteristics: a Qualitative Study of the Health Impact of Immigration Detention, New York City, 2020–2021. Participant characteristics No. (%) N = 16 Age, median (IQR) 35 (31.5, 42.5) Gender Male 13 (81) Female 2 (13) Non-binary 1 (6) Identified as lesbian, gay, bisexual, or queer 5 (31) Country of birth Dominican Republic 4 (25) Jamaica 2 (12.5) Trinidad and Tobago 2 (12.5) Honduras 2 (12.5) Mexico 2 (12.5) Guatemala 1 (6.25) Pakistan 1 (6.25) Russia 1 (6.25) Kazakhstan 1 (6.25) Self-identified as Black 4 (25) Four themes emerged from our analysis: (1) poor conditions and inhumane treatment, (2) a pervasive sense of injustice, (3) structural barriers limiting access to care, and (4) negative health impacts of immigration detention. We created a conceptual model of the negative health impacts of immigration detention (Fig. 1) that highlights the salient themes and their directionality based on our interviews. Figure 1 Health impact of immigration detention: a conceptual model. Poor Conditions and Inhumane Treatment Participants described how the physical conditions of immigration detention (such as nutrition, overcrowding, lack of sanitation) and the inhumane treatment they experienced (including prolonged confinement, isolation, discrimination) impacted their physical and mental health. Participants recounted unpalatable and unhealthy food, crowded dorms that lacked ventilation and facilitated the rapid spread of infections, and freezing temperatures that made it difficult to rest. They reported lack of soap and toilet paper and unclean conditions. Recreation time was limited and there was little or no access to outdoor spaces. One participant reflected, “you’re locked up in a box… you don’t even get sunlight. You’re begging for sunlight, imagine that.” Many of the participants that were detained at the beginning of the COVID-19 pandemic reported that they had little ability to socially distance due the overcrowding, and that they felt more isolated once facilities implemented measures confining them to their cells for most of the day. Participants mourned the loss of what few distractions they had before the pandemic, such as bible study, visitations, and socializing with others inside. “Then they put us in cells, that was even worse, to me it was like torture because there were 2 of us in each box. And do you know how much time we spent outside? Half an hour every 24 hours… I’d call my mom for 15 minutes, take a bath, get my clothes on and cook, all within those 30 minutes.” Some participants had no prior experiences with the criminal justice system and felt unsafe being incarcerated with individuals charged with violent crimes. Participants recounted witnessing and/or experiencing violence, often without intervention by corrections officers. One individual that was frequently targeted by other detainees because of their sexual orientation described: “There were times when the detention officers they would have been helping by giving stuff for the beating.” Another participant sought care after being assaulted by an inmate, only to have their injuries minimized by staff. “I told the doctor… they didn’t check it, they just give me the ice pack. But the officer say, it’s okay, you’re not going to die, and I was really surprised, like he’s really thinking about me like that, like I’m not worthy, like I’m really not [a] human being…” Participants felt that they were treated with a callous disregard because of their immigration status that made them feel less than human. Corrections officers deprived some participants of their rights, for example with retaliatory use of solitary confinement or withholding of commissary funds, and subjected them to humiliating treatment. “Most of the C.O.s [corrections officers] they treat us like we’re garbage. They treat us very bad. And they treat the criminals better than us. We get treated like you have no rights worse than animal and it’s terrible. And they tell you right on your face, ‘Yeah, you ain’t got no rights.’” Pervasive Sense of Injustice Almost every participant described their detention as unjust. This sense of injustice often exacerbated the emotional distress participants experienced due to the chronic/sustained uncertainty of detention. Most participants had no criminal histories, and they often perceived their non-criminal detention as arbitrary and unfair, adding to the psychological burden of being detained. “I was traumatized with all that and I think it harms you morally, psychologically, physically, what immigration officials do to you, there shouldn’t be immigration jail, really, why don’t they let you go through the process while you’re free…” Participants who had served time in prison prior to being in ICE custody described worse conditions in immigration detention where they felt “more imprisoned than prison itself.” “It’s the worst experience that I’ve been through in my life was being detained. Not even the three-and-a-half years that I did [in prison] had me overwhelmed and stressed out the way being detained for 13 months did. And I’ll tell you why, because when I was incarcerated, in my head I convinced myself that I’m in this predicament because I put myself in this predicament…” Participants described a persistent state of uncertainty, both with respect to how long it would take to resolve their immigration case and whether they would be released. The fear of what would happen to them if they were ultimately deported was omnipresent. The uncertainty about both the length and outcome of their detention, as well as the potential for deportation, led to high levels of anxiety. As one participant explained, “You’re on death row… You’re a chicken right, waiting to get slaughtered any second.” Some participants felt that immigration officials and officers in the jails created a false distinction between immigration detention and prison. There was a common perception that the goal of detention was deportation at any cost. Participants felt detention was “designed to… break one down,” exploiting their fears to exert pressure to self-deport. They faced an impossible choice:“I told them, you say that I am not a prisoner here, and they said, you are not because you can go back to your country. And I told them, the problem I have in my country is worse than being here because they will kill me there.” “I think they want people to become desperate… so they’ll sign their deportation and leave… officials from ICE would come and ask how are you doing, and they’d say, the food is awful, I haven’t seen a doctor… and they’d say, okay step in line and sign this deportation form and it’ll all be over.” Structural Barriers Limiting Access to Care Multiple systemic barriers prevented access to needed care and led to delays in medical attention that impacted individuals’ health. Evaluation by the medical team typically meant submitting a written request, which required literacy and fluency in English. Individuals with limited English proficiency were routinely denied access to interpreter services during medical visits. Participants often had to submit multiple sick call requests before they were called, sometimes waiting weeks. “I’d write on the screen, I need to talk to the psychologist, please, it’s urgent, I feel depressed, please help me… when you write sometime like that it’s because you really have a serious problem… 2-3 weeks later they’d say, okay, come over here.” Participants reported barriers to receiving care for more urgent medical needs. One participant with HIV experienced delays in receiving their medication when they entered detention, and frequent unexplained interruptions to therapy. Overall, participants perceived the barriers to getting needed medical and mental health care were insurmountable. “In fact, the doctor told me pretty much, "You're not dead yet, so there's no reason for us to help you yet.” During the COVID-19 pandemic, participants described inconsistent and concerning practices around isolation that increased the spread of infection (such as cohorting symptomatic and asymptomatic cellmates) and increased their risk of poor outcomes from COVID-19. For example, some individuals experiencing COVID-19 symptoms were placed in solitary confinement rather than transferred to hospitals, regardless of the acuity of their illness or underlying risk. “He had the COVID full blown, you could say that it was so bad he couldn't walk. Instead of them sending him to a facility where they could provide better care for him, they kept them in the box, which is solitary confinement, and just fed him aspirins.” Barriers to accessing health care often persisted after release due to lack of health insurance and inadequate discharge planning, particularly for patients with chronic conditions, including serious mental illness. Nonetheless, with navigation support from community-based organizations, several participants were able to enroll in insurance and access health services after release. Some participants felt their health needs were only met after they were released. “After I was released, I spent like 4 months without being able to see a doctor because I didn’t have insurance… I needed psychological help because I felt really bad and so [my social worker] helped me out… the first thing I did, the day after I got my insurance is go in for a physical.” Negative Health Impacts of Immigration Detention Participants reported chronic medical conditions prior to their detention. They described how conditions of confinement, inhumane treatment, distress associated with the injustice of detention, and the multiple barriers they encountered when seeking care negatively impacted their overall health. In addition to the sequelae of violence, treatment interruptions, and COVID-19 infection, previously healthy participants directly attributed the development of obesity, high cholesterol, and/or high blood pressure to the unhealthy food and lack of physical activity in detention. Every participant described how immigration detention deeply impacted their mental health. Their time in detention was characterized by a sense of isolation, overwhelming loss, and emotional pain. Many participants reported developing symptoms of depression and anxiety; some also had thoughts of suicide. Experiencing solitary confinement heightened their distress. “I spent 15 days alone, alone, alone, alone, without even being able to read a book, without being able to read anything, I mean, I was there alone[...] I got desperate and again I wanted to take my own life...” Family separation further contributed to worsening mental health. Some participants described an irreparable harm to familial bonds, including divorce/separation from spouses. Children could not understand why their parent had disappeared. The pain of separation was sometimes worse during visitations, as those detained were forced to maintain physical distance from their loved ones. The economic consequences of detention, whereby families were left in more precarious financial circumstances because a primary breadwinner was detained, added to the psychological distress participants experienced. “I never had suicidal thoughts. I never had depression. I never had anxiety… I did not know what that felt like or what it meant until I was detained— just constantly thinking about my case, and my family, and constantly hearing negative things, my mom going through it, and my wife not having enough to pay the rent one month— just all of that hit me at once and became overwhelming.” Participants that were detained during the COVID-19 pandemic recalled feeling even more isolated from their loved ones and afraid, as they lacked information and personal protective equipment while COVID-19 rapidly spread in the detention facilities. Immigration officials seemingly prioritized incarceration over health and failed to allocate needed resources to reduce the spread of infection. Thus, many were exposed to COVID-19 and developed symptoms, though few were tested for COVID-19. Participants expressed they were only able to protect themselves from COVID-19 after release. “They didn't come and ask people, "Hey, do you feel any symptoms? Are you okay, do you want to get tested?" There were no proper tools given like hand sanitizer, Clorox... [no] masks given out or gloves given… we barely had toilet paper and soap.” Many participants expressed that their time in immigration detention left a “mental scar.” They were dealing with the sequalae of trauma long after their release, which included a persistent sense of insecurity. “Those kind of traumas don't go away overnight… I am walking down the street and I am afraid that immigration could come at any moment to get me... I've just leveled off a little bit mentally, and I've stopped having those dreams of imprisonment, those nightmares. But you keep thinking that immigration may come any time. You don't feel free.” DISCUSSION In one of the only US studies examining the health impact of immigration-related confinement in the current era of mass detention, participants described how systemic features of detention eroded their health. Participants were young and healthy with few chronic medical conditions prior to detention. Yet they described how the physical environment of detention, neglect of basic human needs, and the dehumanization they experienced undermined their well-being. Most were subjected to prolonged detention. During this time, poor access to medical and mental health care created a major barrier to addressing pre-existing health conditions and new medical problems arising from their time in detention. Our data point to several mechanisms by which immigration detention can uniquely increase structural vulnerability. A study of detained immigrants in California found that conditions of confinement cumulatively worsened perceived health status, increased stress symptoms, and were associated with a greater likelihood of a mental health diagnosis.10 Participants in our study also described how food insecurity, lack of recreation, overcrowding, inadequate hygiene, solitary confinement, and discrimination negatively impacted their health. Some participants attributed the development of obesity, hypertension, and prediabetes to lack of nutritious foods and recreation, which is supported by studies on food insecurity.24 Similarly, solitary confinement is known to lead to deterioration of mental health and increases the risk of self-harm,25, 26 and suicide rates are rising in ICE detention.27 Advocates have long documented problematic conditions in immigration detention.28 Prior to the COVID-19 pandemic, frequent outbreaks of infectious diseases were attributed to overcrowding and unsanitary conditions.29 The pandemic led to immigration court shutdowns, facility lockdowns, and suspension of visitation by attorneys, advocates, and families. Our findings describe conditions that facilitated the spread of COVID-19 and placed those detained at increased risk during the early months of the pandemic, including lack of access to personal protective equipment and basic sanitation. These results are consistent with published reports of COVID-19 infections in immigration detention that identified systemic shortcomings in mitigation and containment strategies, and deficient medical care during the pandemic.30, 31 As a result, the death rate among individuals in ICE detention increased sevenfold between 2019 and 2020 while the population decreased by nearly a third.32 Underlying the significant psychological toll of immigration detention was a sense of arbitrariness, injustice, and uncertainty. Though under US law immigrants in detention are considered civil detainees, participants experienced detention as punitive. Thus, a pervasive sense of injustice, or moral injury, emerged as a potent mechanism for traumatic stress. Moral injury refers to the lasting psychological impact that can result from experiencing or “bearing witness to acts that transgress deeply held moral beliefs and expectations.”33 Developed in the military context, the concept has also been applied to refugees.34, 35 Studies suggest that these moral wounds have long-term implications for one’s health.36, 37 A recent study evaluating the psychological impact of US immigration detention on Latinx transgender immigrants found that the dehumanization, abuse, and transphobia they experienced in detention led to trauma, anxiety, depression, suicidal ideation, and a preference to self-deport.38 Similar to our study, they found detention facilities failed to address the mental health needs of participants. Legal scholarship has examined how US immigration laws and policy choices have led to a reliance on immigration detention as a default.39 These narratives demonstrate that detention can be so intolerable that some individuals feel pressured to surrender their right to have their immigration case decided in a court even if they have valid claims to relief from removal. Yet evidence suggests that detention does not significantly deter migration and is not necessary to ensure people appear in court.40–42 Pilot programs testing alternatives to immigration detention in several countries, including the USA, have been found to be cost-saving and effective.43 Clinicians also have an important role to play. Medical-legal-community partnerships exist in the USA that link lawyers and advocates to volunteer clinicians to support clients with serious health issues held in immigration jails.44 Clinicians review medical records and visit detained individuals, writing medical affidavits and reports that are used to advocate for their release or access to care.45 This study has several limitations. First, we interviewed a sample of formerly detained individuals referred by legal partners in NYC, a city with the first program that provides residents guaranteed right to counsel in immigration cases. Therefore, our findings may not be representative of detained individuals in other locations, or those who lack legal representation. We did not ask where participants had been detained to protect confidentiality. Although thematic saturation was reached during the interviews, the relatively small sample size did not allow for comparisons of subgroups of participants, such as across genders or facilities. Though only 13% of our sample identified as female, this is close to the proportion of women detained by ICE.46 Finally, interviews were only conducted in English and Spanish. These findings have significant public health implications for immigrant communities as, on any given day, thousands of immigrants are held systematically and unnecessarily in punitive conditions. Evidence is mounting that the negative consequences of immigration enforcement and detention extend beyond detention, to impacted families and communities.47, 48 An estimated 16.7 million people in the USA share a home with a family member who is unauthorized.49 By depriving individuals of their freedom and ability to work, detention also deprives families of economic stability.3, 50 This study contributes to the growing body of literature demonstrating the health risks associated with immigration enforcement policies.8, 51–53 In conclusion, the participants’ powerful narratives illustrate some of the negative impacts of immigration detention on the health, the lives, and the families of detained immigrants. Overall, the systems put in place to detain immigrants were perceived to be designed to cause harm. Clinicians caring for detained, or formerly detained, immigrants must be cognizant of these health harms. Our work further emphasizes the need for post-detention care that is trauma-informed and focuses on ameliorating the psychological harms caused by detention. Supplementary Information ESM 1 (DOCX 18 kb) Acknowledgements We thank our community collaborators, the Bronx Defenders, the Legal Aid Society, NYC New Sanctuary Coalition, and the New York Lawyers for the Public Interest. This study received support from the Albert Einstein College of Medicine Global Health Center and Department of Medicine, the National Institute of Mental Health (K23 MH114752), and the Agency for Healthcare Research and Quality (K12HS026396). This research was presented at the 2022 Society of General Internal Medicine Annual Meeting. Declarations Conflict of Interest The authors declare that they do not have a conflict of interest. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Budiman A. 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==== Front Amino Acids Amino Acids Amino Acids 0939-4451 1438-2199 Springer Vienna Vienna 36449096 3217 10.1007/s00726-022-03217-6 Original Article l-Arginine is a feasible supplement to heal chronic anal fissure via reducing internal anal sphincter pressure: a randomized clinical trial study Khalighi Sikaroudi Masoumeh 12 Sedaghat Meghdad 25 Shidfar Farzad 3 Talebi Sepide 1 Hosseini-Baharanchi Fatemeh Sadat 4 Masoodi Mohsen [email protected] 2 Farahani Safoura Vasheghani 3 1 grid.411705.6 0000 0001 0166 0922 Department of Clinical Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran 2 grid.411746.1 0000 0004 4911 7066 Colorectal Research Center, Iran University of Medical Sciences, 1445613131 Tehran, Iran 3 grid.411746.1 0000 0004 4911 7066 Department of Nutrition, School of Public Health, Iran University of Medical Sciences, Tehran, Iran 4 grid.411746.1 0000 0004 4911 7066 Department of Biostatistics, School of Public Health, Iran University of Medical Sciences, Tehran, Iran 5 grid.411746.1 0000 0004 4911 7066 School of Medicine, Iran University of Medical Sciences, Tehran, Iran Handling editor: N. Le Floch. 30 11 2022 110 18 8 2022 21 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The hypertonicity of internal anal sphincter resting pressure is one of the main causes of chronic anal fissure. Therefore, the aim of this study was to assess the effect of oral administration of l-arginine on the improvement of the anal fissures by relaxing the internal anal sphincter. Seventy-six chronic anal fissure patients (aged 18–65 years) who were referred to Rasoul-e-Akram Hospital, Tehran, Iran from February 2019 to October 2020 participated in this randomized, double-blind, placebo-controlled trial. Participants were allocated into treatment (l-arginine) and placebo groups. They took a 1000 mg capsule three times a day for 1 month, and then we followed them at the end of the first and third months after the intervention. Clinical symptoms, anal sphincter resting pressure, and quality of life (QoL) were completed at baseline and the end of the study. The analysis of data showed a significant decrease in bleeding, fissure size, and pain for each group; however, in the L-arginine group was more than the control group at the end of the study (P values < 0.001). Following that, a significant increase in QoL was seen just in patients treated with l-arginine (P value = 0.006). In addition, the comparison of anal pressures at baseline and, between groups at the end of the study showed a significant reduction in sphincter pressure in patients treated with l-arginine (P value < 0.001, = 0.049; respectively). The oral administration of 3000 mg l-arginine can heal chronic anal fissures by reducing internal anal sphincter pressure with more negligible side effects. However, we recommend long-term study with more extended follow-up. Clinical trial registry: IRCT20190712044182N1 at Iranian clinical trials, date: 2019-08-27. Keywords l-Arginine Anal fissure Sphincter pressure Clinical symptoms Quality of life ==== Body pmcIntroduction An anal fissure is a condition resulting from a superficial open wound or tear in the anus mucosa with a sharp pain that can extend from the anal canal to the periphery (Schlichtemeier and Engel 2016). It is one of the most common anorectal diseases that equally occurs in men and women and is usually more common in middle age and youth, but can also occur in old age and childhood (Chaudhary and Dausage 2019; Newman and Collie 2019). Anal fissures can be acute or chronic. Chronic fissures are present for more than 6–8 weeks (Newman and Collie 2019). Fibers of internal anal sphincters with hypertrophied anal papilla and a skin tag expose in chronic fissure (Ferri 2021). The etiology of an anal fissure is not precisely clear, but trauma to the anal canal by the passage of hard or large stool is a common fissure cause. Hypertonicity of resting pressure of the internal anal sphincter is seen in patients with fissures as compared with normal controls, which can induce pain and spasm with defecation, and it also has an unfavorable effect on wound healing by reducing blood flow to the traumatized anoderm. Studies have shown abnormal increase of anal contraction in patients with anal fissures, which can explain the sphincter spasm and pain experience with defecation (Nothmann and Schuster 1974; Braun and Raguse 1985). Fissures recurrence, wound infection, or abscesses might occur when ineffective therapeutic measures are taken. Ensuring the soft stools using fiber and laxative, pain controlling by analgesia drugs or short-term use of topical anesthetic, and wound healer ointments, such as glyceryl trinitrate and diltiazem hydrochloride, which can have the side effect profile and recurrence rate for some patients, is the most common management of anal fissures (Medhi et al. 2008; Latif et al. 2013; Stewart et al. 2017). Nonhealing wounds, pain, and bleeding can lead to fecal impaction as patients avoid defecation, which can result in decreased quality of life (Sailer et al. 1998). l-Arginine is a semi-essential amino acid that performs functions in the body, such as improving the immune system, accelerating healing time and repairing damaged tissues, reducing the risk of heart disease by lowering blood pressure, and increasing muscle mass by increasing blood flow (Scibior and Czeczot 2004; Albaugh and Barbul 2017). The metabolites produced by arginine perform many of these actions. Ornithine and polyamines, such as spermine and spermidine are arginine metabolites involved in cell division and, cell growth processes. Proline, another metabolite of arginine, plays an important role in collagen structure, tissue repair, and wound healing (Li et al. 2001; Majumdar et al. 2016). One of the most important metabolites of this amino acid is nitric oxide (NO), which acts as a neurotransmitter and vasodilator in the body (González and Rivas Ferreira 2020). Low amounts of NO increases cell survival and stimulate cell division, in contrast in higher concentrations, can promote apoptosis and cell aging; this action play a crucial role in the immune response, inflammation, and free radicals reduction (Luiking et al. 2012; Kelly and Pearce 2020). Therefore, this hypothesis is raised that l-arginine can improve anal fissure due to its effect on wound healing and increase blood flow to the anal area resulting in reduce the pressure of the internal sphincter. Various studies have investigated the effect of topical l-arginine gel at doses of 400 mg/ml. The results of the studies demonstrated that the use of l-arginine gel significantly reduced anal muscle tone in manometric evaluations, and rectal blood flow increased in laser Doppler flowmetry assessments. However, long-term use of topical l-arginine due to the pH of the drug, osmolality, and its effect on sphincter tonicity are still questionable, and its side effects have not been evaluated (Acheson et al. 2003, 1; Griffin et al. 2002; Gosselink et al. 2005; Fariborz 2007). To date, only a pilot study performed in 2005 examined the effect of 15 g/day oral l-arginine supplementation on eight healthy individuals for seven days via reducing anal pressure, and the results showed that the level of plasma arginine significantly increased, but the anal resting pressures and anodermal blood flow did not show a significant effect (Prins et al. 2005). To the best of our knowledge, this is the first study evaluating the effect of oral L-arginine as a safer method with better performance on clinical symptoms, quality of life, and internal anal sphincter pressure in patients with chronic anal fissure. Materials and methods Study design and participants This was a randomized, double-blind, placebo-controlled trial with parallel design conducted in the 4-week intervention and the 8-week follow-up. We recruited 76 adult men and women (aged 18–65 years) with chronic fissures who were referred to a gastroenterologist in gastrointestinal clinic, Rasoul-e-Akram Hospital, Tehran, Iran. Based on patients’ symptoms like bleeding (bright red) after passing stools without a past medical history of bowel disease, physician diagnosed an anal fissure by physical examination. The study performed from February 2019 to October 2020. Participants who had chronic anal fissure (acute anal fissures classified as last < 6 weeks, whereas chronic fissures last > 6 weeks), without any GI disorder as inflammatory bowel disease, celiac, a history of colon cancer, without history of fissure or hemorrhoid surgery in the last 12 months, other malignancy, absence of diseases that fissures can be complications such as tuberculosis, AIDS, syphilis, herpes, leukemia; no history of heart disease, were not pregnant or lactating women, were enrolled in this study. The study exclusion criteria were specified as follows: a diagnosis of specific gastrointestinal diseases, such as Crohn’s, ulcerative colitis, gastrointestinal cancers, or other malignancies during the study; pregnancy during supplementation; exacerbation of the disease or failure of patients to recover, resulting in the discontinuation of the drug and referral to a colorectal surgeon for fissure surgery during the study, any abnormal reactions to the supplement like gastrointestinal reactions including nausea, vomiting, heartburn, recurrent diarrhea, allergic reactions including itching and rash, and hypotension; consumption less than 80% of supplements; and who were on special dietary pattern; and unwillingness of the person to continue cooperation. Participants completed demographic data, medical history, physical activity, and anthropometric indicators at baseline and the end of the study. Ethics statements The study protocol was approved by the Research Ethics Committee of Iran University of Medical Sciences (IR.IUMS.REC.1398.319), Tehran, Iran, and registered in the Iranian clinical trials (IRCT20190712044182N1-Date: 2019-08-27). The aim of the study was clarified to the participants, and they signed a written informed consent form demonstrating acceptance of study details. Intervention The study participants were assigned to two groups. They received 3000 mg l-arginine, or a placebo filled with Maltodextrin. Capsules were made by Karen company, Yazd, Iran. The shape, color, and packing of placeboes were like l-arginine. Patients received a capsule contained 1000 mg l-arginine or placebo three times a day after each meal for four weeks. The follow-up of patients was continued up to the next eight weeks without any oral anti-fissure agents. In addition to l-arginine or placebo supplementation, during the intervention and follow-up, all patients received once a day Lidocaine-H 4% ointment before defecation and 30 cc/day syrup magnesium hydroxide (in constipate patients) during the 4-week intervention and 8-week follow-up. According to studies conducted, the amount of l-arginine absorption in the intestine is about 20% of a 10-g supplement and depends on various factors, including L-arginine concentration, time of supplementation, and diet. The daily dose of 3 g of l-arginine is safe and has no side effects (McNeal et al. 2016). Randomization, allocation, and blinding For randomization, the permuted block randomization was used with quadruple blocks. According to the sample of size n = 76, 19 blocks were produced using the online site (www.sealedenvelope.com). For the concealment in the randomization process, dedicated codes were generated by the software used on the pharmaceutical box. We performed the randomization process using the relevant software, and also provided the list to the executor in the form of codes A and B for two interventions. Both of patient and the physician who evaluated the clinical and manometric symptoms were blind to the medication received. Other evaluators who filled out questionnaires were also blind. Outcomes The primary outcome was the assessment of the severity of symptoms. Internal anal sphincter pressure, quality of life, anxiety, and depression were the secondary outcomes. Sample size calculation Considering a similar study (Gosselink et al. 2005) for the two primary outcomes of the study (anal pressure and clinical symptoms), the sample size was determined by a probability of type I error of 5% (α = 0.05) and a probability of type II error of 20% (β = 0.2; power = 80%). The reduction in anal pressure in patients receiving arginine was 14 ± 74, which according to the study, was about 15% improvement compared to placebo, and the reduction of clinical symptoms based on VAS after receiving arginine was 1.3 ± 1.5, which according to the results of this study was approximately 80% improvement. In the current study, we calculated the sample sizes of 25 and 32 per group for each outcome, respectively, and also determined the maximum of which as the final sample size. At the end, due to a 20% chance of falling, this number was increased to a total of 76 patients needed. The variance in both groups of placebo and treatment was assumed to be equal. Information gathering tools Physical examination, clinical symptoms At the first visit, patients filled the questionnaires that included bowel habits, duration of suffering from anal fissure, and fissure size, which was determined in millimeters by a gastroenterologist in clinical examination. In addition, the patients were asked to report the symptoms of headache at the beginning and end of the study. To evaluate the clinical signs, we used the Visual Analog Scale (VAS), giving score of 0–100 for each symptom. The patient’s bleeding was questioned and classified into mild bleeding criteria: bloody discharge with mucus unrelated to defecation; moderate type: sometimes there is bleeding with defecation; and in the severe type: defecation is accompanied by bleeding each time. Wound healing examination is also graded based on the appearance of chronic anal fissure including grade 1 includes good and complete healing: no wounds or cracks is seen in the fissure site; grade 2 includes moderate and partial healing: wound or simple longitudinal incision with fibrous bands of the internal sphincter muscle in the wound floor; grade 3 includes poor healing without change: the appearance of the wound is not the same as before, and the deep wound protrudes with a scar; grade 4 includes very poor healing: chronic abscess fissure wound or fistula is added. We evaluated Pain during defecation according to 4 scales graded: score three severe and unbearable pain, score two moderate pain, score one mild pain and score 0 for excretion is painless (Eshghi et al. 2006). All criteria were graded and reported 0 to 100. General health survey short form-36 (SF-36) The 36-item Quality of Life (SF-36) is the most widely used tool for measuring the quality of life and is also used for people with anal fissures (Arısoy et al. 2017). This questionnaire has already been validated in Iran (Montazeri et al. 2005). The SF-36 questionnaire has 36 questions and consists of 8 scales, each consisting of 2–10 items. The subscales of this questionnaire are physical functioning (PF), role limitations due to physical health (RP), role limitations due to emotional problems (RE), energy/fatigue (EF), emotional well-being (EW), social functioning (SF), pain (P), general health (GH). The integration of the subscales also yields two other general subscales, which are as follows:Physical health sub-scale: the sum of the subscales including PF, RP, P, and GH. Mental health sub-scale: a set of subscales of role disorder due to RE, EF, EW, and SF. Hospital anxiety and depression scale (HADs) The HADS questionnaire (Navarro-Sánchez et al. 2021; Zigmond and Snaith 1983) is a 14-item self-rating scale used to assess anxiety and depression symptoms in chronic anal fissure patients. The questionnaire has two parts, consisting of seven questions related to anxiety (HADs-A) and seven to depression (HADs-D). Each item rated between 0 (“not at all”) and 3 (“most of the time”), which the higher scores indicate more significant anxiety or depression symptoms. The total score is calculated by the sum of the concession items, ranging from 0 to 21. The scores from 0 to 7, 8 to 10, and 11 to 21 illustrated a normal scale, borderline, and clinical problems, respectively. Balloon pain score We assessed the severity of the patients’ anal pain and spasm in the initial evaluation in the clinic before the manometry. It was performed with a 20cc volume balloon (with maximum of 6 atm pressure) which was inflated by a 20cc syringe. The balloon is placed in the middle of the anus near the inner sphincter and inflates gently until the patient feels pain. The amount of air injected (0–20) was considered a measure of pain, and spasm in the patient and we compared its amount at the beginning and end of the intervention. Anorectal manometry test Anorectal manometry is a medical test to measure the pressures and the function of the anus and rectum. We performed this procedure using small flexible water perfused catheter with a balloon on end, inserted through the anal opening, past a ring of muscles called the anal sphincter before passing into the rectum. This causes the nerves and muscles in the rectum and anus to squeeze. The end of the tube remains outside of the anus. It is connected to a machine that records the contractions and relaxations of the rectum and anal sphincter. The night before the test, patients should not eat or drink foods. The exam took 10 min to complete. Statistical analysis The quantitative data were described as mean ± standard deviation (SD), and also the qualitative data were reported as frequency (percentage). The normality of the data was checked by the Kolmogorov–Smirnov test and other indicators. If the normality of quantitative variables was satisfied, the distribution between groups and within groups would be performed by independent t test at each point in time of study and paired t test, respectively. To examine the mean outcome at three-time points, we used an ANOVA analysis with repeated measures. If the normality of the data was not satisfied, we would compare them with non-parametric tests, including Mann–Whitney and Wilcoxon and Friedman test. Qualitative variables between the two groups were compared by Chi-square test and also variables within groups were compared by McNemar test. P value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 24. Results Enrollment and study completion Of the 122 eligible participants, 76 patients with chronic anal fissure were enrolled in the study, and 65 completed the study. 11 participants were excluded from the study because of community quarantine due to the COVID-19 pandemic, disease exacerbation, and need to surgery, unwillingness to continue. The flowchart of the study participants is presented in Fig. 1.Fig. 1 The flowchart of enrolled patients in the study Characteristics of the study participants The final analysis was done for participants who completed the study (35 women and 30 men, age: 39.79 ± 11.72 years, BMI: 27.09 ± 4.85 kg/m2). All patients had chronic anal fissure (32.12 ± 51.80 months) and most of them suffered from constipation in both the groups. No significant differences were observed between the two groups in terms of alcohol consumption, smoking, and physical activity at the baseline of the study. Table 1 shows the data.Table 1 The characteristics of the study participants at baseline Characteristics l-Arginine (n = 33) Placebo (n = 32) P value Age (years) 40.64 (10.78) 38.94 (12.66) 0.563 Height (cm) 168.24 (10.50) 167.06 (12.13) 0.677 Weight (kg) 75.85 (15.88) 76.26 (14.75) 0.913 BMI (kg/m2) 26.78 (4.88) 27.41 (4.83) 0.601 Duration of fissure (months) 36.39 (53.62) 27.86 (49.99) 0.509 Sex Male 15 (45.5%) 15 (46.9%) 0.909 Female 18 (54.5%) 17 (53.1%) Alcohol consumption status Yes 5 (15.2%) 5 (15.6%) 0.958 No 28 (84.8%) 27 (84.4%) Smoking Yes 7 (21.2%) 7 (21.9%) 0.948 No 26 (78.8%) 25 (78.1%) Bowel habit Normal 15 (45.5%) 9 (28.1%) 0.156 Constipation 18 (54.5%) 21 (65.6%) Diarrhea 0 (0.0%) 2 (6.3%) Activity Very light 18 (54.5%) 21 (65.6%) 0.260 Light 13 (39.4%) 7 (21.9%) Moderate 2 (6.1%) 2 (6.3%) Heavy 0 (0.0%) 2 (6.3%) Quantitative data are shown as mean ± SD and qualitative data are reported as number (%) P values are statistically significant (< 0.05) Clinical symptoms The significant wound heal was seen in both groups, and the size of the fissure was reduced in the treatment group from 7.52 ± 3.01 to 2.42 ± 2.68 mm and from 7.81 ± 2.76 to 5.16 ± 3.22 mm in the placebo group, where more improvement was demonstrated in the L-arginine group compared to the placebo group (P < 0.001). As shown in Table 2, the VAS questionnaire analysis to assess the alteration of the severity of clinical symptoms demonstrated that all factors in both groups, including bleeding, wound healing, pain, and VAS-total score, were significantly improved from the baseline compared to the control group at the end of the study. We observed more significant improvement in the group receiving l-arginine. Minor fissure recurrence was seen in the control group at the 3-month follow-up. We did not see new ulcer and tag on the anus in both groups. At the beginning of the study, headache was observed in l-arginine and control groups (30.3% and 34.4%, respectively). After taking the supplements, the headache improvement was observed in 7 and 1 patients in the intervention and control groups, respectively, indicating that there was a significant difference between groups at the end of the study (P = 0.026). Data are presented in Table 2.Table 2 Comparisons of severity of symptoms, anal pressure, and balloon pain score before and after intervention in l-arginine and placebo groups in patients with anal fissure Symptoms l-Arginine (n = 33) Placebo (n = 32) P value Fissure size (mm) Before 7.52 (3.01) 7.81 (2.76) 0.680 After 2.42 (2.68) 5.16 (3.22) < 0.001 P value < 0.001 < 0.001 VAS-bleeding Before 39.09 (25.66) 39.38 (29.40) 0.013 After 15.45 (12.52) 24.38 (22.99) Follow up 12.73 (11.26) 26.72 (23.13) P value < 0.001 < 0.001 VAS-wound healing Before 51.52 (13.49) 54.06 (15.00) < 0.001 After 21.82 (17.22) 39.37 (21.10) Follow up 20.76 (17.68) 44.06 (15.63) P value < 0.001 < 0.001 VAS-pain Before 64.09 (30.50) 72.66 (28.68) 0.010 After 32.73 (24.14) 59.06 (33.25) Follow up 29.55 (26.29) 59.69 (31.67) P value < 0.001 0.018 VAS-total score Before 154.70 (49.02) 166.09 (54.22) < 0.001 After 70 (38.41) 122.81 (61.02) Follow up 63.03 (45.79) 130.47 (53.92) P value < 0.001 < 0.001 Wound site Before Posterior 27 (81.8%) 22 (68.8%) 0.221 Anterior 6 (18.2%) 10 (31.3%) After Posterior 25 (75.8%) 22 (68.8%) 0.528 Anterior 8 (24.2%) 10 (31.3%) P value 0.500 1.00 Tag Before Yes 23 (69.7%) 22 (68.8%) 0.934 No 10 (30.3%) 10 (31.3%) After Yes 21 (63.6%) 22 (68.8%) 0.663 No 12 (36.4%) 10 (31.3%) P value 0.625 1.00 Anal itch Before Yes 26 (78.8%) 22 (68.8%) 0.357 No 7 (21.2%) 10 (31.3%) After Yes 9 (27.3%) 17 (53.1%) 0.033 No 24 (72.7%) 15 (46.9%) P value < 0.001 0.063 Bleeding Before Yes 26 (78.8%) 26 (81.3%) 0.804 No 7 (21.2%) 6 (18.8%) After Yes 9 (27.3%) 18 (56.3%) 0.018 No 24 (72.7%) 14 (43.8%) P value < 0.001 0.021 Pain Before Yes 28 (84.8%) 28 (87.5%) 0.757 No 5 (15.2%) 4 (12.5%) After Yes 14 (42.4%) 22 (68.8%) 0.033 No 19 (57.6%) 10 (31.3%) P value < 0.001 0.031 Headache Before Yes 10 (30.3%) 11 (34.4%) 0.466 No 23 (69.7%) 21 (65.6%) After Yes 3 (9.1%) 10 (31.3%) 0.026 No 30 (90.9%) 22 (68.8%) P value 0.039 1.00 Anal pressure (mmH2O) Before 70.15 (15.15) 64.72 (16.94) 0.178 After 50.61 (14.38) 57.97 (15.21) 0.049 P value < 0.001 0.053 Balloon pain score Before 10.24 (4.57) 11.78 (5.60) 0.230 After 16.12 (4.19) 14.00 (5.02) 0.070 P value < 0.001 0.055 Quantitative data are shown as mean ± SD and qualitative data are reported as number (%) P values are statistically significant (< 0.05) Balloon pain score Evaluation of patients’ tolerance to pain as measured by a balloon showed that patients who received l-arginine less sensed pain (P < 0.001), although this effect was not significant between the groups (P = 0.070) (Table 2). Anorectal manometry test Internal anal sphincter resting pressure was significantly decreased in l-arginine-treated patients compared to the baseline values (P < 0.001). As shown in Table 2, sphincter resting pressure was significantly reduced in the treatment group as compared with the placebo group at the end of the study (P = 0.049). Quality of life Comparing the total physical and mental health scores at the end of the study with the baseline, we observed a significant increase in quality of life in l-arginine receiving group. However, there was no significant difference between groups at the end of the study. In addition, subgroup analysis demonstrated that emotional well-being, social function, pain, and general health significantly improved in the l-arginine-treated group and also the score of pain significantly relieved compared with the control group at the end of the study as expected. The data are shown in Table 3.Table 3 Comparisons of mean ± SD HADs-Anxiety, HADs-depression, and quality of life subscales before and after intervention in l-arginine and placebo groups in patients with anal fissure Variables l-Arginine (n = 33) Placebo (n = 32) P value HADs—depression total score Before 5.45 (3.87) 3.62 (3.16) 0.041 After 3.79 (3.39) 3.91 (3.57) 0.891 P value 0.004 0.451 HADs—anxiety total score Before 9.76 (5.14) 7.50 (4.41) 0.062 After 6.52 (4.35) 7.72 (4.55) 0.280 P value < 0.001 0.636 Physical function Before 79.55 (21.37) 70.16 (31.35) 0.165 After 81.06 (25.88) 68.91 (31.36) 0.094 P value 0.644 0.648 Role disorder due to physical health Before 70.45 (40.24) 52.34 (45.51) 0.095 After 80.30 (35.22) 71.09 (40.22) 0.330 P value 0.243 0.042 Role disorder due to emotional health Before 64.65 (42.44) 55.20 (47.60) 0.403 After 77.78 (37.88) 69.79 (40.04) 0.412 P value 0.102 0.104 Energy/fatigue Before 53.64 (38.65) 52.03 (27.41) 0.847 After 61.06 (23.04) 54.37 (24.94) 0.266 P value 0.238 0.513 Emotional well-being Before 45.09 (25.16) 50.67 (25.31) 0.376 After 62.18 (28.74) 52.00 (23.41) 0.123 P value 0.001 0.699 Social function Before 65.53 (31.10) 71.87 (30.62) 0.410 After 79.92 (22.08) 69.14 (25.20) 0.710 P value 0.004 0.486 Pain Before 67.88 (22.04) 59.77 (28.68) 0.205 After 77.73 (24.54) 62.27 (24.75) 0.014 P value 0.014 0.478 General health Before 53.33 (28.96) 60.16 (27.58) 0.335 After 64.84 (29.30) 61.72 (24.38) 0.641 P value 0.001 0.532 Total score (physical health) Before 271.21 (90.35) 242.42 (100.75) 0.230 After 303.94 (97.07) 263.98 (87.54) 0.086 P value 0.042 0.154 Total score (mental health) Before 228.90 (100.65) 229.79 (103.72) 0.972 After 280.94 (87.95) 245.31 (88.34) 0.108 P value 0.006 0.331 Anxiety and depression The analysis of the HADs questionnaire showed that the significant mean change of anxiety and depression scores of treated patients at baseline were 3.24 ± 3.75 and 1.66 ± 3.13, respectively. However, this improvement was not statistically significant between the groups (Table 3). Discussion In this randomized clinical trial, we showed that the supplementation with l-arginine could relieve clinical symptoms, especially pain and bleeding. Following that, we observed a significant increase in quality of life of the patients with chronic anal fissure. In addition, analysis of anal internal sphincter pressures evaluated by manometry and balloon showed the significant reduction of sphincter pressure in these patients. The exact mechanism of the pathophysiology of anal fissures has not established; however, anorectal manometry testing and laser Doppler flowmetry demonstrated hypertonicity of anal sphincter and reduction of anal blood flow in patients with anal fissure (Schlichtemeier and Engel 2016). The most critical mechanisms of l-arginine in the body are to increase blood flow due to the production of nitric oxide and to decrease the time of wound healing (Scibior and Czeczot 2004). Previous studies have shown that topical l-arginine gel significantly reduces anal resting pressure, increases anal blood flow, and significantly heals persistent fissure without any side effects (Acheson et al. 2003, 1; Griffin et al. 2002; Gosselink et al. 2005; Fariborz 2007). However, a small study conducted on eight healthy volunteers showed that oral administration of 15 g l-arginine had no significant positive effect on anal resting pressure (Prins et al. 2005). In contrast to this study, we observed a significant wound healing, improvement in symptoms, and reduction in anal resting pressure. l-arginine orally or topically improved wound repair; however, the oral administration showed a higher NOS and TGF-β expression, and a significant decrease in inflammatory response. In addition, some studies showed that systemic l-arginine was more efficient than topical l-arginine in wound healing (Zandifar et al. 2015; Jerônimo et al. 2016; Witte et al. 2002; Heffernan et al. 2006). For this reason, the oral supplement was our option to treat the patients. l-Arginine can heal wound by two mechanisms. The first one is the NO pathway. Arginine is only substance for NO production, which has vasodilation and anti-inflammatory effects (Patel et al. 2017). The second is the production of ornithine that plays a crucial role in collagen formation and wound repairing (Albaugh et al. 2017). Most of the studies conducted on the effect of l-arginine on wound healing were pressure ulcers, which the majority of them administered it in combination with other supplements, such as vitamin C and zinc (Kl and N 2019). However, some studies evaluated the effects of different dosages of l-arginine supplements on post-surgery wound healing, especially cancer patients and in diabetic foot ulcers. They have supplemented patients with 2 gr for 30 days to 30 gr l-arginine for 5 days and most of them showed ulcers improvements (Arribas-López et al. 2021; Zhou et al. 2021). Our results demonstrated that a supplementation of 3 g of l-arginine for 30 days was a safe and sufficient dose to heal anal ulcers. In addition, compared to other treatments, such as diltiazem and glyceryl trinitrate, leading to side effects like headaches (Latif et al. 2013; Stewart et al. 2017), treatment with l-arginine not only did not cause headaches, but the headache symptoms were improved in some participants, which can be considered as one of the advantages of using l-arginine as compared with other treatment methods. In addition, digestive complications in some patients should be considered. Anal fissure disease widely affects the quality of life of patients due to impaired social, work, and personal relationships, which are directly associated with the duration of stricken and severity of symptoms (Navarro-Sánchez et al. 2021; Griffin et al. 2004). In addition, patients who succeeded in treating chronic anal fissures reported an improvement in bodily pain, health-perception, vitality, and mental health (Tsunoda et al. 2012). As mentioned earlier, a significant quality of life improvement was seen in l-arginine treated group, incredibly more relieved feeling of pain was observed in the treated group than the control group. However, quality of life is one of the factors that need a longer time to improve than our study. On the other hand, anxiety and depression are the main factors that may contribute to the quality of life (Hohls et al. 2019). In addition, from another point of view, the quality of life of the patients with chronic anal fissures, was negatively correlated with severe depression and anxiety, and symptoms were worsened (Arısoy et al. 2017). Administration of anti-anxiety medications in these patients could relieve pain along with standard anal fissure treatment (Mirsadeghi et al. 2021). However, in the present study, l-arginine supplementation did not significantly change the scores of anxiety and depression compared to the control group due to the short time of intervention and assessment, and the score of these indicators significantly improved in the intervention group at baseline. It might be due to the lower numerical scores in the placebo group at the beginning of the study. Given the strengths of our study, it is, therefore, the first study that has shown positive effect of oral administration of l-arginine with low side effect. The other strength point is the use of anal manometry and balloon pain score for evaluating sphincter pressure. On the other hand, in contrast to the control group, none of patients in the intervention group experienced the exacerbation of disease and needed surgery. However, this study has some limitations. Because of the COVID-19 pandemic and quarantine, we lost to follow-up of six patients, and there was a time lag to restart the project. Due to the chronicity of the disease, it would be of benefit to follow the patients for longer duration to make a better examination of the condition of the patients in long term. In addition, due to limited financial resources and equipment, we cannot determine the serum levels of L-arginine as vagaries of absorption, metabolism, and the tissue availability, if possible, it is, therefore, recommended that they to be measured in future studies to increase the accuracy of the results. Conclusion The present study revealed that oral supplementation of l-arginine in patients with chronic anal fissure could heal the wound and improve clinical symptoms due to reducing resting anal sphincter pressure without side effects. Following that, the quality of life, anxiety, and depression were improved in treatment group. Fissure repair was also seen in control group, but improvement in the intervention group was more than that in controls. However, further studies with long-term follow-up are recommended. Acknowledgements We would like to thank the patients who contributed to this study. We are so grateful for the cooperation of Zeynab Sadat Ahmadi and other Rasoul-e-Akram hospital staff to accomplish this project. Author contributions Designed the study: MKS, MM and FS; contribution in sampling: MKS, MM, SVF and MS; analysis and interpretation of the data: MKS and FSH-B; writing the original draft: MKS, FS, ST, and MM; reviewing and revising the paper: MKS, FS, and MM. All the authors read and approved the final manuscript. Funding This study was funded by the Colorectal Research Center with grant number 98-1-49-14857. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. Conflict of interest The authors declared that there were no conflicts of interest. Ethical approval The study was carried out according to the guidelines and approved by the Ethics committee of Iran University of Medical Sciences. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Acheson AG Griffin N Scholefield JH Wilson VG l-Arginine-induced relaxation of the internal anal sphincter is not mediated by nitric oxide Br J Surg 2003 90 9 1155 1162 10.1002/bjs.4182 12945087 Albaugh VL Barbul A “Arginine”. 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==== Front Urban Rev Urban Rev The Urban Review 0042-0972 1573-1960 Springer Netherlands Dordrecht 649 10.1007/s11256-022-00649-y Article Emotions as Both a Tool and a Liability: A Phenomenology of Urban Charter School Teachers’ Emotions http://orcid.org/0000-0002-4975-3826 Stark Kristabel [email protected] 1 Cummings Chauntea 2 1 grid.164295.d 0000 0001 0941 7177 Department of Counseling Higher Education and Special Education, University of Maryland at College Park, 3942 Campus Drive, College Park, MD 20742 USA 2 grid.65456.34 0000 0001 2110 1845 Florida International University, Miami, USA 1 12 2022 127 31 10 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Teachers’ emotional experiences at work have important implications for both teachers and students, particularly during challenging behavioral interactions. Understanding how teachers conceptualize the role of their workplace emotions can help school leaders and researchers develop policies and interventions to support teachers in navigating the emotional dimensions of their roles. In this phenomenological study, we examined how nine teachers working at two urban charter schools made sense of the role of their emotions in their work. We found that teachers conceptualized their emotions as both a tool and a liability for themselves and their students. We provide implications for charter school leaders working to improve teacher-student interactions and promote equitable practices in their schools, and for researchers aiming to understand the affective aspects of teachers’ work. Keywords Teacher development Qualitative research Charter schools Affect Emotion ==== Body pmcIntroduction An emerging body of research points to the importance of teachers’ emotions in shaping the nature and quality of their interactions with students (Frenzel, 2021). Because teacher-student interactions impact students’ academic success (Allen et al., 2013; Cadima et al., 2010) and their social-emotional development (Luckner & Pianta, 2011; Poulou, 2017), both teachers’ internal emotional appraisals and attributions, and their external emotional expressions have important consequences during teacher-student interactions. The external emotions that teachers express during interactions with students, via facial expressions, body language, or verbal communication, can directly influence students’ emotional states in the classroom (Becker et al., 2014; Frenzel et al., 2009; Keller & Becker, 2018). At the same time, teachers’ internal emotional attributions, e.g. their causal appraisals of perceived stimuli, can influence their judgment and decision-making during interactions (Angie et al., 2011). Teachers’ implicit biases for and against students based on their race, disability identification status, and other identities often influence their emotional attributions during interactions (Arcia et al., 2000; Nelson 2018; Reed, 2020; Westling, 2010). These attributions regarding the nature of students’ behavior may impact how teachers feel about the student (Gaier, 2015; Weiner, 2010), and, in turn, inform how they respond to students (Andreou & Rapti, 2010; Poulou & Norwich, 2002; Wang & Hall, 2018). Teachers’ emotional appraisals and attributions hold particularly strong consequences during teacher-student interactions regarding challenging behavior (Hart & DiPierna, 2017; Lucas et al., 2009). When teachers attribute a challenging behavior to something beyond the student’s control, teachers may feel more sympathetic and engage in more supportive responses; conversely, teachers are more likely to feel frustrated, and thereby less likely to respond from an empathetic standpoint, when they believe a student can control the behavior (Wang & Hall, 2018). Teachers’ emotions thereby inform the extent to which they perceive behaviors as intentionally defiant or disrespectful, and the extent to which their responses to students are punitive or restorative. Most student suspensions occur because of subjectively defined offenses, such as perceived disrespect (Girvan et al., 2017; Department of Education and Secondary Education 2020a). Thus, teachers’ emotions play an important role in the persistence of exclusionary discipline practices in many schools today (e.g., Gregory et al., 2014; Ritter & Anderson, 2018; Sullivan et al., 2014). Because teachers are socialized within the cultural environments of their school institutions, organizational norms shape their expectations regarding student interactions and the types of emotions they believe they should be experiencing and expressing during interactions with students (Atiles et al., 2017; Stark & Bettini, 2021). For example, many schools’ “zero tolerance” policies criminalize behaviors that do not align with the forms of communication and emotional expression considered standard by white staff and/or administrators (Hirschfield, 2008; Osher et al., 2012). Thus, to support positive teacher-student interactions, particularly during challenging behavior, it is crucial to critically examine these underlying emotional norms, and teachers’ internal and external emotional experiences. Understanding teachers’ emotional experiences during challenging behaviors should be of particularly urgent concern for charter school leaders: charter schools’ rates of suspension are, on average, 16% higher than rates at public non-charter schools (Losen et al., 2016). Although charter schools are sometimes positioned as leaders in innovation (Gawlik, 2016), and, by nature of their unique accountability structures, charged with the opportunity to engage in creative solutions to educational problems (Pandit et al., 2016), charters often reproduce problematic practices and policies instead (Baker, 2016; Sondel et al., 2019). Data on charter schools’ school suspension rates raises questions regarding the question of why “innovation'' is desired in the first place, and who is best equipped to lead this charge. Often, the creation of a charter school results in the displacement of educators from the same culture and community as the school’s students, by racial and economic outsiders (Buras, 2011; Lipman, 2009). Within this process, there is not only a reorganization and redistribution of the school’s physical space, but its emotional space as well (Buras, 2011). Just as the physical structures of classrooms, hallways, and entrances shape equity and access, so also do the emotional structures within these spaces. Thus, if we aim to support positive outcomes for teachers and students within charter schools, we must critically examine charter schools’ emotional spaces. Our goal in this paper is neither to absolve or indict charter schools categorically; there is both diversity within this category of schools, and there is already a wealth of rich research on the political implications of the charter movement. However, in recognition of the widespread current operation of charters, our aim in this paper is to conduct an exploratory examination of how a sample of urban charter teachers makes sense of the role that their own emotions play in the culture, policies, and practices enacted within their schools. In doing so, we aim to understand teachers’ perceptions of the role of emotion within these contexts. To our knowledge, researchers have not directly asked urban charter teachers to engage in reflection regarding the overall place of emotion in their work. To meet this gap in the literature, we conducted a phenomenological thematic analysis of nine urban charter teachers’ emotional experiences. Our research question was: How do teachers in urban charter schools conceptualize the role of their own emotions in their work? Theoretical Framework: Sensemaking Theory In this study, we draw on sensemaking theory to demonstrate the importance of research regarding teachers’ perceptions of the affective dimensions of their work. Originating in the work of organizational psychologists, sensemaking theory asserts that the ways in which individuals within organizations make sense of their workplace experiences have important implications for how policy is enacted within the organization (Weick et al., 2005). In previous education research, sensemaking theory has been used to demonstrate how the implementation of school or district wide policies, such as reading policies (Coburn, 2001, 2005) or disciplinary practices (Dhaliwal et al., 2021; Russell & Crocker, 2016), is informed by teachers’ interpretation of policy change, and their ability to incorporate it into existing schemas regarding their professional roles and responsibilities. Such research suggests that when policies threaten teachers’ preconceived notions of their professional identities, they are not likely to be effectively implemented. For example, in a study of teachers’ sensemaking regarding disciplinary reform, Russell and Crocker (2016) explain,[Restorative justice] in schools cannot be mandated as a policy change or implemented through training in practices isolated from changes in organisational rules and formative context….[doing so] will disrupt the sensemaking process, [and] unsettle teachers' sense of themselves, resulting in no real change (p. 210). We posit that an important aspect of teachers’ “sense of themselves” is the role of emotion in their work. Understanding teachers' sensemaking about their emotional experiences at work will offer important insight into the potential for restorative justice policies to be implemented. Given the importance of positive student–teacher interactions for both students and teachers, we assert that for interactions to be positive and restorative, policy initiatives must be grounded in a deep understanding of how teachers conceptualize the role of their own emotional experiences during teacher-student interactions. In this study, we explore how nine teachers in urban charter schools make sense of the role of emotions in their professional work. Method Participants and Context After obtaining IRB approval for this study, we used state administrative data to identify charter schools with relatively high rates of exclusionary discipline. After reviewing administrative data, we requested permission from administrators in seven charter schools to recruit teacher participants during the summer and fall of 2021. We received research approval from administrators in three of these schools. Of the administrators who declined participation, several stated that while they supported the project conceptually, they felt the need to protect teachers from additional responsibilities due to the added stressors of the COVID-19 pandemic. We assured participating administrators that teacher participation was completely voluntary and confidential. In schools with administrator approval, teachers were invited to participate in this study through an all-staff email and a follow-up email, sent approximately one month later. Teachers from two of the schools responded to our research invitation. Both schools’ student population was predominantly students of color and both schools were located within the same metropolis area. The first school was part of a larger charter network, established in 1995, and had approximately 350 students in grades 9–12; the second school was a stand-alone charter school, established in 2005, serving approximately 360 students in grades 6–12. Overall, we obtained a final sample of 9 middle and high school teachers, which fits well within recommendations for phenomenological research (Creswell, 2018; Polkinghorne, 1989). Table 1 exhibits our participants’ demographics. Mirroring the teaching staff in many American schools, our sample was predominantly composed of white female teachers.Table 1 Participants Pseudonym Content Area Years of Experience Gender Identity Racial Identity Brittany High school special education 1 Female White Heidi High school science 1 Female White Luke High school ELA 1 Male White Lydia High school math 1 Female White Michael Middle school humanities 1 Male White Rebecca High school ESL 4 Female White Sharon High school history 5 Female African American Mikey Middle school special education 5 Female White Melinda Middle & high school reading specialist 10 Female White Data Collection After providing informed consent, teachers who expressed interest in the study participated in a semi-structured interview conducted on Zoom; interviews lasted between 45 and 60 min. During the interview, we first sought to understand which emotions were most salient in each teachers’ professional experience broadly—not just those experienced during challenging student behavior. In the tradition of phenomenological research, our interview protocol was designed to help teachers consciously attend to their experiences and explore the meaning of them (Creswell, 2018), in this case, their experiences during emotionally-salient interactions with students. In phenomenology, the study of a phenomenon consists both of what is experienced and how it is experienced (Moustakas, 1994). As such, we were interested not only in the valence and intensity of teachers’ emotions, but also in how they interpreted them, including how they made sense of the contexts and circumstances associated with particular emotions. In our interview with each teacher, we asked a series of open-ended questions that addressed our overarching research question. Our full interview protocol is provided in Appendix A. Each interview was transcribed via Zoom, and each transcription was verified by a member of the research team. Each teacher who participated received a $25 gift card. Data Analysis In the tradition of educational phenomenology, we aimed to glean both an understanding of what was experienced and the circumstances under which it was experienced (Creswell, 2018). To do so, we used iterative rounds of coding and thematic analysis to understand our data. Throughout the analytical process, we approached the data from a social constructivist lens, drawing on the work of previous scholars of emotions in education, such as Zembylas (2007) and Boler (2004), who posit that norms regarding what constitutes professional emotional regulation, display, and experience are socially constructed within school organizations. We used both deductive and inductive coding during our analysis. To help orient ourselves to the data, we completed an initial round of structural coding to organize and categorize the data (Saldaña, 2013). During this round of coding, we used deductive codes to identify two types of excerpts relevant for phenomenological analyses: textual and structural excerpts. Textual excerpts would inform our description of teachers’ experiences of emotions during interactions (e.g. the phenomena itself); structural excerpts would inform our understanding of “the conditions, situations, or context in which they experienced the phenomenon” (Creswell, 2007, p. 254). To this end, we first used the codes of emotional interaction and general emotion to bucket excerpts informing the textual description of the phenomenon; that is, excerpts in which teachers described the role of their emotional experiences in their work. Emotion has been defined in many ways, and a precise scientific definition remains up for dispute (Dixon, 2012; Izard, 2010). In this study, we aimed to code very broadly; we included anything that the participants described as a feeling, affective experience, or physiological response to a stimulus in their environments in our coding of “emotion.” Then, we used the codes of teacher identity and school context to bucket excerpts informing our structural description of the phenomenon (i.e., excerpts regarding the factors which shape how teachers’ experience the role of their emotions). These two overall deductive codes were created based on existing literature demonstrating how aspects of teachers’ identities and aspects of workplace conditions and culture can both influence emotional experiences. Using the structural codes, we created summaries for each participant to reflect what they shared about their own identities and how they characterized the conditions in which they worked. Once familiarized with the participants’ contexts, we then returned to the transcripts to conduct open, inductive coding of their emotional experiences (Saldaña, 2013). We considered the types of emotion(s) teachers described, the types of professional interaction or activity within which the emotion was experienced, and the teachers’ perceptions regarding how specific emotions impacted that interaction or activity. In doing so, we sought to understand the essence of the emotional experience (Creswell, 2007). Throughout this process, we recognized that teachers described a wide range of emotions as serving both positive and negative roles during student interactions. We noticed four themes regarding the role of teachers’ emotions during interactions with students: emotions serving a positive role for teachers, positive role for students, negative role for teachers, and negative role for students. Across participants, we found that various aspects of their identities, as well as their perceptions of workplace conditions, affected each of these four potential roles of emotions. As the themes emerged, we considered the boundaries, coherence, and meaningfulness of each potential theme (Braun & Clarke, 2012). We reviewed each transcript individually and documented additional quotes relevant to each theme. Finally, we built a figure to visualize our findings. During all stages of the data analysis, we took steps to increase credibility and trustworthiness. For example, an important aspect of phenomenology is epoche, or the suspension of judgment. The intention of epoche is to arrive at the essence of participants’ experience, rather than our own experiences. To do so, we actively sought not to judge teachers’ attributions for their emotions during student interactions or interrupt their telling of stories to “correct” their terminology, but to understand why and how they came to those attributions. We considered how our own positionalities influenced our analysis, discussing both the ways in which we aimed to bracket (Girogi, 2009; Moustakas, 1994) our own experiences during analysis, as well as the ways in which our own personal and professional identities could support our work (Esteban-Guitart & Moll, 2014). The first author is a white female who was previously a special education teacher in public and charter schools in a large Midwestern city, and in several public institutions in the Northeast; she is now employed as a researcher at a large university. The second author is a Black, female school psychologist currently working in a large, Southeastern school district and pursuing a doctorate in special education. As we engaged with the data, we met on a regular basis to share our experiences with the data and what we were uncovering within the data throughout the coding process. Given the topic of the study, we discussed the emotions we experienced as we encountered the data, what was potentially missing from the data, and how our own personal and professional identities might influence our analysis. Finally, to support the credibility of our findings, we use thick descriptions of our participants and frequent, direct quotations. In the following section, we provide a description of our participants’ phenomenologies of the role of emotions in their work; we aim to refrain from our own commentary or analysis to report the participants’ own experiences. We then situate these phenomenologies within the broader literature on charter schools, emotions, and teacher- student interactions in our discussion. Findings We found that all nine teachers conceptualized their own emotions as highly salient to their work, and as influential in their responses during interactions with students. Although teachers idealized their emotions as a professional tool during student interactions, they recognized that sometimes their emotions functioned as a professional liability. Thus, as shown in Fig. 1, teachers conceptualized their emotions during student interactions as falling on a continuum ranging from liability to tool. In addition, we found that teachers experienced both their own identities and the characteristics of their workplaces as important factors in determining whether their emotions functioned as a liability or a tool during specific teacher-student interactions.Fig. 1 Conceptualizing the Role of Teachers’ Emotions During Teacher-Student Interactions Conceptualizing Emotions as a Professional Tool Across the sample, teachers conceptualized the role of their emotions during teacher-student interactions as potentially serving as a professional tool that they could actively and strategically use to support both their students’ success as well as their own. In regard to student success, teachers intentionally used their emotions during interactions with students to engage students in instruction, to provide appropriate academic support, and to build relationships. With regard to their own success, teachers experienced the role of their emotions during student interactions as a motivator, a catalyst for professional innovation, and a source of data that reinforced their self-efficacy as an educator. Emotions as a Tool to Support Students First, teachers used emotional displays strategically during interactions with students to support students’ engagement with academic content and to motivate them to persist in learning activities. For example, Melinda, a special education teacher, described using her own emotions to challenge a student and encourage him to succeed. She explained,I have the power to change the way that they are seeing themselves in this context…[by] getting very excited, ‘YES!’…or giving a big ‘snaps!’...you keep going, building that positive momentum for them….both of us are super excited and super proud and impressed…we got through this challenge, and it was together. Like Melinda, other teachers described being able to use their natural emotions as a tool. Heidi, a first-year science teacher, stated, “I get really excited about things, and I hope that the students can sense that, or at least…my excitement about what I’m teaching gets me through it, hoping that at least one student’s going to like it.” In fact, sometimes teachers’ naturally experienced negative emotions even served as a tool. For example, Lydia, a first-year math teacher, described how her emotions were a useful source of data regarding the gravity of a situation. She explained how, when a student with a disability in her classroom was having difficulty self-regulating, she immediately called for support rather than trying to de-escalate him on her own, since she didn’t have sufficient training regarding his disability category. She noted, “I was actually really happy with how it went….we got into a more calm place… I think my fear might have positively affected my response because I knew how serious it was.” Lydia believed her decision allowed for the student to be effectively supported and prevented a situation in which she may have responded ineffectively due to her lack of training. Other times, however, teachers strategically expressed specific types of emotions that they knew would benefit the student(s) during the interaction, even if they didn’t naturally feel those emotions inside. For example, Luke, a first year ELA teacher, explained, “[On some days,] you're really tired, but you're gonna pretend to be motivated and excited about this grammar.” Michael, a first-year humanities teacher, explained, “[The students are] just endlessly entertaining and they just do goofy things all the time…so I really try and hold onto [that joy] and try and spread [it back] to them as well, and make school somewhere they're excited to be my classroom, where they're excited to step into.” In these cases, teachers generated emotions they believed would serve students. Teachers also noted that that one specific type of feeling, empathy, functioned as a tool during their interactions with students–whether they experienced it naturally or after emotional re-appraisal. Mikey, in her fifth year of teaching special education, explained,Emotions work for me... when I lead with empathy and when I do lead with inquiry…. this kid is dealing with something, let me pause, let me consider what they're going through, let me consider the fact that they would not be acting this way if they had a better way of communicating… they wouldn't be acting this way if they were able to help it…how can I help? Similarly, Brittany, a first-year special educator, recalled a time when her own empathy served as a tool to help her de-escalate two students. She explained, They're swearing up and up and down a tree [but] instead of me saying, Hey, watch your language, I was like, all right, let me, you know, let me take it back, let me look at them as humans. My empathy definitely took over and I think it was for the better of everyone in the situation… they were able to return to the next class after the bell. Emotions as a Tool to Support Teachers A second way in which teachers conceptualized the role of their own emotions during student interactions was as a tool for their own benefit; that is, they experienced their emotions as reinforcing their own professional success. Experiencing positive emotions during student interactions served as an indicator of their professional competencies, or as a “reward” for their investment. For example, Mikey described seeking out emotional rewards throughout the day, explaining,I feel like: [I’m] in a video game where you're jumping to get a star…whenever there's an interaction, I hit that star… you see the goal and you're kind of trying to tap it… it's this rising up feeling... joy is the emotion I feel again: I'm like ooh, like I feel this burst of strong emotions, like serotonin. Experiencing positive emotions during student interactions motivated teachers to continue to invest in their work. For example, Lydia stated, Feeling I'm making a difference…feeling fulfilled in my job, really helps me to get through those long days you know? It helps me kind of reset at the beginning of each day and try to take each new day as it comes. Because I know that if I do that, I'll have those positive emotions of feeling fulfilled and feeling validated in my hard work. Similarly, Brittany stated, “It's just so rewarding for me to help these kids, to be an advocate for them…to create relationships with those who might not have advocacy at home.” Teachers noted that compared to other professionals, these emotional rewards were particularly important in their role as educators. Heidi explained, “On days where you feel like you have nothing left to give, it's nice to have a purpose… these kids are depending on me, rather than… some other job, where you might just say I’m going to call out.” Like Heidi, many teachers viewed emotions as positive reinforcement for their work. Across the sample, teachers conceptualized the role of their emotions as potentially facilitating both their own success, as well as that of their students. However, despite the potential for emotions to benefit teacher-student interactions, teachers also recognized that their emotions sometimes functioned as a professional liability during student interactions, compromising the success of their students and themselves. Conceptualizing Emotions as a Professional Liability Teachers described how their emotions, when functioning as a liability, had the potential to harm relationships with students, detract from the quality of students’ learning opportunities, and lead to their own burnout from the profession. Teachers’ Emotions as a Liability for Students Teachers described how the emotions they experienced during student interactions sometimes compromised the quality of the learning opportunities to which students were exposed. For example, Heidi described having to pause a lesson and step outside the classroom to compose herself because she felt like she was about to cry. Luke described how his emotions during interactions make him more passive and less engaged with students. He explained, I guess [my negative emotions] will manifest in…more of a checking out…I've never really blown up on a student or anything… I think sometimes my own emotions with this kind of lead me to just being like, I don't want to deal with this right now, I don't want to have to go talk to the student about something so yeah sometimes I'll be a little bit more withdrawn than I would like to be. Several teachers noted how feeling defensive, embarrassed, or vulnerable during student interactions made it difficult for them to understand the underlying causes of student behavior. For example, Michael recalled an interaction in which his feelings of frustration with a student’s lack of progress could have prevented him from identifying new ways to explain the content to her. He explained, “I’m getting very frustrated, but getting frustrated is not going to help the situation, I have to present [as] having patience.” Teachers described having to actively put their emotions aside, or enact different emotions, in order to appropriately interact with students. For example, Rebecca, an ESL teacher with four years of experience, explained, You do have fear [of some students], so you kind of want to just be friendly and get rid of those fears that you may have. But I think part of our work, it is hard and you have to be brave with emotions and… it's kind of about meeting each other halfway. Another emotion that often functioned as a liability during interactions was anxiety, particularly anxiety regarding the ability to manage the classroom. This sometimes resulted in harm during teacher-student interactions: for example, two teachers described witnessing teachers’ fears that they had lost control of their classrooms manifest in racially-biased ways. Mikey explained, There was a lot of conflict…at that school between white teachers and students of color because of…something as basic as how am I expressing my emotions, and what's an appropriate way of expressing my emotions versus for me versus an appropriate way for you….but one [form] of it is this [perceived] need to control the students… these power dynamics. She explained that the shift to restorative justice practices is sometimes challenging for teachers because, “We’re used to this power dynamic where you see the sense that we need to be in control and I think that's why [with the shift to] restorative practice, there is so much skepticism.” Some teachers recognized that their emotions were more likely to be a liability later in the school year when their emotional resources were more drained. Brittany explained, “I think at the beginning of the year, I was a lot better at managing those frustrations. And then now I feel like I've noticed that I've become more [of a] short fuse.” Similarly, Melinda explained, “I'm still in the part of the year where I have enough energy to flip the narrative,” recognizing that later in the school year she often struggled more. Teachers’ Emotions as a Liability for Themselves In addition to potentially compromising student relationships and learning during interactions, teachers recognized that the emotions they experienced during interactions sometimes harmed themselves as well. In particular, when teachers experienced negative emotions during student interactions, this often resulted in subsequent negative emotions about their work, and reduced their confidence, self-efficacy, and motivation. For example, Michael noticed that his confidence dropped when he felt frustrated about an interaction. He explained, There are definitely feelings of insecurity; Wow I was so bad today…I might get another shot to teach more years, but the kids I'm teaching only to do sixth grade once…it really makes me think about [whether] I'm the best person to be in the spot. Similarly, Lydia recalled a time when, I did not have control over the class …[afterwards,] I felt pretty embarrassed, I think, because of all these messages we've been taught and then as a first year teacher…I felt kind of inadequate…I felt vulnerable….in moments where I've been really frustrated and shown it in my class, I've thought afterwards that I was a little embarrassed about that or, I'm glad that my principal wasn't there for that moment! Brittany also described feeling embarrassed about how her emotions manifested during an interaction with a student: “Those frustrations, especially the feeling of disrespect, completely took over me. I raised my voice and I was honestly embarrassed afterwards.” Several teachers shared another way in which their emotions sometimes functioned as a liability for them: because the emotional rewards of positive teacher-student interactions were so compelling and motivating, they struggled to have boundaries about what they were willing to emotionally invest in the job. For example, Rebecca felt that because her job was so emotional and her energy was depleted after work, it became her “whole life.” Since the emotions teachers experienced related to their work led them to work around the clock, many participants described a lack of investment in the self-care and relaxation needed to sustain themselves in the career. For example, Brittany described having to choose between therapy sessions for herself or lesson planning. She viewed self-care as a heavy ethical decision, noting the[It’s a] moral [question] of: do I do something for myself, but then [making sure] it doesn't jeopardize what I'm able to do for the kids. And there's that balance between, while I can't save the kids, I'm here to give them resources and put them through the right path in life and teach them. Sharon, who had been teaching for four years, noted how she had to learn to take care of herself after the emotions of the job during the first few years resulted in her getting to an unhealthy point,My biggest mistake was thinking my job would be my fulfillment…This job is not your sense of life, your job is not that. You are here to serve people, but these people don't make you who you are. Contextual Influences: Teacher Identity within Workplace Conditions As described above, teachers conceptualized the role of their emotions during interactions with students as falling on a continuum, ranging from their emotions functioning as a tool to their emotions functioning as a liability. In explaining where on this continuum their emotions fell during a specific interaction, the contextual influences associated with teachers’ own identities and their workplace conditions became evident within our data. First, teachers recognized how their identities influenced the role that their emotions served during specific interactions. Many of the teachers described how their identities as learners, (i.e., identities related to their own educational experiences as students), shaped the role of their emotions during interactions. They noted that both similarities and differences between their own learner identities and their students’ learner identities shaped the function of emotions during the interaction. For example, Mikey explained how her identity supported the role of her emotions as a tool,Part of my life history and why I specialized in [special education] in the first place is because …of my past as a learner… which then gives me my own perspective…Students with disabilities struggle emotionally. There's always going to be that emotional piece, you're not able to show a bit of your vulnerability and be like yeah, I get frustrated too. Sharon recognized how her own experience of ADHD as a learner helped her to be a more patient and creative problem solver during interactions in which her students exhibited behaviors and emotions associated with ADHD. She explained,The impulsive speech one is really hard, because I have impulsive speech issues, so I try really hard to use the impulsive speech moments to keep the class moving forward and instead of it being a moment of, be quiet. Sometimes, it has to be but, for the most part, I try to be like, Okay that's a good point, let's keep going with that, instead of making someone ashamed for calling out. Luke described how his own experiences with anxiety and depression make him especially empathetic toward students whose learning experiences were impacted by mental health challenges. He explained,I was the kid who was hanging out in my English teacher’s classroom during lunch…being sad. So I feel like there are some kids…who have issues with anxiety and they will end up to talking to me and you know, [I] tell them about how I've also had issues with anxiety and depression personally… that helps me, I think kind of both to know a little bit of what they're going through and kind of relate to them. Other teachers recognized that differences between their students’ learner identities and their own learning identity had the potential to positively or negatively shape the role their emotions played during interactions. For example, some teachers mentioned how the expectations that had been set for them as a learner sometimes impacted the emotions they experienced during interactions with their students. Michael explained, “I went to private schools growing up, so there were just not as many kids with learning disabilities, and it was just a culture of being high achieving.” He described being aware of the ways that his natural emotions about what it meant to be “high achieving” could function as a liability, and he was actively engaging in efforts to be “more understanding, more patient of… kids who really struggle because of the learning disabilities they have, not because of a lack of motivation.” Similarly, Rebecca described how part of her learner identity had included the assumption from a young age that she would go to college; this caused her to experience disappointment during interactions with students who didn’t want to go to college. She recognized that this disappointment could function as a liability for her students, negatively impacting her relationships and rapport with students. Another aspect of teachers’ identities that shaped the function of their emotions was their racial identity. Several of the white participants described their whiteness as an emotional liability during interactions with students. For example, Heidi described how her students repeatedly called her racist when she would tell them to go back to their seats and start working on an assignment. She noted that the first time this happened she had felt “disarmed.” However, when it continued to happen, she learned to pause, re-appraise her initial emotions, and conclude that although students’ comments were directed at her, they were often grappling with racial injustices within the broader educational system, of which her classroom was just one small piece. Yet, despite this being a common experience, she did not directly acknowledge or explicitly address the systemic roots of these racial dynamics with her students or with her colleagues. She explained, “Sometimes I still get really like, Wow that was super personal attack on me, which hurts, but they’re kids, so I’m mostly trying not to take things personally.” Similarly, Michael recalled a time when a student had called him racist for asking the student to be quiet. He explained how in the moment, he wanted to defend himself, but he decided not to pause the lesson or show emotion. He explained, “Being called racist is…really uncomfortable, you’re like, Oh what did I say?! But I have to respond calm and even-keeled because anything else, I think, would have just resulted in making the situation worse.” Like Heidi, he did not mention any direct attempts to address these racial dynamics with his students or with colleagues. Melinda, another white teacher, noted that when students made race-based comments during interactions, she would feel “an initial shock …a paralyzed sort of…the fight or flight freeze, like more like a freeze mentality of, like I know I need to respond and do something, and I feel like I don't have words or I don't have something to say.” These white teachers experienced their emotions surrounding race, as influenced by their own racial identities, as a potential liability during interactions with students. They noted how such interactions raised a variety of complex emotions, including discomfort, guilt, fear, and frustration. They recognized that such emotions might lead the teacher to try and re-establish their own authority in oppressive ways, or end the interaction preemptively, rather than addressing the causes and complexity of students’ experiences of racism. Sharon, an African American teacher, described how she had invested significant time and effort in developing and implementing new social studies curriculum that was more inclusive of diverse and critical historical perspectives, informed by both her own experiences and her desire to continue learning about other cultures. Her pride in implementing this curriculum was a key source of motivation to invest in her work and in her students. However, despite this pride and motivation, she experienced an uphill battle against pervasive whiteness in her school. She explained, It’s just really hard. It just seems, no matter where I go, there's a favorite kind of teacher and that's usually a young, unwed, white, type-A person, who can take on lots of responsibilities and doesn't need any extra support or guidance. That format of choosing a leader or choosing people who have talent, to me, is kind of ableist, to only focus on people who perform in the way that you think is worthy. So, it's added to a lot of imposter syndrome that I'm working through right now. As shown in this quote, Sharon’s emotional experiences were informed not just by her individual identity, but by the positionality of her identity within the broader context of her schools’ organizational conditions and cultures; in this case, a culture characterized by pervasive whiteness. Across the sample, all nine teachers described their workplace conditions as increasing the likelihood that their emotions functioned as a liability, not a tool. Many of the teachers described feeling stressed or anxious daily due to not having the time or resources they needed to fulfill their professional responsibilities. Because of the overwhelming demands of their work and the stress they were experiencing, their emotional resources were limited, and they weren’t always able to regulate their emotions consistently during student interactions. When teachers interacted with students, the amount of cognitive and emotional resources they could bring to bear on these interactions was sometimes limited. They recognized that if their emotional resources were drained, interactions with students were less likely to be restorative. For example, Sharon explained,[When teachers] seem to not have good emotional control…that's indicative of the lack of self-care that they're able to do, or the amount of powerlessness that they feel. Overall, teachers recounted toxic workplace cultures in which teachers were exhausted, isolated, and had few moments during the day to plan, reflect, or take care of themselves. For example, teachers described how being required to take on responsibilities for which they had not been trained cost them valuable emotional resources. Both Luke and Brittany mentioned being responsible for consistently covering Spanish classes due to teacher absences, even though neither spoke Spanish. Luke noted that teachers were also taking on additional emotional demands because “our school has been really short staffed this year…we haven't had any counselors this whole year.” Heidi explained, “We are the subs, we are everything because we don't have those... you don't even get your planning periods [to] kind of breathe and reset.” Teachers across the sample experienced their emotions during specific student interactions as informed by a backdrop of stress and overwhelm due to this imbalance between resources and demands in their workplaces. Teachers also noted that establishing restorative and inclusive practices within their schools was emotionally demanding. Sharon shared how challenging it was to create inclusive systems within the cultural norms and working conditions of her school: In this culture, our very white Anglo-Saxon Protestant culture, the idea that a child's heard while you're talking is [considered] ridiculous. And so I'm trying to move away from the urge to associate silence with learning. And I didn't come from that kind of background so it's an adjustment for me, I think, to get more comfortable with [the idea] that there might be murmurs, there might be talking…the challenge and the difficult emotion [of this shift] is: how do I handle [situations if] students aren't using the talk/work time properly, how do I address it? Lydia, a white teacher, described the additional emotional demands of restorative practices: As a school wide thing, they have moved away from merits, demerits, things like that… to completely restorative justice model, which I think is really great…I can see how it's going to work. I think it can work, but this is a lot on me! It feels like I'm supposed to be the math teacher and the counselor at the same time. I just, I don’t know how to do that. Although she wanted to engage in restorative justice practices, it was not always easy to do so. Because she did not feel fully equipped by her school to respond to all her students’ needs, her emotional overwhelm could sometimes function as a liability during challenging student interactions. Discussion In our investigation of how teachers in urban charter schools conceptualized the role of their emotions while teaching, we found that teachers made sense of the role of their emotions as highly salient and impactful in their interactions with students. From a phenomenological standpoint, we found that the “essence” of teachers’ emotional experiences was neither inherently good nor bad: they made sense of emotions as functioning in both useful and problematic ways in teacher-student interactions. Often, teachers experienced their emotions as a tool: teachers were able to use their emotions to motivate and encourage their students' learning in the classroom, build relationships with students, and reinforce their own commitment to their work. Conversely, teachers recognized that their negative emotions may have harmful or punitive effects on students’ experiences in school, resulting in disengagement or punitive responses to students, reduced self-efficacy, and emotional exhaustion. Furthermore, both teacher identity and workplace conditions shaped how teachers’ emotions functioned within specific interactions. By employing a phenomenological perspective, we first sought an understanding of how teachers made sense of their own emotional experiences; like any of our experiences, our participants’ experiences were deeply shaped by their contexts, life experiences, and identities. Now, as researchers, we reflect on our participants’ phenomenologies, through the lens of our own life experiences and identities, including our understanding of existing research on the role of emotions, identity, and workplace conditions in the work of teachers. We organize our discussion around three reflective questions. Which Emotions Actually Function as Tools and Liabilities? First, we reflect on how to support teachers in recognizing when their emotions are serving or harming their students, and themselves. While we recognize our participants’ understanding that emotions have the power to function as a tool or liability, we notice that many of the participants held assumptions about the nature of certain types of emotions—e.g., that anger is a liability or that empathy is productive. However, research on teachers’ emotions suggests that emotions that teachers in this study characterized as a tool may actually function as a liability, and vice versa. For example, feelings of care or empathy from white teachers about students of color may not serve students or teachers, if rooted in deficit notions of students’ experiences (Matias & Zembylas, 2014); these emotions may lead to reduced inclusion, reduced instructional rigor, or the reinforcement of biases (Ruppar et al., 2018; Neary, 2020). Similarly, the feelings of pride that some white teachers recognize as serving their work may in fact reinforce concepts of white saviorism that are ultimately harmful for their students (Grant, 2019, Matias, 2016). On the other hand, while many teachers recognize that expressing feelings of frustration/anger could function as a liability, such emotions may also serve as a tool when they reorient teachers to their values and moral responsibilities, or motivate them to act against injustices experienced by their students or themselves (Liljestrom et al., 2007). Teachers in our sample aspired for their emotions to serve as a tool during interactions, and white teachers noted how their own biases and the biases of their colleagues had harmful effects on students. However, they did not always feel comfortable or equipped to address these issues. As researchers, we see this as an urgent call to explicitly support white teachers in recognizing which emotions are actually a tool or a liability, and in regulating their emotional expression accordingly. Instructional leaders need to establish learning opportunities to help white teachers understand and acknowledge the role of their own emotions in reinforcing White privilege within their schools (Matias, 2014). White teachers can deepen their understanding of their position of privilege by engaging in interrogation and examination of how whiteness manifests in their own emotional patterns (Boucher, 2016). Critical reflection on the role of emotions is particularly urgent for any teacher who choses to work in an urban charter communitys where their identity carries racial, linguistic, or economic privilege; without reflection, assumptions about the nature of emotions may result in harm inflicted on students or communities. And, given the cultural diversity within racial groups, it is crucial for teachers of all racial identities to critically examine how their own racial identity shapes their emotional responses to students, even if they share the same racial identity of their students. Regardless of their racial identity, when teachers become more transparent and proactive in acknowledging how their own identity impacts their emotions, proactive dialogue with their peers can emerge. With an increase in knowledge of, and engagement in, culturally responsive teaching practices and cultural competence, teachers communicate respect and value towards the sociocultural experiences with which students enter the classroom. In this context, teachers can achieve solidarity, which Boucher (2016) defines as allyship with students built through advocacy and connectedness with the students and the community. Rather than avoiding the topic, teachers who are able to critically reflect on the role of their emotions are more likely to allow the types of conversations about race and culture with students, families, and colleagues that promote their own learning and self-efficacy (NCTSN, 2017). These exchanges might take place in structured or unstructured ways such as parent/student–teacher conferences, parent nights, community events, and staff team building activities. Can Teachers Navigate Emotions Effectively in Unjust and Unsustainable Working Conditions? This need for critical reflection leads to the second question raised by our analysis: can teachers navigate emotions effectively in unjust and unsustainable working conditions? Jupp and Slattery (2012) describe teachers’ own “professional identifications” as “social and historical processes of self-authorship” (p. 287). That is to say: teachers’ own narratives can be changed through critical self-reflection, and successful charter school teachers engage in reflection on the role of emotions in their work (Boucher, 2016). But while teachers have agency to make ethical choices about how they interact with students, regardless of where they are, there are also system-level constraints that impact teachers’ emotional interactions with students. First, although norms regarding what emotions are appropriate to express or not appropriate vary culturally (Mesquita & Walker, 2003), teachers’ emotional expression in schools is often reinforced by implicit emotional display rules, e.g. unspoken norms about what emotions are professionally acceptable to display and what emotions are not (Stark & Bettini, 2021). Because teachers are socialized into these norms by observing their peers, usually without explicit training, the ways that teachers’ own identities intersect with the broader cultural norms of the school to shape their perceptions of emotion are often uninterrogated. Thus, if a teacher works in a school organization that only permits emotional expression aligned with emotional norms deriving from whiteness, gaslights or undermines the range of emotions that teachers experience, or ignores the need for teachers’ own emotional development, teachers are more likely to engage in the emotional demands of their work in ways that are not conductive to themselves or their students. It is therefore crucial that school leaders address organization-level emotional norms in their schools, and engage in work to understand which types of emotional displays (or suppressions) serve as a tool or liability within their communities. A key aspect of such work is acknowledging and reducing the unique emotional responsibilities and demands placed on teachers of color in schools. Previous research has clearly exemplified the wide range of disproportionate emotional responsibilities placed on teachers of color, including expectations that they champion efforts to implement culturally relevant pedagogy appropriately (Borrero et al., 2016), address stereotype threat for students and teachers of color (Milner & Hoy, 2003), translate for white teachers (Achinstein & Ogawa, 2011), serve as a student role model (Maylor, 2009), or respond to the behavior of white teachers’ students (Bristol & Goings, 2019; Bristol & Mentor, 2018). In addition to addressing norms regarding emotional expression, school leaders need to actively acknowledge these other forms of emotional demands, and shift responsibilities among staff proactively. School leaders and teacher educators may also consider providing affinity spaces/groups, which can facilitate vulnerability and trust among teachers of color and help them navigate the different emotional demands they may experience associated with their identity (Kohli, 2012). Regardless of their individual identities, all the participants in our study experienced the working conditions in their schools as stressful; this general level of emotional burden left teachers with few resources to navigate emotions arising from individual interactions with students. Without systemic change in the balance of demands and resources to address them, teachers will have no bandwidth for individual or collective critical reflection and development regarding the role of emotions in their work. We recognize that some workplace conditions, such as staffing shortages, may be beyond the control of school-level administrators, and may be exacerbated by COVID-19. However, it is important that as school leaders fill positions within their schools, they set a clear vision for each person’s roles and responsibilities within the organization and acknowledge emotional responsibilities within this vision. And, as leaders consider the implementation of new policies, initiatives, and “innovations,” it is important that they explicitly discuss the emotional demands associated with the types of student–teacher interactions addressed by these policies and initiatives. For example, if school leaders aim to implement policy aimed to reduce suspensions due to perceived disrespect, school leaders should consider holding professional development sessions geared towards building all teachers’ capacity for emotional regulation and emotional de-escalation. Given the widespread experience of stress and burnout evidenced within this sample, professional development opportunities that support teachers’ stress management may be useful, as they provide teachers with time and space to center themselves and connect with each other. For example, CARE for Teachers, a mindfulness-based professional development program designed to support teachers’ positive interactions with students, has demonstrated evidence of effectiveness in randomized trials (Jennings et al., 2017, 2019). Providing opportunities to learn about such practices communicates to teachers that school leaders value their emotional health: it shows that they recognize teachers’ engagement in emotional regulation as an important aspect of their professional work, and they appreciation teachers’ efforts to engage in this work. Professional learning opportunities can also serve as a maintenance tool by reinforcing or supporting ongoing school initiatives such as restorative practices, rather than introducing a new concept each time. Because teachers in this study indicated a need for ongoing support with changes made at the school level, it is important for school leaders to follow up initial professional development sessions with ongoing opportunities for professional learning, as suggested in Darling-Hammond et al.’s (2017) recent review of effective PD practices. Does The Call for Innovation Undermine the Complexity of this Work? Finally, we come to our third and final reflection question, which is how we can effectively unpack the complexity of emotions within cultures created with a mandate to “innovate” and to do under accountability systems with fast timelines. Both schools in this study were in the process of shifting toward more restorative practices; like these two schools, leaders across many states and districts have demonstrated a commitment to increasing positive, restorative teacher-student interactions, through reforms to suspension policies (Department of Education and Secondary Education, 2020b), investments in professional development on trauma-informed practices (e.g., McIntyre et al., 2019), and initiatives to increase restorative practices (Velez et al., 2020). Yet teachers in both schools noted the messiness and complications of these transitions. Thus, our findings suggest that taking the time to understand how teachers experience their work is crucial to designing and implementing effective policies, as policy changes on their own are likely insufficient to improve teacher-student interactions (e.g., reducing disproportionate disciplinary decisions, McIntosh et al., 2018, 2020). Teachers are what Lipsky (2010) called “street-level bureaucrats:” public servants whose experiences and interpretation of policy is essential to its implementation, and sensemaking theory explains that educators’ subjective interpretations of their experiences inform how they implement policies (Coburn, 2001, 2005; Spillane 2001). As such, unless informed by research exploring how teachers make sense of their own experiences within schools, attempts to innovate school practice and policy may not have the intended consequences we seek. If we expect charter schools to innovate in ways that are ethical for students, teachers, and families, we have to recognize that shifts in teacher-student interactions, and the movement toward restorative practices, may not be easy to measure on quick timelines or in standardized ways; we need to continue to use a wide variety of methodologies to understand teachers’ experiences within schools, and their implications for policy. In the following section, we provide recommendations for future research in this area. Limitations and Directions for Future Research As an exploratory, but limited, study about how teachers conceptualize the role of their emotions, our study points to several important directions for future research. First, although our sample size fits within recommendations for phenomenological research, we had a relatively small sample size. A larger study with more participants could help researchers identify further nuances in how teachers conceptualize the role of their emotions, and how workplace conditions and identity factor into these roles. In addition, teachers in this study represented two different urban charter schools, each with a unique culture and governing body. We did not gather information regarding the establishment of these charter schools, or the nature of the politics of their establishment or presence within their neighborhoods. These spatio-political histories likely inform the emotional cultures of the school, and the nature of interactions among teachers and students in the schools; future research could involve longitudinal data collection or better situate teachers’ current experiences within the histories of their schools. Importantly, the juxtaposition of teachers’ external displays of emotions versus their internal reality represents conscious choices teachers made to avoid the liability of emotion during interactions with students. Future research could focus more closely on how emotional and social norms within specific charter schools impact teachers’ choices to display or hide specific emotions (e.g., the display rules of the school, Stark & Bettini, 2021). Research could also examine whether teachers in multiple school settings within one district or charter network (e.g., teachers across multiple schools who follow a single set of policies and procedures) have similar or distinct conceptions of the role of their emotions during teacher-student interactions. Our study was also limited in that we only collected data from teachers at one point in time—this was due to budget constraints as well as the challenges of collecting data from teachers during the COVID-19 crisis. In future research, scholars could conduct longitudinal interviews or surveys to see how their experiences shift over time. Finally, future research should explore how intersectional aspects of teachers’ identities impact the function of their emotions during specific interactions. Although teachers shared rich data with us, participants likely experienced a broader range of socially undesirable emotions, such as fear, disgust, embarrassment, and anger, than they shared with us, due to social desirability bias. Although our data provided examples of how teachers’ learner identities served as both a tool and a liability during interactions, future research could examine how the intersection of teachers’ learner identities and other identities, such as gender, age, or socioeconomic status, impacts the role of their emotions during interactions. Despite these limitations, this exploratory study adds to the growing body of research regarding the importance and relevance of teachers’ emotional experiences at work, for both teachers and students. Given the implications of such research for the intersection of policy and practice, we urge future researchers to continue examining how teachers in urban charter schools made sense of the role of their emotions in their work. Understanding how teachers conceptualize the role of their workplace emotions helps school leaders and researchers develop policies and interventions to support teachers in effectively navigating the emotional dimensions of their work. Appendix A: Semi-Structured Interview Protocol Intro: Thanks so much for taking the time to meet with me today. I want to start by introducing myself. I just finished my doctorate at [university] and now I work at [university] as a researcher. I was previously a special ed teacher in [city]. In my research, I look at how all the emotions that teachers experience as part of their work affect them and their students. As you know, teaching raises a lot of emotions and these emotions can really influence our work, but sometimes we don’t have the time or space to reflect on them. I’m interested in understanding how educators can use those emotions to serve their students and themselves. So, some of these questions might be things you think about every day, and some you might never think about. There’s no wrong answers, I just want to understand your experiences.First of all, can you tell me what your current role is, and what year of teaching this is for you? When you think about your job as a teacher, what emotions come to mind? Can you give me an example of when you think your emotions supported your work as a teacher? Can you give me an example of when you think your emotions got in the way of your work as a teacher? Thank you so much for sharing these experiences with me. I really appreciate hearing these examples, because emotions are so important, and they impact so much of the work we do as educators, but there’s not a ton of research about them. In this study, because of my own background in special education, I’m particularly interested in teachers’ emotions when interacting with students with disabilities. So first of all, can you tell me whether you interact with students with disabilities in your role? How so? Can you think about a time when you had an interaction with a student with a disability which made you feel a strong emotion?Tell me about that situation? What were you feeling? Why do you think you were feeling that way? What happened as a result? Now, I want you to think about a different incident with a student where you also felt a strong emotion, but a different kind of emotion… [same questions] [If none of the examples are about behavior]: One of the complex aspects of working with students with disabilities is responding to challenging behavior. Can you think of a time when the behavior of a student with disabilities raised strong emotions for you? What emotion did you experience? Why do you think you felt that way? What happened as a result? You could also share about any other student interaction that brought up strong emotions for you. Sometimes educators have to display different emotions externally than what they actually feel internally, for professional purposes. This is called emotional labor. Can you think of a time when you were interacting with a student with a disability and you had to show a different emotion externally than you were feeling inside?Tell me about that situation? What were you feeling? Why do you think you were feeling that way? What happened as a result? At the beginning of the interview, you said you’ve been teaching for ____ years. Do you think the way you express your emotions with students has changed since you started teaching? How long have you been at your current school? When you think about your school in general, how do you think emotions play the ways that staff respond to the behavior or students with disabilities at your school? Can you give an example? One of the aspects of teaching that raises a lot of emotions for many teachers is managing challenging behavior. Charter schools like the one you work at are known for having very rigid behavior response systems, which are designed to be very consistent. Do you think teachers’ emotions ever affect how staff respond to students’ behaviors at your school? Can you give me an example from your own experience, or your observations of a colleague? What advice would you give to new teachers in regard to navigating their own emotions? The interview is confidential, but if I publish I need to include information about the sample. Usually researchers report the gender and race of their participants. So, I would like to ask you, how would you identify your gender and race? Finally, would you like to see a copy of the transcript or the manuscript before I publish it? I like to know so that I can be sure to follow up. Thanks! Declarations Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Achinstein, B., & Ogawa, R. T. (2011). Change (d) agents: School contexts and the cultural/professional roles of new teachers of Mexican descent. Teachers College Record, 113(11), 2503–2551. 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==== Front Comput Math Organ Theory Comput Math Organ Theory Computational and Mathematical Organization Theory 1381-298X 1572-9346 Springer US New York 9362 10.1007/s10588-022-09362-3 S.I. : Ground Truth: in silico Social Science (GTIS3) Does big data serve policy? Not without context. An experiment with in silico social science Graziul Chris 1Chris Graziul is a data scientist for the Urban Resiliency Initiative in the Department of Comparative Human Development at the University of Chicago. His research integrates diverse theoretical approaches and methodological techniques to better understand the mechanisms that structure social systems, especially the co-constitution of physical context and social behavior. Belikov Alexander 1Alexander Belikov is a visiting scholar at Knowledge Lab at the University of Chicago (and the Head of Data Science at Hello Watt, Paris). In his research he merges computational techniques, such as network science, natural languages processing, neural networks and graphical models to identify universal patterns in social phenomena. Chattopadyay Ishanu 1Ishanu Chattopadyay is an Assistant Professor of Medicine at the University of Chicago, specializing in Machine Learning, AI and data science. Professor Chattopadhyay’s research focuses on the developing core algorithmic principles for modeling complex systems. As the Principle Investigator of several DARPA programs and the winner of the 2020 DARPA Young Faculty Award, Dr. Chattopadhyay’s interests span complex phenomena in biology, clinical decision-making, epidemiology, and social interactions. The Chattopadhyay laboratory focuses on the design of learning algorithms that push the limits of the current data science revolution. His work resides at the cusp of artificial intelligence, statistical theory, formal languages, dynamical systems, and machine learning; aiming to formulate modeling approaches that work in the absence of subject matter experts, hopefully answering questions that we have not yet thought to ask. Chen Ziwen 1Ziwen Chen is a MA student in the Computational Social Science program at the University of Chicago. Her research focuses on using large-scale data and advanced computational methods to study complex human behaviors, including urban mobility, digital culture, and business innovation. Fang Hongbo 2Hongbo Fang is a PhD student in the Institute for Software Research at Carnegie Mellon University. His research focuses on the empirical study of online collaboration and social capital in the context of online interaction, and aims to understand the logic of internet-based voluntary participation and collaborative behaviors. Before joining CMU, he obtained his bachelor degree in computer science at Zhejiang University in 2019. Girdhar Anuraag 1Anuraag Girdhar is an MA student in the Computational Social Science Program at the University of Chicago. His research uses discussion-based games to explore the emergent properties of social network structure, including political polarization, social theory of mind, and the wisdom of the crowd. Jia Xiaoshuang 3Xiaoshuang Jia is a Ph.D. student in the School of Sociology and Anthropology at Sun Yat-sen University. She was a visiting scholar at Knowledge Lab at the University of Chicago during December 2017—December 2018. In her research, she uses computational methods such as machine learning and social network analysis to understand social stratification and social structure. She also explores advanced techniques to do causal inference while taking the heterogeneity of population into account. Krafft P. M. 4P. M. Krafft is a Senior Lecturer and MA Internet Equalities Course Leader at the University of the Arts London Creative Computing Institute. Dr. Krafft’s research, teaching, and organizing aim to bridge computing, the social sciences, and public interest sector work towards the goals of social responsibility and social justice. Dr. Krafft pursues multiple programs towards this end. Much of Dr. Krafft’s research centers around using mixed-methods social data science and participatory action research to study beliefs, ideology, and institutions in the information society, with a special focus on these topics in relation to artificial intelligence (AI) and other information systems. Dr. Krafft also conducts research in computer science and cognitive science with contributions to the areas of distributed AI, multiagent systems, human–computer interaction (HCI), and Bayesian modeling. Kleiman-Weiner Max 56Max Kleiman-Weiner is Co-founder and CEO of Common Sense Machines. He was previously a Data Science Institute Fellow at Harvard and completed his Ph.D. in Brain and Cognitive Sciences at MIT in 2018. His research focuses on reverse-engineering the way the people build and use mental models of objects, places, and agents. He has focused on social aspects of human cognition and machine learning including cooperation, coordination, competition, social learning, and morality. Lewis Candice 1Candice Lewis is the Assistant Director of Knowledge Lab at the University of Chicago. She completed her Ph.D. in genetics from Penn State University in 2010. Dr. Lewis has worked on broadening participation and development of education programs at the University of Chicago for a decade. Liang Chen 1Chen Liang is a MA student in the Computational Social Science Program at the University of Chicago. She graduated from the University of Michigan with a major in public policy. Her research focuses on using social network analysis and natural language processing techniques to understand the political polarization among policy experts in the United States. Muchovej John 56John Muchovej is a Research Assistant in the Computation, Cognition, and Development Lab at Harvard, where he focuses on how people develop their common-sense understanding of the world. Previously, he was an undergraduate at the University of Central Florida and an RA at MIT. Past and current research focuses on using computational tools to better understand how we summarize extract intent from language and action observation and how people drastically narrow solution spaces in question-answering and creative thinking. Vientós Alejandro 57Alejandro Vientós is a PhD student at Rutgers-Newark working in the CoDaS Lab. Past and current work centers around social inference, trust, and cooperation in multi-agent, incomplete information settings. Research interests include the design of games or mechanisms where cooperation is both a stable and optimal strategy and systems for incrementally converging to such strategies in said domains. Young Meg 8Meg Young is a postdoctoral fellow at Cornell Tech as part of the Digital Life Initiative in New York City. She uses ethnographic methods to study government technology. Much of her work explores artificial intelligence and public policy, with a focus on public–private partnerships, procurement, and accountability. Her other work explores artificial intelligence research and development through an organizational lens. She completed her PhD from University of Washington Information School. http://orcid.org/0000-0001-9838-0707 Evans James [email protected] 19James Evans is Max Palevsky Professor of Sociology, Director of Knowledge Lab, and Faculty Director of Computational Social Science at the University of Chicago and External Faculty at the Santa Fe Institute. He is Editor of the new Journal of Social Computing (IEEE). His research uses large-scale data, machine learning and generative models to understand how collectives think and what they know. This involves inquiry into the emergence of ideas, shared patterns of reasoning, and processes of attention, communication, agreement, and certainty. Thinking and knowing collectives like science, Wikipedia or the Web involve complex networks of diverse human and machine intelligences, collaborating and competing to achieve overlapping aims. Much of Evans’ work has investigated modern science and technology to identify collective biases, generate new leads taking these into account, and imagine alternative discovery regimes. Evans also explores thinking and knowing in other domains ranging from political ideology to popular culture. 1 grid.170205.1 0000 0004 1936 7822 University of Chicago, Chicago, USA 2 grid.147455.6 0000 0001 2097 0344 Carnegie Mellon University, Pittsburgh, USA 3 grid.12981.33 0000 0001 2360 039X Sun Yat-sen University, Guangzhou, China 4 grid.4991.5 0000 0004 1936 8948 University of Oxford, Oxford, England 5 grid.116068.8 0000 0001 2341 2786 MIT, Cambridge, USA 6 grid.38142.3c 000000041936754X Harvard University, Cambridge, USA 7 grid.430387.b 0000 0004 1936 8796 Rutgers University, New Brunswick, USA 8 grid.5386.8 000000041936877X Cornell University, Ithaca, USA 9 grid.209665.e 0000 0001 1941 1940 Santa Fe Institute, Santa Fe, USA 30 11 2022 132 24 5 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The DARPA Ground Truth project sought to evaluate social science by constructing four varied simulated social worlds with hidden causality and unleashed teams of scientists to collect data, discover their causal structure, predict their future, and prescribe policies to create desired outcomes. This large-scale, long-term experiment of in silico social science, about which the ground truth of simulated worlds was known, but not by us, reveals the limits of contemporary quantitative social science methodology. First, problem solving without a shared ontology—in which many world characteristics remain existentially uncertain—poses strong limits to quantitative analysis even when scientists share a common task, and suggests how they could become insurmountable without it. Second, data labels biased the associations our analysts made and assumptions they employed, often away from the simulated causal processes those labels signified, suggesting limits on the degree to which analytic concepts developed in one domain may port to others. Third, the current standard for computational social science publication is a demonstration of novel causes, but this limits the relevance of models to solve problems and propose policies that benefit from the simpler and less surprising answers associated with most important causes, or the combination of all causes. Fourth, most singular quantitative methods applied on their own did not help to solve most analytical challenges, and we explored a range of established and emerging methods, including probabilistic programming, deep neural networks, systems of predictive probabilistic finite state machines, and more to achieve plausible solutions. However, despite these limitations common to the current practice of computational social science, we find on the positive side that even imperfect knowledge can be sufficient to identify robust prediction if a more pluralistic approach is applied. Applying competing approaches by distinct subteams, including at one point the vast TopCoder.com global community of problem solvers, enabled discovery of many aspects of the relevant structure underlying worlds that singular methods could not. Together, these lessons suggest how different a policy-oriented computational social science would be than the computational social science we have inherited. Computational social science that serves policy would need to endure more failure, sustain more diversity, maintain more uncertainty, and allow for more complexity than current institutions support. Keywords Computational social science Simulated societies Policy Quantitative social science Machine learning Deep learning Simulation http://dx.doi.org/10.13039/100000185 Defense Advanced Research Projects Agency HR00111820006 ==== Body pmcLet the expedition begin The DARPA Ground Truth (GT) project provided an abundance of novel and unexpected opportunities for participants to select and implement similarly novel methodological approaches. Its stated purpose was to test the viability of using simulated social systems to conduct productive social scientific research. To advance this goal, hundreds of researchers and key support personnel participated in the GT project in various capacities. The project was highly structured in how researchers were organized into groups, how these groups did (or did not) communicate, what information was (or was not) provided to each group, and what problems each group had to solve. Otherwise, participants were free to choose how to achieve their goals. See Fig. 1 for an overview.Fig. 1 DARPA ground truth project collaborative research strategy Four worlds The formal structure of the GT project, in terms of research teams and their roles, involved the creation of one simulated social system by each of four simulation teams (TA1A-D), which our team affectionately titled “Urban World”, “Power World”, “Disaster World” and “Conflict World”. These four “worlds” were then explored and studied by two different research teams (TA2A-B) The research teams who explored and studied these worlds had no knowledge about the simulator teams who created them nor, for the first year, each other. Our team—one of the research teams—employed a pluralistic approach that sought to engage many possible methods and models. The other research team focused on a mixed methods design, applying and extending advances in causal analysis, conducting sociologically informed modeling, and using agent-based modeling to replicate phenomena of interest. A seventh testing and evaluation team (T&E) provided guidance to those building the simulations about the substantive features of each world, determined what data could be shared by simulation teams with research teams, and evaluated the performance of both simulators and researchers. Within this structure, T&E was understood to represent the research interests and intended goals of DARPA as the organization funding this research. Finally, an eighth team from the Pacific Northwest National Laboratory was assigned to replicate results submitted by the research teams during the final phase of the GT project. As can be read in other papers of this issue of CMOT, the four worlds were varied in substance and mechanism. “Urban world” was created by a simulation team of quantitative geographers from George Washington University on the street grid of a modern city and was powered by an agent-based model that balanced personal preferences and exigencies of locations, such as work, recreation sites, restaurants and home. Individuals had money, made friends, and could eventually contract disease, and research tasks surrounded the creation of more (or less!) income equity, more social connections, and lower morbidity. “Power world” was created by an industry-led engineering team at Raytheon in collaboration with social scientists based on principles of social groups and complex collective behavior. Also powered by an agent-based model, Power World involved regional elections and policies, group competition, and group-acquired income. Individuals exhibited levels of happiness, and research tasks involved the creation of policies that increased happiness while achieving particular election outcomes and other group success criteria. “Disaster World” was created by machine learning and artificial intelligence researchers from USC’s Institute for Creative Technologies who developed an agent-based model of human behavior in response to risks associated with a hurricane, which was driven probabilistically with partially observable Markov decision processes (POMDPs). Disaster World inhabitants maximized their “reward” (i.e. personal health status, the health status of family members, etc.) by reacting to perceived risks and realized outcomes associated with different courses of action vis-a-vis hurricane impact. “Conflict World” was created by a team from Wright State research institute in partnership with experienced intelligence analysts to generate an agent-based model that reflected a state in crisis, with civil conflict, violent insurgency, food shortages and popular unrest. Their model represented the synthesis of multiple types of perspectives about the state of the world, possible courses of action, and the role of path dependency. Its defining feature was a simulation where actors’ preferences and choices responded to a dynamic shared environment. The GT project timeline consisted of 30 months of activities organized around three project phases that each consisted of three tasks. These tasks were formally labeled Explain, Predict, and Prescribe, corresponding to the type of questions or problems research teams were expected to answer or solve. While these tasks were uniform in character across phases, it was understood that simulation teams would make their worlds more complex with each phase. Aside from the common use of agent-based models to generate data, the content and structure of the GT project’s four worlds differed vastly and in fundamental ways. For example, agents in Power World sought to maximize their utility, simulating the familiar Homo economicus located in an unfamiliar landscape of opportunity (Granovetter 1985). Conversely, agents in Conflict World engaged in stigmergic behavior (Padgett and Powell 2012), actively shaping and responding to their simulated environment in a manner characteristic of Homo sociologus (Dahrendorf 1973), an agent driven by personally acquired or cultivated values in a landscape of dynamic, shifting possibilities. This range in simulated characteristics of social systems, from market-based utility maximization to culture-based co-constitution of agents and their environment, was not known to research teams during the GT project but reflects extreme variation in assumptions made about human behavior within social systems. This variety affected not only the properties of agents, such as their motivations, but other properties of the simulations, such as the kinds of social processes that existed in each world. Within this framework, research teams sought to validate existing quantitative tools and test novel methodologies to conduct social science on these simulations, DARPA’s stated goal of the GT project. These efforts spanned a range of approaches encompassing traditional social science methods, bespoke and novel methods reliant on sophisticated computational strategies, and bleeding edge neural models from computer science, which we discuss in the final section of the manuscript. Our team also tested the efficacy of crowdsourcing for completing tasks in Phases 1 and 2. This work sought to realize the stated goals of the GT project through any means necessary. It also seemed plausible to our team the GT project might serve other valuable purposes for DARPA, such as the creation of better wargames or the evaluation of standard social science methodologies’ ability to identify a novel process not previously found in the real world but built into the simulation. The ambiguity of potential uses for project outputs created a unique situation where decisions by T&E to restrict or allow the flow of certain kinds of information between simulation and research teams (a) had definitive impacts on how and what research could be conducted and, due to these impacts, (b) inspired discussions about the metagame that may be structuring these decisions. This feature of the GT project is notable as most large-scale social science research involves proposing a specific, highly detailed research plan with clear aims that align with the funding organization’s goals. While we were provided specific tasks, our performance was evaluated using known metrics, and project goals were documented/discussed explicitly, we perceived ambiguity in project goals (i.e., the potential existence of an undocumented and undiscussed metagame) which led to real ambiguity about what we believed to constitute productive versus unproductive effort within strict project timelines. The combination of strict timelines, restricted information flows, and the assumed known unknown of the GT project’s potential “true” goal(s) presented a novel problem-solving environment that negatively affected our team yet inspired substantial reflection about the basic nature of social scientific research tasks such as survey data collection and ethnography. Along with strict timelines, teams participating in the GT project experienced other constraints which shaped lines of inquiry and thus generation of knowledge about each world. First, simulation teams created initial data for analysis by research teams. In Phase 1 and 2, these initial data were incomplete, intentionally excluding the vast majority of data generated for each world since data collection was a major part of research teams’ tasks. To complete data collection tasks, research teams posed well-constructed questions and requests for data. Within this framework, questions were used as a way to learn about the existence (or properties) of objects in a simulated world, while requests for data, termed “research requests,” were used to simulate the implementation of social scientific research methods to collect data for analysis. Submitted questions and research requests had to adhere to strict guidelines regarding their structure, content, and framing. In Phase 3 this constraint was removed, with simulation teams providing all generated data to research teams, precipitating the inverse problem of data management (i.e., analysis of hundreds of gigabytes of high frequency simulation data). In both cases, the novelty of the problem-solving environment again inspired self-reflection about the idiosyncratic nature of social scientific inquiry as it has evolved in the real world, particularly its tendency to expect certain inputs for analysis. Specifically, we learned tacit disciplinary knowledge about human behavior led to the expectation that (a) certain features existed in the simulation because they were so “fundamental” to understanding agent behavior or, conversely, (b) certain features of human experience were so basic (e.g., feeling hungry) they could be ignored, both of which revealed how difficult it was for us to decontextualize simulated human behavior. A second constraint faced by simulation and research teams was the fact that the research request system was managed by the T&E team, a third-party mediating how teams interacted. Research requests (RRs) generated by research teams were expected to reflect actual methods of research used within the social sciences, such as fielding a survey asking a series of questions about agents’ age, current status, and preferences. In this context, each world had a temporal component similar to sampling frequency, which sometimes meant that well-formed RRs inadvertently led to the generation of massive amounts of data. For example, Urban World’s agents were observed in 5-minute increments over a period of multiple simulated years. Not knowing the temporal scale of (sub)-mechanisms that governed these agents’ behavior meant it was often safest to generate RRs with maximum sampling frequency. Related, when simulation teams received RRs they had to both (a) interpret the intended meaning of the proposed data request and (b) conform to limits placed on the kind and amount of data they could provide according to the T&E team. This system enabled “blinding” of simulation/research teams by making T&E the intermediary between both. This constraint introduced yet another novel feature of the problem-solving environment in the form of “blinded” data collection which required self-reflection about the biases we held about the data collection process. We found through trial and error that biases about which features to try to measure (and further study) could, again, turn assets into liabilities, expertise into folly, if we failed to accept certain seemingly natural lines of inquiry were unproductive. Within these constraints, the GT project consisted of three distinct tasks during each phase. Each task focused on producing a particular kind of knowledge: The “Explain” task required research teams to develop as full an understanding of the causal relationships within each simulated world (i.e., a directed causal graph) as possible. This task required identification of nodes consisting of features within the simulation, such as agent age and current location, and edges consisting of causal relationships between features, such as the tendency for older agents to visit particular sites. Fulfilling this task required discovering all features within each world and performing an exhaustive search for all causal relationships between these features. The “Predict” task required research teams to make an out of sample prediction of agent behavior—individually, in aggregate, or as an observable property of the simulated world—based on data provided by or solicited from simulation teams. This task always followed the Explain task and so, while identification of additional features and causal relationships sometimes continued, efforts often focused on converting a working knowledge of each simulated world into a model of each social system. The primary requirement of this model was fidelity to the ground truth of each simulated world. Fulfilling this task required discovering methods that could accurately reproduce observed outcomes, such as agent behavior, within each world. Nevertheless, some prediction tasks were counterfactual—if X happened tomorrow, what would happen next year?—demanding a rich understanding of causal relationships that were supposed to be uncovered during the “Explain” task. The “Prescribe” task required research teams to optimize a set of possible interventions for each world to maximize or minimize a specific property of the world (e.g., average number of friendships, total number of casualties, etc.). As in the Predict task, team performance was evaluated via out of sample observations: simulation teams implemented the interventions provided by research teams then observed their effects, recording relevant metrics for a period of time following the time series observable by research teams. Fulfilling this task required developing a counterfactual understanding for each simulated world, specifically (a) submission of RRs to provide data on the holistic effect of interventions on various aspects of the social system and (b) development of an optimization strategy. The latter logically focused on the features and causal relationships that most strongly impacted the evaluation metrics before considering secondary and tertiary aspects of the world that perturbed these metrics. Here we report how the structure of the GT project and the nature of its activities contribute to our understanding of knowledge production about social systems via our experiences studying the simulated worlds generated by simulation teams. This is from the perspective of our research team, initially branded as Social Machine Intelligence for Novel Discovery (or Social MIND) to reflect our commitment to explore the connection between emerging artificial intelligence techniques and familiar social science goals. Many of our insights are best characterized in terms of ontological, epistemological, and methodological issues raised and accompanying lessons learned. We believe these issues are relevant to knowledge production in the social and behavioral sciences broadly, especially within collective problem solving settings where rapid development of effective public policy is the primary goal, such as during the COVID-19 pandemic. Ontology of in silico worlds The type and extent of ontological issues associated with the GT project were unexpected. Some issues, such as discovery of features and causal mechanisms, stemmed directly from the structure and stated goal of the project. We briefly discuss these expected challenges but focus discussion on broader questions with substantial implications for collective problem solving. These questions arose from parallels between the GT research setting and real world problems, like political decision making about complex social issues or environmental challenges. We generally conclude that ontological questions about the nature of social systems can pose significant risks to the ability of disparate groups to arrive at a consensus about how to solve complex problems, such as racial disparities in public health and appropriate local responses to a global pandemic. We organize these questions according to the type of “game” being played when we study social systems, the ambiguity around what constitutes a social system, and the process of data collection given limited resources. Expeditions into the unknown: a game within a game? Limited initial knowledge about simulation teams’ worlds presented a unique opportunity to test the efficacy of different problem-solving strategies. One novel aspect of this extraordinary ambiguity was the fact that we had no sense of the type of “game” (i.e., rules used to build simulations, goals of each simulation, etc.) being played by simulation teams nor the potential metagame being played by DARPA, though the assumption of an unknown metagame was unique to our team alone. Thus, there was little reason to believe any one disciplinary perspective or methodological skill set would be better suited to achieving GT project tasks than any other. To this end, we tested the efficacy of approaching each task as a lone explorer, as two explorers, and as a team or community expedition into the four worlds. Multiple worlds were studied as single explorer expeditions where we tested if the most effective way to complete a task was for one individual (or one methodological approach) to direct the bulk of our activities. In our team, each world had either one or two primary “explorers” with additional individuals providing support. A single explorer approach meant the use of a novel methodological strategy whose execution was led by a single individual, sometimes supported by others acting at their direction. For example, for Urban World we used Granger causal networks to complete tasks in Phase 1 and Phase 2 (Chattopadhyay 2014; Li et al. 2019). This novel method was content free in terms of being broadly applicable to the type of data associated with Urban World regardless of what these data represented. We found that this flexibility sometimes helped ensure certain tasks could be completed, but that it relied on a small handful of experts to produce results which were not always interpretable within our broader understanding of the world. Single explorers were good at finding a way to play the game using their own rules but the idiosyncratic nature of their efforts could limit the generalizability of their modeling strategy even among those with domain knowledge. Another approach to GT project tasks was to treat these tasks as a two explorer cooperative expedition. A two explorer approach meant that two individuals cooperatively explored and tested different methods for completing tasks. Cooperative exploration and problem solving allowed us to leverage both individuals’ perspectives and skill sets to iteratively uncover effective solutions to the problems faced in each phase. We expected that pairing researchers with differing skill sets, differing levels of experiences, or both could be effective for completing GT tasks (Hall et al. 2018). Overall, work on Urban and Disaster Worlds supported this hypothesis about the nature of the GT project’s challenges and the best way to handle inherent uncertainties about simulated worlds. In each case, insights from both explorers contributed significantly to completing tasks depending on the underlying ground truth of simulated relationships. For example, in Urban World one player was well-versed in GIS and spatial analysis, which proved more salient to performing certain tasks, while the second player was versed in social theory and network analysis, which helped provide initial directions for analysis. We elaborate below how this role-taking worked in practice. We observed that even when empirically grounded, theory-based analysis proved uninformative it still allowed us to rule out common theories of social behavior or common modes of social scientific analysis (e.g., the use of exponential random graph models to study friendship networks between agents). Our experience with the GT project suggests that two heads are, indeed, better than one when presented with a complex research task whose parameters are not fully known—which reflects the vast majority of research tasks (Tambe et al. 1999; Denzinger 1995; Xyrichis and Ream 2008). The final approach we tested for effectively completing GT tasks was to assume each task represented a team or community expedition. In this context, a team expedition involved many individuals working to independently complete a task using their personal understanding of the task and the most suitable methods for completing the task. The “team” aspect of this approach is embodied by shared striving toward a common destination given identical information and constraints. Team expeditions were operationalized through use of crowdsourcing competitions to solicit solutions to the challenges associated with GT tasks. For Phase 2, we worked with TopCoder.com, which maintains a vast community of “solvers” who engage with data science, programming, and other scientific challenges to win prizes for the top solutions. TopCoder represents a large global network of technologists, boasting approximately 1.5 million community members. Up to 450,000 members of this community are data scientists with backgrounds in computer science, physics, mathematics, and engineering. In Phase 2 of the project, we involved TopCoder in a four tournament Grand Challenge through which we solicited community assistance to: (1) suggest possible social entities, forces, and causal relations in each of the four worlds to stimulate our requests for data, as well as (2) accomplish the explain, predict, and prescribe tasks alongside our teams. We scheduled completion of these challenges a week before each of our solutions were due, with our independent or cooperative explorers intensively engaging with their results to improve our final submissions. Across all three competitions we had 686 registered participants, from 15 countries; 64% from India, 7% from Russia, 5% from Indonesia, 4% from the United States and less than 2% were from Kenya, Iran, China, Philippines, South Africa, Italy, Canada, Romania, Brazil, Mexico and Egypt. For the first of our four challenges, we received a barrage of suggestions that broadened our scope of questions and research requests to simulation teams . These included using more of the research methods available to us, such as experimentation, social media analyses or use of government records, and the proposition of out-of-the box causal possibilities (e.g. boredom; importance of cultural diversity, public safety, presence of public transit; innate intelligence, actively blogging, having a criminal background, participating in ongoing criminal behavior, experience of bullying, alcohol/drug use, number of languages spoken, fatigue, emotional state, materialism, global warming, pollution, drug addiction, volcanic eruption, etc.). For the remaining challenges, TopCoder solvers had a more difficult time, at least in part because they had to work with an evolving dataset in a compressed time frame without the benefit of direct feedback on the success of their strategies. We found this problem solving context made the creation of a leaderboard, and the accelerating competition it engenders at the end of a competition, infeasible. This finding illustrates how the features of such contexts shape how effectively crowdsourcing can be leveraged as a form of collective problem solving. Moreover, the tasks were complex and involved many continuously evolving forms of data to produce many different required predictions and prescriptions. We observed it was difficult for solvers to put together a complete solution despite efforts to share code that integrated and cleaned relevant data. In other cases, community solutions misunderstood the data or questions, or added nothing new to own analyses. Other times, community members proposed methods like Bayesian Networks for causal discovery (Cheng et al. 2002) or categorical boosted forests, implementing these methods via working python code that successfully uncovered world dynamics. We then extended these with new data and applied them to those same worlds and others. Consider Urban World’s Phase 2 Prescribe task as an illustration of how these different problem solving strategies worked in practice to help triangulate the best possible submission. The goal of this task was to select a subset of 200 agents in the world who will, over a 30 day period and in isolation (i.e. all other agents in the world are removed), collectively exhibit higher average daily friendship network degree over the final week of this period than any other subset of 200 agents subject to the same conditions. As part of this task, we were provided a sample evaluation metric (i.e. 5.05) in the initial data package representing the outcome of drawing a uniform random sample of 200 agents. We were then allowed to submit up to four mock test sets of 200 agents where the simulation team would then provide the corresponding value of the evaluation metric based on each test set of agents. Table 1 summarizes the results of these mock tests and relative change in the evaluation metric versus the sampling strategy and metric included in the initial data package.Table 1 Comparison of evaluation metrics in urban world phase 2 prescribe Source Average degree (08/08–08/14) Change Initial data package 5.05 – Mock test #1 6.15 + 22% Mock test #2 5.87 + 16% Mock test #3 6.36 + 26% Mock test #4 15.04 + 298% Final submission 14.10 + 279% Each of the four mock tests in Urban World’s Phase 2 Prescribe task represented a systematic approach to the selection of 200 agents. The first mock test drew a stratified random sample of agents designed to reflect the age, education, and income distributions of all agents, under the belief that these agent-based characteristics, including homophily (McPherson et al. 2001), might systematically affect friendship networks. The second mock test used a matched sampling approach designed to maximize insight into how other features of the world affected friendship networks, such as the role of location. The third mock test focused on the effect of geographic propinquity on friendship networks (e.g. Holzhauer et al. 2013). The fourth and final mock test represented an entirely new perspective on the selection of agents. The first and second test sets were drawn based on input from a team member trained in social theory and social network analysis, the third test set was developed between this team member and another team member trained in GIS methods, but the fourth set was generated based on intuitions about spatial processes as understood by the GIS-trained team member. The fourth set was derived by applying a clustering algorithm to the location of agents’ homes weighted by the “popularity” of each home (i.e. how many friendship ties were linked to a home through its residents). The substantial improvement in evaluation metric over the other three mock tests was exciting and moderately unexpected. However, the overall utility of this clustering approach became apparent when we learned that one of the TopCoder submissions used clustering in a similar yet more intricate manner. This work inspired us to pursue essentially the same strategy used in fourth mock test for our final submission, tweaking our approach based on input from the TopCoder submission that also used clustering. Ultimately, this fine tuning led to a slightly lower final evaluation metric. However, without the TopCoder submission we would have felt more uncertain about the exclusive use of clustering and might have made more substantial changes to our strategy that would have resulted in a much lower evaluation metric. In this case, the two explorers experimented with strategies based on analytic perspectives cultivated in two distinct fields of study, but the value of one explorer’s perspective (i.e. GIS-based clustering) became unambiguous via use of crowdsourcing to externally validate its efficacy. Simulated social systems still require real definitions Ontological questions about what constituted a social system presented both the most difficult challenges to accomplishing GT tasks as well as, arguably, the most insight into social science as a practice. The basic ontological questions that plagued both simulation and research teams was “What exists in the simulated social system?” and “How do objects in the simulated social system interact with each other?” when it was made clear to all involved that these simulations were not meant to be realistic representations of social behavior; the GT project recreated the basic features of “first contact” with an unknown civilization (and, at times, used language to suggest that research teams should assume they are studying an alien planet). The setting, then, was intended to be functionally similar to a real world social system where individuals’ roles, motivations, and behaviors are not known to the observer. Determining what objects exist and how these objects interact was the most fundamental task in the GT project; without this information it is impossible to explain/predict behavior or prescribe interventions. Because GT worlds were simulated social systems whose features were artificially generated and intentionally not reflective of real social systems, and because research teams had to communicate with simulation teams to request additional data, who in turn had to interpret these requests in order to provide the requested data, the interpretation of these requests proved to be fundamental for making sense of each world. Basic ontological questions about what objects exist and how these objects interact were thus complicated by each team’s use of words in naming data attributes. For example, the team behind Urban World explicitly renamed variables in Phase 2 to prevent observed cases in Phase 1 where research teams had inferred erroneous information based on variable names (e.g., assuming that “has Child” meant the existence of family units when, in fact, “has Child” was only meant to indicate that agents had increased expenses if they had a child). Another ontological question raised by GT project tasks was the overall purpose of each world or, more properly, the win condition(s) of each game embedded within these four worlds. For example, Conflict World presented unique challenges to our team because we assumed that all simulated agents followed a basic utility maximization principle. While more or less true in other worlds, the stigmergic basis of Conflict World meant that agents pursued shifting goals in reaction to their experiences as well as changing motivations in response to these experiences and available options (Heylighen 2016; Dorigo et al. 2000). Put simply, we found that the existence of a stable human nature and stable social system was itself an assumption that we should not have made. Making this assumption led to significant misinterpretation of data and a poor understanding of the world in question, despite success in some tasks. This finding lends novel support to the idea that even certain forms of social science inquiry (e.g., identifying utility maximizing behavior) can be effectively blind to coherent, rule-based behavior if agents’ understanding of their world differs from researchers’ understanding. Such an idea has gained prominence in discussions about systemic inequality, especially racial inequality, where scholars dispute what constitutes agentic versus structural sources of inequality (Royce 2018; Wilson 1987; Massey and Denton 1993). Our experience with the GT project indicates it is possible for agents to be embedded in a social structure perfectly navigable to themselves but so alien to scholars that the latter cannot conceive its existence. Moreover, the social ontology of Conflict World was relatively intuitive once revealed, suggesting that access to agents’ own understanding of their social system (e.g., through interviews, ethnography, etc.) would have been key to understanding it correctly and thus making accurate inferences. Data collection versus data generating processes: the language of observation A final constraint that applied across simulated worlds was, again, the role of language, but this time in the context of data collection. Research teams requested information from simulation teams in order to test hypotheses about each simulation. These requests were a focal point of frustration due to (a) the desire to test the full range of social science research methods for studying simulated social systems and (b) the reliance of research teams on the resulting data to both construct a basic understanding of each simulation and complete GT tasks. The former is best illustrated by persistent attempts to employ qualitative methods, in particular ethnography. We suggest that such dissonance between stated GT project goals and the specific goals of our research team reflect distinctions between data collection as often practiced in the social sciences and data generating processes foundational to simulated social systems. In short, it was effectively impossible to conduct ethnography in these simulated worlds. This finding was not self-evident at the time, nor do we believe it intentional on the part of simulation teams or the T&E team. Rather, the natural value of ethnography is its ability to leverage human perception to identify gaps in our understanding of social phenomena (Becker et al. 2004; Jessor et al. 1996; Small 2009; Pacewicz 2020), often through discovery of persistent, multi-dimensional configurations. For example, in Urban World if an agent enters a site and we can determine how that agent knows what time of day it is then we may better understand why agents spend less time at one type of site compared to another type of site—an important factor in human behavior exploited by casino designers. An ethnographic account of agents visiting each type of site could reveal that one type of site always has a clock prominently displayed on the wall while the other type of site never does. However, we ultimately learned that agents simply “know” what time it is, and that time spent at sites was an inherent property of site type. Similar instances of perfect information and just-so features of each world could be found in other simulations. For example, in Disaster World, hurricane dynamics were governed by fixed parameters with deterministic impact on the risk posed to regions/individuals. Yet our experience with this ontological question (i.e. “What observations are possible?” regardless of their truth value) provided an important lesson learned that complemented the lesson learned from Conflict World’s radically different ontology compared to the other simulations: Regardless of the type of social system being studied, the accessibility of one type of data does not negate the need for access to additional types. Because we knew the simulations were simulations, we understood that they lacked the complexity of real life, but uncertainty around what was observable compounded uncertainty around the objects and causal relationships being simulated. This doubly shifting landscape is a common feature of real social systems and represents the social scientific equivalent of the “state of nature” where one has no knowledge of what is or is not socially meaningful. Historically, empirical social science was more qualitative in nature and made extensive use of interviews, participant observation, and ethnography. Over time, quantitative social science developed in parallel, emerging as soon as suitable methods were developed (Hacking 1990). Our experience with the GT project suggests that the initial use of qualitative methods as a tool for uncovering ontological properties of social systems logically precedes quantification of those properties. The GT simulated worlds represented an anachronistic case where we began with quantification then attempted to understand how a social system works in order to develop effective interventions. However, we did not have the benefit of human perception to identify meaningful properties and/or their configurations in each world. Ultimately, this parallels the hyper-quantification of social behavior we observe today (Lazer et al. 2009; Edelmann et al. 2020) and suggests that without the ability to observe the social system in vivo we risk developing a working model of behavior that excludes key properties of the world. For example, in Urban World there existed an entire process for “eating” that we never uncovered because we thought it was self-evident from other behavioral data, but this process proved crucial for understanding simulated disease transmission in Phase 3. Ontological lessons learned In addition to the specific lessons learned noted throughout, we identified two broad lessons learned from the ontological questions raised by the GT project. First, successful quantitative social science requires well-posed questions that use well-defined terms. Data does not, in itself, induce understanding, and descriptions of data can impart significant bias, even when it is known that such bias exists and could substantively affect analysis. Second, collective problem solving without a shared ontology about the object of study is extraordinarily difficult. Both have real world implications, but we will focus briefly on the latter as the former may be an artifact of the GT project’s design. The lack of a shared ontology concerning what objects exist in a social system and the causal relationships between these objects generated significant challenges for resource allocation. Specifically, cognitive responses to GT tasks (i.e., problem solving) required some level of certainty concerning this ontology, but without this certainty many team members could not intuit how to proceed. For example, consistent lack of progress in learning how agents behaved in the Conflict World simulation made it difficult to justify devoting additional resources to associated tasks. Conversely, the expansive nature of the Urban World simulation meant that it was difficult to gauge whether progress had been made at all since we never knew if we had uncovered all relevant properties of the world. Both situations led to ambiguity about the entities and relationships under study which often resulted in pursuing an exhaustive understanding of causal relationships (i.e., perfecting the “Explain” task during each phase). However, this ambiguity has implications for real world problem solving in the form of issue advocacy and public policy recommendations. A limited understanding of salient social entities (e.g., agents, processes, contexts) limited our ability to conduct experiments within these worlds. Experimentation requires control in the form of sufficient knowledge about relevant forces shaping behavior and the variation of one or a few factors. Without this knowledge, experiments can become fishing expeditions where other research methods may produce more insight with less effort. It was not until we approached the end of the project that we came to understand enough about these worlds to cultivate expectations about which features would be most insightful to systematically vary for the direct identification of causes and useful policies. This delayed understanding, combined with the significant turnaround time of research requests, made experimentation a high-risk endeavor during most of the project. Future simulation studies of this kind might consider how to support a sufficient ontological framework for posing meaningful experiments. The GT project constituted a highly structured yet cooperative team-based assessment of agent based simulations as a platform for productively testing different methods for studying social behavior. Despite prolific uncertainty around the form and content of these simulated social systems, project participants committed to achieving the same goal under the belief that this goal was, generally speaking, beneficial to advancing social science and its applications. Most complex policy debates involve similar levels of uncertainty about the true nature of social problems and policymakers must typically make decisions based on an incomplete understanding of these problems (Head 2019; Ney 2009; Yung et al. 2019). However, policymakers are also often subject to a barrage of information from issue advocates, popularly termed lobbyists (Bok 2001; Nelson and Yackee 2012). These advocates advance a particular understanding of a social problem so that policymakers can address this problem in the manner they believe most effective. Yet it is rarely clear which policies will be most effective because advocates must begin with their own assumptions about the social system they intend to influence (Markusen and Venables 1988; Schneider and Ingram 1990; Pielke et al. 2008). Competing assumptions will naturally produce disparate policy solutions. Thus, uncertainty regarding assumptions in real world policy debates, to some extent, mirrors the ontological uncertainty experienced by GT participants. In the case of the GT project, participants’ commitment to rigorous scientific inquiry and shared striving toward a common goal were not enough to overcome the effects of this uncertainty in many cases. More broadly, we tentatively conclude that ontological differences in how issue advocates understand the same social problems will constrain the ability of policymakers to identify compromise solutions to these problems. Further, our experience with Conflict World suggests it is possible for good faith actors to be incapable of identifying key features of social systems if they rely on a limited array of evidentiary sources. It is unclear how this issue might be addressed in the short term except to acknowledge its effects. Our experience with the GT project, however, suggests that long term efforts to formalize ontological assumptions about social systems must be supported to avoid inefficient, ineffective, or counterproductive public policies enacted by elected politicians. Because there is much we do not understand about social systems, and representative political systems rely on popular understandings of social problems and their solutions, it is important that social scientists highlight this barrier to consensus building and begin adopting a means of communicating the social ontologies used within their work. Epistemology All work that overlaps neighboring fields, such as we occasionally undertake and which the sociologists must necessarily undertake again and again, is burdened with the resigned realization that at best one provides the specialist with useful questions upon which [they] would not so easily hit from [their] own specialized point of view. ~ Science as a Vocation, Weber (1958) The type and extent of epistemological issues associated with the GT project were often predictable and provided significant insight regarding fundamental tensions associated with (a) doing social science research versus (b) applying findings from social science research. Issues, such as choice of problem solving strategy for each task, stemmed directly from the structure and stated goal of the project. We do not discuss the specifics of these issues but, as with our examination of ontological issues, focus on broader questions with substantial implications for collective problem solving. These questions arose from the nature of the GT tasks performed during each phase of the project and speak to what Weber termed “science as a vocation” in reference to the external economic forces associated with and the lack of intrinsic value characteristic of scientific inquiry in practice. We thus organize our discussion of these issues and related lessons learned according to each type of GT task. A clear pattern emerged over the course of the GT project around the types of knowledge that were necessary to complete GT tasks. Each phase of the project was identical insofar as research teams had to complete the same three tasks for each world: explain, predict, and prescribe. While social systems differed in their structure and content according to the simulation teams’ models, explaining each social system, predicting out-of-sample properties of each social system, and prescribing interventions for each social system represented loosely related yet distinct epistemological goals. The first epistemological goal during each phase of the GT project was to explain the simulated social system. Explanation was defined as the development of a directed acyclic graph constituted by all relevant features (i.e., nodes) and causal relationships between features (i.e., directed edges). Relevancy was defined through prompts—such as “explain how agents form friendships”—that served to anchor inquiries for additional information. This GT task required coping with the ontological uncertainties noted above to produce an acyclic graph effectively reproducing the agent-based rules and/or agent-based model parameter relationships used to generate the simulation data provided to research teams. A defining characteristic of this task was its holistic framing. While prompts provided a way to help research teams begin studying each phase’s simulated world, research teams were evaluated according to their ability to uncover nodes and directed edges. This evaluation approach presents an egalitarian epistemology where all knowledge had approximately equal value regardless of the relative importance of individual pieces of knowledge for understanding the social system. This egalitarianism incentivized investigation of potentially second and third order effects that were, at best, tangentially related to properties of interest. Any knowledge was good knowledge. A secondary characteristic of this task was significant slippage between what research teams believed they were investigating and what simulation teams had identified as relevant features. Specifically, the ground truth under study was an assortment of data generated by simulation teams and analyzed by research teams. The terminology used by simulation/research teams was, as noted, ill-defined and referred to simulated social processes. To “explain” these social systems research teams had to name causal nodes and provide a written explanation of how each node influences another if a directed edge existed. However, evaluating these explanations required simulation teams to interpret nodes and directed edges according to their internal understanding of the data generating processes they had developed. This situation meant it was possible that research teams could describe a simulated social process but, without further clarification, simulation teams infer research teams were referring to a different simulated social process than intended, a phenomena reminiscent of boundary objects (Star and Griesemer 1989) that attract shared attention despite being understood and used in very different ways. Once the ground truth of each simulation was revealed, it was clear that there were many cases where this applied, such as agents’ affinities for particular sites in Urban World. While our understanding of site visitation was based on the desire to form and maintain friendships with similar agents, the Phase 2 Urban World simulation replicated this agent-based form of homophily through a process where agents chose to visit preferred sites based on both agents’ and sites’ characteristics. However, our understanding of the ground truth causal diagram for Urban World’s Phase 3 simulation suggests that agents of a similar type formed friendships by choosing to visit similar sites rather than explicitly choosing similar agents. This minor distinction had serious implications for our ability to understand the simulated social system in the predict and prescribe tasks and highlights the fact that simulation/research teams could believe they are referring to the same social processes verbally (i.e., homophily in friendship formation) but are actually referring to different ground truths. Our experience with this epistemological issue suggests that clearly defined, shared referents are vital for constructing an accurate ground truth understanding of a social system, and we recommend future simulations testing the utility of different social science methodologies remove this ambiguity to ensure the robustness of results. Conversely, terminological ambiguity also meant that research teams could refer to features using different words or phrases than simulation teams yet still reference the same ground truth processes. For example, it is unclear how much our failure to identify the nodes and directed edges associated with the process of food consumption in Urban World affected the evaluation of our explanation of this world in Phase 3. Without additional information, this lack of clarity cannot be resolved since we assumed data referring to relevant agent behavior (e.g., entering a restaurant) were sufficient to understand the process of quelling hunger and we could not know this explanation was deficient since we, as a research team, did not have access to the ground truth (i.e., the simulation) or other feedback indicating that our understanding was incomplete (i.e., knowledge about unobserved features). Our experience suggests it is vital to identify such measurement issues before attempting to construct an exhaustive causal model of a social system. This finding supports multiple arguments made in the social sciences about the role of measurement theory and highlights their applicability to data-driven analysis where measurement is assumed to be error free (or correctable with sufficient data) (Shultz et al. 2013; Bandalos 2018; Leplège 2003; Goertz and Mahoney 2012). The second epistemological goal of each phase of the GT project was to predict out-of-sample characteristics of the simulated social system. For each world, simulation teams provided data for a discrete period of time. The primary goal of research teams was to then make predictions about what happens immediately following this time period. Evaluation metrics were typically based on predictions made over a discrete period of time (e.g., 7 days of simulated behavior). This GT task avoided many of the ontological issues noted above since the simulated data was itself the ground truth for each world and research teams did not need to explain the set of causal relationships used to generate predictions. A defining characteristic of this task was a data-centric approach to modeling each simulated social system. Because the goal was to predict outcomes based on the data provided it was not necessary to construct any broader understanding of the social system than was needed to accurately predict future behavior. This feature of the predict task provided both natural scoping conditions for analysis and clear priorities in terms of analytic effort. The most efficient strategy was to identify the primary causal relationships governing the outcome of interest, such as the average number of friendships in the world or agents’ responses to a natural disaster. Other aspects of the simulation could be ignored to the extent that they did not affect this outcome. In some ways, the prediction task was the most familiar to our research team members as it most accurately reflects both current expectations around effective data science and historic efforts in the social sciences to identify the features most important for understanding social phenomena. For example, residential segregation is a well-known phenomenon in the United States which could allow simple and often correct guesses about your neighbors’ race given your own (Massey and Denton 1988; Charles 2003; Reardon et al. 2015). Even if predicting your neighbors’ race based on your own race is not something social scientists typically do, the fundamental epistemological issue remains reproducibility via prediction. In some ways, data scientists are more familiar with this issue given that many applications of machine learning involve generating out-of-sample predictions. Our experience with the GT project suggests that, when interpretation of features is irrelevant, even basic data science approaches, such as clustering, can yield a huge payoff in terms of predictive accuracy. Notably, however, the explain task and predict task were only loosely related. Knowledge production in the former required a robust understanding of the social systems’ ontology in order to exhaustively test possible causal relationships, but knowledge production in the latter only required a “good enough” understanding of each social system to accurately reproduce and then predict agent behavior. Emphasis on explicit directed causal relationships and predictive accuracy by those studying causal inference in the social sciences would suggest both tasks are important, but our experience with the GT project suggests the opposite: Fully understanding how a social system operates does not necessarily reveal which parts of this system are most important for outcomes of interest, and the ability to predict outcomes of interest does not indicate a full understanding of the social system. One can even imagine a GT task between explanation and prediction in which a causal weight is assigned to each edge in the graph. There are three potential implications from this observation. First, if the goal of generating new knowledge is not clear, then it is easy to fall into a trap where researchers examine either a small part of a social system or attempt to fully explain the social system when one or the other will suffice. Second, marginal advances in our understanding of a social system need to be put into context relative to an outcome of interest. If no outcome is specified, then the value of these advances cannot be judged. Third, when the social system is unintuitive, as in the case of Conflict World, then it may be unclear whether a full or partial understanding has been attained. This last implication is the most serious as it means we can have a working understanding of one part (or even multiple parts) of a social system that produces good predictive accuracy but that does not capture key causal relationships operating essentially out of sight. Consider Captain Cook’s fateful encounter with indigenous Hawaiians: he believed he understood enough about Hawaiian culture to play god, but was killed when he and his men failed to meet Hawaiian expectations about how gods behave. It was not that Cook could not predict the average reactions of the Hawaiians, but that he did not fully understand the social system generating those reactions (Sahlins 1995). The third and final epistemological goal of each phase of the GT project was to optimize a set of policy prescriptions to maximize or minimize an outcome of interest. As with the predict task, research teams were evaluated based on out-of-sample prescriptions where interventions had been introduced into the simulation as prescribed. The primary goal of research teams was to construct counterfactual predictions of agent behavior based on their understanding of how each world operated. In this respect, the predict and prescribe tasks were tightly coupled, largely avoided ontological issues noted above, and reflected a pragmatic approach to knowledge production. The prescribe task differed from the predict task in that the effects of interventions had to be estimated and, once known, optimized to elicit the “best” possible outcome in each world. This task was akin to data-driven policymaking where the effectiveness of policies are empirically tested after the fact. Given the practical implications for policymakers, the purpose of this knowledge production was clear and the problem (i.e., optimization) well-defined. As with the predict task, however, only a pragmatic understanding of each social system was required to generate high quality prescriptions. Our experience with the prescribe task during each phase of the GT project again led us to conclude that the types of knowledge generated during each phase were loosely coupled, at best. Identifying the optimal timing and implementation of prescriptions for each social system did not require a complete understanding of its causal structure. It also did not necessarily require high predictive accuracy, only high impact prescriptions leading to the best outcomes possible. The latter was illustrated in Urban World during Phase 3 when efforts to develop an accurate predictive model of the simulation fell short of our expectations, yet we had sufficient knowledge of the world to develop effective prescriptions based on little more than logic and basic estimates regarding the relative efficacy of each type of prescription available. Other epistemological issues came to the fore during the GT project, such as what constituted ground truth and how to evaluate it, but we have organized our experience by task since each task had unique requirements that led to fairly discrete forms of knowledge production. Performing well on each task required a slightly different type of knowledge. Due to the fast-paced nature of the GT project, these differences manifested in our problem solving (i.e., knowledge production) strategies. As noted, the epistemological approaches used for each task were only loosely related: explaining the social system had marginal benefit for predicting behavior, and predicting behavior had marginal benefit for developing an optimal set of prescriptions. While fine-grained causal information mostly distracted from the largest factors impacting outcomes of interest, knowing the largest factors impacting outcomes of interest mostly distracted from the relative impact of each potential intervention and thus the task of formulating an optimal portfolio of interventions. Our experience with epistemological issues during the GT project suggests three possible lessons learned of use to an array of stakeholders ranging from non-profit organizations to academic researchers to public office holders. First, understanding a social system is not the same as learning about a social system. It is possible to learn more than enough about a social system to accurately predict behavior and generate effective policy prescriptions without a thorough understanding of the system as a whole. Yet this pragmatic focus, which creates a natural scope condition for data collection and analysis, can miss pivotal features of the system while still performing well on predictive/prescriptive tasks. Our tentative conclusion is that the need to balance holistic understanding with pragmatic inquiry emerges precisely because the knowledge produced by each strategy speaks to different epistemological goals. Alternatively, it is possible that real world social systems exhibit more tightly coupled relationships between these goals, as in cases of heterogeneous treatment effects that imply a more complex set of causal relationships than originally assumed when designing an intervention. A second lesson learned was that knowledge production in an academic setting, specifically quantitative research in the social sciences, often, but not always, focuses on marginal improvements in our understanding. This focus means an academic mode of inquiry, which involves the demonstration of new social objects, forces, or relationships, will tend to be less effective at generating policy relevant findings. It is not that scholars lack the tools to produce such findings, but that the epistemological goal of social science differs from that of policymaking. However, pragmatic and holistic approaches to policy development and implementation are not antithetical to the practice of academic research insofar as scholars can demonstrate that their marginal improvement in our understanding has a substantive effect on outcomes of interest. In fact, this approach is often adopted as a model for evidence-based policymaking and major grantmaking organizations often demand that proposals explicitly identify concrete implications for policy (e.g., through requiring randomized clinical trials of interventions). A final lesson learned was that, even if knowledge produced by quantitative academic researchers in the social sciences has identified the primary causal relationships associated with an outcome of interest, it is very difficult to uncover this information for reuse. To find and apply this work requires not just methodological competency (e.g., ability to distinguish between high quality and low quality studies) but also (a) domain knowledge of the field/sub-field and its internal debates, (b) willingness to identify and attempt to overcome disciplinary biases both in the field/sub-field and as individuals, and (c) access to diverse sources of research, including not just a wide range of peer-reviewed journals but also respected, if informal, repositories for research, such as the National Bureau of Economic Research (NBER) working papers, and high quality studies produced by non-academic institutions run by academic researchers. The amount of time and energy necessary to effectively search the literature is thus prohibitive even for the best trained, most well-read social scientist, suggesting that devolving that burden onto policymakers, which is often the tacit strategy of social science, is unrealistic. In fact, our experience with the GT project suggests that the inability for social scientists to move beyond their own disciplinary biases can lead to significant wasted time and effort when those biases strongly suggest a course of action inapplicable to the situation at hand. Methodology With the ontological and epistemological issues above in mind, we now turn our focus to methodology. It was expected that methodological issues would be both prolific and highly productive. We found this to be true but not always in ways we anticipated. The variety of approaches employed reflected a diversity of perspectives about which methods might be most applicable in each world during each phase and task. While not always followed, we found the strategy of working backward from the goal of each task helpful for constraining the set of methods to be tested. When we found a particular method was useful in one world/phase/task we often attempted to deduce its relevance in another world/phase/task. Before discussing an example of each strategy, it is useful to review both the variety of methods applied and the immediate methodological issues they often raised. During the GT project, members of our team applied a menagerie of methods ranging from information visualization and bespoke Google queries (e.g., number of webpages on which a pair of Conflict World labels appeared) to standard statistical models to machine learning approaches for agent-based models of complex systems to emerging artificial intelligence techniques to simple searches of the Internet. These included geographic information visualization, correlation, linear regression, logistic regression, Shapley regression, Granger causality estimation, auto-regression and time series analysis, sparse regression, survival models, markov models, clustering, Bayesian graphical models, decision trees and random forests, ensemble methods (e.g., boosting, bagging), support vector machines, k-nearest neighbors, Hawkes process analysis and simulation of spatially interdependent point processes with probabilistic finite-state machines, agent-based models, probabilistic programming models (Wood et al. 2014), along with a wide array of deep learning approaches from standard, feed-forward artificial neural networks (Fine 2006) and recurrent neural networks (Rumelhart et al. 1986), to auto-encoders (Hinton and Salakhutdinov 2006), sequence-to-sequence neural networks (Sriram et al. 2017), graph convolutional networks (Kipf and Welling 2016), theory of mind neural networks (Rabinowitz et al. 2018), and world models (Ha and Schmidhuber 2018). We also used network analysis (Wasserman and Faust 1994) and methods for analysis of GIS data when applicable. Choice of method was, ideally, based on its suitability to a particular task and/or form of data. We first review general approaches to method selection and their efficacy before discussing three approaches that showed the most promise—Granger causal networks, probabilistic programming, and neural networks. Given the varied backgrounds of team members, bias towards particular habits in problem solving sometimes led to (over)-reliance on a favored method. For example, regression analysis and clustering algorithms were commonly employed if only because they were familiar tools for exploring unfamiliar data. This meant that choice of method was not always ideal given the task/data, but the relative suitability of a method would often quickly become apparent. One clear case of this situation was when we attempted to study a social network in Urban World with more than 1000 nodes using exponential random graph models (ERGMs) only to find one of the most robust packages for studying ERGMs struggled to estimate even simple properties of the network (Handcock et al. 2008). Conversely, there were times when clustering algorithms outperformed more refined models based on our working understanding of the social system. In this regard, pragmatism was the rule and deliberate planning the exception. Related to bias in habits of problem solving, domain specific knowledge affected both the collection and interpretation of data. Each world tended to encompass an overarching set of social processes that could be described in succinct and coherent terms. For example, we came to understand the first simulation as Urban World because it involved agents living in a geographic space and moving between points within this space to achieve a variety of goals, in the process entering and exiting sites where they performed actions according to site type. This conception arose out of our team’s intuition about the features within the simulation and their relationship to each other. In response, analysis of Urban World was primarily left to an expert in GIS methods and an urban sociologist. However, the Urban World simulation team was primarily composed of geographers. The result was that Urban World was aptly named, and this heuristic often helped us to intuit the existence of basic features, but that differences in domain expertise between geography and sociology led to focus on different social processes. This disparity was most evident in the fact that agents did not directly pursue relationships with similar agents but formed relationships with similar agents by seeking out sites of shared interest where they could then meet and, potentially, form a friendship. The former is foundational in sociological understandings of friendship formation (McPherson et al. 2001), while the latter is foundational to geographic understandings of travel patterns (Liu et al. 2015). To grossly exaggerate this distinction, we might say those who built “Urban World” viewed it as a social system where people often focused on the process of choosing where to go next while our team viewed it as a social system where people often focused on the process of choosing friends. Misalignment in domain expertise, in this case, taught us an important lesson: Similar understandings of a social system can be equally grounded in empirical research but methodological strategies aligned with the ground truth (i.e. a geographic perspective on urban social systems) may better catalyze knowledge production. This illustrates and underscores the No Free Lunch theorems in machine learning proved by David Wolhpert and William Macready (Wolpert and Macready 1997), which “state that any two optimization algorithms are equivalent when their performance is averaged across all possible problems” (Wolpert and Macready 2005)—or all possible worlds. If a method works well in some social world, it will work poorly in another. Our understanding and methodological strategies worked well when they aligned, and poorly when they did not. An alternative to applying domain specific knowledge to study a social system is to adopt a Bayesian approach to knowledge production. Specifically, domain expertise generates strong priors about the form and function of a social system, but it is always possible to consider semi-informative priors to tentatively adopt then test. A significant amount of basic analysis involved this kind of work. This often meant iterative testing of relationships between features of each social system with basic constraints on what relationships were thought to be possible. We found that this could produce results where all features were related to all other features but, when the approach was properly specified, could also help advance our understanding of each world significantly. While making too many assumptions could prove problematic and systematically testing a set of assumptions could prove useful, a third methodological strategy was to begin from the tabula rasa of no (or as few as possible) assumptions about each social system. Methods that embody this strategy were highly data driven, which translated into a need for high levels of technical expertise unrelated to social science. Under the circumstances, these methods often showed the most promise for use in social science research precisely because they were not beholden to relationships we expected to find and focused on uncovering or modeling the relationships we did find in the data. This strategy was especially useful given the ontological issues we faced. For example, we explored whether we could construct causal predictors from observation sequences of variables alone. Designing an efficient causality test, that may be carried out in the absence of restrictive presuppositions on the underlying dynamical structure of the data at hand, is non-trivial. Nevertheless, ability to computationally infer statistical prima facie evidence of causal dependence may yield a far more discriminative tool for data analysis compared to the calculation of simple correlations. On this line of thought, we devised a non-parametric test of Granger causality for quantized data streams realized from the variations of the observed variables in the world simulations. In contrast to state-of-the-art binary tests, this approach computes the degree of causal dependence between data streams, without making any restrictive assumptions, linearity or otherwise. Additionally, without any a priori imposition of specific dynamical structure, we were able to infer explicit generative models of causal cross-dependence, which may then be used for prediction. These explicit models are represented as generalized probabilistic automata, referred to crossed automata, and are shown to be sufficient to capture a fairly general class of causal dependence (Chattopadhyay 2014). The proposed algorithms are computationally efficient in the probably approximately correct (PAC) sense (Valiant 1984); i.e., we find good models of cross-dependence with high probability, with polynomial run-times and sample complexities. The causality network inferred from this dataset revealed non-trivial relationships, and laid the groundwork for such deep data-driven interrogation of complex social phenomena in the future, particularly in situations where sequential observations on many interacting variables are available. Another data driven approach that enabled the explicit incorporation of social theoretical intuition was probabilistic programming (e.g. Salvatier et al. 2016). Probabilistic programming languages (PPLs) allow stochastic elements to be included in deterministic models by treating statistical distributions as objects on which we may perform basic logical/mathematical operations. This functionality further allows us to create a generative model of behavior within which we may embed prior information about the social system (Goodman et al. 2012). For example, a Bernoulli random variable, such as a coin toss turning up heads, may determine whether a person in Urban World contracts a disease given exposure, and the severity of the disease for that person represents a second random variable, perhaps normally distributed such that it typically causes discomfort and the potential to infect others but not lasting disability. In extreme cases, however, the disease may cause death, or spread much more rapidly than normal, like COVID-19 at a super spreader event (Althouse et al. 2020). Within a PPL framework, the coin toss determines whether the disease is passed then contraction itself has a chance (e.g., based on a different statistical distribution) of leading to transmission and loss of health or loss of life. If we wanted to understand the effect of contraction on severity and transmission, we would observe the distribution of these events, conditional on one another, then tune the probabilities of our program in order to generate the appropriate distribution of outcomes, which would later be available for us to determine whether or not a new disease had emerged from the same “world” as the last. Within the GT project, early stages of Power World used PPL as a means to answer questions about patterns in the data, trying to support or disprove potential hypotheses about the way the world works (e.g., “Does the team with the highest productivity always win conflicts?”). A sketch of the overarching structure of the world was built using information provided in briefings and communiques. We knew the general outline of the program we were modeling but only general things about what happened at distinct points in time. We hard-coded the things we did know and set a parameterized distribution over the space of programs consistent with behavior expected at the unknown regions. PPLs are able to search this space of programs (i.e., potential worlds) to find the one most consistent with observed data. Many of the hidden processes we wanted to model, such as conflict between two groups, had binary outcomes. To figure out which features contributed to a given outcome, we mapped each state of each feature to a value and then used linear combinations of these values to produce the weighting of a coin, the flip of which stood-in for the process we were modelling. This approach of searching through the space of weights for factors helped guide data analysis. For example, if the inferred program for determining outcomes for group conflicts weighed group sizes heavily, then we would know to try to look at the data to see if indeed a large group size was reported shortly before a conflict and whether it was correlated with victory. Once features had been narrowed down to those contributing most strongly to certain outcomes, we could hand-craft competing models that only considered those features. PPLs iterate over a “program trace” (Cusumano-Towner et al. 2019) or snapshot of various states during the execution of a probabilistic program. However, our program was written such that it could also provide us with a likelihood of actually observing a particular trace. Given that we know that at a particular time the target system was in a particular state, we could force our program to make those same choices and change any other unconstrained choices in our program such that it maximized the likelihood of the observed data. One such choice might be the weight given to a particular feature. The challenge arises when trying to propose a new value for a particular choice, especially when choices are tightly coupled. If a change we proposed to a variable made the trace less likely we were cautious about accepting it. One simple but common problem occurred when variable A only takes on a particular state when the states of variables B and C agree (i.e., correlate). It is possible to detect such cases and handle them appropriately with PPLs, but it currently requires explicit knowledge of the underlying system as well as expert-level understanding of the MCMC algorithms employed. Furthermore, under certain conditions, probabilistic programs are guaranteed to converge, but they are not guaranteed to converge quickly. Reasonable convergence time comes down to well-designed model spaces informed by knowledge of the target system. In this regard, PPLs may be well-suited to doing social science in domains where experts are readily available, well-informed, and forthcoming about plausible and implausible mechanisms of behavior. A final promising methodological approach we found for studying agent behavior was deep learning in general, and graph convolutional networks (GCNs) in particular (Kipf and Welling 2016). GCNs are a subset of graph neural network (GNN) models that characterize nodes by including features of neighboring nodes (Wu et al. 2020). We also tested the efficacy of attention-based approaches, weighting neighboring nodes according to their “importance” for ego nodes. To capture the time-varying nature of the networks involved we explored the use of long short-term memory (LSTM) propagation for constructing successive GCNs over time (Hochreiter and Schmidhuber 1997), one of many ways GNNs can be constructed to suit specific use cases (Zhou et al. 2018). Finally, we tested an approach to dynamic graphs (EvolveGCN) designed to reduce computational complexity by focusing on temporal dynamics over node representations (Pareja et al. 2020) and the use of hyperbolic GCNs that preserve scale-free or hierarchical graph structures (Chami et al. 2019). The most salient use case for these approaches was Urban World, where all three phases/simulations included multiple dynamic social networks in the form of friendships, work relationships, and site co-location. In essence, GNN methods assign latent states to graph nodes by embedding these nodes in a geometric space. GCNs assign latent states based on the states of a node’s neighbours (identified from an adjacency matrix) and can be propagated using an LSTM-like mechanism. Indeed the aforementioned methods seem to adequately capture the structure of social science problems in question: Agents can be represented by nodes, their associations by edges, and time evolution corresponds to the evolution of agents' states. These approaches seemed a natural fit during, for example, Phase 3 of Urban World where tasks focused on understanding, predicting, and intervening in disease transmission networks. For instance, we expected that the embedding of agent features using latent states would prevent human bias in feature selection while still retaining maximal information. However, these methods only partially solved the problem of disease evolution because almost all focus either on node state prediction or link prediction separately. Only the EvolveGCN framework purported the ability to do both simultaneously, but this feature was novel and its implementation brittle. In the case of these synthetic worlds, many types/levels of associations existed: node states had discrete properties, and we had to predict the evolution of the whole system, not just the state of nodes, for example. All of these are complications we believe had yet to be addressed by methods at the time but represent ready targets for the future. Perhaps the most notable aspect of applying GCNs in the GT project was their persistent inability to reproduce macrosocial properties of social systems based on microdata. That is, disease transmission is a fundamentally network-based process and so we expected GCNs to perform well when modeling the variety of factors influencing this process. However, we were never able to reproduce the accuracy obtained from applying simple compartmental models of disease evolution, such as the susceptible, infected, and recovered (SIR) model. These models consist of basic differential equations whose parameters determine the overall distribution of disease states within the population at an aggregate level. We expected that GCNs would at least be able to reproduce (if not improve on) compartmental models but, despite significant effort, we found that they could not. Given the structural similarity between the GCN architecture and network-based mechanisms of disease transmission we are forced to conclude that either (a) our implementation of GCN was conceptually flawed, or (b) the use of GCN fails to capture a fundamental property of network evolution. We discovered later that disease spread in Urban World was modeled with SIR-like models and so our models may have been too precise for the coarse-grained spread of disease in data. In either case, GNNs (and especially GCNs) appear to be a promising new method for network analysis in the social sciences but may require further development before scholars can realize their full potential in complex social settings. The methodological issues we faced during the GT project suggested three basic lessons learned. First, the level of analysis and type of social process involved are critical for selecting the appropriate method. This lesson is almost remedial in nature given that it amounts to a reminder to select the right tool for the right job. However, the second lesson was methods that may seem intuitively applicable can fail spectacularly (e.g. GCNs), but that openness to alternative approaches can allow for a process of self-correction. Sometimes the latest and greatest method seems like it should work but does not, and that failure to perform as expected can be useful for thinking about less complex but similarly applicable methods with a proven track record and which still embed unarticulated understandings about the world in question. Finally, we learned that imperfect knowledge about a social system can be good enough to find effective methods. Beginning from a tabula rasa typically does not imply beginning from a state of total ignorance. Rather, acknowledging some level of ignorance can help guide the use of methods that have few if any assumptions and may thus “enlighten” our thinking about a problem. Discussion The ambitious DARPA Ground Truth project led to the simulation of four social worlds in which social science could be evaluated in silico. Because these worlds were based on simulations, simulation teams knew the causal ground truth—they had designed the programs themselves—but the research teams did not. Our experience attempting to crack puzzles of these worlds reinforced what AI pioneer Allen Newell stated about research: “You can't play 20 questions with nature and win” (Newell et al. 1972). And we couldn’t play 20 questions about in silico social worlds and win consistently. Stochastic elements of the simulations resulted in a Bayes error rate or irreducible error far greater than 0, and natural limitations on certain forms of data gathering like ethnography and other qualitative methods in the in silico setting were awkward and limited the context research teams were able to achieve. Nevertheless, we and the other research teams were able to do better than random change on most tasks in most phases, and we improved over time and with additional data. Moreover, by confronting tasks with distinct ontological, epistemic and methodological requirements, we gained deep insight into the limits of quantitative social science, especially with respect to informing social policy. Faced with unfamiliar simulated worlds, we struggled to identify their underlying ontology. This highlighted the crucial role of grounded, qualitative insight from insider views of any social system, which cannot not be substituted with quantitative censuses or digital trace data. Why? Because data labels did not provide enough context. They became boundary objects, passed from simulators to researchers through T&E without a shared certainty of reference. This was not a flawed property of the GT program but reflects the limits of ungrounded quantitative social science—data science—where variable names disseminate with interpretations that shift with context. Without a tighter sense of not only the ontology of GT worlds, but what was salient, we struggled to construct experiments despite their availability as a sanctioned data gathering approach because, until the end, we did not know which critical factors to vary, holding others constant. This underscored the challenges of problem solving under conditions of extreme existential uncertainty that contribute to many complex societal challenges. The policy relevance of quantitative social science is also conspired against by the current epistemic standard for publication. Demonstration of novel entities and causes is expected in science, but this narrow exhibition can work against the ability to make meaningful interventions on problems and propose robust policies—from above or below. Finally, we attempted to use a vast menagerie of methods. Some of the most promising emerging methods included detailed bespoke descriptive data analysis, probabilistic programming, deep neural networks of many flavors, and systems of predictive probabilistic finite state machines, which we developed alongside robust statistical and machine learning approaches to supervised and unsupervised learning. Through this exploration, we learned that imperfect knowledge about the most important factors can be sufficient to generate robust predictions and policies. Moreover, applying competing approaches via distinct subteams, including at one point the vast TopCoder.com global community of program solvers, enabled us to discover relevant structure underlying worlds that singular investigators and methods could not. Collectively, these lessons suggest how different a policy-oriented quantitative social science would be than the quantitative social science and data science most commonly practiced to date. Data science and quantitative social science that serves policy will need to endure more failure, sustain more diversity, tolerate more uncertainty, and allow for more complexity than current institutions are well-positioned to support. Acknowledgements The authors gratefully acknowledge DARPA grant HR00111820006 for the Ground Truth program, for Adam Russell, the architect of that program, and other participants in the program (and authors of articles in this special issue) for their inspiration as fellow travelers and contributors to this project. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Althouse BM, Wenger EA, Miller JC, Scarpino SV, Allard A, Hébert-Dufresne L, and Hao H (2020) "Superspreading events in the transmission dynamics of SARS-CoV-2: Opportunities for interventions and control." 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==== Front DNP Die Neurologie & Psychiatrie 2731-8168 2731-8176 Springer Medizin Heidelberg 5580 10.1007/s15202-022-5580-6 Medizin Aktuell Neue Demenztherapien dank COVID-Forschung? M. Heim Thomas Freier Medizinjournalist, 79100 Freiburg, Germany 1 12 2022 2022 23 6 1415 © Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. issue-copyright-statement© Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 ==== Body pmcMöglicherweise spielen Autoimmunprozesse bei neurodegenerativen Erkrankungen eine größere Rolle als bisher angenommen. Neben den üblichen Verdächtigen wie HSV, EBV oder Influenza scheint auch SARS-CoV-2 die Bildung antineuronaler Autoantikörper zu begünstigen. Nach Einschätzung von Prof. Dr. Harald Prüß, Neurologie, Charité, Universitätsmedizin Berlin, hatte die umfangreiche Forschung rund um die COVID-19-Pandemie positive Nebeneffekte auch außerhalb der Virologie. So habe sie Einblicke in das Zusammenspiel von Autoimmunprozessen und Neurodegeneration gewährt, die die Behandlung neurodegenerativer Erkrankungen in Zukunft revolutionieren könnten. Stetig zunehmendes Demenzrisiko Eine COVID-19-Erkrankung geht nach Prüß nachweislich mit einem erhöhten Risiko einher, kognitive Einschränkungen bis hin zur Demenz zu erleiden. Das belege etwa eine systematische Metaanalyse von 290 Personen, die nach einer durchgemachten COVID-19-Erkrankung im Montreal Cognitive Assessment (MoCA) im Mittel um rund einen Punkt (-0,94; 95 %-Konfidenzintervall [KI]: -1,59 bis -0,29); p = 0,0049) schlechter abschnitten als gesunde Kontrollpersonen [Crivelli L et al. Alzheimers Dement. 2022;18(5):1047-66]. Eine retrospektive Analyse von Gesundheitsregistern in Großbritannien habe zudem anhand von 1,5 Millionen Genesenen nach einer COVID-Erkrankung in einem Referenzzeitraum von zwei Jahren gezeigt, dass diese im Vergleich zu Personen mit anderen Atemwegsinfekten ein erhöhtes Risiko für psychotische Störungen (Hazard Ratio [HR]:1,27; 95 %-KI: 1,18-1,37; p < 0,0001), kognitive Störungen und Demenz (HR:1,33; 95 %-KI:1,26-1,41;p < 0,0001) sowie für epilpetische Anfälle oder Epilepsie aufwiesen. Das Risiko für eine Demenz nahm nach einer COVID-19-Erkrankung im Langzeitverlauf sogar weiter zu, während sich das Risiko für affektive Störungen wieder dem Niveau nach anderen Atemwegsinfektionen anglich (Abb. 1) [Taquet M et al. Lancet Psychiatry 2022;9(10):815-27]. Elimination von Antikörpern Prüß und Mitforschende fanden im Liquor von Post-COVID-Betroffenen ein bestimmtes Spektrum an Autoantikörpern, die sich laut Immunfluoreszenzuntersuchungen an Mäusehirnpräparaten gegen jeweils verschiedene neuronale Strukturen richten. Man wisse bei vielen dieser Antikörper noch nicht genau, an welchen Antigenen sie andocken, habe sie aber als Risikokonstellation zusammengefasst. Tatsächlich schnitten Personen, bei denen diese Antikörper im Liquor nachweisbar waren, im MoCA signifikant schlechter ab. Prüß hält es für denkbar, dass hier das Potenzial für eine neue Behandlung von Post-COVID liege. Ähnlich wie bei anderen Autoimmunerkrankungen gelte es, die Antikörper gezielt zu entfernen, etwa mittels Apherese, um die Neurodegeneration so aufzuhalten. "An der Charité nutzen wir derzeit unsere experimentelle Pipeline, um diese Antikörper zu klonieren," führte er aus. So habe man im Liquor von Post-COVID-Betroffenen bereits einen Antikörper gegen Myelin identifiziert, nachgebaut, kloniert und ins Gehirn von Mäusen infundiert, die daraufhin charakteristische Verhaltensänderungen gezeigt hätten. Durch Immuntherapie früher in Krankheitsprozesse eingreifen? Der Neurologe vermutet zwischen autoimmunologischen und neurodegenerativen ZNS-Erkrankungen ein Spektrum mit fließenden Übergängen. Am einen Ende seien Erkrankungen, die neurodegenerativ erscheinen, aber tatsächlich klassische Autoimmunenzephalitiden sind. Am anderen Ende befänden sich Erkrankungen, die fast ausschließlich auf der Fehlfaltung und dem chronischen Abbau von Proteinen basieren. Bei der Demenz gäbe zwar viel versprechende antikörperbasierte Therapieansätze mit Medikamenten, die gegen β-Amyloid oder Tau-Protein gerichtet sind. Mit der Immuntherapie tue sich aber möglicherweise die Chance auf, bereits viel früher in den Krankheitsprozess einzugreifen. Neuroinflammation bei neurodegenerativen Demenzen, 4.11.2022, DGN-Kongress/Neurowoche, Berlin	0	PMC9713147	NO-CC CODE	2022-12-02 23:22:08	no	DNP. 2022 Dec 1; 23(6):14-15	utf-8	null	null	null	oa_other
==== Front Mol Biotechnol Mol Biotechnol Molecular Biotechnology 1073-6085 1559-0305 Springer US New York 36451062 616 10.1007/s12033-022-00616-8 Original Paper Incubation Temperature and Period During Denarase Treatment and Microfiltration Affect the Yield of Recombinant Adenoviral Vectors During Downstream Processing Sonugür Fatma Gizem 1 Babahan Cansu 1 Abdi Abgarmi Samira 1 http://orcid.org/0000-0003-1631-5739 Akbulut Hakan [email protected] 12 1 grid.7256.6 0000000109409118 Department of Tumor Biology, Cancer Research Institute, Ankara University, Ankara, Turkey 2 grid.7256.6 0000000109409118 Department of Medical Oncology, School of Medicine, Ankara University, Ankara, Turkey 30 11 2022 111 25 9 2022 16 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Adenoviral vectors (AV) are commonly used as vaccine and gene therapy vehicles because of their ease of construction, ability to grow to high titers in the large-scale production process, and safety for human applications. However, the efficiency rate of downstream processes for adenoviral vectors still varies greatly. In the current study, we aimed to investigate the effect of the downstream treatment protocol and microfiltration of the harvested upstream material on viral vector yield. We compared the performance of the repeated freeze–thaw (RFT) and the Tween-20 detergent lysis (DLT) methods. In addition, the effects of the cell lysis method, incubation temperature, and time on viral yield were investigated. The samples were incubated at either room temperature or 37 °C for 1-, 2-, and 4-h periods. Samples were filtered with PES and SFCA membrane. Virus yield and infectivity were assayed by qPCR and immuno-titration. In conclusion, our results suggest that 2-h incubation gives the best results when incubated at 37 °C for denarase activity when Tween-20 is used for virus recovery. If the room temperature is preferred, 4-h incubation could be preferred. A phase 1 clinical trial (NCT05526183, January 21, 2022) was started with the recombinant adenovirus used in the study. Keywords Downstream processing Bioprocess development Viruses Vaccines SARS-CoV-2 http://dx.doi.org/10.13039/501100004410 Türkiye Bilimsel ve Teknolojik Araştırma Kurumu TUBITAK-1004-18AG020 Akbulut Hakan ==== Body pmcIntroduction Viral vectors such as adenoviruses, adeno-associated viruses, or retroviruses are becoming increasingly crucial in vaccines and gene therapy to deliver genetic material to the target cell [1]. Adenoviral vectors have several properties that make them ideal candidates as gene delivery systems in gene therapy and recombinant vaccine design. Contrary to lentiviral vectors, adenoviruses do not integrate into the host genome, with the viral genome remaining episomal [2, 3]. This property makes adenoviral vectors safe for mass vaccination against viral diseases. In addition, many features of adenoviral vectors make them advantageous in gene therapy, including their ease of construction, their ability to grow to very high titers in cell culture, the availability of certified cell lines for large-scale production and purification, and their safety for human applications. They also induce high levels of transgene expression and high levels of antigen-specific humoral and cell-mediated immune (CMI) responses by activating innate immunity [4, 5]. Adenoviral vectors also have the advantage of systemic or mucosal administration. Adenoviruses (Ad) are a diverse group of double-stranded DNA viruses with more than 50 distinct human adenoviral serotypes [6]. The most common human adenoviral vector serotypes used in clinical studies are Ad serotype 5 (Ad5), Ad26, Ad35, and Ad4 [7–9]. Human Ad serotype 5 (Ad5) has a non-enveloped icosahedral nucleocapsid containing a linear, double-stranded DNA genome consisting of “early” and “late” regions of approximately 36 kb. Typically, gene insertion into an Ad vector occurs in any early region (E), mainly in the E1 region. E1, E2, and E4 regions critical for virus replication can be deleted to increase transgene insertion capacity. E3 genes are not required for virus replication. Adenoviral vectors with only the E3 gene are replication-competent; vectors with deletion of E1, E2, E4, or any combination are replication-defective [10]. Therefore, a cell line expressing gene cassettes capable of providing viral replication functions is needed [11]. Ad5 has been widely investigated as a gene delivery vector because it can be easily produced at high titers. A wide variety of studies are available to improve its efficacy and safety for clinical use [12–15]. With the COVID-19 pandemic, four adenoviral vector-based vaccines were approved for emergency use [16]. In addition, many adenoviral vector-based COVID-19 vaccines are being tested in clinical trials [3, 17, 18]. Improvement studies are still ongoing to reduce production cost and increase the stability of the vaccine. The adenoviral vector production process consists of two steps: upstream and downstream. First, the upstream process of the adenoviral vectors is amplified in suitable cell lines (HEK293 or PERC.6) [19]. The downstream process mainly includes the viral particle release, clarification, and ultra/diafiltration steps. All the steps in the downstream process affect the yield of the final product. Likewise, the downstream processes cause the most loss of vectors in the adenoviral vaccine production process [20]. Replicated viruses in the cells release into the supernatant by a freeze–thaw method in small-volume productions and detergent-mediated methods in large-scale productions. Cell lysis using detergent is easy to perform in the laboratory and at the production scale. In this step, most commonly, non-ionic detergents such as Tween-20 and Tween-80 and ionic detergents such as sodium deoxycholate can be used to solubilize cell membranes and release viruses [21, 22]. Tween-20 detergent, also known as Polysorbate-20, is frequently used for protein solubility, protein–lipid, lipid–lipid aggregation, and enveloped virus clearance in the pharma industry [23, 24]. The release of intracellular proteins and cellular genomic nucleic acids during the viral particle release step may compromise the clarification steps, including microfiltration. DNA is known to mediate particle aggregation, including the aggregation of viruses [25]. To prevent the aggregation of adenovirus with nucleic acids, incubation with a nuclease enzyme, especially denarase endonuclease (between 1 and 1000 U/ml), is used to reduce the DNA concentration rapidly [26, 27]. In addition, denarase endonuclease facilitates the elimination of nucleic acids in later steps by reducing the nucleic acid chain length and lowering the viscosity by reducing nucleic acid-mediated aggregation [25]. However, the incubation temperature and time for cell lysis affect the virus purification efficiency and need to be optimized. In downstream processes of adenoviral vectors, incubation times vary from half an hour to 4 h during the release of viruses from the cell [22, 28]. The incubation temperature varies between room temperature and 37 °C [21, 22, 28]. However, the effects of incubation periods and temperature on vector yield and stabilization are not well known. The clarification efficiency directly affects downstream process performance. A poorly optimized clarification step may adversely affect the sterilizing-grade filter capacity or shorten the life of the chromatography resin [29, 30]. The clarification step includes low-speed centrifugation, microfiltration, tangential flow filtration (TFF), and normal flow filtration (NFF) or a combination of these for the purification of adenoviral vectors [31, 32]. Low-speed centrifugation allows the removal of cells and cell debris by sedimentation. Microfiltration with membrane pore sizes in the 0.4–1.2 μm range has been successfully used to remove cells, cell debris, and other large contaminants [33]. Filters with a pore size of 0.45 µm are typically used for the microfiltration of rAd5 [34]. The type of membranes used and the pore diameters are other important parameters affecting the yield. The most commonly used virus filtration membranes are polyethersulfone (PES), Surfactant-free Cellulose Acetate (SFCA), Polyvinylidene Fluoride (PVDF), and Regenerated Cellulose (RC) membranes [35, 36]. PES membranes are known to have low protein-binding properties [37, 38]. Adenoviral particles are expected to pass through these membranes with high efficiency if the concentration of adenoviral particles is kept below 5 × 1011 particles/ml [37]. Nestola et al. showed that PES-based membranes have a low recovery rate of 23–58% in total virus particles [39]. There is no small-scale study comparing the PES and SFCA membranes. While the cell lysis method performed at the beginning of the downstream process reveals the AV particles, Denarase applied in this process also reduces the host cell DNA. During cell lysis and enzyme incubation, protease enzymes released in the medium can affect the number of viral particles released from cells and their infectivity, depending on temperature and time [40–42]. Many viral products are less stable at culture temperatures (typically 37 °C) as compared to lower temperatures (between 4–25 °C) [43]. There is no consensus regarding lysis temperature and enzyme treatment time [29, 43]. Therefore, in this study, we aimed to compare the performance of the repeated freeze–thaw method as a physical lysis method and the chemical cell lysis method with Tween-20 detergent and determine the optimum incubation temperature and period with denarase endonuclease. In addition, we also studied the performance of microfiltration with SFCA and PES membranes. Materials and Methods Adenoviral Vector Amplification and Study Groups HEK293 (human embryonic kidney) cells were purchased from Germany (DSMZ, ACC305). These cells adapted to suspension culture were amplified in Acti-Pro (Cytiva, MA) cell culture medium supplemented with cell boost 7A (Cytiva, MA). After cell density was reached at 1 × 106 cells/ml, cells were infected with a recombinant human type 5 replication-deficient adenoviral (rAd5) COVID-19 vaccine vector carrying spike gene of SARS-CoV-2 [44] at the multiplicity of infection (MOI) of 5 and incubated at 37 °C and 5% CO2 with shaking at 130 rpm in a shaker flask for three days. The development of suspended HEK293 cells’ cytopathic effects (CPEs) was observed daily. The cells were harvested when the ratio of cells with CPE was 85% on the third day of incubation and kept at − 80 °C till the downstream process. The harvested rAd5 vaccine vector was thawed on ice before starting the treatment protocol. The harvested sample was divided into 12 separate study groups with 40 ml each for treatment (Fig. 1).Fig. 1 Schematic of the process steps used to produce and clarify the adenovirus Cell Lysis and Nuclease Digestion Two cell lysis protocols were used: The Repeated Freeze–Thaw method (RFT) and the Tween-20 Detergent Lysis method (DLT) [45]. Due to the very low viral particle concentration of the supernatant, the harvested material was first kept at − 80 °C immediately following the harvesting procedure. As a reference treatment, the repeated freeze-thawing method was used. For freeze-thawing, 6 tubes from the harvested sample were frozen in a − 80 °C freezer for 10 min, followed by thawing at 37 °C. This procedure was repeated twice. Following the RFT cycles, MgCl2 at a final concentration of 1 mM and 30 U/ml of Denarase (cLEcta GmbH, Germany) was added to the samples. For the detergent lysis method, Tween-20 (0.5% v/v) was added to the other 6 tubes in addition to MgCl2 and Denarase. The digestion proceeded for 1-, 2-, and 4-h either at room temperature (25 °C) or 37 °C in the incubator on a shaker with 100 rpm. Experiments are conducted at both temperatures. After the incubation, samples were taken to analyze pH, cell viability, and remaining intact cells. Then, the samples were centrifuged at 5000×g for 5 min, and supernatants were collected for microfiltration. Microfiltration Clarification of the harvested material was performed by normal flow filtration (NFF), using syringe filters with a membrane diameter of 28 mm and a pore size of 0.45 µm, with Surfactant-free Cellulose Acetate (SFCA) (Corning, NY) or Polyethersulfone (PES) (Sartorius, Germany) membranes for removal of cell debris and initial impurity reduction. Before use, filters were equilibrated with growth medium. Half of the samples (15 ml) from the same incubation were filtered with a PES membrane and half (15 ml) with an SFCA membrane using a 20 ml syringe with constant pressure for 60 s. Characterization Assays To test the cell viability, the trypan blue exclusion method was performed just after harvesting, following the first thawing, repeated freeze–thaw cycles, and denarase treatment of RFT and DLT. Viral DNA Quantification For the determination of rAd5 vector genome copies, quantitative Real-Time Polymerase Chain Reaction (qPCR) was used. According to the manufacturer's instructions, the viral DNA was extracted using a commercial kit (PureLink™ Viral RNA/DNA Mini Kit, Thermo Scientific, USA). The viral genomes were quantified in triplicate by qPCR using the Adenovirus type 5 specific primers (forward: 5′-CCACCGATAGCAGTACCCTT-3′, reverse: 5′-GACCAGTTGCTACGGTCAAA-3′) and TaqMan probe (5′-FAM-TGCCCAAGCTACCAGTGGCAGT-TAMRA-3′). PCR reaction was performed in CFX96 Touch Real-Time PCR Detection System (Bio-Rad) under the following conditions: hold at 95 °C for 12 min, and 40 cycles at 95 °C/15 s (denaturation) and 60 °C/1 min (hybridization). Titration results are given in viral DNA/ml (vDNA/ml). A standard sample of known titers was used for quantification. Infectious Particles Infectious particles were quantified using the Immuno-titration assay kit (Cell Biolabs, CA). HEK293 cells in DMEM supplemented with 10% FBS were incubated in 24 well plates in an incubator with 5% CO2 at 37 °C for 1-h. The cells were then infected with diluted viral samples and incubated for two days. Each sample was performed in duplicate. Positively stained cells (brown) were counted for at least five separate fields per well using a light microscope with a 10X objective. Viral titer was calculated using the formula Viral Titer (Infectious Unit (ifu)/ml) = (average positive cells/field) × (79 fields/well) × (dilution factor). The methods used in the current study are standard in the literature [46, 47]. The methods were repeated three times with similar results. Statistical Analysis Statistical analyses were conducted with the Statistical Package for Social Sciences (SPSS) software, version 10.0. Mann–Whitney U test and Kruskal Wallis H test were used for non-parametric comparisons, and one-way ANOVA was used for appropriate parametric comparisons. Results The Cell Lysis Method and Incubation Conditions Affect the Viral Particle Yield The effects of the cell lysis method, incubation temperature, and period on total viral particle yield were investigated. For cell lysis, freeze-thawing and treatment with the 0.5% Tween-20 methods were used. The ratio of viable cells was assessed by trypan blue assay at the end of each incubation period. The ratio of the viable cells by trypan blue test was 15% at the harvest time. The ratio of the viable cells on the trypan blue test was 2% following the gradual thawing of the harvested material and was less than 1% in the first hour of treatment protocols. There was no significant difference between the treatment methods. No viable cell was detected after 2-h of incubation of the samples in any treatment methods. The viral genome copies in the harvested material were determined by the qPCR method. It was found that the total copy number of vDNA (3.75 × 108 vDNA/ml) in the repeated freeze-thawed samples significantly increased compared to pre-treatment samples (2.5 × 108 vDNA/ml, p ≤ 0.001). The more lysed the cells, the more the viral genomes increased. To investigate the effect of incubation temperature and period on the total viral genome copies, the samples were incubated at either room temperature or 37 °C for 1-, 2-, and 4-h and virus yield was assayed by qPCR before filtration. As shown in Fig. 2, the percent viral genome copies (vDNA) obtained by qPCR in the RFT method decreased at the 1st-hour of incubation at room temperature (p ≤ 0.001). At the end of the 2nd-hour incubation, the percent of vDNA yield in the RFT sample showed a statistically non-significant increase compared to the 1st-hour incubation. However, at the 4th-hour RFT incubations, the vDNA yield increased significantly compared to the 1st-hour (p = 0.003) and the 2nd-hour (p = 0.029) incubation periods.Fig. 2 The viral genome (vDNA) yield of two different cell lysis methods (Repeated Freeze–Thaw and Tween-20 Detergent Lysis) at different incubation periods and temperatures. The percent vDNA yield was calculated as the percentage of the final yield to yield before the treatment. In general, higher viral DNA concentration was obtained in the results of the DLT method compared to the RFT method. Higher vDNA concentrations were observed in the samples at the 2- and 4-hour incubation compared to the 1st hour. Data are expressed as mean ± standard deviation, p ≤ 0.05; p ≤ 0.01; p ≤ 0.001; ** p ≤ 0.0001 A significant decrease was observed in RFT samples following 1-h incubation at 37 °C compared to the pre-incubation (p ≤ 0.0001). Similar to the incubation results at room temperature, a slight increase was observed for RFT in the 2nd-hour incubation compared to the 1st-hour incubation (p = 0.069). The 4th hour incubation results showed a statistically non-significant change compared to the 1st and 2nd-hour incubations at 37 °C. In the DLT method, the percent vDNA yield in the cell suspension decreased significantly at the first hour of incubation compared to pre-treatment samples at room temperature (2.5 × 108 vDNA/ml, p ≤ 0.001). However, a statistically significant increase in the 2nd-hour incubation compared to the 1st-hour incubation at room temperature (p ≤ 0.0001). In addition, a significant increase in the vDNA yield at the 4th hour incubation was observed in the DLT sample compared to the 1st-hour (p = 0.0001) and the 2nd-hour (p = 0.001) incubation period at room temperature (Fig. 2). A slight decrease was observed in DLT samples following 1-h incubation at 37 °C compared to the pre-incubation values (p = 0.141). In contrast, the vDNA yield increased significantly in the 2nd-hour Tween-20 incubation compared to the 1st-hour incubation (p = 0.0001). At the 4th-hour incubation at 37 °C for DLT, while the vDNA yield increased significantly (p = 0.005) compared to the 1st hour, a significant decrease was observed compared to the 2nd-hour incubation (p ≤ 0.0001) (Fig. 2). Microfiltration with SFCA Membrane Yields More vDNA After low-speed centrifugation, clarification was performed by filtrating the sample through PES and SFCA membranes to remove the cell debris from the virus suspension. The viral genome amplification after filtration was determined by qPCR (Fig. 3). The filtering efficiency was calculated by dividing the vDNA concentration before and after filtration. Also, the filtering efficiency was calculated for different incubation temperatures and periods using different methods for cell lysis.Fig. 3 The percentages of vDNA yield following microfiltration. The percent vDNA yield was calculated as the percentage of the final yield to yield before filtration. The SFCA membrane was detected to be more efficient than the PES membrane. Data are expressed as mean percentage ± standard deviation. p ≤ 0.05; *p ≤ 0.01 While the efficiency of the PES membrane was between 10 and 50%, the efficiency of the SFCA membrane was between 20 and 55% (Fig. 2 and 3). The filtration efficiencies of the PES membrane with the RFT method after 1-, 2-, and 4-h of incubation were 18.13%, 19.72%, 17.60% at room temperature, 28.07%, 10.02%, and 23.27% at 37 °C, respectively. The SFCA membrane filtration efficiencies of the same group of samples were 40.28%, 26.12%, and 32.94% at room temperature and 41.87%, 27.14%, and 20.76% at 37 °C, respectively. The PES membrane filtration efficiencies of the samples incubated with Tween-20 detergent were 80.36%, 49.75%, 21.37% at room temperature, and 76.02%, 22.81%, and 25.02% after 1-, 2-, and 4-h of incubation. However, the SFCA membrane filtration efficiencies of the same group of samples were 81.05%, 55.09%, and 34.73% at room temperature, while it was 65.49%, 35.24%, and 47.17% at 37 °C. Although the filtration efficiency over the incubation period did not change significantly, a slight decrease was observed with longer than 1-h incubation with PES and SCFA membrane filters. Cell Lysis Method and Microfiltration Affect the Infectivity of the Vector The effects of the cell lysis method, incubation temperature, and incubation period on virus infectivity were investigated by the immuno-titration method. The results of the immuno-titration as infectious units or IFU performed in the study groups are shown in Fig. 4.Fig. 4 Infectious viral particles (IFU/ml) were obtained after cell lysis using repeated freeze–thaw and detergent-based methods. Infectious viral particles were determined by immuno-titration assay. In the DLT method, more infectious particles were obtained than in the RFT method. However, better results were obtained in samples filtered with PES membrane in both methods. Data are expressed as mean ± standard deviation In the RFT method incubations at room temperature and 37 °C, the incubation period in the samples filtered with PES membrane caused a slight but not significant increase in the infectivity of the virus. In contrast, though not significant, a decrease in the 2nd hour and an increase in the 4th hour were observed in the samples filtered with SFCA (Fig. 4). The insufficient degradation of host cell DNA and free vDNA from lysed viral particles at 2 h of incubation with Denarase might decrease the infectivity of the vector in HEK293 cells [46]. When cell lysis was performed with the DLT method, the highest IFU value was obtained with 1-h incubation of PES membrane filtration at room temperature. At the same time, the infectivity of the virus decreased as the incubation period extended (Fig. 4). Similarly, for the SFCA membrane filtration, the highest infectivity ratio was achieved during the 1st-hour incubation at room temperature. A progressive decrease of virus infectivity was found at the 2nd and 4th-hour incubations. The incubation at 37 °C with an increasing incubation period decreased the infectivity of the virus during the 1-, 2-, and 4-h incubations after PES membrane filtration. The highest immuno-titration result in SFCA membrane filtration was obtained in the 2nd-hour incubation (Fig. 4). In the DLT method, more infectious vectors were obtained than in the RFT method (Fig. 4). However, both methods obtained better results in samples filtered with PES membrane. The pH of the samples did not change significantly during the incubation and filtration processes. While the pH was 6.84 before the cell lysis process, it increased to 7.08 and 6.98 at room temperature and up to 7.04 and 6.90 during incubations at 37 °C in the RFT and DLT groups. When the vDNA/IFU (viral DNA copy number / infectious units) ratios were compared after cell lysis by the RFT method, the most consistent and lowest rates were in samples filtered with PES membrane after 1-, 2- and 4-h incubations at room temperature (Table 1). However, filtering the samples with SFCA increased the rate by almost double (Table 1). In addition, the PES membrane had better vDNA/IFU ratios than the SFCA membrane in the clarification of harvested vector material, in which cell lysis was performed either by RFT or DLT methods (Table 1).Table 1 The ratio of Viral Genomes to Infectious Units (vDNA/IFU) vDNA/IFU Repeated Freeze–Thaw Tween-20 Room temperature 37 °C Room temperature 37 °C 1 h 2 h 4 h 1 h 2 h 4 h 1 h 2 h 4 h 1 h 2 h 4 h PES 3.95 4.08 4.00 9.64 3.69 6.19 9.62 33.91 7.77 10.90 8.35 12.95 SFCA 10.36 8.10 8.60 11.19 11.43 6.11 21.72 21.90 20.20 13.72 10.45 17.09 Discussion The efficiency of downstream processing of adenoviral vectors varies significantly between 2 and 60% among different protocols [48, 49]. The cell lysis method, DNase-I and MgCl2 concentrations, pH, incubation period, and incubation temperature are important parameters affecting the virus purification efficiency. Therefore, the current study investigated the effects of the cell lysis methods and microfiltration of the harvested upstream material on viral vector yield. Furthermore, the effect of different incubation temperatures (room temperature and 37 °C) and times (1-, 2-, and 4-h) in these processes were investigated. The treatment protocols, including repeated freeze–thaw cycles and detergent lysis methods, have been used widely. Previously, it has been reported that the virus yields from host cells using the detergent lysis method were higher than that of the repeated freeze–thaw method [50, 51]. The repeated freeze–thaw method is the oldest method for cell lysis; however, it is only suitable for lysing small amounts of harvest and cannot be linearly scaled up to an industrial scale, unlike the detergent-based lysis method [22, 52]. Benzonase and Denarase have been routinely used in virus manufacturing studies to eliminate the host cell DNA and RNA from the recombinant vector product. The temperature and time during the treatment may affect the viral vector yield. Total vDNAs and infectious particles are critical parameters for comparing production processes [43, 53, 54]. In the current study, we compared the efficiency of the Tween-20 and the traditional repeated freeze–thaw method. The viral genome yields obtained by qPCR in both the RFT and DLT methods decreased significantly at the 1st-hour of incubation at room temperature (Fig. 2, p < 0.001). The decrease of vDNA yield in the first hour of incubation may result from the degradation of the vDNA outside the capsid of the vector by the denarase caused by osmotic shock during the gradual thawing of the frozen-harvested material [55]. We found a significant increase in the vDNA yield at the 4th-hour incubation of the RFT protocol compared to the 1st-hour and the 2nd-hour incubation periods (Fig. 2). Complete lysis of the cells remaining intact during the process and virus release seem to cause the increase of virus yield. Likewise, a significant increase in vDNA yield during the 4-h incubation was observed in the DLT samples compared to the 1st-hour and the 2nd-hour incubation periods. Incubation with the Tween-20 sample group at room temperature resulted in a higher vDNA yield than the samples in the RFT sample group (2nd and 4th-hour). The highest vDNA yield was achieved after 4-h of incubation at room temperature with the DLT method (Fig. 2). Accordingly, we found a slight decrease in the viral DNA yield of samples in the first hour of incubation at 37 °C. Then a significant increase with 2-h and 4-h incubations was observed (Fig. 2). In both methods used for the cell lysis step to obtain viruses, there was lysis of the cells and the lysis of the viruses. The qPCR results of viral DNA released into the cell suspension and genomes isolated from viruses are shown after 1-h of incubation. The purpose of using the Denarase enzyme in these incubations is for the degradation of vDNA from lysed viruses. Therefore, after 1-h of incubation, the vDNA released into the medium by Denarase enzyme activity was degraded, and a decrease in the total vDNA yield was observed. The vDNA yield was significantly decreased at 37 °C compared to room temperature in both treatment methods. The vDNA yield of the DLT method was significantly higher than the RFT method at 2- and 4-h incubation periods (Fig. 2). 4-h incubation at room temperature yielded significantly more viral genomes than the 2-h incubation at room temperature and 37 °C (Fig. 2). However, the yields of 2-h incubation at both room temperature and 37 °C were similar. Our results show that the DLT treatment for 4 h at room temperature yields the best vDNA. By previous results (vDNA, IFU, and vDNA/IFU), a 2-h incubation period with Tween-20 at 37 °C seems the optimum period to lyse the host cells and release the virus completely [29]. Previously, it has been shown that virus viability decreased when the incubation temperature was above room temperature [56–58]. It has been reported that 2-h incubation is sufficient at 37 °C [54], which is the optimum temperature for denarase enzyme activity to eliminate host cell DNA better. In the study by Jardon and Garnier, they showed that low temperature increases cell viability, and the virus yield obtained at 35 °C is three times higher than at 37 °C [58]. Accordingly, we observed that the viability of the adenovirus type 5 vector decreased with increasing temperature (room temperature vs. 37 °C). Clarification has received little consideration and is performed as an obligatory step before other DSP steps to reduce bioburden and increase the capacity of further downstream steps [59, 60]. The cell debris in the virus fermentation broth can easily be removed by filtration to attain the purpose of clarification using low-speed centrifugation to clarify the virus feed [52]. However, releasing intracellular content by cell lysis complicates the purification of viral vectors [38]. In membrane filtration, factors such as the structural properties of the membrane and the diameter of the membrane might affect the efficiency. One of the critical points in optimizing the DSP is the loss of viability and infectivity of the virus during filtration. Generally, PES, SFCA, and PVDF filters with 0.2 µm to 0.45 µm pore diameters are used for clarification [61]. There is no uniform consensus on the type of membrane used for adenoviral vector clarification. It has been shown that Tween-20 detergent is absorbed into the PES membrane surface and can reduce the throughput of adenoviral particles [22, 62]. Nestola et al. showed that PES-based membranes have a low yield recovery rate of total viral genomes (23–58%) [39]. In the current study, we have tested two widely used filters with SFCA and PES membranes. The filter efficiencies obtained in our study were also in the range of previous reports [39]. The vDNA yields of microfiltration with the SFCA were higher than the PES membrane in both RFT and DLT groups (Fig. 3). More vDNA yield was obtained in DLT samples filtered with the SFCA membrane compared to the PES membrane. SFCA caused the lysis of viruses in the filtered virus suspension due to the properties of the membrane, and the vDNA yield in the medium was determined by the qPCR method used. The results of the immuno-titration, in which the number of live viruses was determined, reflect this fact. On the other hand, the Denarase enzyme breaks down the viral DNAs released from the degraded viruses in the suspension. Figure 3 shows a higher vDNA yield at room temperature since room temperature (25 °C) is insufficient for the Denarase enzyme's activity. Although the vDNA yield quantification is mainly used for the formulations of final drug products of recombinant vectors, the virus infectivity is the most critical parameter of the biological activity of those preparations. Therefore, we also studied the vector infectivity rates of the processed samples with an immuno-titration assay (Fig. 4). Incubation temperature and period, pH of the harvested material, shear forces from stirring, centrifugation of fermentation broth, and filtration can damage the structure of adenoviruses, thereby reducing the infectivity of adenoviruses [63]. In the current study, we found that the infectivity of the rAd5 vector decreased with the increase in temperature (Fig. 4 and Table 1). The pH values at different incubation temperatures and periods in our study were very close to the optimum pH (7.2) value [58, 64]. On the other hand, biological factors, including various proteases, are primarily released from host cells. These proteases can hydrolyze adenovirus surface proteins, leading to structural damage and reduced infectivity of adenovirus particles [65]. This explains the difference between total viral genome yield and immuno-titration results (Fig. 3 and Fig. 4). The denarase treatment used in the study did not destroy the ability of the infectivity of viruses [66, 67]. The most exciting and surprising effect of incubation with Tween-20 detergent is the production of rAd5 vectors with higher yields and better infection efficiency than vectors obtained by the classical cell lysis method (Fig. 4 and Table 1). Using several cycles of freezing/thawing, the physical cell disruption method can alter the integrity of viral capsids without destroying the genome-full particles that can still be quantified by quantitative PCR. In other words, some of these vectors could be released in supernatant long before the end of the production phase. The progressive acidification of the culture medium during the upstream process, resulting from the cell metabolism, and the presence of proteases originating from producer cell lysis both provide an unfavorable environment for released rAd5 particles and lead to structural alteration of the supernatant-derived vectors [38]. These findings can have a significant impact on large-scale production. The results of the current study suggest that 2-h of incubation gives the best results when incubated at 37 °C for denarase activity when Tween-20 is used for virus recovery in the cell suspension. If the room temperature is preferred for this suspension, 4-h incubation could be used. Although the methods of cell lysis and clarification affect the virus yield, the purity of the product depends on the chromatography and ultrafiltration steps, which are relatively more standard methods in the later stages. This study mainly targeted the detergent lysis method, incubation temperature and period, and then the microfiltration method. The methods investigated in our study are those used on an industrial scale. However, the effects of incubation temperature, period, and membranes used in microfiltration on adenovirus yield before moving to further purification steps have yet to be well known. HEK293 cells or their derivatives are generally used to produce rAd vectors. We used the RFT and DLT methods in our study of vector production that can be effective in all mammalian cells [46, 54]. Especially the DLT method used in this study as a bench scale is also used in large-scale production. The data we obtained on the bench scale regarding incubation temperature and period can also be applied on a large scale. The data we obtained on the bench scale for microfiltration, especially the parameters such as sample volume, flow rate, and membrane diameter, can also affect the filtration efficiency at a large scale. The lack of purification steps after microfiltration in this study is a limitation. In addition, empty to full ratio and agglomerates affect virus infectivity. Another limitation is that microfiltration is not carried out at different pressures. However, the pressure applied during microfiltration during large-scale production might affect the virus yield. Therefore, the microfiltration step should be optimized for large scale. Author Contributions FGS: Conceptualization, methodology, data curation, writing—original draft preparation. CB: Data curation, software. SAA: Visualization, ınvestigation. HA: Conceptualization, supervision,writing—reviewing and editing. Funding This research is supported by Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (Grant No. TUBITAK-1004-18AG020). Data Availability All data generated or analyzed during this study are included in this article. Declarations Conflict of interest The authors declare no commercial or financial conflict of interest. Ethical Approval Our study did not require an ethical board approval because it did not contain human or animal subjects. Consent for Publication Not applicable. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Morenweiser R Downstream processing of viral vectors and vaccines Gene Therapy 2005 12 103 110 10.1038/sj.gt.3302624 2. Lukashev A Zamyatnin A Viral vectors for gene therapy: Current state and clinical perspectives Biochemistry (Moscow) 2016 81 700 708 10.1134/S0006297916070063 27449616 3. Mendonça, S.A., Lorincz, R., Boucher, P., & Curiel, D.T. 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==== Front J Med Humanit J Med Humanit The Journal of Medical Humanities 1041-3545 1573-3645 Springer US New York 9772 10.1007/s10912-022-09772-z Article As Gods: A Moral History of the Genetic Age, by Matthew Cobb. New York: Basic Books, 2022 http://orcid.org/0000-0002-8793-6412 Chapman Carolyn Riley [email protected] grid.137628.9 0000 0004 1936 8753 Center for Human Genetics and Genomics; Department of Population Health, NYU Grossman School of Medicine, NY New York, USA 1 12 2022 13 7 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcDoes our ability to manipulate genes give humans godlike powers? And if so, are we worthy of the accompanying responsibility? These are the implicit questions asked by Matthew Cobb in his newest book, on the history of genetic engineering. Titled As Gods: A Moral History of the Genetic Age in the United States, the book is perhaps more fittingly named The Genetic Age: Our Perilous Quest to Edit Life, as it is titled in the United Kingdom. To be sure, Cobb raises ethical, social, and cultural issues and questions throughout the book, but it is the stories about genetic advances and their implications—and the humans involved in these stories—that really take center stage. Cobb, a professor of zoology in the School of Biological Sciences at the University of Manchester, has authored several books on the history of science. In 2021, the Genetics Society honored Cobb with the JBS Haldane Lecture, an annual award given to “an individual for outstanding ability to communicate topical subjects in genetics research, widely interpreted, to an interested lay audience” (The Genetics Society n.d.). As Gods confirms Cobb’s talent in this area. By focusing on the scientists, Cobb effectively conveys the positive and negative emotions—excitement, satisfaction, disappointment, hope, and fear—associated with the science. Cobb was motivated to write the book to explore his own misgivings about three technologies made possible by genetic engineering: heritable human genome editing; the use of gene drives to alter characteristics of living organisms, species, and ecosystems; and so-called gain-of-function research on disease-causing pathogens. Accordingly, many of the chapters concentrate on these issues. However, Cobb spends a lot of time covering other topics, including commercial uses of recombinant DNA technology, the history of somatic gene therapy, the discovery of CRISPR (a genome editing technology that has emerged in the past decade), and the development of—and political reaction to—genetically modified foods. Written during the COVID-19 pandemic, each chapter of the book is thoroughly researched and referenced and clearly benefits from information Cobb gained from interviewing many prominent researchers directly involved in the events. The book provides such rich description that even the most knowledgeable readers, those who are familiar with the field and history of genetics, will learn something new. For example, Cobb reveals fascinating backstories related to the 1975 Asilomar conference, which led to the development of safety guidelines for recombinant DNA research: Janet Mertz, then a graduate student in biochemistry at Stanford, shared her plans for a cutting-edge, but potentially dangerous, experiment involving recombinant DNA with Bob Pollack, a postdoctoral fellow at Cold Spring Harbor, in the early 1970s. Pollack called Mertz’s distinguished supervisor, Paul Berg, to express concerns but was initially rebuffed. However, further consideration of Pollack’s concerns ultimately led Berg to organize the Asilomar conference. In another chapter, Cobb informs readers that Bob Swanson, a recently unemployed venture capitalist, cold-called a list of Asilomar attendees and convinced one of them, Herb Boyer, to start a company focused on commercialization of recombinant DNA technology (Genentech). Later in the book, Cobb shares that, unbeknownst to the American organizers of Asilomar, three Russian scientists who attended were involved in establishing a bioweapons program for their country. Readers without scientific training may find the book less accessible as it does get technical and detailed at times, but it will reward those who are interested in the topic with a deeper appreciation of the power, limitations, and potential dangers of genetic engineering. Three significant, particularly thought-provoking high-level themes emerge from Cobb’s book. The first is the allure of genetics as compared to alternative, less “sexy” methods of improving human lives. Second is the balance between pessimistic and optimistic perspectives on the potential benefits and risks of genetic engineering. And third is the tension between unrestricted pursuit of scientific knowledge and the need for regulation. At several points in As Gods, Cobb emphasizes scientists’ attraction to understanding and leveraging genetics to solve problems and contrasts it with the tendency to discount other types of solutions. For instance, Cobb describes the development of and opposition to Golden Rice, which is genetically engineered to have higher levels of β-carotene to address Vitamin A deficiency in certain populations. Notwithstanding all the attention on Golden Rice, Cobb notes that non-genetic solutions to Vitamin A deficiency, such as public education and capsule supplementation, have been successful in the Philippines. Regarding human heritable genome editing, Cobb shares prominent genome editing researcher Fyodor Urnov's assessment that it is a solution in need of a problem, adding his own appraisal that “it is a distraction from the problems we have now, on planet Earth” (282). In the chapter on bioweapons, Cobb reminds readers that releases of organisms such as anthrax and Salmonella can wreak significant havoc and destruction in the absence of any genetic engineering. Cobb questions “why genetic engineering solutions are often seen as a priority” (361) when these solutions elicit ethical issues and may not appropriately address complex, socially driven problems. Whether to take a pessimistic or optimistic viewpoint on the power of genetic engineering is another issue that Cobb contemplates: he seems to identify strongly with both perspectives at various times in the book. Clearly, Cobb recognizes the power and potential of genetic engineering. However, he also stresses that hopes for the technology often exceed reality: “Real-world problems, most of them social and not amenable to simple technofixes, have repeatedly brought a sharp dose of reality to the dreams of the genetic engineers” (348). In a discussion about CRISPR, Cobb points out that some scientists believe that metaphors such as “editing” and “targeting” bely this underlying complexity: experiments don’t always go as planned. Cobb recognizes CRISPR as a powerful tool but stresses that it isn’t a panacea for human disease—and it does not give humans absolute control over genomic DNA. Cobb agrees with one of his interviewees, Sheila Jasanoff, a prominent technology studies scholar who speculates that, with respect to genetic research and the power of science, both “warriors” (“hope people”) and “worriers” (“fear people”) are needed in society to keep these perspectives in check and in balance (363). Finally, Cobb also explores the pros and cons of restrictions on genetic engineering. Describing the mood at the Asilomar conference, in which approximately 140 scientists convened with the aim of developing “biosecurity guidelines that would allow [recombinant DNA] research to recommence” (78), Cobb notes that many of the attendees felt that “‘academic freedom’ effectively gave them the right to do whatever they wanted” (78). Although Cobb highlights four moments in history, including Asilomar, in which scientists voluntarily chose to pause their work to carefully consider risks, he advocates for broader public involvement—and regulation—going forward: “it is clear to me that the model of self-regulation adopted in the 1970s cannot meet the needs of the discoveries of the twenty-first century” (365). The issues are “too important to be left to the scientists” (13). After reading his comprehensive book on the history and promise of genetic engineering, Cobb’s point is well taken. Humans are human, after all. Author contribution Carolyn Riley Chapman wrote this book review. Data Availability Data sharing not applicable to this article as no datasets were generated or analysed for this book review. Declarations Financial or non-financial interests None. Ethical approval N/A Informed consent N/A Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs Reference The Genetics Society. n.d. “JBS Haldane Lecture.” The Genetics Society (website). Accessed October 31, 2022. https://genetics.org.uk/medals-and-prizes/genetics-society-medals-and-lectures/jbs-haldane-lecture/.	36454352	PMC9713152	NO-CC CODE	2022-12-02 23:22:08	no	J Med Humanit. 2022 Dec 1;:1-3	utf-8	J Med Humanit	2,022	10.1007/s10912-022-09772-z	oa_other
==== Front Can J Public Health Can J Public Health Canadian Journal of Public Health = Revue Canadienne de Santé Publique 0008-4263 1920-7476 Springer International Publishing Cham 36449223 712 10.17269/s41997-022-00712-x Special Issue on Sociocultural and Behavioural Factors Affecting Communities' Responses to Public Health Measures: Implications for the COVID-19 Pandemic and Beyond: Qualitative Research Aunties, WhatsApp, and “haldi da doodh”: South Asian communities’ perspectives on improving COVID-19 public health communication in Ontario, Canada Bhalla Manvi Boutros Helana Meyer Samantha B. [email protected] grid.46078.3d 0000 0000 8644 1405 Faculty of Health, School of Public Health Sciences, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1 Canada 30 11 2022 18 23 5 2022 7 10 2022 © The Author(s) under exclusive license to The Canadian Public Health Association 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Objective To identify, from the perspective of South Asian communities, areas for improvement in public health communication. Methods Focus groups were conducted with individuals (N=24) who could converse in English and self-identified as South Asian adults (18+) residing in Ontario. Participants were asked to share how, if at all, their identity as South Asian shaped their experiences during the pandemic and acceptance of public health measures put in place to mitigate the spread. Data were interpreted through the lens of intersectionality. Results Participants perceived a lack of culturally relevant and linguistically accessible health messaging, leading to the proliferation of misinformation. Peer-to-peer knowledge sharing filled a critical gap but created opportunities for misinformation to spread. Conclusion Improving equity in health communications should be informed by structural changes to the public health sector in Ontario. Résumé Objectif Identifier, à partir de la perspective de membres de communautés sud-asiatiques, les aspects à améliorer dans les communications de la santé publique. Méthodes Des groupes de discussion ont été réalisés auprès de participants (N=24) vivant en Ontario, âgés de 18 ans et plus, capables de tenir une conversation en anglais et s’identifiant comme originaires de l’Asie du Sud. Les participants ont été invité à partager si et comment leur identité sud-asiatique avait influencé leurs expériences vécues durant la pandémie et leur adhésion aux mesures de santé publique mises en place pour limiter la propagation du virus. Les données ont été interprétées à travers le prisme de l’intersectionnalité. Résultats Les participants ont noté le peu de messages de santé culturellement et linguistiquement adaptés, ce qui a laissé place à la prolifération de la désinformation. Le partage des connaissances entre pairs a permis de combler ces lacunes, mais a créé des opportunités de propagation de désinformation. Conclusion L’amélioration de l’équité dans les communications sur la santé devrait être éclairée par des changements structurels dans le secteur de la santé publique en Ontario. Keywords COVID-19 Health communication South Asian Qualitative research Mots-clés COVID-19 communication en santé Asie du Sud recherche qualitative CIHRgrant #420096 Meyer Samantha B. ==== Body pmcIntroduction Within Ontario, areas with higher numbers of racially and ethnically minoritized residents reported three times higher rates of COVID-19 infection, four times higher rates of hospitalization and intensive care unit admissions, and a two time higher rate of death as compared to those residing in neighbourhoods with higher proportions of white residents in 2020 (Public Health Ontario, 2020). The top three health units with the highest caseloads also consisted of Ontario’s greatest racially and ethnically diverse populations (Public Health Ontario, 2020; Al-Jaishi, 2021). For example, the Region of Peel reported that between April 2020 and January 2021, racially and ethnically minoritized peoples accounted for 63% of the population but 81% of COVID-19 cases, with South Asians being among the most overrepresented (Region of Peel, 2021). This study is conceptually grounded in the theory of intersectionality (Crenshaw, 1990); an analytic framework that recognizes how layers of one’s identities (including but not limited to race, ethnicity, gender, sex, sexuality, class, religion, (dis)ability, and neurodiversity) interact, intersect, and compound to manifest one’s unique lived experiences. Through an intersectional analytic lens, a white man’s experiences within the COVID-19 pandemic differ from those of a South Asian man, which then differ significantly from those of a South Asian woman, and so forth. Each of these identities intersects with multiple sources of societal and systemic oppressions alongside privileges. Aligned with this theory, existing data suggest the aforementioned inequities related to COVID-19 experienced by South Asians in Ontario may in part be explained by reports that racially and ethnically minoritized peoples—particularly new immigrants—often lack access to culturally safe and linguistically accessible risk communication materials, experience greater difficulty in navigating the healthcare system, and have higher rates of poverty and as such, greater predisposition to several risk factors for severe COVID-19 (Greenaway et al., 2020). There remain gaps in the provision and accessibility of health promotion and risk communication materials for South Asian communities. Given this, investigating community-based perspectives and solutions that promote public health measures within these communities remains a critical role for public health—particularly in light of South Asians reporting greater COVID-19 vaccine hesitancy as compared to the general population in Canada (Arora, 2021). As such, the aim of the present work was to identify, from the perspective of South Asian communities, areas for improvement in public health communication. Through the use of intersectionality, we identify the unique lived experiences of subpopulations within the South Asian community. Methods Study population and recruitment Individuals who could converse in English and self-identified as South Asian adults (18+) residing in Ontario, Canada, were recruited using posters shared on Instagram and Twitter and shared with community groups with South Asian audiences. Interested parties emailed the study email address and a demographic survey was administered to screen for eligibility. Socio-demographic information was also collected to identify contextual factors that might shape COVID-19 experiences and perceptions of public health measures. Study design Semi-structured focus groups were facilitated by a young South Asian woman researcher residing in Ontario fluent in Hindi, Punjabi, Urdu, and English. Participants were asked to share how, if at all, their identity as South Asian shaped their experiences during the pandemic and acceptance of public health measures put in place to mitigate spread. Individuals were intentionally allotted into certain focus groups, aiming for diversity across gender and age in two of three focus groups (Table 1); however, for one of the focus groups, we centred upon creating a safe space for only youth (under 30 years old). This decision was informed by work we were concurrently doing for a larger study centred upon youth experiences/perspectives with COVID-19. Table 1 Age and gender characteristics of focus groups Focus group Mean age Gender* Sample size (n) 1 21.7 6 women, 2 men 8 2 36.7 5 women, 2 men 7 3 31.7 8 women, 1 man 9 Total 24 *There was no non-binary/third gender representation Study sample Individuals were aged 19–64 and identified as South Asian, with noted representation from Sri Lankan, Tamil, Pakistani, Punjabi, Indian, and mixed-East African/Indian and Pakistani communities. There were a total of 19 women, 5 men, and no non-binary individuals. Thirteen of 24 (54%) of focus group participants reside directly within the jurisdiction of the public health units reporting the greatest number of cases in Ontario. Data collection In April 2020, 1-hour-long focus groups were facilitated (via WebEx or Zoom) using a semi-structured interview guide. Participants were encouraged to utilize the chat feature, especially if it felt more comfortable than responding verbally, with the moderator reminding participants to engage throughout. After audio files were auto-transcribed using Otter.ai, the transcripts were anonymized with pseudonyms, verbatim checked, and revised by a research assistant and then checked/edited for accuracy again by the focus group facilitator, particularly for any language translation needs. During transcription, we aligned transcribed audio with the real-time chat text which we saved after each focus group. We analyzed data (written and verbal) together as one continuous transcript. Data analysis Borrowing from a grounded theory approach, transcripts were coded using inductive, bottom-up line-by-line initial coding, followed by focused coding (Miles et al., 2014). Two analysts independently coded the same transcript for rigour. Differences, additional or ambiguous coding, were discussed before all transcripts underwent focused coding. Interrater reliability was 84%, exceeding the recommended standard by Miles and Huberman (1994). Data were interpreted through the lens of intersectionality (Crenshaw, 1990). Results Findings speak to perceptions regarding public health failing to reach South Asian communities and consequences of poor access to health information. Data also speak to how intersecting identities shape experiences with COVID-19 and in turn, engagement/involvement with health information. South Asian communities underserved by public health messaging Most participants expressed that South Asian-led education efforts were necessary due to a lack of trust in government and health officials, and/or accessibility to what official, mostly Anglo-centric health messaging was communicating. As such, participants who identified as community leaders, those fluent in English, those working in health-related or public sector jobs, and youth often said that they felt they had no choice but to fill that gap and become messengers navigating the health directives to be relayed back to their family and community. For example, Keerat (23) expressed, “the way that these changes are being communicated is pretty ineffective and especially in the case of language barriers -- people who might not have family members to share this information with them.” All youth in our study—who are all fluent in English—uniformly expressed that given the severity of COVID-19, they felt they must advocate for the health of their parents and elders, because they perceived the system as failing to do so. Commonly, due to the language and cultural barriers, newer immigrants and elderly individuals were cited as using news sources from South Asia to inform themselves on the latest news regarding the pandemic and best public health measures to take, even if the information was not Canada-specific. This put an onus on younger South Asians, particularly elder daughters, to be fact-checking actively for their loved ones. Shreya (28) shared, “My parents and family love listening to Punjabi media for news. I made sure to vet these sources. I know there a channel called ‘Y Channel’ that does programming in Punjabi and they have physicians come in and talk so I trust this source and let them watch this channel for news.” Along these lines, Mohammad summarized the importance and efficacy of the practice of peer-to-peer knowledge-sharing, as it is common in South Asian communities, and how it addressed the issue of mistrust as the information was then being filtered through someone from their community.One of the things that I wanted to ensure in my own circle was that if it comes to me, or if it’s going through me, it has to be verified. So, at the start of the pandemic for about 6 months, the daily conferences that Trudeau would do… I listen to that conference every single day and summarize in notes with citations, and then circulate through the WhatsApp groups and be like: ‘this is it; this is straight from the horse’s mouth- here are the sources.’ Because that would give me a level of control and also address a certain level of like, anxiety that I had with the fact that elders-- people in my own circle that are disseminating this information. But like this is where it stops, you know. Proliferation of misinformation in the absence of culturally relevant, accessible health messaging Although many reported that they felt there is an increased likelihood of adherence to public health measures if advised/recommended by a trusted South Asian person (e.g., family, respected community leader, or South Asian medical authority), these engagements were also identified as an opportunistic means to perpetuate misinformation. The most cited platform used for spreading information (but also proliferating misinformation) across all focus groups was WhatsApp.Deepika (20) to Everyone (9:02 PM) my mom uses whatsapp for everything, but it gives a basis for information and it makes it easier to communicate with her (even if half of it is fake news) information she finds Iqbal (21) to Everyone (9:03 PM) honest my dad using WhatsApp too Bhavna (19) to Everyone (9:04 PM) lool my brother calls it WhatsApp University Participants felt that WhatsApp was most popular with middle-aged and older individuals and was in many cases one of the only places where elderly individuals were getting their news over Anglo-European health experts. Participants felt that members of the community with misconceptions surrounding public health countermeasures or misunderstandings surrounding COVID-19 were also more likely to make health decisions that centred or aligned with their spirituality, religion, intergenerational ancestral and cultural knowledge systems, and/or lived experiences. Many felt that these individuals often also strongly factored in perspectives, experiences, and opinions from peers and community leaders within their community with whom they expressed a greater sense of trust for their holistic well-being. Geeta explains that this unfortunately meant their sometimes buying into misinformation surrounding home remedies for preventing or treating COVID-19.My mom is addicted to Facebook and WhatsApp, so she’s the one who forwards anything and everything like, “here is a cure for COVID; I will forward it to you and your entire family”. That’s where I feel like a lot of the fake news is coming from and where a lot of the South Asian community has failed. There has just been so much of spreading complete misinformation like drinking ‘haldi da doodh’ (Translation from Punjabi to English: milk with turmeric in it) is not gonna cure COVID, I promise (laughs). I feel like there’s no actual like, “I’m not gonna think before I send this, I’m just gonna send it.” If it’s forwarded many times, there are definitely mistakes. I promise you—and I mean a lot. It’s a misinformation, but they see it, they read it and they believe it, which sucks, but that’s just what it is now. In the chat during this focus group of mostly youths, there was wide support for this statement, with murmurs of people expressing similar experiences, laughing and nodding in agreement. Keerat (23) to Everyone (9:04 PM) Geeta every auntie across ontario is screaming rn In a different focus group, WhatsApp and turmeric came up again organically. Amina (21) said, “I know a lot of WhatsApp group chats had that message going around that ‘COVID isn’t real - all you need is a couple of things like, turmeric and home remedies… [to] prevent getting it or [it is] what to do if you do get it’, which is really not the case.” Intersecting identities and experiences of COVID-19 health messaging From an intersectional perspective, we identified added responsibilities and expectations—and as a result, added stress—upon young South Asian women due to gender roles. Leila (19) expressed, “me being the only girl in my house, I’m the only advocate. I’m like, ‘okay guys, everyone mask up; we’re going outside, okay. we’re getting our vaccine.’ I had to sign up my parents with the vaccine.” Others in different groups mentioned that the eldest girl in the family often takes on a lot of added responsibility and care work in South Asian households. In addition, many newer South Asian immigrant participants also recognized the lack of job safety parameters and COVID-19 harm reduction measures (e.g., PPE) within the type of work that is more readily accessible to this population. There were multiple mentions of the intersectional experiences of new immigrant racialized women, and the lack of support offered to them despite working precarious jobs often associated with caregiving which increased their risk of exposure. Devi (43) shared, “I have been working in long term care home since the outbreak. [A] majority of caregivers are immigrant racialized women. Socio-economic background is a determinant that contributes to this disproportionate representation [in case load]. Non-paid sick days prompt [us] to neglect or not reveal initial symptoms if any, and also [encourage us to] get back to work immediately post-COVID.” Beyond the individual level, participants mentioned community-led health communication and advocacy initiatives created by South Asians to advocate for their own health. For example, Aliyah (23) explained the critical need for such initiatives.I work for a nonprofit in Peel with South Asians... We’ve been asking the provincial government for months, like since even before June to give us the funding to send out community health ambassadors to hot spot areas in Brampton. If you went to a grocery store there [at the time], the language or the poster would be completely in English. There were no South Asian languages, like there wasn’t that messaging that needed to go out. The funding itself came so late—there was a high-priority initiative that funded our nonprofit in addition to some other ones, like the Punjabi Community Health Services and [so] we launched this program and [its name] says (laughs) that the government didn’t do it and you know, individual like, grassroots and other initiatives have ... Basically, what we created was a hotline that people can call into to get culturally appropriate information… (emphasizes) the biggest issue that we felt was that international students over here are basically being scammed because they’re living in basement apartments, some of their passports have been taken, they have no access to supports, they cannot go back to their countries, their colleges or educational institutions haven’t really supported them, a lot of them are working at Amazon right now and other warehouses where they’re being terribly exposed to the virus... The big Canada-wide distribution centre was shut for 2 weeks because the cases there were astounding and because there’s like 10 people in a house and the landlords are not paying attention. So, there’s just so many systemic issues that we discovered along the way and through this program, we were able to help people with grocery supports, food supports— if they needed transport to a vaccine clinic or a testing clinic or, if they needed information in their language if they didn’t speak English. So, there were a lot of definitely gaps that nonprofits had to step in and fill that the government didn’t. They just didn’t even translate documents correctly; we had to get people to do that. In a different focus group, Geeta (23) described another initiative, Apna Health, a website designed to specifically serve the South Asian community by centring the communities’ collectivist values.Apna Health teamed up with a bunch of other nonprofit organizations - the ones that usually are associated with like the Punjabi community, the Hindu community and the Muslim community… they’re actually handing out [COVID-19 PPE] for free at Mosques, Gurdwaras and… they’re also teaching the elderly [and] international students– that may not know enough English— to understand the impacts of COVID… Their website is also multilingual, they have it in every single language. If you contact them, they will actually give you money either for groceries or if your whole family is isolating with COVID, they will literally drop off groceries to your house like every single day. Discussion Our findings demonstrate the limits of public health communications in reaching South Asian communities in Ontario, and the impacts of underservice upon this population. Identified as a systemic issue at its core, a key recommendation emerging from this work is that improving health communications to better serve communities such as these will require structural changes to the Ontario public health sector. This underscores the parallel necessity for individual actors to challenge the public health sector’s intentional and/or unintentional discriminatory practices—at minimum, surrounding cultural competence—to set forth a “new normal” that centres upon equity. As a microcosm of the larger problem of white, western-centric worldviews and practices dominating public health and policy-making environments, it was only after race-based data began to be collected, and the dire realities of the pandemic as expressed by the lived experiences of racialized communities in Ontario (Thompson et al., 2021) that equity-centred approaches began to take centre stage in the public health response. Our data too suggest that discrimination was experienced by this community as they reported consistent underservice by government and public health officials, forcing the fostering of a culture of self-advocacy among South Asian community members to help protect their disproportionately vulnerable community and mitigate their risk of COVID-19 transmission. Participants in our study cited initiatives created by South Asian community members, including one called “Apna Health,” meaning “our health” when translated to English. A community-led research report focused on Brampton, one of the hardest hit regions within Ontario, was produced by Social Planning Council of Peel (SPCP) and Punjabi Community Health Services (PCHS) (2021) which offered major recommendations that are well aligned with perspectives and suggestions offered by our study’s participants. In brief, there is a need for increased culturally and linguistically accessible risk communication resources, actively working towards clearing misinformation using a culturally sensitive approach, culturally responsive mental health support with a particular focus on women and the elderly, community-led support for health services, and increased translation services. Our data regarding the disproportionate burden of care assigned to young, racialized women lend further support for considering intersectionality in the design and implementation of public health policies and practices (Bowleg, 2020). Interestingly, many young participants mentioned “aunties” as being the people who frequently share information out of concern for others in the community, commonly through WhatsApp. Culturally, this refers to any woman who is older than you regardless of having a direct relation. Within a collectivist community, overlapping with the intersectional implications concerning gender roles, this presents an opportunity for further exploration of aunties as a subgroup within the South Asian population who might be beneficial to target for health promotion and risk communication to minimize the spread of misinformation within the community. Our data also demonstrate the importance of developing and disseminating culturally compatible, linguistically accessible health promotion and risk messaging, an approach said to respect and promote health equity (Sabatello et al., 2021). To operationalize culturally competent community engagement, Sabatello et al. (2021) recommend increasing meaningful involvement of diverse peoples inclusive of race, ethnicity, (dis)ability, gender, and other dimensions of identity who can help steer decisions towards the best interests of their respective communities. Accordingly, our data suggest the need for public health to engage with, understand, and include the perspectives of South Asian community members with various intersecting identities—in our case, class, ethnicity, age, and sex. Increasing the cultural competence of health professionals regarding more appropriate communication and explanatory styles would also facilitate health promotion efforts (Miconi et al., 2021). For example, the framing of messages should also be given express consideration to ensure that communication materials are inclusive and promote solidarity instead of perpetuating isolation (Miconi et al., 2021). However, while the above recommendations are important, our findings and the broader literature underscore the critical role of addressing upstream systemic issues above all else. To this effect, the reactive work of improving the cultural competency of public health professionals will need to be usurped by minimizing disparities in health created by broader, systemic factors such as discrimination and stigma. Shahi et al. (2019) suggest that we might begin by addressing the presently inadequate consideration for and practice of equity, diversity, and inclusion within the internal structures of the public health sector in Ontario. Sabatello et al. (2021) also postulate that meaningful engagement with truth and reconciliation commissions (TRCs) to recognize the harm inadvertently inflicted by way of systemic inequities and discriminatory policies upon communities of colour is important to help invest in genuine relationship building with communities where trust has been lost as a result of continued underservice. In terms of limitations, our study sample consisted of a greater proportion of young adults with a majority of the sample being under the age of 29. This is likely due to the study being advertised and primarily conducted in English as well as it being advertised primarily on social media platforms. Given the necessity of having access to a computer and Internet to be able to participate in this study, there is the potential for an overrepresentation of participants with class privileges. However, our data do reflect perspectives beyond those present in the focus groups. For example, all the participants could at very least speak to a wider range of experiences of the South Asian community beyond their own. Participants often acknowledged relevant privileges afforded to them (e.g., being employed in a remote job) while sharing stories of family members, neighbours, and community members who did not have the same privileges. Importantly however, given the intersectional implications, it would be beneficial to capture more diverse perspectives with regard to class, as well as non-binary and gender-fluid peoples. Beyond the empirical data, we offer a novel contribution to a methodological approach in conducting online focus groups. Through this work, we developed a novel method to facilitate semi-structured focus groups that relied on dual data collection from both verbal responses and simultaneous engagement in the online chat of the video call. This allowed us to capture more data than would have otherwise been possible during a 1-h focus group, online or in-person. We found that individuals were sharing perspectives in the chat in response to those answering interview questions verbally out loud in real time and vice versa, allowing for an increased sense of engagement. It also promoted accessibility for introverted individuals, those with social anxieties, those with weaker Internet connections, and those who could not speak as freely or distraction-free as they would like to in response to the questions verbally (e.g., due to computer access being in a shared space). It was observed that even if only one participant vocalized their response, often four to six other participants would indicate in the chat whether they agreed or disagreed, stating their rationale in the chat. This fostered a sense of group camaraderie and made it less intimidating to participate and share one’s opinions and experiences. Individuals would also have concurrent conversations about related points, and prompt each other with follow-up questions which led to the emergence of more inductive themes than what we would have found otherwise. For example, the exchange that took place involving “haldi da doodh” and WhatsApp in the youth focus group led us to ask meaningful follow-up questions surrounding misinformation and helped elicit our third key theme of how underservice by public health creates opportunities for misinformation to spread through trusted channels, such as peer-to-peer discussion (between the “Aunties”) via WhatsApp. Conclusion Participants perceived a lack of culturally relevant and linguistically accessible health messaging, leading to the proliferation of misinformation. The practice of peer-to-peer knowledge sharing and self-advocacy filled a critical gap, but certain contexts, particularly as it concerns the use of unregulated messaging platforms such as WhatsApp, created opportunities for misinformation to spread. Importantly, it was identified that the design and implementation of public health messaging and countermeasures need to be more aware and considerate of intersectional experiences given our finding that South Asian women were disproportionately underserved and dually tasked with a greater responsibility for ensuring the health and safety of their loved ones. Contributions to knowledge What does this study add to existing knowledge? Health messaging in Canada is largely informed by western science and Anglo-European norms—which can make it incompatible with other cultural/ancestral ways of knowing and foster mistrust. In the absence of effective public health messaging, South Asians in Ontario commonly employed peer-to-peer learning to promote acceptance, confidence, and uptake of countermeasures designed to mitigate COVID-19 transmission for their communities. What are the key implications for public health interventions, practice, or policy? There is a need to acknowledge the inequitable harms experienced by these communities as a result of underservice by health officials, but also the toll on those proficient in English who have had to be hypervigilant in screening information to promote evidence-informed health information for those who are underserved. Public health officials can and should work on building trust with, and follow the guidance of, community leaders, grassroots initiatives, and local non-profit organizations to ensure the creation of culturally safe, linguistically accessible health communication materials, alongside effective dissemination strategies. Acknowledgements We would like to thank the members of the South Asian community who chose to participate in this research and share their stories, experiences, thoughts, and expertise to better inform public health practices. We are grateful for your emotional labour in engaging with this conversation during these trying times. Author contributions Manvi Bhalla led the data collection and analysis and conceptualized, wrote, and revised the manuscript. Samantha Meyer conceptualized the study, obtained study funding, contributed to data collection and analysis, and provided mentorship in writing this manuscript. Helana Boutros aided with transcription and second coder analysis. All authors read and approved the final manuscript. Funding This study was funded by the Canadian Institutes of Health Research (grant #420096). Availability of data and material All data required to make the conclusions reached in this manuscript are included here. Code availability Not applicable. Declarations Ethics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of Waterloo’s Research Ethics Office (#42160) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent for publication Participants signed informed consent regarding publishing de-identified excerpts from the interview data. Conflict of interest The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Al-Jaishi, A. A. (2021). COVID-19 in Ontario. https://www.covid19inontario.com Arora, A. (2021). COVID-19 in Canada: A one-year update on social and economic impacts - Delivering insights through data for a better Canada (March 2021). 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Journal of Travel Medicine, 27(7). 10.1093/jtm/taaa113 Miconi D Li ZY Frounfelker RL Venkatesh V Rousseau C Socio-cultural correlates of self-reported experiences of discrimination related to COVID-19 in a culturally diverse sample of Canadian adults International Journal of Intercultural Relations 2021 81 176 192 10.1016/j.ijintrel.2021.01.013 Miles MB Huberman AM Qualitative data analysis: An expanded sourcebook 1994 Sage Publications, Inc. Miles MB Huberman AM Saldana J Qualitative data analysis: A methods sourcebook 2014 Sage Publications, Inc. Public Health Ontario COVID-19 in Ontario – A focus on diversity: January 15, 2020 to May 14, 2020 2020 Queen’s Printer for Ontario Region of Peel. (2021). COVID-19 in Peel dashboard: Social determinants of health. https://www.peelregion.ca/coronavirus/case-status/. Accessed Nov 2022. 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==== Front Int J Energy Environ Eng International Journal of Energy and Environmental Engineering 2008-9163 2251-6832 Springer Berlin Heidelberg Berlin/Heidelberg 552 10.1007/s40095-022-00552-y Original Research Green and renewable resources: an assessment of sustainable energy solution for Far North Queensland, Australia http://orcid.org/0000-0002-3096-7664 Islam M. K. [email protected] 1 Hassan N. M. S. 1 Rasul M. G. 2 Emami Kianoush 1 Chowdhury Ashfaque Ahmed 3 1 grid.1023.0 0000 0001 2193 0854 School of Engineering and Technology, Central Queensland University, Abbott Street, Cairns, QLD 4870 Australia 2 grid.1023.0 0000 0001 2193 0854 School of Engineering and Technology, Central Queensland University, Yaamba Rd, Rockhampton, QLD 4701 Australia 3 grid.1023.0 0000 0001 2193 0854 School of Engineering and Technology, Central Queensland University, Bryan Jordan Dr, Gladstone, QLD 4680 Australia 30 11 2022 129 10 5 2022 13 11 2022 © The Author(s), under exclusive licence to Islamic Azad University 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Remote communities, which do not have a connection to the national grid in Far North Queensland (FNQ), depend on dirty and costly diesel generators to meet their energy demands. The cost of power generation is considerable in those areas, because the diesel fuel must be carried by truck or ship and a fuel reserve must be held on-site in case of expected demand or weather closure. Moreover, Australia has an energy security issue in relation to liquid fuels. Australia is reliant on imported fuel such as diesel to fill the shortage, as domestic production and supply are unable to fulfil domestic demand. As a result, by deploying hybrid integrated renewable energy systems in remote areas, isolated communities may lower their power prices, enjoy a more secure and dependable source of electricity and minimise their carbon footprint by eliminating or reducing the usage of diesel. In this study, an extensive literature review has been conducted focussing on renewable resources for Australia and Far North Queensland, different hybrid energy systems including energy storage, and finally highlights the alternative clean and renewable energy options for Far North Queensland (FNQ) remote communities. In addition, this study has performed an assessment of renewable energy available from solar and wind resources considering climatic, geographical and economic aspects for FNQ. The literature review and the assessment show that solar and wind resources including hydrogen storage have significant potential for energy solution of FNQ. The assessment results indicate that selected regions of FNQ have suitable land area of 142,294.86 km2 (55.94% of total selected areas) for solar and 144,563.80 km2 (56.83% of total selected areas) for wind. The total calculated potential power can be 14,448 GW from solar PV and 1040.97 GW from wind energy. This study provides a significant pathway for parties interested in investing in renewable energy in FNQ. Moreover, knowing a land’s suitability will increase confidence and hence speed up the renewable energy investment. Keywords Renewable energy Hydrogen Solar farm Wind farm Electrical power potential GIS ==== Body pmcIntroduction The rapid socio-economic uplift, including population growth, technology upgradation, trade, increased production and consumption, has made the relations between Earth and human beings deeper than ever. Consequently, fossil fuel reserves are depleting so quickly to meet the energy demands, which are the globe’s current issue. The gathering of greenhouse gases in the atmosphere, causing global warming and the urgent requirement of renewable and eco-friendly energy resources has been a great concern. Apart from that, energy demands are growing every day with the globe’s population which may grow up to roughly 9.9 billion by 2050 [1]. At the same time, developing countries will have to enhance energy expenditure intensely because of their spreading economy [2]. To be sustainable, a society needs high and constant amounts of energy with limited environmental impact. There are many remote areas in the FNQ, and they have to rely on diesel power generation for fulfilling their basic energy requirements [3]. However, diesel is one kind of fossil fuel which has limited stock. In addition, the diesel power generation is costly [4] and harmful to the ecosystem because of its greenhouse gas emissions [5]. To keep the globe environmentally sustainable, so that the civilisation can continue for a longer time, the resources of the earth should not be changed from the equilibrium levels which help to sustain the ecosystem for thousands of years. Simultaneously, the world has to limit carbon emissions. The incoming energy resources, the solar and wind, should be trapped, not the energy system innate in the globe. Solar energy can be exploited in several ways: utilising solar cells to convert solar to electricity [6], and wind energy to generate electricity [7]. Wind energy is, in reality, a secondary effect consequencing from solar energy [7, 8]. FNQ has an abundance of solar and wind resources that have bright potential towards the fruitful sustainable pathways. However, the intermittency or unsteady nature makes solar and wind less secure, incapable of providing energy at all times [9]. There is one that sounds to be consistent with promoting energy security and cleanliness, that is hydrogen. Hydrogen is an energy carrier, convenient to transform from different energy sources, and possesses the highest amount of energy by weight compared to any common fuels [10, 11]. Hydrogen leverages the utilisation of solar and wind, as they can be used to generate hydrogen, which can then be utilised at any time for fulfilling energy needs [12]. Furthermore, if hydrogen is generated through water splitting and used in fuel cell for power production, it will produce only water as a by-product, with no hazardous emissions [11]. This is called green hydrogen. Therefore, solar and wind including green hydrogen, with their promising clean features, could be the best option for the future energy system of FNQ and Australia in general. The motive of this study is to find out the solution for reducing the impact of diesel generation so that the remote communities can sustainably lead their life in a sustainable way. In this study, an extensive literature review has been conducted. The review focuses on renewable resources in an Australian context, emerging hybrid renewable energy systems, hydrogen storing options, renewable generation in Australia and the renewable energy potential in FNQ. In addition, a potential assessment of solar and wind resources has been conducted in terms of climatic, geographical and economic to see the suitability for the growth of solar and wind projects. The main objectives of this study to find a sustainable solution for FNQ’s energy system can be summarized as follows:Survey on renewable resources and its’ potentiality, renewable generation in Australia; Explore suitable resources for FNQ’s energy system; Investigate suitable lands to install solar and wind power stations, using the multi-criteria Geographical Information System (GIS) modelling technique. To date, this is the first elaborate study where FNQ has been explored to identify potential resources as well as to investigate the suitability of places for solar and wind analysis using GIS. Finally, as this study covers the potential of renewable resources, especially the two most mature technologies, namely solar and wind including hydrogen storage, and location suitability for the installation of solar and wind farm, hence it can be expected that it will give valuable insights for hybrid versions of solar and wind including hydrogen energy applications. The present article is structured into six sections, including introduction (Sect. 1), global weather pattern (Sect. 2), global energy demand and consumption (Sect. 3), literature review that presents overview on renewable resources of global and Australian perspective, electricity energy value chain and different hybrid energy systems, potential of hydrogen energy storage, renewable generation in Australia and renewable resource potential in FNQ (Sect. 4), assessment of solar and wind resources presenting climatological, geographical and economic potential including discussion about the importance of solar and wind energy development for FNQ’s energy system (Sect. 5). The last section presents the main conclusions and future perspectives. Weather pattern The weather pattern has been changing; for example, the global surface temperature increment, narrowing the cryosphere extensively including weight loss from ice sheets and glaciers, depletion in snow cover and Arctic Sea ice extent and thickness [13]. Global warming is responsible for extreme events, such as more extended periods of heat, heavy rainstorms and oceans acidification because of absorbing carbon dioxide [14]. All weather-related problems are happening because of the greenhouse gas (GHG) emissions in the atmosphere. Global surface temperature increment may cause more drought and damage of agricultural land. In addition, extremely hot weather may push people from their motherlands and compel them to move to other areas, eventually driving them to compete for depleting resources in their areas of arrival. Coastal regions can be invaded by multiple natural hazards such as tornado, extreme sea levels and flooding. The ocean temperature increment will also pose marine lives at possible food insecurity risk. Ultimately, a change in the weather pattern will result in changing ecosystem structure and functioning, which may cause losing the globe’s unique biodiversity [13, 15]. Figure 1 highlights some indicators of global warming that were examined over the past decades [16, 17]. The globe’s climate system comprises the land surface, atmosphere, oceans and ice. As seen in Fig. 1, white arrows indicate rising trends and black arrows indicate declining trends.Fig. 1 Ten indicators of the global warming [16, 17] Global warming is augmented at an average rate of 0.08 °C per decade since 1980 and over twice that rate since 1981. In 2020, the global surface temperature augmented at an average of 0.98 °C, which is the second highest record in the last 141 years [18]. If the present trend continues, the global temperature may reach to 1.5 °C between 2030 and 2052 [18, 19]. The Intergovernmental Panel on Climate Change (IPCC) [20] reported that the global mean sea level was augmented from 1.4 mm per annum (1901–1990) to 3.6 mm per annum (2006–2015); the 0–700 m and 700–2000 m layers of the ocean were warmed at rates of 6.28 ± 0.48 ZJ and 3.86 ± 2.09 ZJ, respectively, from 1993 to 2017. In addition, the continual ocean acidification because of carbon uptake (the ocean surface water pH level has been falling at a range of 0.017–0.027 pH units per decade since the 1980s) including oxygen vanished with a loss of 0.5–3.3% between 1970 and 2010 from the ocean surface to 1000 m [20]. Numerous peer-reviewed literature has already been reported about the weather extremes around the world, such as droughts in South Africa [21], extreme heatwaves in Sweden [22], excessive 6-day rainfall in Bangladesh [23] and hurricanes in the Caribbean [24]. These extreme weather events are due to the global surface temperature increment which are likely to be the results of the accumulation of GHG (carbon di-oxide, nitrous oxide, methane, halocarbons) in the atmosphere [25]. The global atmospheric concentration of carbon di-oxide has raised from a preindustrial (1750) level of 280 ppm to 417.64 ppm (March 2021), nitrous oxide from 270 to 333.6 ppb (Nov 2020), methane from 715 to 1892.3 ppb (Dec 2020) [26, 27]. As seen in Fig. 2, maximum greenhouse gas (GHG) emissions come from fossil fuel sources that are used in different economic sectors [28]. Among the GHG, carbon di-oxide is the most abundant in the atmosphere, hence the main contributor to global warming. Figure 3 depicts the trend of the increment of the global annual mean temperature anomaly with the concentration of carbon dioxide in the atmosphere [29, 30].Fig. 2 Global GHG emissions by economic sectors in 2016 [28] Fig. 3 Global annual mean temperature anomaly and carbon dioxide concentration [29, 30] Australia’s climate is changing in response to global warming, with an average temperature increment of 1.44 ± 0.24 °C since the Australian observations commenced in 1910 [31]. The most warming has been happening since 1950, and since then, Australia is getting warmer and warmer every decade. For instance, Australia suffered 43 exceedingly hot days in 2019, more than three times as many as in any year before 2000. High monthly maximum temperatures, around 2% during 1960–1989 and over 4% during 1990–2004, have recently been over 12% during 2005–2019. [31]. This number is more significant than a sixfold increment over the 60 years. Climate change is observed across the whole Australia. All areas of Queensland are warming since 1910, with an average annual temperature increment of 1.5 °C. Rainfall has increased in most parts of Queensland during the summer or humid season [32]. The number of days including threatening weather conditions for bushfires has augmented in all regions across the state. Sea levels are predicted to rise by nearly 26 cm along the coast of Queensland. Queensland may face more extreme rain events in the near future [32]. The FNQ region is specifically in danger because of the impact of climate change. Alteration in temperature or rainfall may impact significantly on the tourism, agriculture, dairy, cane and fisheries sectors [33]. Local community will also be affected, as climate change may augment heat-related health problems and potentially increase catastrophic occurrences, such as cyclones and floods, with endangering lives and infrastructure. Healthy reef and rainforest environs are the core for the tourism industry. These ecosystems are particularly at risk because of the adverse effect of climate change. Raising temperatures may constantly cause coral bleaching in the Great Barrier Reef, and this impact will become more severe if the temperature continues to increase [33]. The deterioration of the reef will be a destroy of the distinguished innate value for FNQ, which will ultimately adversely affect the tourism industry. Furthermore, sea water acidification has been occurring due to the continuous absorption of carbon dioxide. Continual acidification can affect coral formation [33, 34], which would further exacerbate the vulnerability of the Great Barrier Reef. The globe is warming continuously, and if the GHG emissions are not controlled immediately and strong development of fossil fuel plants continues, then the globe’s surface temperature may rise by 4 °C, or more, within 2100 [35], including terrible impacts on the globe’s ecology and substantial demolition of the world’s major coastal parts [36]. A maximum of 2 °C of global mean surface temperature deviation from pre-industrial level is the widely accepted climate policy target, but the temperatures above 2 °C will cause the ecosystems to be highly vulnerable [37]. In addition, without taking any action against GHG emissions, climate change might cost as high as 5–20% of the global gross domestic product (GDP) per year [36]. Specially, developing countries like Africa may face more difficult situations with the GDP losses as high as 26.6% per year [38]. Instead, taking action might cost 1% of the global GDP per year [36]. So, it is from an economic point of view that worldwide investment is inevitable now for the drawdown of GHG emissions and fossil fuel utilisation by alternative approaches to energy production that could potentially arrest the current climate trend. Energy demand and consumption Energy is the crucial and fundamental element for the global civilisation. There is a close and firm relation among the energy supply, national and international security, human basic needs and economic growth and the ecological pollution. Hence, energy is a complicated issue now, while the global energy demand continues to rise. Energy, as a production input or as a direct component of human well-being, is the key component of economic development. Its uninterrupted supply with increased global demand continuously poses a significant challenge to society. The global energy demand may rise by 30% within 2035, driven by emerging economies such as China, Brazil, Russia and India [39]. But the demands are fulfilled mainly by fossil fuels. The ongoing fossil fuel utilisation is posing a threat to the planet by emitting GHG [39]. Figure 4 depicts the energy consumption of different fuels in 2020, where it is seen that fossil fuels are dominating the energy regime. However, the pandemic Covid-19 causes global energy demand to decline by 4.5% in 2020, which is the biggest fall since World War II [39].Fig. 4 Global energy consumption by fuel in 2020 [39], note: CIS-Commonwealth of Independent States The downfall in demand in 2020 did not influence all fuels evenly. Oil was the hardest hit, with restrictions on transport resulting in demand dropping by a remarkable 9.3%—the biggest drop in history [39]. However, the oil demand bounced back by 5.7 mb/d in 2021 [40], which is faster than any other fuel. Coal demand declines globally by 4% in 2020, but the coal utilisation for power generation in advanced economies dropped down by 15%, more than a half of coal’s global decline. Low power demand increased renewable power generation and low gas prices squeezed coal utilisation in power generation. However, coal demand rebounded strongly in 2021, although with vast, diverse geography [39]. In contrast, natural gas showed far greater resiliency in 2020, with demand dropping only by 2.3%. Due to low prices and rapid growth in economies across Asia and the Middle East, in 2021, global gas consumption rebounded by 4.6%, the most substantial rebound among all fossil fuels and double the decline that occurred in 2020 [41]. In 2020, the low gas prices caused a gas generation to obtain a share in the US power market as well as sustain in the European Union (EU) [39]. Global power consumption experienced a smallest drop, 0.9% in 2020. Despite the drop in overall power consumption, renewable generation (wind, solar, geothermal energy, bioenergy, and excluding hydroelectricity) boomed to 358 TWh, the highest ever increase. This achievement was gained by the strong growth in both wind (173 TWh) and solar (148 TWh) generation. The continued deployment of renewables including power demand falls hurt coal utilisation in 2020, losing competitiveness especially in the USA and EU. However, ‘more than doubling’ in the wind and solar power generation over the last 5 years has not made even a tiny dent in total coal production. Essentially coal production level in 2020 was unchanged from that’s level in 2015, since last year’s decline just counterbalanced the previous few years’ increments [39]. Although the continued deployment of renewable, it cannot keep up with the rising demand. The world will need more than a just strong growth in renewable energy to banish coal from the power sector. It is still long to put coal out of the power sector. Global power demand rebounded strongly in 2021, boosted by more than 6%. 2021 have experienced the biggest ever annual rise, over 1500 TWh [42]. Coal served more than half of the additional demand in 2021, raising in absolute terms faster than renewable energy for the first time since 2013. Global power demand is expected to grow by around 3 to 4% in 2022, 2.6% in 2023 and above 2% in 2024 [42], as energy efficiency measures start showing effects. However, fossil fuel-based power generation may grow by 0.2% annually from 2022 to 2024, but still is expected to serve 58% of total power generation in 2024, with coal-based power generation to serve 34% in 2024 [42]. Hence, the world will be still affected by fossil fuels including GHG increment in the upcoming years. The increment of GHG should be minimised as soon as possible to avoid severe ecological damage [43]. Therefore, time is crucial now and an urgent energy transition is pivotal. The German Advisory Council on Global Climate Change [44] has drawn attention to the energy transition that it could benefit a double dividend: ‘Not only will it prevent a fatal degradation of the global environment, but it could also create the basis for a new economic dynamism, with positive effects on employment, prosperity and equity’. To narrate it more pointedly, there are many indications that the renewable energy transition is an opportunity to reshape the prevalent energy regime to sustain the natural life support system, which will rescue the global economy. Decarbonisation is at the centre of the path of transition towards sustainability to battle against climate change, which can be succeeded by massively expanding renewable energies while giving access to modern energy for the billions of people living in energy poverty. The remote communities of FNQ are leading their lives in a very unsustainable way. Currently, for their basic energy needs, they are heavily reliant on the diesel generator [3] which is associated with limited resource diesel including higher, unstable fuel prices [4] and greenhouse gas emissions, damaging the biota including trees, vegetation and marine lives. In addition, the diesel fuel must be shipped by truck or ship to the remote areas. Fuel reservation on-site is necessary in case of higher power demand than expected or the area being cut-off by any weather event. Therefore, this worse situation underscores the requirement for developing a new energy system with the minimal environmental impacts. By utilising hybrid renewable energy systems at isolated locales, remote communities can minimise their power costs and can have a more secure and consistent power supply with diminished carbon emissions because of no or less diesel. FNQ region, with an area of 380,748.3 square kilometres [45], is one of the most attractive tourist destinations in Australia. The region has a number of World Heritage Sites, including the Great Barrier Reef, the Wet Tropics of Queensland and Riversleigh, Australia’s largest fossil mammal site [33, 46]. But climate change, mainly due to the use of fossil fuels, is exacerbating extreme weather events that threaten FNQ’s unique features and tourism industry [33]. Literature review Renewable resources Energy resources are categorised into three types: (1) fossil fuels, (2) renewable and (3) nuclear. Renewable resources are non-depletable sources that emit very low or no greenhouse gases. A superabundance of renewable resources exists, namely: wind, solar, hydro, biomass, geothermal, tidal and ocean—all these resources are vastly available and easily exploitable in Australia’s geographical and political context. Solar and wind power generation have been proved to be the most logical and easily harvested option of all renewable resources available. Solar is vast, and the globe receives solar irradiation on an average 1.6 MWh/m2, which is potent to fulfil the annual world energy requirements [47]. The red sea regions including Saudi Arabia and Egypt are receiving the highest amount of solar irradiation, while Australia and the USA receive above average solar irradiation [49]. Figure 5 reports that the largest solar radiation greatly blesses the north-west and central regions of Australia. Australia is receiving on an average 35 megajoules per square metre per day or 58 million petajoules per annual of solar irradiation (around 10,000 times Australia’s annual energy consumption), the world’s highest solar radiation [50]. There is also notable solar potential in areas with access to the electricity grid. The annual solar irradiation fallen onto the areas within 25 km of existing transmission lines is around 500 times larger than the yearly Australian energy consumption [50].Fig. 5 Australian average daily solar exposure [48] With the prospective and rapid growth in solar energy exploitation, wind energy is the fastest-growing source in Australia. Australia is one of the best continents in the world for having high wind resources that primarily are located in south-western, southern and south-eastern regions and extending hundreds of kilometres inland and including highland areas in south-eastern regions [51, 52]. Wind has already been proven as one of the least-cost power options. Small-scale wind turbines are sufficient to serve the remote community power needs, as well as large-scale wind farms could be a feasible option instead of fossil fuels [53]. Hydropower has been utilised for 135 years to convert the energy of water to electrical energy. Hydropower was developed in Australia in the nineteenth century within the areas of high rainfall and elevation such as Tasmania and New South Wales. Hydropower, including installed capacity of 8790 MW, is the 2nd largest renewable resource in Australia, and Australia is the world’s fourth-largest producer of hydropower [54]. However, Australia’s hydropower development trend is relatively slow due to a notable lack of viable on-river locations, variable annual rainfall, high temperature, very high evaporation rates [55]. Ocean renewable energy from ocean waves, tidal and ocean currents, has significant potency in Australia. The first ocean power patent was reported in Australia in 1909; after that the twenty-first century has seen significant government investment and private venture capital-funded developments [56]. Australia’s wave energy resource is the most significant source on earth [57]. Australia’s best wave energy resources are located along the southern and western coastlines. The total wave energy on entire Australia is on average, approximately 3.125 petajoules (PJ) [57], and it could meet a maximum of 11% of Australia’s total energy demand by 2050 [58]. Australia also has good opportunity to exploit mechanical energy from tides, current and waves, and ocean thermal energy from the sun’s heat. The Australian government has funded projects aiming to make this inborn energy as a major contributor to the country’s energy mix by 2050. The Australian Wave Energy Atlas (as shown in Fig. 6) is one of the first outputs of this agenda which forms the Australian Renewable Energy Mapping Infrastructure [59]. However, ocean energy technologies are relatively new and still need to be proven in pilot and demonstration plants. Also, these technologies are significantly more expensive than other, more mature forms of variable renewable energy.Fig. 6 The Australian wave energy atlas, displaying the annual mean wave energy flux (in kW/m) and the Australian Renewable Energy Mapping Infrastructure (AREMI) [59] Australia has notable hot rock geothermal resources, capable of producing superheated water or steam suitable for base load electricity generation. There are also potential lower temperature geothermal resources in a number of sedimentary basins for power generation or direct-use applications. The global electricity energy value chain and emerging hybrid renewable energy systems The electricity energy value chain comprises all functions required to generate, distribute and consume power. Figure 7 presents the electricity energy value chain, which can be segmented into five major parts: fuel procurement, electricity generation, transmission, distribution and end user [60]. Due to the irresistible challenges of increasing power demand, depletion of fossil fuels, the emergency for decarbonisation and power accessibility, the energy communities [4] are reshaping the energy value chain:A paradigm shift-decentralised or distributed generation network has appeared as an alternative to highly centralised architecture; decentralisation is observed and reflected through the envelopment of micro-grids which comprise distributed renewable resources, battery storage, load and controlling systems; The deployment of hybrid integrated renewable energy system is continuously increasing for maximising the usage of locally distributed and dispersed renewable resources; The scope of hybrid energy system for grid connection or Power-to-X applications. Fig. 7 The electricity energy value chain [60] A hybrid energy system constitutes more than one energy source: either renewable or non-renewable sources so that one source being unavailable can be substituted by another available sources to ensure sustainable power supply. This is a worthy option to meet the power demand from locally available energy sources for regions where grid extension is expensive or power transmission from centralised utility is difficult. Utilising only locally available renewable resources to provide power is a sustainable option for human beings. A hybrid system constituting only renewable resources has benefits where fuel cost inclines, fuel transport is expensive, and globe’s ecological system is worsening. But due to the unpredictable, seasonal and time-dependent natures, renewable resources are not entirely reliable options. In this regard, including an energy storage system is one kind of approach for hybrid systems [61]. Solar PV is dominating in off-grid installation among all renewable systems [62, 63]. Lower maintenance and more straightforward implementation make PV a common and more suitable option in many off-grid applications [64]. Along with the PV, wind power has already shown itself as a very low-cost and promising option. Albeit solar PV and wind can supplement each other to handle intermittent nature and can enhance overall reliability [65], an energy-storing stuff, such as batteries, ultra-capacitors and fuel cells, is normally an essential option to manage renewable intermittency, enhance energy efficiency and assure secure and good-quality power supply [66, 67]. Diesel generators are sometimes used as back-ups to reduce power loss probability in off-grid hybrid systems [68, 69]. Widespread implementation of solar and wind energy generation system accredits to the global reachability of these resources and naturally complements characteristic of solar and wind resources. Research regarding complementing effects of different renewable resources has been conducted in recent studies [70, 71]. In addition, integrating hydro [72, 73], geothermal [74], biomass [68, 75, 76] and tidal [77] energy resources alongside solar and wind resources, instead of diesel generator, which is related to higher fuel and maintenance cost, have been suggested in recent research work. Recent research indicates that HRES can be implemented not only in remote areas or in off-grid conditions such as sites away from the national grid or at satellite earth stations [78], but also in a grid-connected conditions or Power-to-X applications such as direct charging of electric vehicles (EV) [79], renewable hydrogen generation [80], chemical production [81], desalination [82, 83] and multi-generation [84]. Hydrogen: a new opportunity towards the decarbonisation The ecological damage due to global warming and excessive energy needs has put unprecedented pressure on the world to seek clean alternative energy options. The most important and immediate factors to be considered in seeking alternatives are their prospective curtailment of high levels of GHG and other emissions hazardous to the ecosystem. Clean renewable energy sources have become a global concern in this context. Recently, along with the available renewable resources, hydrogen is regarded as the major option in this sustainable journey of seeking. Hydrogen has far reached significance for energy security, emission mitigation and green economic development. Theoretically, hydrogen could be imagined as an infinite supplement, if renewable energy is implemented to generate hydrogen through water electrolysis and then discharge the chemical energy through the reaction of hydrogen gas and oxygen gas to water. It is the greatest attraction of hydrogen to be present in water, which covers about 71% of globe’s surface [85]. If hydrogen can be generated from water economically, it can be a future energy provider as well as can concurrently diminish emissions in various sectors such as the power sector, transportation and industry [86]. Moreover, compared to solar, wind and geothermal resources, which have limitation of their own volatile and intermittent nature, hydrogen with no toxic emissions and greater energy content (higher energy density: between 120 and 142 MJkg−1, around 3 times greater than fossil fuels) can be an excellent option for future energy systems [10, 11, 87]. The world was experienced energy transition a long time ago. Wood was the main source of energy since several 1000 years ago. The usage of coal did energy transition to fossil fuel, while the industrial revolution happened in the eighteenth century [88]. Then the transition from coal to petroleum occurred by 1930. The invention of the internal combustion engine accelerated oil utilisation, which peaked in 2000 to meet the world’s energy needs. Since 1970 natural gas utilisation enhanced gradually and is hoped to reach a climax by 2050 [88], when the hydrogen energy carrier may take the global lead in fulfilling energy needs. However, shifting from one fuel to another has not wiped out the previous ones; rather, their exploitation has been superimposed with much higher amounts. Wood, coal, oil and natural gas are providing energy concomitantly and recently in complement to that modern windmills and solar PV are supplying energy also. A noticeable fuel transition has happened. Wood contains more complex chemical structure and lower specific energy (20.6 MJkg−1) than coal (23.9 MJkg−1) [89]. Similarly, coal has more complex chemical structure and lower specific energy than oil (45.5 MJkg−1), which continues this trend with natural gas (52.2 MJkg−1) [89]. Additionally, this shifting seems to be the continual decarbonisation of fuels. Because, the carbon quantity declined from wood to coal, to oil, to natural gas. It is also surprising that the hydrogen quantity enhances continuously from wood, to coal, to oil, to natural gas, ultimately to arrive at the perennial, carbon-free hydrogen. Figure 8 depicts the trend towards the decarbonisation of the global energy supply, from one based on mainly carbon to one that is based on hydrogen. This trend reflects the hydrogenation of the global energy supply, which means the global movement towards the hydrogen energy carrier. It is noteworthy that clean hydrogen or ammonia has no relation with any carbon emission.Fig. 8 Carbon/hydrogen ratio of common fuels [89–93] The transition to the hydrogen energy era has already been started worldwide with marked participation of renewable resources. Australia is not out of this hydrogen race. Clean hydrogen production at low cost is one of the priority stretch goals under the Australian government’s 2020 Low Emissions Technology Statement [94]. FNQ needs clean, versatile, flexible, storable and safe fuels to support the energy needs with mitigating carbon emissions. Hydrogen possesses all of these features. When hydrogen is generated using renewable energy through water electrolysis, and fuelled into fuel cells to generate power, it produces only water, with no carbon emissions [11]. In addition, renewable energy’s inherent intermittency can be complemented well by the production and storage of hydrogen. Thus, hydrogen can be a mode of reserving renewable energies for utilising at a later time when it is required, resulting in a yearly, renewable and sustainable circular cycle. Storing hydrogen can act as a buffer for enhancing the resiliency of the energy system of the remote parts of FNQ, thereby stabilising the regional electrical network. Eventually, utilising renewable resources to produce hydrogen is of immense potential for the remote parts of FNQ, with their own local specificities in terms of raw materials and energy sources. Renewable energy generation in Australia Australia has a rich, diverse renewable resource ranging from solar, wind, bioenergy, geothermal to ocean energy. The exploitation of solar and wind energy has been increasing since 2010 and continues rapidly. Before 2019, hydro energy was the most significant generator of renewable electricity among all renewable resources. In 2018, hydro energy provided 35.2% of generation (17,002 GWh) of total renewables. 2019 is the best year for wind, which has taken over the mantle as Australian’s clean energy leader, accounting for 35.4% renewable power generation (as shown in Fig. 9). Australia has demonstrated record in 2019, that renewable power generation accounts 24% of total electricity generation (as shown in Fig. 10), an increase of 2.7% on 2018 [52].Fig. 9 Renewable energy generation in Australia by different technology [52] Fig. 10 Total electricity generation in Australia in 2019 [52] The completion of the Large-scale Renewable Energy Target (LRET) was the largest achievement for 2019, which is more than a year ahead of the 2020 deadline. Australia has attained the LRET in September 2019 following the 148 MW Cattle. Hill Wind Farm [52]. This remarkable milestone has transformed renewable energy from one of the most expensive energy generations to the cheapest. The contribution of different renewable sources to national power generation is presented in Table 1.Table 1 Power generation by renewable resources in Australia [52] Resources Generation (GWh) Percentage of renewable generation (%) Percentage of total generation (%) Equivalent number of households powered over course of the year Wind 19,487 35.4 8.5 4,240,013 Hydro 14,166 25.7 6.2 3,082,150 Small-scale solar 12,269 22.3 5.3 2,669,440 Large-scale solar 5141 9.3 2.2 1,118,596 Bioenergy 3314 6.0 1.4 7,21,005 Medium-scale solar 716 1.3 0.3 155,867 Total 55,093 100.0 24.0 11,987,070 Australia has six states: New South Wales, South Australia, Tasmania, Victoria, Queensland and Western Australia with two mainland the Australian Capital Territory and the Northern Territory. Figure 11 illustrates the contribution of renewable generation to supply consumers in each state in 2019.Fig. 11 Renewable power penetration in Australia by state in 2019 [52] Tasmania is in the leading position for using renewable energy with a penetration level of 95.6%. Due to the shutting down of Northern coal-fired power stations [95], renewables provide 52.1% of South Australia’s electricity, special thanks to hydro and wind energy. Still, Queensland has the largest fossil fuel power generation and is very low in renewable generation, while Tasmania has the biggest total renewable generation [95]. Renewable energy potential in FNQ Queensland is the fastest-growing and most energy-intensive state in Australia. The GHG emissions in Queensland are approximately 43 tonnes per capita, greater than that in any other state [96]. With the strong growth in power demand, Queensland is facing the challenge of mitigating the rise in GHG emissions. Because of the vast geographical area and highly decentralised population, Queensland faces challenges in ensuring cost-effective and reliable power supply to remote and sparsely populated regions [96]. The remote parts of Queensland still meet their power demand by mainly the diesel power generation, which needs fuel with fluctuating prices and maintenance. Renewable energy could be an option in addressing all these challenges, and FNQ has a vital role to play in this regard. The FNQ has a powerful combination of solar, wind, hydro and bioenergy resources. The region has dozens of sites suitable for off-river pumped hydro to store and release clean power on demand. Solar and wind farms in FNQ can generate 20–50% more electricity per unit than most other countries. Even wind generators in the region often can be operated at times when southern wind farms are idle [97]. However, hydropower plants have geographical constraints including harmful dams for marine species. Biomass resources are not entirely green as they emit some GHG. They are also associated with transportation and processing costs. On the other hand, solar and wind have less impact than hydro and biomass. In this study, the main concern is about the remote areas of FNQ such as Cook shire, Doomadgee, Burke, Pormpuraaw, Northern Peninsula, Umagico, Mapoon, Torres shire, etc. There are some renewable solutions including community-scale solar PV in few remote parts such as Doomadgee, Mapoon, Pormpuraaw, the Northern Peninsula Area, but diesel power generation is still dominating in the remote areas of FNQ [3]. These areas are full of abundant solar and wind resources. Solar irradiation and wind speed are considered high in these regions which is the proof of potency for the development of renewable energy systems, that can replace the present diesel power generation system [3]. Assessment of solar and wind resources Climatological potential Solar irradiation in Central and North Queensland including FNQ can be considered as one of the highest amounts in the globe, only the Northern and the Southern African desert and the Southwestern United States receive a comparable amount [98]. The combined sunny climate and latitude of FNQ exhibit potentiality for solar electricity generation. Any area receiving solar irradiation of greater than 4 kWh/m2/day can be geographically potential for harnessing solar energy [99]. FNQ receives average daily solar irradiation, that is greater than 5.5 KWh per square metre per day [98], more than enough with annual average daily sunshine hours of 7 to 8 (as shown in Fig. 12). Figure 13 presents monthly mean daily global horizontal irradiation for some selected remote areas of FNQ, which varies between 4 and 7.5 kWh/m2/day [101], which is the proof of FNQ’s promising potential to harness solar energy. It is also seen that Mornington Island has peaked in November recording 7.3 kWh/m2/day, followed by Cook at 7 kWh/m2/day, Lockhart at 6.8 kWh/m2/day and Burke at 5.9 kWh/m2/day. On the other hand, Aurukun, Carpentaria and Kowanyama have recorded a maximum of 7.2 kWh/m2/day in October, followed by Doomadgee and Pormpuraaw at 7.1 kWh/m2/day, Napranum and Torres shire at 7.0 kWh/m2/day, Mapoon and Northern Peninsula area at 6.7 kWh/m2/day.Fig. 12 Annual average daily sunshine hours in Australia including FNQ [100] Fig. 13 Monthly mean daily solar irradiation in selected regions of FNQ [101] Similarly, wind resource in FNQ is another potential option for power generation, with annual average wind velocity at 80 m above ground level ranging from 5.6 to 10 m/s (as shown in Fig. 14). Previous studies considered the minimum mean daily wind velocity 4 m/s [103] and 5 m/s [104] for installing wind farm. In addition, the mean operational cut in velocity for the Vestas V117-3.45, horizontal axis wind turbine, which is used in Mount Emerald wind farm in Arriga (FNQ), is 3 m/s [105, 106] and this indicates that FNQ is the best suited to wind energy.Fig. 14 Mean wind speed at 80 m above ground level in Australia including FNQ [102] Geographical potential Suitable place identification to install solar and wind farm is a complicated task. Moreover, issues such as meteorological needs, ecological concerns and financial gains, also need to be considered for plant installation and operation [107, 108]. Area with the abundant resources such as solar irradiation and wind velocity may not be the only feasible thing. Some other factors that can also be crucial for the evaluation of suitable places, such as social, economic and environmental constraints [109, 110]. Recently, Geographical Information System (GIS) has appeared as the most convenient and proficient tool, and is being employed by many countries for assessing renewable energy potentiality [111, 112]. GIS tool can digitise, convert, analyse and visualise spatial data [113]. GIS can handle a range of environmental, financial, social and regional aspects for planning renewable energy development. It has inbuilt abilities to investigate the territories, generate and sort data, capture the geographical information, manage commands and visualise the output [113]. GIS data give an appropriate path to ascertain a suitable place considering location-specific circumstances (social and environmental limitations and resource availability) [114–116]. GIS includes various built-in tools; the present study principally utilises data management, conversion and spatial analysis tools. In this study, to perform GIS-based analysis, several remote areas from FNQ have been selected, namely Cook, Carpentaria, Burke, Doomadgee, Mornington Island, Kowanyama, Pormpuraaw, Aurukun, Injinoo, Torres, New Mapoon, Umagico, Mapoon and Lokhart river. All topographical data are extracted from different digital databases that are given in Table 2. Unsuited locations have been omitted utilising different tools of ArcGIS. Firstly, data management toolbox is used to make projection of all GIS data layers (vector and raster) on a similar coordinate system, project tool for vector data and project raster tool for raster data. Then different GIS activities have been executed for each layer to fulfil the renewable energy development criterion, as described below:Extraction of land cover and digital elevation models of selected areas of FNQ from the land-use land cover and global digital elevation models, respectively, by utilising Extract by Mask tool. Extraction of slope model from the elevation model by Slope tool. Reclassification of raster model of a slope by Reclassify tool. Identification of suitable and unsuitable land cover from land cover data by Weighted Overlay tool. Conversion of all raster models into vector data set by Raster to Polygon tool. Implementation of a suitable buffer by Buffer tool on each layer that further expands the omitting criterion according to the assumptions considered in this study and merging unsuited layers by Merge tool. Removal of merged unsuitable areas from the suitable land cover (vector data) by Erase tool. Finally, the Clip tool is utilised to extract the administrative boundary of selected regions with the aid of the administrative boundaries of Australia from the final suitable layer. Table 2 GIS data set for identifying suitable locations Thematic theme Type of data Source Spatial resolution Administrative boundary Vector GADM, version 1.0 [117] – Water bodies Vector DIVA-GIS [117] – Protected areas Vector WDPA [118] – Airports Vector Data Share [119] - Urban built-up area Vector SEDAC [120] – Rail network Vector DIVA-GIS [117] – Road network Vector DIVA-GIS [117] – Land-use land cover Raster GlobCover [121] 10 arcsec Digital elevation model Raster CGIAR SRTM [117] 30 s At last, the total suitable places for each administrative boundary of the selected regions are acquired by computing their geometry in the attribute table. Based on the appropriate land-use factors, the maximal limits of solar and wind power generation capacity are evaluated for each selected region that illustrates the geographical potential of renewable energy. Several exclusion criterion included for the assessment is outlined below:Land-use land cover (LULC): In this assessment, UN global land cover data are utilised. As per the criterion of a previous study [122], irrigated (class 11), rain-fed croplands (class 14), mosaic cropland (class 20), shrubland (class 130), grassland (class 140), sparse vegetation (class 150) and bare areas (class 200) are considered suitable for wind plants. For solar, similar suitability criterion which is implemented for wind, is used, except irrigated (11), rain-fed croplands (14). Other land categories such as mosaic vegetation (class 30), broad-leaved deciduous (classes 50, 60) or semi-deciduous forest (class 40), needle-leaved ever green forest (classes 70, 90), mixed forest (class 100), mosaic forest (class 110) and grassland (class 120), woody wetlands (classes 160, 170), artificial areas (class 190), water bodies (class 210), snow or ice (class 220) are considered unsuitable for any power plant installation. Water bodies: In some studies, to conserve natural resources, reservoirs such as seas, rivers and lakes were omitted with a buffer of 100 m [123] and 400 m [109] for wind application; 400 m for both solar and wind application [103]. In this study, 400 m is considered for both solar and wind analysis. Protected area: Previous studies [124, 125] applied 1 km buffer for protected areas in wind application to preserve the protected areas such as world heritage sites, national parks and sanctuaries, which are not suitable for power plant installation. This study has adopted 1 km buffer for wind analysis. Urban and rural built-up area: In deploying renewable energies at a large-scale, densely populated and urbanised areas are not practicable for avoiding inconvenience to human life. Previous studies applied a buffer of 5 km [124] for wind applications, 500 m [126, 127] for solar and wind applications. This study has adopted 5 km buffer for wind analysis. And no buffer zone is applied for solar analysis. Rail and road network: Rail and road networks are not also practicable for renewable energy installation. Previous studies applied a 300 m [124] buffer to road and rail networks considering their future expansion and a 500 m [128] buffer for reducing visual disturbance and ensuring electrical safety. In this study, 500 m buffer is maintained with existing rail and road networks for solar and wind analysis. Airports: In some studies [114, 129, 130], distance to airport is used for wind analysis, as wind turbines may disturb air traffic control by muddling the airport surveillance radar signals. Previous studies have implemented a buffer of 2500 m [104, 123] and 3000 m [129], and in this study, 3000 m is considered as a safer option. However, consultation is inevitable for installing any wind farm within a range of 30 km [131] of an airport. In the case of solar, glimpse and glint [132, 133] from panels may mystify pilots’ vision and may also affect the radar systems. Hence, a 1000 m buffer is applied between airports and solar plant for the present study. Slope and elevation: Slope and elevation are the elementals of topography. Almost all studies recommend land with low slopes and low elevations for developing solar and wind projects [114, 126, 134, 135]. For wind projects, the allowable range of slope may vary within 10 to 30 degrees [134] and for solar can vary between 3 and 5 degrees [126, 134]. The elevation above sea level varies from place to place, and previous studies proposed elevation of 2000 m for solar and wind farms [126, 136], 4500 m for solar farms [104], 2000 m for wind farms [128]. This study considers slope up to 5°for solar analysis and maximum 15° for wind analysis. From the slope map (as shown in Fig. 19), it is found that the selected regions of FNQ have maximum slope 14.82°. So, the slopes of the areas are fully suitable for wind power installation. Figure 18 represents the elevation of the regions. The maximum elevation is 1276 m, which is considered suitable for both solar and wind projects development. Results The current study is the first ever work to explore suitable locations in FNQ for the development of solar and wind projects using GIS multi-criterion decision making. ArcMap 10.8.1 is used for GIS-based analysis. The assessment is conducted using GIS-based multi-layer approach. The outcome of different GIS activities executed for location suitability analysis, is displayed in Figs. 15, 16, 17, 18, 19, 20, 21. Figure 15 represents land cover information for the selected regions of FNQ such as classes 14 (rainfed croplands), 20 (mosaic croplands), 30 (mosaic vegetation), 40 (semi-deciduous forest), 60 (broadleaved deciduous forest), 110 (mosaic forest), 120 (mosaic grassland), 130 (shrubland), 140 (grassland), 150 (sparse vegetation), 170 (woody wetlands), 190 (artificial areas), bare areas (200) and water bodies (210). Figures 16 and 17 represent the suitable land cover for solar and wind, respectively, which are found after excluding unsuitable land cover. Figure 18 shows the elevation map. The elevation map shows that maximum areas of the selected regions are within − 4 m to 66 m. Other areas have different ranges of elevation such as 66–153 m, 153–319 m, 319–633 m and 633–1276 m. It is noticeable that the selected regions have a maximum elevation of 1276 m, which is well below previous studies’ adopted elevation of 2000 m [126, 136]. So, the selected whole regions are considered suitable for both solar and wind. Figure 19 represents the slope map. From the slope map, it is seen that maximum areas of the selected regions have slope below 1.4° that means that maximum areas are almost flat and very much suitable for installing power plant. Few areas are within the range of 1.4° to 4.94°. Very few areas are within the range of 4.94–14.82°. The slope map is reclassified within the range of 0–5° and 5–14.82°, which is presented in Fig. 20. This figure has given more better understanding that almost all areas have a slope within 5°, which is suitable for solar. In addition, the maximum slope is 14.82°, so all the selected regions are fully suitable for wind, as this study adopts maximum 15° slope for wind analysis. Figure 21 represents the unsuitable GIS layers that include urban areas, protected areas, rail and road networks, water bodies and airports. Excluding these unsuitable layers with suitable buffer and considering suitable land cover, elevation and slope, final suitable places for solar and wind farms installation in selected administrative areas have been generated that are presented in Figs. 22 and 23, respectively. Suitable locations for solar and wind in every administrative region are calculated in the attribute table that are presented in Tables 3 and 4, respectively.Fig. 15 GlobCover land-use land cover for selected regions of FNQ Fig. 16 Suitable land cover for solar in selected regions of FNQ Fig. 17 Suitable land cover for wind in selected regions of FNQ Fig. 18 Elevation of selected regions of FNQ Fig. 19 Slope map for selected regions of FNQ Fig. 20 Reclassified slope map for solar of selected regions of FNQ Fig. 21 Unsuitable GIS layers for selected regions of FNQ Fig. 22 Final suitability map for solar power installation in selected regions of FNQ Fig. 23 Final suitability map for wind power installation in selected regions of FNQ Table 3 Theoretical solar power potential Selected site Annual solar mean radiation (G) (kWh/m2/day) Suitable area (m2) Theoretical power potential (MW) Cook 5.8 61,940,133,432.00 6,107,297.16 Carpentaria 6.1 45,689,555,002.00 4,738,006.85 Burke town 6.2 22,407,859,618.00 2,361,788.40 Doomadgee 6.1 1,049,736,835.00 108,857.71 Mornington Island 5.9 632,097,742.50 63,399.40 Kowanyama 6.1 1,546,394,148.00 160,361.07 Pormpuraaw 6.0 2,733,477,907.00 278,814.75 Aurukun 5.9 5,948,460,436.00 596,630.58 Injinoo 5.6 105,737,663.10 10,066.23 Torres 5.8 48,331,606.32 4,765.49 New Mapoon 5.7 1,264,375.11 122.52 Umagico 5.6 244,564.76 23.28 Mapoon 5.5 191,562,294.40 17,911.07 Lockhart River 5.6 521,935,891.28 347,818.08 Table 4 Theoretical wind power potential Selected site Suitable area (m2) Theoretical power potential (MW) Cook 62,514,737,696.00 450,154.65 Carpentaria 46,115,274,994.00 332,065.78 Burke town 22,636,366,913.00 162,999.42 Doomadgee 1,035,985,866.00 7,459.90 Mornington Island 811,595,814.20 5,844.12 Kowanyama 1,581,502,920.00 11,388.05 Pormpuraaw 2,806,975,362.00 20,212.40 Aurukun 5,993,704,069.00 43,159.32 Injinoo 137,748,431.80 991.89 Torres 80,323,315.77 578.39 New Mapoon 1,288,902.50 9.28 Umagico 191,127.54 1.38 Mapoon 251,693,470.70 1,812.39 Lockhart River 596,438,879.30 4,294.82 The suitable land evaluation process has been conducted for solar and wind separately. Initially, the selected regions are assumed only for being solar project and later only for wind project. Finally, the total land area suitable for solar power has been found to be 142,294.85 km2 and for wind 144,563.83 km2. The solar energy potential can be evaluated considering mean horizontal solar irradiation, suitable land area and solar panel efficacy. Solar power potential can be calculated by the following equation [137]:1 Psolar=G×SA×AF×η where Psolar is the solar power potential, G is annual mean horizontal solar irradiation in kWh/m2/day, SA is the suitable land area (m2), AF is the area factor (%), and η is the solar panel efficacy (%). Here, the area factor reveals the maximal places covered by the solar panels with minimal shadow effect. Area factor is considered 70% as used in the previous studies [113, 137]. Daily average solar irradiation data for selected regions are collected from Bureau of Meteorology. The study has used the First solar series 4™ PV module (advanced thin-film solar module), used in Kidston solar farm on the site of Kidston gold mine of FNQ. The maximum efficiency, 17%, of the module is used in this study to get the maximum possible solar power. Table 3 represents calculated solar power potential for the selected sites. The biggest area Cook is suitable for 6,107.30 GW solar power production, whereas the smallest area Umagico is suitable for 23.30 MW solar power production. Wind power potential can be evaluated by the following equation [134]:2 Pwind=SA×AF where Pwind is the wind power potential (MW), SA is the suitable land area (m2), and AF is the area factor (kW/m2). In this study, wind turbines have been arranged at a distance of 7D × 5D [113], where D is the rotor diameter. Area factor (AF) can be calculated as follows [134],3 AF=Capacity/7D×5D The study has used Vestas V117-3.45 wind turbine specification to calculate wind power potential. The turbine capacity and rotor diameter are 3.45 MW and 117 m, respectively [138]. Table 4 represents calculated theoretical wind power potential for the selected regions. The biggest area Cook can be suitable for 450 GW wind power production, whereas the smallest area Umagico is suitable for 1.38 MW wind power production. Economic potential Another important thing is that renewable power generation cost needs to be cheap or cost competitive. According to Roam consulting Pty Ltd, compared to fossil fuel power generation, renewable generation could be cheaper in the remote communities that would be advantageous and crucial for local economic growth. The Australian Climate Council [139] has reported that renewable power generation is the cheapest option now compared to new built coal and gas power generation, even with all of the existing subsidies associated with coal and gas. Table 5 depicts the power generation cost by sources [139].Table 5 Cost of new built power plants [139] Power technology Levellised cost of energy (LCOE) $(aus)/MWh Wind $50–65 Solar PV $78–140 Solar thermal plant $78 Gas combined cycle $78–90 Coal $134–203 Coal with carbon capture system (CCS) $352 Recently, Queensland solar and wind projects show possibility to be the cheapest power generation option in Australia. For instance, the Coopers Gap wind farm of 453 MW in Queensland can deliver power with cost bellow $60/MWh [139]. Large-scale solar PV in Queensland has already reached bellow $80/MWh [140]. However, remote communities of FNQ have access to electricity, but emissions, cost and reliability are the major concern. They have to rely on expensive imported diesel. In 2018–19, the Queensland government [141] paid $465 million to Ergon Energy Retail for ensuring remote customers paid similar electricity prices to other users. Hence, many remote places are not paying the real cost of power supply. In addition, the utilisation of diesel-based power generation is contributing to environmental pollution such as noise, carbon emissions and oil spillage, high supply cost and increased road maintenance costs [141]. On the other hand, continued fall in the cost of solar and wind generation, including storage, can lessen the power supply cost and the need for diesel power generation [141]. The cost competitiveness of solar and wind open up the prospect of utilising local renewable energy resources in place of the existing diesel power generation system of FNQ. In addition, a vast deployment of renewable energy can fuel economic growth in FNQ, with creating new job opportunities, enhancing human well-being and eventually can contribute to a climate-safe future. Queensland has a high ambitious target for installing renewable energy in FNQ, by expanding especially solar and wind energy resources. However, the installation of renewable power plant is very slow compared to their targets. The possible barriers may include lack of evidence of energy resources, poor infrastructure, high investment costs, limited planning and lack of political motive. However, the results of the study can help various government agencies, policy and decision makers, researchers and parties in bringing renewables into the FNQ’s energy system. The present study has performed a GIS-based analysis to assess favourable locations to develop two eminent resources, namely solar and wind. From the analysis, it is found that the FNQ has significant potential for exploiting solar and wind. Then the study has highlighted the LCOE or investment for solar and wind energy including conventional power systems, which proves that solar and wind resources have significant economic potential. The present study can give worthy guidance to the research community as well as potential parties regarding the location suitability to install solar and wind power stations. The current study assesses suitable places for solar and wind projects, including power potential, and presents an overview on economic potential. Hopefully, the results of the study will assist in attaining future renewable energy targets. Conclusion Since the world progressively incorporates renewable power generation and switching away from expensive or emissions-intensive technologies, the demand is rising for hybrid renewable energy assets that combine multiple forms of generation and storage. Renewable energy sources are promising for eliminating ecological damage. From the technical and economic context, different methodologies have been developed to size and analyse techno-economic characteristics regarding renewable energy adoption. This study has presented a global survey on the renewable energy availability, development and an essential to implement renewable energy system within remote regions of Far North Queensland in Australia. This study has performed potential assessment for solar and wind resources in terms of climatic, geographical and economical. The assessment shows significant potential for solar and wind development in every aspect. This study concludes that the selected areas from Far North Queensland are highly potential for solar and wind. The total maximum potential areas are found at 142,294.86 km2 (55.94% of total selected areas) with power potential of 14,448 GW for solar and 144,563.80 km2 (56.83% of total selected areas) with power potential 1040.97 GW for wind. Along with the solar and wind, hydrogen presents a clear perspective for a clean and affordable energy supply in the remote areas as well as deep decarbonisation which is the global target, needed to be reached by 2050 for limiting global surface temperature increment to 2 °C. In addition, the possibility to store hydrogen opens the opportunity for the integration of high renewable resources (solar and wind) shares with positive effects on Australia’s sustainable development goals through reduced GHG emissions. Hence, utilisation of renewable resources: solar and wind with water splitting hydrogen is the ultimate solution for energy system and sustainable ecology. This study has presented a potential overview to the complexity of decision making in the renewable energy sector and a scientific basis for selecting efficient, cost-effective clean solutions for the energy system of remote communities of Far North Queensland, Australia. This knowledge will increase stakeholder confidence in investing in renewable energy, which will be integral to Australia’s efforts to reduce reliance on dirty and costly diesel-based energy systems. Data availability Data will be made available on request. Declarations Conflict of interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. IISD: World population to reach 9.9 billion by 2050. (2020) Accessed 12 Sep 2021 2. Mandal TK Gregory DH Hydrogen: A future energy vector for sustainable development Proc. Instit. Mech. Eng. Part C J. Mech. Eng. Sci. 2010 224 3 539 558 10.1243/09544062JMES1774 3. Ergon Energy: Renewables in Far North Queensland. (2020) Accessed 20 April 2021 4. 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==== Front Folia Microbiol (Praha) Folia Microbiol (Praha) Folia Microbiologica 0015-5632 1874-9356 Springer Netherlands Dordrecht 1021 10.1007/s12223-022-01021-z Original Article Ribotyping of Clostridioides difficile in the Liberec Regional Hospital: a tertiary health care facility http://orcid.org/0000-0001-9643-2067 Kracík Martin [email protected] 123 http://orcid.org/0000-0001-9635-3473 Dolinová Iva [email protected] 1 http://orcid.org/0000-0002-3868-0545 Žemličková Helena [email protected] 345 1 grid.447961.9 0000 0004 0609 0449 Department of Genetics and Molecular Diagnostics, Liberec Regional Hospital, 46001 Liberec, Czech Republic 2 grid.447961.9 0000 0004 0609 0449 Department of Microbiology and Immunology, Liberec Regional Hospital, 46001 Liberec, Czech Republic 3 grid.4491.8 0000 0004 1937 116X Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Kralove, Charles University, 50005 Hradec Kralove, Czech Republic 4 grid.425485.a 0000 0001 2184 1595 National Reference Laboratory for Antibiotics, Centre for Epidemiology and Microbiology, National Institute of Public Health, 10000 Prague, Czech Republic 5 grid.4491.8 0000 0004 1937 116X Department of Microbiology, 3rd Faculty of Medicine, Charles University, University Hospital Kralovske Vinohrady and National Institute of Public Health, 10000 Prague, Czech Republic 1 12 2022 16 22 9 2022 21 11 2022 © Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The ribotyping of Clostridioides difficile is one of the basic methods of molecular epidemiology for monitoring the spread of C. difficile infections. In the Czech Republic, this procedure is mainly available in university hospitals. The introduction of ribotyping in a tertiary health care facility such as Liberec Regional Hospital not only increases safety in the facility but also supports regional professional development. In our study, 556 stool samples collected between June 2017 and June 2018 were used for C. difficile infection screening, followed by cultivation, toxinotyping, and ribotyping of positive samples. The toxinotyping of 96 samples revealed that 44.8% of typed strains could produce toxins A and B encoded by tcdA and tcdB, respectively. The ribotyping of the same samples revealed two epidemic peaks, caused by the regionally most prevalent ribotype 176 (n = 30, 31.3). C. difficile infection incidence ranged between 5.5 and 4.2 cases per 10,000 patient-bed days. Molecular diagnostics and molecular epidemiology are the two most developing parts of clinical laboratories. The correct applications of molecular methods help ensure greater safety in hospitals. Keywords Clostridioides difficile Toxinotyping Ribotyping Health care facility Clonal spreading http://dx.doi.org/10.13039/100007543 Grantová Agentura, Univerzita Karlova SVV UK LFHK No. 260544 ==== Body pmcIntroduction Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, spore-forming anaerobic bacterium that is widely distributed in the intestine of humans and animals and in the environment (Nyc et al. 2015; Janezic et al. 2016; Lawson et al. 2016). Its spores are usually found in the human or animal intestine as part of the microbiome (Czepiel et al. 2019), but some strains of C. difficile are capable of producing toxins (toxin A, toxin B, and binary toxin) that damage the intestinal mucosa and cause diarrhea. This ability is more important when there is an imbalance in the intestinal microbiota caused by previous antibiotic treatment (Smits et al. 2016; Czepiel et al. 2019). A reduction in microbiome complexity creates space for C. difficile to proliferate. Since 2000, hospital strains of C. difficile are common hyperproducers of toxins (ribotypes 027, 176, and 001) (Persson et al. 2011). These strains often colonize elderly polymorbid patients, complicating their primary diagnosis and causing life-threatening infections (Beneš et al. 2012). Laboratory diagnostics of C. difficile infections (CDI) is based on examination of the patient´s stool. The two-step algorithm consists of the highly sensitive detection of glutamate dehydrogenase (GDH), which is a Clostridioides antigen, followed by toxin detection. The detection of toxigenicity is essential for the diagnosis. Non-toxigenic strains are not considered to be pathogenic (Beneš et al. 2012; Crobach et al. 2016; Krůtová and Nyč 2018). Therefore, closer identification of strains by toxinotyping, ribotyping, or whole-genome sequencing is important. (Huber et al. 2013; Krutova et al. 2018). Toxinotyping is the basic characterization of a C. difficile strain. It describes which toxins the strain produces—toxin A, toxin B, binary toxin, or none. This can be done by enzyme immunoassay or PCR. PCR detects the cdtA and cdtB genes for toxins A and B, respectively, and the tcdA and tcdB genes for binary toxin (Delmée et al. 2005; Persson et al. 2008; Rupnik and Janezic 2016). Ribotyping is based on the number and length of intergene sequences between the 16S and 23S rDNA. PCR focused on this region produces several DNA fragments with characteristic lengths. The resulting profile can be assigned to a specific ribotype (Huber et al. 2013). Whole-genome sequencing is the most powerful tool for molecular epidemiology, but it is expensive for tertiary health care facilities. Rep-PCR-based fingerprint DNA testing systems are also commercially available but are not widely used for the diagnosis of C. difficile (Corbellini et al. 2014). Whole-genome sequencing and ribotyping in the Czech Republic is preferably carried out by university hospitals (quaternary medical facility). Tertiary health care facilities, such as Liberec Regional Hospital, had few resources (personnel, financial, and instrumentation) for molecular epidemiology (ribotyping and whole-genome sequencing). This was changed by the SARS-CoV-2 pandemic, which caused a significant development of regional PCR laboratories. The majority of regional laboratories are now able to analyze C. difficile strains in more detail. In our study, we use ribotyping to detect outbreaks of CDI in Liberec Regional Hospital. Material and methods Hospital Liberec Regional Hospital is a tertiary health care facility providing specialized care in the Liberec Region, in the north of the Czech Republic, which is inhabited by approximately 500,000 people. The hospital has about 1,200 beds, with 200 beds for follow-up care. As a nonuniversity hospital, it does not have state-funded research activities but has been listed as a research organization since 2019. The average number of patient-bed days in Liberec Regional Hospital in the years 2017–2018 was 224,039. Sample collection and demographics Samples for our study were collected over 13 months—from June 2017 till June 2018. In this period, 556 stool samples from hospitalized patients (n = 493) were sent to the microbiological laboratory of Liberec Regional Hospital. In this group, 46.9% (n = 261) of samples were from males and 53.1% (n = 295) from females. The 70–79 age group was the most represented, accounting for 32.7% (n = 182) of the sample. Internal medicine was the most represented specialty with 35.3% (n = 196). A more detailed overview is presented in Table 1. A duplicate sample, which was defined as a sample collected within 10 days of the first collection, was not included in the study.Table 1 Number of samples by sex, age and medical specialty Medical expertise/age 0–9 10–19 20–29 30–39 40–49 50–59 60–69 70–79 80–89 90–99 Total Females 5 8 5 7 8 19 63 94 69 17 295 Internal medicine 1 2 5 5 25 35 28 6 107 Infectious medicine 1 3 3 12 8 12 3 42 Surgery 2 1 1 2 11 16 6 39 Subsequent inpatient care 6 11 5 22 Rheumatology 1 7 7 1 16 Oncology 4 3 6 1 14 Anesthesiology and intensive care 2 2 1 3 1 2 1 12 Others < 10 samples 4 4 2 0 2 2 10 14 4 1 43 Males 8 5 5 7 15 23 67 88 37 6 261 Internal medicine 3 3 5 9 23 35 9 2 89 Surgery 1 1 3 3 6 11 17 9 1 52 Infectious medicine 1 1 1 8 8 5 24 Anesthesiology and intensive care 1 4 4 4 2 15 Subsequent inpatient care 1 4 3 5 1 14 Pediatric medicine 7 3 10 Cardiology 1 1 4 4 10 Others < 10 samples 1 0 0 0 6 1 13 17 7 2 47 Total 13 13 10 14 23 42 130 182 106 23 556 Screening and cultivation QuikChek Complete (TechLab, USA) was used for CDI screening and basic toxinotyping C. difficile. After the CDI screening, GDH-positive samples were selected for stool cultivation. The stool samples were exposed to 96% ethanol (1 mL or a small pea-sized portion of stool in 1:1 mixture with alcohol) for 30 min. Two drops (approximately 50–75 µL) of deposit were inoculated onto Brazier’s Clostridium difficile Selective agar (Oxoid, Czechia) and spread to obtain single colonies. The inoculated plates were immediately transferred to an anaerobic environment and incubated at 37 °C for 48 h. The identification of C. difficile colonies was performed according to the characteristic color and shape of the colonies (irregular with grey to white color) and by Gram-stained microscopy and subsequent toxinotyping by PCR. PCR toxinotyping DNA was isolated from the C. difficile suspension (1–3 colonies in 500 µLµ nuclease free water) using a PathogenFree DNA Isolation Kit (GeneProof, Czechia) and used for PCR reaction focused on the genes encoding the toxins (cdtA, cdtB, tcdA, and tcdB). Mastermix was 12.5 µL Hotstart Mastermix (Qiagen, Netherlands), 5 µL primer mix (see Table 2 for more details), 5 µL nuclease free water, and 2.5 µL sample DNA. The cycling program was activation for 15 min at 94 °C, followed by 35 cycles of denaturation for 45 s at 94 °C, annealing for 45 s at 50 °C, and elongation for 1 min at 72 °C, with the final elongation step for 30 min at 72 °C. The obtained amplicons were detected by capillary electrophoresis in a MultiNa (Shimadzu, Japan) instrument.Table 2 Ribotyping and toxinotyping primers (Bidet et al. 1999; Persson et al. 2008) Name Target Sequence Concentration (µM) Size (bp) Toxinotyping primer mix tcdA-F tcdA gene 5′-GCATGATAAGGCAACTTCAGTGGTA-3′ 3.00 629 tcdA-R 5′-AGTTCCTCCTGCTCCATCAAATG-3′ 3.00 tcdB-F tcdB gene 5′-CCAAARTGGAGTGTTACAAACAGGTG-3′ 2.00 410 tcdB-RA 5′-GCATTTCTCCATTCTCAGCAAAGTA-3′ 1.00 tcdB-RB 5′-GCATTTCTCCGTTTTCAGCAAAGTA-3′ 1.00 cdtA-FA cdtA gene 5′-GGGAAGCACTATATTAAAGCAGAAGC-3′ 0.25 221 cdtA-FB 5′-GGGAAACATTATATTAAAGCAGAAGC-3′ 0.25 cdtA-R 5′-CTGGGTTAGGATTATTTACTGGACCA-3′ 0.50 ctdB-F cdtB gene 5′-TTGACCCAAAGTTGATGTCTGATTG-3′ 0.50 262 cdtB-R 5′-CGGATCTCTTGCTTCAGTCTTTATAG-3′ 0.50 PS-F 16S-rDNA 5′-GGAGGCAGCAGTGGGGAATA-3′ 0.25 1062 PS-R 5′-TGACGGGCGGTGTGTACAAG-3′ 0.25 GluD-F gluD gene 5′-GTCTTGGATGGTTGATGAGTAC-3′ 0.50 158 GluD-R 5′-TTCCTAATTTAGCAGCAGCTTC-3′ 0.50 Ribotyping primers FAM-16S 16S-23S intergene 5′-GTGCGGCTGGATCACCTCCT-3′ 10.00 fragment profile 23S rev 5′-CCCTGCACCCTTAATAACTTGACC-3′ 10.00 PCR ribotyping This was based on the amplification of an intergene locus between the 16S and 23S rDNA genes. Each ribotype has a specific number of intergene locus with a different length. Amplification thus produced multiple fragments with different lengths. This fragment profile is specific for each ribotype. For ribotyping, the PCR mastermix was 25 µL Hotstar Mastermix (Qiagen, Netherlands), 0.3 µL of each primer (see Table 2 for more details), 22.4 µL nuclease free water, and 2 µL sample DNA. The cycling program was activation for 15 min at 95 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 60 °C, and elongation for 1 min at 72 °C, with the final elongation step for 30 min at 72 °C. PCR fragments were then analyzed in an ABI310 automatic genetic analyzer (Thermo Fisher Scientific, Waltham, MA, USA) in a 50 cm capillary loaded with POP4 polymer (Thermo Fisher Scientific, Waltham, MA, USA). LIZ600 was used as the size standard (Thermo Fisher Scientific, Waltham, MA, USA). The length of each fragment was determined using the GeneMapper software (Thermo Fisher Scientific, Waltham, MA, USA). The resulting profile was then uploaded to the WEBRIBO database (https://webribo.ages.at/). Results Between June 2017 and June 2018, 556 stool samples from hospitalized patients were sent to the laboratory of Liberec Regional Hospital for CDI screening. GDH positivity was detected in 124 (22.3%) samples. Toxin positivity was confirmed in 70/124. Fifty-four GDH-positive samples and all 432 (77.7%) GDH-negative samples were toxin negative. Based on GDH positivity, the incidence of CDI in Liberec Regional Hospital was 5.5 cases per 10 000 patient-bed days. The monthly incidence of CDI ranged from 3 to 13 cases, with a median of 10 and mean of 9.5 cases. It was not possible to estimate any epidemiological trend from the number of CDI occurrences over time. Stool cultivation was performed on 98 GDH-positive samples. The remaining 26 GDH-positive samples could not be cultured due to lack of material. Cultivation yielded 96 (98.0%) viable cultures. Only 10 GDH-positive samples came from patients without antibiotic treatment. The most commonly used antibiotic in the remaining 88 patients was amoxicillin/clavulanic acid (n = 45, 45.9%), followed by cephalosporins (n = 31, 31.6%). PCR toxinotyping revealed that 43 (44.8%) strains carried the genes for toxins A and B (tcdA and tcdB) only, 33 (34.4%) strains carried the genes for both major toxins and the binary toxin (tcdA, tcdB, cdtA, and cdtB), and only 2 (2.1%) strains only carried the genes for toxin B and the binary toxin (tcdB, cdtA, and cdtB). Fourteen (14.6%) strains were non-toxigenic, and in 4 (4.2%) cases, toxinotyping failed or the presence of C. difficile was not confirmed. Based on PCR confirmation, the incidence of CDI in Liberec Regional Hospital was 4.2 cases per 10 000 patient-bed days. PCR ribotyping was performed on 96 strains from cultivation. The most abundant ribotype was 176 (n = 30), followed by 012 (n = 10) and 014 (n = 5). The remaining ribotypes (n = 24) were found in less than 5 cases. See Table 3 for a more detailed overview. In 5 cases, ribotyping failed. Ribotype 176 was most commonly seen in the internal medicine department, primarily between August 2017 and November 2018 (n = 10). In March 2018, ribotype 176 was detected simultaneously in three departments (2 × internal medicine, 2 × pulmonary medicine, and 2 × surgery). The pulmonary and internal medicine departments share one building in Liberec Regional Hospital.Table 3 Occurrence of individual C. difficile ribotype by month 176 012 014 010 002 001 078 413 017 596 449 043 440 629 483 051 046 005 667 081 AI-72 AI-60 651 400 020 023 New ribotype Failed Total 2017 VI 1 1 1 1 4 VII 3 1 1 1 1 7 VIII 2 1 1 1 2 1 1 9 IX 4 1 1 6 X 5 1 1 1 1 1 10 XI 4 2 1 1 8 XII 1 1 1 1 1 1 1 7 2018 I 2 1 1 1 5 II 1 2 1 1 1 1 7 III 6 1 1 1 9 IV 1 1 1 1 1 1 6 V 2 1 2 1 1 7 VI 1 3 1 1 1 1 1 1 1 11 Total 30 10 5 4 4 4 3 3 3 3 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 5 96 The strains of ribotype 176 most frequently carried genes for toxins A and B and binary toxin (n = 24). In contrast, all strains of ribotypes 012 and 014 only carried genes for the major toxins (A and B). The most frequently detected non-toxigenic ribotype was 010 (n = 4) followed by 596 (n = 3). Other non-toxigenic ribotypes were only found once. Discussion Our study found ribotype 176 to be the most frequent in the reporting period. This hypertoxigenous ribotype is common in the Czech Republic and is also specific for Central Europe (Czech Republic, Poland, and Slovakia; see Table 4). It is also called 027-like ribotype because of its quite similar profile to ribotype 027, the hypertoxigenous ribotype widespread in Western Europe (Krutova et al. 2014, 2016; Beran et al. 2017).Table 4 Occurrence of individual C. difficile ribotype by country Czech Republic Poland Slovakia Presented data Beran et al. (2017) Vaverková et al. (2021) Krutova et al. (2016) Aptekorz et al. (2017) Novakova et al. (2020) 176 (31.3%) 176 (60.9%) 001 (20.0%) 176 (29%) 027 (82.4%) 001 (59.0%) 012 (10.4%) 014 (9.4%) 014 (13.0%) 001 (24%) 176 (2.8%) 176 (29.5%) 014 (5.2%) 002 (6.3%) 078 (7.5%) 014 (9%) 014 (2.8%) 017 (3.8%) 010 (4.2%) 078 (3.1%) 176 (6.5%) 012 (5%) 010 (1.9%) 020 (1.3%) 002 (4.2%) 043 (3.1%) 020 (6.5%) 020 (4%) 027 (1.3%) 001 (4.2%) 015 (1.6%) 017 (4%) 049 (1.3%) 078 (3.1%) 017 (1.6%) 070 (1.3%) 413 (3.1%) 103 (1.6%) 017 (3.1%) 174 (1.6%) 596 (3.1%) 449 (2.1%) 043 (2.1%) Ribotype 176 was the main one that caused local outbreaks in internal medicine and adjacent departments of Liberec Regional Hospital. Table 3 shows two outbreaks in 2017 and 2018. The small outbreak in June 2018 was caused by a different ribotype—012. All other ribotypes represented a maximum of 5% and had no significant peak in the reported period. The proportion of individual ribotypes in Liberec Regional Hospital is quite different from those detected within the same period in Hradec Kralove University Hospital. There, the most frequent ribotype was 001 (20.0%), followed by 014 (13.0%), 078 (7.5%), 176 (6.5%), and 020 (6.5%) (Vaverková et al. 2021). Both hospitals are located in neighboring regions of the Czech Republic, but patient transfers between them are relatively minimal. The combination of the ribotype spectrums of Liberec and Hradec Kralove is very close to the ribotype spectrum of the Czech Republic as a whole (176–29%, 001–24%, 014–9%, 012–5%, 020–4%, and 017–4%) in 2014 (Krutova et al. 2016). Our observation suggests that the proportions of C. difficile ribotypes are probably highly connected to the specific hospital, and the transmission of C. difficile strains from one hospital to another is not common. A different view was reported by Beran et al. (2017), when they found two genetically related strains captured in two hospitals. However, they worked with hospitals belonging to the same organization or from the same region of the Czech Republic. In our cohort, 89.8% of GDH-positive patients received antibiotic treatment. The most commonly used antibiotic was amoxicillin/clavulanic acid (45.9%) followed by cephalosporins (31.6%). High rates of prior treatment with antibiotics have been described in the literature (Beran et al. 2014). The incidence of C. difficile infections in the Liberec Regional Hospital in 2017 and 2018 ranged from 5.5 to 4.2 cases per 10,000 patient-bed days. Both incidence rates correspond to the CDI incidence in the Czech Republic (6.1 to 4.5 cases per 10,000 patient-bed days) (Krutova et al. 2016) and in Europe (4.1 to 3.98 cases per 10,000 patient-bed days) (Bauer et al. 2011). Molecular diagnostics and molecular epidemiology are one of the most rapidly developing parts of clinical laboratories, helping to make hospitals safer. In our case, it helped to detect local outbreaks of ribotype 176, which could not be detected when evaluating the overall prevalence of CDI due to the high variability of the detected ribotypes. Until recently, tertiary hospitals in the Czech Republic have not had the capacity to use these methods. However, the COVID-19 epidemic changed this. New laboratories have been established, and old ones have been expanded. Spatial and instrument capacities have been increased. The only thing lacking is personnel capacity. If they can be scaled up, molecular methods can change the way hospitals operate. Author contribution M.K.: data curation, investigation, formal analysis, methodology, writing original draft. I.D.: methodology, writing—review and editing. H.Z.: methodology, project administration, writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding The study was supported by Charles University, project GA UK (SVV UK, LFHK, No. 260544). Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. 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==== Front J Happiness Stud J Happiness Stud Journal of Happiness Studies 1389-4978 1573-7780 Springer Netherlands Dordrecht 605 10.1007/s10902-022-00605-3 Research Paper The Psychological Costs of the COVID-19 Pandemic and Heterogeneous Effects in South Korea: Evidence from a Difference-in-Differences Analysis Kim Jinho [email protected] 12 Park Sujeong [email protected] 1 Subramanian S. V. [email protected] 3 http://orcid.org/0000-0002-7754-3105 Kim Taehoon [email protected] 4 1 grid.222754.4 0000 0001 0840 2678 Department of Health Policy and Management, Korea University, Seoul, Korea 2 grid.222754.4 0000 0001 0840 2678 Interdisciplinary Program in Precision Public Health, Korea University, Seoul, Korea 3 grid.38142.3c 000000041936754X Department of Social and Behavioral Sciences, Harvard T.H. Chan School of Public Health, Boston, MA USA 4 grid.289247.2 0000 0001 2171 7818 Department of Economics, Kyung Hee University, Seoul, Korea 30 11 2022 122 15 11 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. This study estimates the effects of the COVID-19 pandemic on life satisfaction and stress and examines whether these effects vary across different sociodemographic groups using a nationally representative sample in South Korea. We estimate the causal effects of COVID-19 on psychological well-being by exploiting regional variation in the spread of the pandemic in South Korea. While the number of confirmed cases was very small in other provinces in the first half of 2020, the coronavirus spread rapidly in Daegu after an outbreak in one church. We employ a difference-in-differences approach that compares changes in people’s life satisfaction and stress before-and-after the initial surge of COVID-19 cases in Daegu and other provinces. Our results show that the proportion of people who are dissatisfied with life increased by 2.8–6.5 percentage points more in Daegu than in other provinces after the COVID-19 outbreak. During the same period, the proportion of people who reported feeling stressed increased more in Daegu than in other provinces by 5.8–8.9 percentage points. Our results also suggest that the negative impact of the COVID-19 outbreak on psychological well-being is significantly greater for men, young adults, middle-aged adults, self-employed workers, and middle-income individuals. On the other hand, the proportion of people who report feeling stressed among the highest-educated (a master’s degree or higher) and high-income individuals decreased after the onset of the COVID-19 outbreak. Keywords COVID-19 Life satisfaction Stress Heterogeneous effects Sociodemographics ==== Body pmcIntroduction The coronavirus (COVID-19) pandemic has substantially disrupted the lives of individuals around the world since its onset in 2019 (Ammar et al., 2020). The COVID-19 pandemic has threatened the physical and mental health of individuals. Because the pandemic has persisted for an extended period, the consequences that it has had on population well-being and health are gradually intensifying. A large body of literature documents that the pandemic lowers psychological well-being as well as quality of life (Ammar et al., 2020; Le & Nguyen, 2021; Zhang & Ma, 2020), and increases suicidal ideation (Gunnell et al., 2020; McIntyre & Lee, 2020) and suicide attempts (Tanaka & Okamoto, 2021). The pandemic may directly impact one’s well-being due to fear and anxiety of contracting and/or transferring the virus (Xiong et al., 2020). Such concerns are driven in part by a rise in stigma and prejudice against people who have the virus (Carmassi et al., 2020; Fiorillo & Gorwood, 2020). Beyond its direct impacts, the virus has indirect effects on psychological well-being. There have been significant changes to people’s daily lives as governments around the world have issued public policies that encourage and/or directly enforce social distancing, self-quarantining, and lockdowns to slow the spread of the virus (Farkhad & Albarracín, 2021; Groeniger et al., 2021; Sobol et al., 2020). Disruptions in physical contact with family members, friends, and colleagues make individuals feel socially isolated, leading to an increase in depression, stress, and anxiety (Tang et al., 2020). In addition, the pandemic creates substantial psychological burdens for individuals with children, as parenting roles have intensified. The escalation of interpersonal conflict and stress at home has led to a considerable increase in domestic abuse and family violence around the world (Mazza et al., 2020; Usher et al., 2020). New realities of working from home and home-schooling of children increase stress and stress-related well-being problems, particularly among working couples with school-going children (Lee et al., 2021). Another important pathway through which the pandemic impacts well-being is increased uncertainty about future employment, income, and wealth (Witteveen & Velthorst, 2020). The International Labour Organization (ILO) reported that about 74 percent of the global workforce were impacted severely by full or partial lockdowns in 2020 (Monitor, 2020). The pandemic has led to a sudden, extraordinary amount of job loss (del Rio-Chanona et al., 2020). Global unemployment rates have reached double digits, with millions applying for unemployment benefits, disaster payments, or other social safety net programs (for Economic Co-Operation & (OECD), 2020). Studies suggest that economic uncertainty during the pandemic negatively affects one’s psychological well-being and health (Arthi & Parman, 2021). This study estimates the effects of COVID-19 on people’s life dissatisfaction and stress. Given the complexity and diversity of the mechanisms that underly the impact of the pandemic on psychological well-being, estimating the net impact of the pandemic is an important empirical issue. We conduct the analysis using the Social Survey, which includes a large nationally representative sample that allows for the estimation of heterogeneous effects across diverse sociodemographic groups. We estimate the effects in a causal manner by exploiting regional variation in the initial surge of COVID-19 cases. We employ a difference-in-differences approach that compares changes in life satisfaction and stress before-and-after the outbreak of COVID-19 between Daegu, a province where the coronavirus spread extensively, and other provinces with limited coronavirus cases. We argue that regional variation in the spread of COVID-19 is plausibly exogenous. This is a critical requirement for the identification of causal impacts using a difference-in-differences approach. This approach is based on the fact that the large number of infections in Daegu were caused by an unexpected group infection in a church. We believe that our approach is differentiated from previous studies using a type of time series analysis, which regards a change in psychological well-being before and after COVID-19 as a effect of COVID-19. We also investigate whether the effects of COVID-19 on life satisfaction and stress differ by sociodemographic characteristics (gender, age, education, income, and employment type). An individual’s social location likely influences the extent to which they are affected by and the resources they have to respond to the aforementioned factors and circumstances that can affect psychological well-being. Thus, heterogeneous effects of the COVID-19 pandemic on psychological well-being are worth exploring. The investigation of heteregeneous effect by demographic group would also allow us to analyze whether and how the COVID-19 pandemic has deepened inequality in psychological well-being. In particular, we reveal that the differential economic impact of COVID-19 by socioeconomic group is an important cause of the differential effect on psychological well-being by socioeconomic group. Theoretical Expectations on the Heterogeneous Effect of COVID-19 by Demographic Group In this study, we focus on differential impacts of COVID-19 across gender, age, education, income, and employment type. As explained in section "Introduction", COVID-19 can affect people’s life satisfaction and stress through multiple pathways. We discuss how the effects of COVID-19 on life satisfaction and stress can be different by demographic group by examining the possible pathways. First, the pandemic’s impact on psychological well-being may differ between men and women (Mooi-Reci & Risman, 2021). It is well-documented that women tend to have greater concerns about and fear of risk as well as uncertainty compared with men (Gustafson, 1998). Compared to men, women are more likely to be concerned about contracting the coronavirus and being unable to obtain medicine or medical treatment in case the need arises (Brooks & Saad, 2020). Moreover, women are more concerned than men about feeling isolated or being unable to reach family and friends in times of need (O’Connor et al., 2020). Women also bear disproportionate burdens that come with stay at home orders due to the closure of schools and daycares during the pandemic (Garnett et al., 2021). Women tend do additional care work partly because of the persistence of traditional gender roles and partly because of the fact that women are more likely to be part-time workers, have more flexible work arrangements, and receive lower wages (Gustafson, 1998). While the majority of prior research shows that women are more likely to lose jobs during COVID-19 than men (Albanesi & Kim, 2021; Alon et al., 2021; Dang & Nguyen, 2021), some studies report contrasting patterns(Aum et al., 2020). Men might also be at higher risk of death due to COVID-19 (Rabin, 2020) and this would contract their outdoor activities. Second, the pandemic’s impact on psychological well-being may operate differently across age groups. Older adults tend to be less concerned about the threat of the pandemic and perceive a lower risk of the infection due to higher levels of coping efficacy (Klaiber et al., 2021). Other studies, however, suggest that older adults may be more psychologically vulnerable than their younger counterparts because disruptions to see, care for, and receive support from family, friends, and community members may particularly worsen the psychological health of older adults (Moreno et al., 2020). In addition, it was well known that the elderly are more susceptible to the coronavirus. It has been shared through various reports from the government and media since the early days of the pandemic that the elderly are at higher risk of developing more severe illness from COVID-19. This would promote anxiety and fear and further reduce social activities of the elderly, which can affect their psychological well-being. Conversely, young or middle-aged adults may feel more stressed and depressed than older adults because of greater economic pressures caused by widespread unemployment and income loss brought on by the pandemic (Morrow-Howell et al., 2020; Pierce et al., 2020), though older adults also confront economic challenges due to declines in financial support from adult children (Li & Mutchler, 2020). It is also well known that young people who enter the labor market when economic conditions are bad have high unemployment rates and low wages and the effects are persistent (Bae & Kim, 2022; Kahn, 2010). It is likely that young people have a difficult time finding a job during the pandemic, and this would increase their stress level. Increased parenting stress may be another source of the pandemic-led threats to psychological well-being, particularly for younger married and cohabiting couples with children (van Ballegooijen et al., 2021). Third, socioeconomic status (SES) factors, such as education and income, may moderate the impact of the pandemic on psychological well-being. Individuals with a higher SES may have ample social and financial resources to address both the physical and mental health risks associated with the pandemic (Bu et al., 2020; Kim, 2021; McLeod & Kessler, 1990). For example, the pandemic may pose a greater threat to lower SES individuals because they are less likely to receive timely medical care and treatment as well as social and material support from others in case of infection (Arthi & Parman, 2021). Moreover, low-SES individuals may be more concerned about their job insecurity during the pandemic and the accompanying economic downturn (Moreno et al., 2020). Therefore, low-SES individuals may have disproportionately poorer psychological well-being than their high-SES counterparts (Vieira et al., 2020). Alternatively, low-SES individuals may be less responsive or sensitive to economic and social disruption caused by the pandemic (Ronzani et al., 2018) because they may have already experienced and suffered similar disruptions due to their socioeconomically disadvantaged backgrounds. If this is the case, changes to daily routines at work and home may have a greater impact on high-SES individuals compared with low-SES individuals. Fourth, the psychological impact of the pandemic may vary by a person’s employment type. The well-being of the self-employed may be most severely affected by the pandemic for several reasons. Self-employed workers, mainly concentrated in the service industry in South Korea, may be psychologically vulnerable because they tend to be exposed to a greater risk of contracting the virus (e.g., high-risk working environments can increase risk of infection if they have decreased sanitation, are overcrowded, and have limited opportunities for workers to physically distance) (Cetrulo et al., 2020). Moreover, it is often the case that the self-employed are hardest hit by public health measures that encourage social distancing in local communities. Therefore, self-employed persons are more likely to face economic hardships compared with individuals who work for others (Hupkau & Petrongolo, 2020). For example, in the United States, the number of active small businesses plummeted by 3.3 million or 22% between February and April 2020, and this was the largest drop on record (Fairlie, 2020). The number of small establishments also declines substantially after the COVID-19 pandemic in South Korea (Kim & Kim, 2022). Therefore, economic uncertainty related to the pandemic presents unique challenges for self-employed workers, from revenue losses to temporary or permanent closures, which may harm their psychological well-being (Ruffolo et al., 2021; Vandoros et al., 2019). In sum, the negative impacts of the COVID-19 pandemic on life satisfaction and stress are expected to be greater for people with low socioeconomic status, such as low-educated and low-income people. On the other hand, it is difficult to predict clearly the differential effect by gender and age because there are opposing factors. Estimating heterogeneous effects by demographic group is, therefore, an interesting empirical problem because the net effect of various mechanisms is ambiguous. It is also important to estimate the magnitude of the effect and the difference in the effect by the group, even when the expectation is rather clear. The COVID-19 Outbreak in South Korea In South Korea, the widespread COVID-19 outbreak began in late February after the first COVID-19 case was confirmed on January 20, 2020. Prior to February 18, there had only been 31 total confirmed cases in South Korea. The situation changed in late February when an outbreak was brought on by a group infection in a church in Daegu. The number of confirmed cases began to surge after a large number of unspecified people had contact with and infected individuals in a church. The cumulative number of confirmed cases increased to 9786 on March 31, and most of them were in Daegu. From mid-April to the end of May, the spread of COVID-19 was greatly suppressed as the effects of social distancing began to appear. The number of new confirmed cases increased by 877 from April 15 to May 31. Figure 1 presents trends of the number of confirmed cases per 1000 people separately by province from January 20 to May 31. This time frame captures the time in which the survey data used in this study was collected. South Korea has 17 provinces in administrative districts. Figure 1 shows that most confirmed cases in South Korea by the end of May occurred in Daegu. The province with the second-highest number of confirmed cases is Gyungbuk, which is adjacent to Daegu. The increase in the number of confirmed cases in Gyungbuk was influenced by the increase in neighboring Daegu. Even in Gyungbuk, however, the extent of the coronavirus spread was much lower than in Daegu. The fear and risk of having contact with an infected person may have also been much lower in Gyungbuk even though there were quite a few confirmed cases because the area of Gyeongbuk is the largest among 17 provinces in South Korea (population density in Daegu: 2757.64/km2; that in Gyungbuk: 144.24/km2). With the exception of these two provinces, the number of confirmed cases remained very small until the end of May. The cumulative number of confirmed cases per 1000 people was 2.86 in Daegu on May 31. This is approximately 32 times higher than the average cumulative number of confirmed cases per 1000 people in other provinces. We exploit this huge difference in the spread of COVID-19 between Daegu and other provinces to estimate the impact of COVID-19 on people’s psychological well-being. We also would like to briefly explain the South Korean government’s quarantine policies in response to the pandemic. The South Korean government did not implement different quarantine policies by region. The government did not also implement restrictions on movement or lockdown of regions. The government instead responded with a diagnostic kit that quickly finds the confirmed case, isolates the infected people, identified the movement of the confirmed people, traced the infection route, and tested people who had come into contact with the confirmed people. Schools were closed as face-to-face classes were switched to non-face-to-face classes. Although there were not many confirmed cases in regions other than Daegu, non-face-to-face classes were implemented at most schools across the country. The workplace was not closed, and only confirmed people did not go to work until they tested negative. Data This study relies on data from the Social Survey. The Social Survey is a cross-sectional data set that occurs every two years. We use data from 2010 to 2020. Each survey captures approximately 38,000 household members aged 13 or older in approximately 19,000 nationally representative sample households. The survey collection occurs from mid-May to late May. The Social Survey investigates individuals’ subjective perceptions about health, education, crime, safety, family, and living conditions. The survey’s purpose is to grasp the public’s social interests and subjective perceptions related to their quality of life. The data gathered from this survey are suitable for this study because they contain psychological well-being measures and demographic characteristics of participants. We restricted our sample to participants who were between 19 to 70 years old. Because testing whether the labor market impact of the pandemic is a major pathway influencing people’s psychological well-being is one of our main tasks, we exclude the elderly over 70 or those under the age of 19 from the sample. We analyze two dependent variables: life dissatisfaction and stress. Regarding the first variable, the Social Survey asked how satisfied an individual is with his or her life. There were five response items to the question: (1) very satisfied, (2) slightly satisfied, (3) neutral, (4) slightly dissatisfied, and (5) very dissatisfied. If a respondent chooses item (4) or (5), we judged that the respondent is dissatisfied with life and made a dummy variable of life dissatisfaction. Regarding the second variable, the survey asked how stressed an individual was in his or her daily life over the past two weeks. The response items are: (1) I felt very stressed, (2) I felt stressed, (3) I did not feel stressed, (4) I did not feel stressed, (5) I did not fee stressed at all. If a respondent selects item (1) or (2), we judged that the respondent was stressed at some point over the past two weeks. We use these transformed binary variables as dependent variables for convenience in the interpretation of estimation results. We, however, make it clear that even if the Likert scale itself is used as the dependent variable, the result that COVID-19 worsened people’s life satisfaction and stress is maintained. The main explanatory variable is a measure of the spread of COVID-19. Considering the substantial difference in the number of confirmed cases in relation to population size between Daegu and other provinces, we created a dichotomous variable equal to one if the province of residence is Daegu and the survey year is 2020 and zero if the province of residence is not Daegu or the survey year is not 2020. This measure was also used in a prior study that examined the effect of COVID-19 on employment in South Korea (Aum et al., 2020). All models control for age, gender, 8 dummies of education level (no education, elementary school, middle school, high school, 2-year college, 4-year college, master’s degree, doctor’s degree), 3 dummies of employment type (wage worker, self-employed, non-worker), 4 dummies of marital status (single, married, divorced, bereavement), 3 dummies of household income (high income, middle income, low income), province fixed effects, and year fixed effects. The Social Survey divides household income into eight categories and asks individuals in which category the household income falls. We classify these categories into three groups (low, middle, and high income groups) and analyze trends by income group. In subgroup analyses, the sociodemographic characteristic on which the classification was based is not controlled for. Table 1 shows summary statistics for all provinces, Daegu, and provinces other than Daegu. It also reports differences in the mean values between Daegu and other provinces and their t-test results. The cumulative number of confirmed cases per 1000 people on May 31 was 2.86 in Daegu and 0.09 in other provinces. This is a sizable difference. The psychological well-being measures, the proportion of individuals who are dissatisfied with life and feel stressed, are greater in Daegu than in other provinces both before and after COVID-19. However, the differences substantially increase after COVID-19. Except for the psychological well-being measures, summary statistics are calculated for all periods. In terms of sociodemographic characteristics, the proportion of men is smaller in Daegu than in other provinces. The mean age and the proportion of married people are similar in Daegu and in other provinces. The share of individuals with high levels of education (with college or higher education levels) is higher in Daegu. The proportion of high income households is lower in Daegu than in other provinces, while the share of low and middle income households is greater in Daegu. With respect to employment, the proportion of individuals who do not work is higher in Daegu than in other provinces, while the share of wage workers and self-employed individuals is lower in Daegu. Graphical Analysis In analyses that use the difference-in-differences approach, comparing trends of an outcome variable in treatment and control groups is important because the key identifying assumption of the difference-in-differences approach for causal inference is that the trends of the outcome variable before an event of interest are parallel. In this section, we graphically illustrate trends of the proportions of people with life dissatisfaction and stress in Daegu and other provinces. We also show trends of the proportions by sociodemographic characteristics in the treatment and control regions. Figure 2 provides strong visual evidence that individuals in Daegu have worse psychological well-being after the COVID-19 outbreak than individuals in other provinces. Panel (a) of Fig. 2 depicts the time trends of the proportion of people who are dissatisfied with life in Daegu and other provinces. The vertical line separates the periods before and after the COVID-19 pandemic. While the proportion decreased more rapidly in Daegu than in other provinces from 2014 to 2018, it suddenly increased more in Daegu in 2020. Panel (b) of Fig. 2 depicts the trends of the proportion of people who reported feeling stressed. The proportion of people who reported feeling stressed decreased more rapidly in Daegu than in other provinces from 2010 to 2018. In 2020, the proportion of individuals who reported feeling stressed declined in other regions, while the proportion substantially increased in Daegu. The time trends of the two psychological well-being outcomes measures consistently show that the psychological well-being of individuals in other provinces did not deteriorate significantly, while the psychological well-being of individuals in Daegu significantly worsened in 2020. We infer that the disproportionately large spread of COVID-19 in Daegu after the end of February 2020 drove the worsened psychological well-being of people in Daegu. The trends of the psychological well-being outcomes before 2020 are not parallel, but tend to improve more in Daegu than in other provinces. Without taking into account the different trends across provinces, we may underestimate the impact of COVID-19 on psychological well-being. We allow for differential trends of psychological well-being outcomes, as shown in equation (1). Another main objective of this study is to explore the heterogeneous impacts of COVID-19 on psychological well-being among individuals with different sociodemographic and socioeconomic backgrounds. To examine the heterogeneous impact of the spread of COVID-19 on psychological well-being across different subgroups, we first graphically compare the changes in the psychological well-being of people in Daegu and other provinces by sociodemographic characteristics. Panel (a) of Fig. 3 shows the time trend of the difference in the proportion of people who are dissatisfied with life between Daegu and other provinces by gender. The proportion of men and women who are dissatisfied with life increased significantly after the spread of COVID-19, but the magnitude of the increase is slightly larger for men. Panel (b) of Fig. 3 presents the time trend of the difference in the proportion of people who reported being dissatisfied with life between Daegu and other provinces by age group. It clearly shows that the proportion of people dissatisfied with life in Daegu increased significantly more than in other regions after COVID-19. Panel (c) of Fig. 3 shows the time trend of the difference in the proportion of people reporting stress between Daegu and other provinces by gender. The increase in the proportion of people reporting feeling stressed after COVID-19 is significantly greater for men than women. The proportion of men and women feeling stressed decreased faster in Daegu, but this proportion increased significantly quicker in Daegu after the spread of COVID-19. This trend holds for all age groups. As shown in Panel (d), the proportion of people feeling stressed increased more in Daegu after the onset of COVID-19 across all ages, with the exception of individuals 65 years and older. Figure 4 shows time trends of the difference in the psychological well-being measures between Daegu and other provinces by the following three socioeconomic status variables: education, household income, and employment type. Panels (a) and (d) of Fig. 4 present the time trends of the proportion of people who reported being dissatisfied with life and feeling stressed by education level. Notably, the psychological well-being of people with college or lower education in Daegu deteriorated more than that of individuals in other provinces. Psychological well-being improved more for people with a master’s degree or higher in Daegu than for similarly educated persons in other provinces. Panels (b) and (e) depict time trends of the difference in the psychological well-being measures by household income. The most interesting results are that the deterioration of psychological well-being due to COVID-19 is greatest in the middle income group, while psychological well-being improves in the high income group. Psychological well-being worsens slightly in the low-income group. Panel (c) shows the time trends of the difference in the proportion of people who are dissatisfied with life in Daegu and other provinces. Life dissatisfaction increased the most among self-employed individuals after the COVID-19 outbreak. It also increased for wage workers and individuals who do not work albeit the magnitude of the increase is less in each of these groups compared with the self-employed. Panel (f) of Fig. 4 shows the time trends of the difference in the proportion of people with stress between Daegu and other provinces. The difference in the proportion also increased the most for self-employed people after the spread of COVID-19 in 2020. The difference in the proportion between Daegu and other provinces for wage workers increased the second greatest, and the gap for non-workers increased by the smallest amount. Figures 3 and 4 show that individuals in most sociodemographic groups analyzed in this study who also lived in Daegu experienced greater deterioration in their psychological well-being after the outbreak of COVID-19 compared with individuals who lived in other provinces. On the other hand, the psychological well-being outcomes improve more in Daegu after the outbreak of COVID-19 for high socioeconomic groups, such as those with a master’s degree or higher and those with a high-income. Figures 3 and 4 also show that men, prime working age groups, and self-employed people experienced heightened deterioration of their psychological well-being. We infer that part of the deterioration in psychological well-being after the onset of COVID-19 is related to economic damages and the shrinking economic activities due to COVID-19. Motivated by these figures, we quantitatively analyze how the extensive spread of COVID-19 in Daegu affects the psychological well-being of people in Daegu and how the effect differs across sociodemographic groups. Empirical Strategy We analyze the effect of the COVID-19 outbreak on life satisfaction and stress by estimating the following difference-in-differences model:1 Yist=β0+β1TreatsPostt+Xistβ2+δs+λt+Trendst+ϵist where Yist is an outcome variable (life satisfaction or stress) of individual i in province s in year t. Treats is a dummy variable that indicates residence in Daegu metropolitan city, and Postt is a dummy variable that is equal to one if year t is 2020 and zero otherwise. Xist is a vector of sociodemographic variables/characteristics of individual i. δs and λt represent province fixed effects and year fixed effects, respectively. Trendst represents province-specific time trends. ϵist is an error term. The key parameter of interest in the model is β1, which is the effect of the COVID-19 outbreak on psychological well-being. In the difference-in-differences framework, this is estimated by comparing changes in life dissatisfaction and stress between persons in Daegu, where the coronavirus rapidly spread during the survey period, and persons in other provinces after controlling for individual characteristics. The key identifying assumption in the difference-in-differences estimation is that trends of the outcome variables in treatment and control groups are parallel. In models with province-specific times trends, it is required that trends of the outcome variables after partialling out the province-specific times trends are parallel. Our preferred model is a model with province-specific linear times trends because it may be more plausible to assume that time trends of the dependent variables are different across different provinces and quadratic trends may overfit in the data without long pre-periods. Since it is not known which model is the most appropriate, however, we report the estimation results for several models and analyze whether the results are robust to the models. Another important objective of this study is to analyze the heterogeneous impacts of COVID-19 on psychological well-being across different sociodemographic groups. For this analysis, we estimate the following model:2 Yist=γ0+γ1TreatsPostt+∑k=1mγ2kTreatsPosttGroupki+Xistγ3+ϕs+ζt+Trendst+νist Compared to equation (1), equation(2) adds on triple interaction terms, TreatsPosttGroupki. Groupki is a dummy variable that indicates a sociodemographic group k. If individual i belongs to sociodemographic group k, then Groupki is equal to one and zero otherwise. In the model, there are m+1 different groups based on sociodemographic characteristics. γ1 represents the effect of COVID-19 on psychological well-being for the base group. γ2k represents the additional effect of COVID-19 on psychological well-being for group k compared to the effect on the base group, which means γ1+γ2k is the effect of COVID-19 for group k. Results Table 2 provides difference-in-differences estimates of the impacts of COVID-19 on life dissatisfaction and stress. These are estimates of the coefficient β1 in equation (1). Panel (a) shows that the proportion of people who are dissatisfied with life in Daegu increases by 2.8–6.5 percentage points more than in other provinces after COVID-19. It also shows that the proportion of people reporting feeling stressed in Daegu increases by 5.8–8.9 percentage points after COVID-19 compared to other provinces. All estimation results consistently show that the psychological well-being of individuals in Daegu worsened significantly more than the psychological well-being of individuals in other provinces after 2020. The cause of the deterioration of psychological well-being among people in Daegu is possibly due to the prevalence of COVID-19 in Daegu as other provinces had few cases. Our preferred model, a model with province-specific linear time trends, shows that the ratios of people with life dissatisfaction and stress increase 4.0 and 8.5 percentage points more in Daegu after COVID-19, respectively. Considering the ratio of people with life dissatisfaction and stress were 15.3% and 57.0% in 2018 in the nation, the estimates can also be interpreted that COVID-19 increases the proportion of people with life dissatisfaction by 26.1% and that with stress by 14.9%. We believe that the magnitude of the effect is substantial. Panel (b) of Table 2 shows the results for dependent variables with Likert scale points. Column (1) shows the estimation results from the model that does not control for province-specific time trends. Columns (2) and (3) present results from models that control for linear and quadratic provincial time trends, respectively. Panel (a) shows that the Likert scores of life satisfaction and stress increases more in Daegu than in other provinces after COVID-19. The relative increase in the Likert score in Daegu after COVID-19 are 0.080–0.144 points for life satisfaction and 0.084–0.154 points for stress. Again, these show that the results are maintained qualitatively even when the Likert scale scores are used as the dependent variables. From now on, only the results for the dummy dependent variables are reported due to space constraints. Table 3 provides estimates of the impacts of COVID-19 on psychological well-being by gender and age. These are estimation results for equation (2). Columns (1)–(3) present results for life dissatisfaction by model specification and columns (4)–(6) show results for stress by specification. Panel (a) of Table 3 shows that the proportion of women who are dissatisfied with life increases 2.0–5.7 percentage points more in Daegu than in other provinces after COVID-19. The estimated coefficient of the interaction between the COVID-19 dummy and the male dummy shows that the proportion of men in Daegu who are dissatisfied with life increases 1.6 percentage points more than the proportion of women with life dissatisfaction in Daegu after COVID-19. These estimates are statistically significant at 0.1%. Columns (4)–(6) show that the proportion of women feeling stressed in Daegu increases 4.7-7.8 percentage points more than in other provinces. The impact is 2.1 percentage points higher for men than women. Results in Panel (a) of Table 3 consistently show that men experience greater psychological well-being problems related to COVID-19 than women. Panel (b) of Table 3 shows that the proportion of people aged 19–34 who report being dissatisfied with life increases 6.6-10.4 percentage points more than that in other provinces after COVID-19. The impact of COVID-19 on life dissatisfaction is smaller for older age groups. In addition, the extensive spread of COVID-19 in Daegu increases the proportion of people aged 19–34 who report being dissatisfied with life by 5.2–8.5 percentage points. The impact is 2.4 percentage points greater for people aged 35–49, and it is 9.2 percentage points smaller for people 65 years or older. Results in Table 4 show estimates of the impact of COVID-19 on psychological well-being by education, household income, and employment type. Columns (1)–(3) in Panel (a) of Table 4 show that the proportion of people with middle school or lower education who are dissatisfied with life increases by 0.3–4.0 percentage points more in Daegu than in other provinces after COVID-19. The impact of COVID-19 on life dissatisfaction is statistically significant when regional time trends are controlled for. Compared to people with middle school or lower education, the magnitude of the increase is 2.7 percentage points greater for high school graduates and 3.1 percentage points greater for college graduates. There is no statistically significant difference in the magnitude of the increase between middle school graduates and people with master’s degree or higher education. Columns (4)–(6) show that the proportion of people who report feeling stressed among those with middle school or lower education increases 0.7–3.8 percentage points more in Daegu than in other provinces. The impact of COVID-19 on the probability of feeling stressed is 7.1 percentage points higher for high school graduates and 7.2 percentage points higher for college graduates than people with middle school or lower education. On the other hand, the effect of COVID-19 is 11.6 percentage points lower for people with master’s degree or higher education than people with middle school or lower education. Panel (b) presents estimates of the impact of COVID-19 on psychological well-being by household income. The estimated impact of COVID-19 on the proportion of people with life dissatisfaction in low-income households ranges from −0.2 to 3.6 percentage points, as shown in columns (1)–(3). In the model without province-specific time trends and that with province-specific linear time trends, the estimated impact is not statistically significant. On the other hand, the spread of COVID-19 in Daegu increases the proportion of people who report being dissatisfied with life in middle-income households by 5.3–9.1 percentage points. These estimates are statistically significant at 0.1%. The impact of COVID-19 on the proportion of people who are dissatisfied with life in high-income households is not statistically significantly different from that in low-income households. Columns (4)–(6) show results for stress. The widespread diffusion of COVID-19 in Daegu increases the proportion of people with stress in low-income households by 3.4–6.7 percentage points. The magnitude of the impact is 8.3–8.4 percentage points for people in middle-income households. On the other hand, the proportion of people who report feeling stressed in high-income households decreases by 3.0–6.3 percentage points in Daegu after COVID-19. Results of the pandemic’s impact of on psychological well-being by employment type are reported in Panel (c) of Table 4. Columns (1)–(3) show that the proportion of non-workers who are dissatisfied with life increases by 0.4–4.2 percentage points more in Daegu after the onset of the COVID-19 pandemic than in other provinces. The proportion of wage workers with life dissatisfaction increases by 2.3–6.1 percentage points more in Daegu than in other provinces. The impact of COVID-19 on life dissatisfaction is the greatest for the self-employed. The proportion of self-employed people who are dissatisfied with life increases by 11.2–15.0 percentage points more in Daegu than in other provinces. Columns (4)–(6) show the estimation results for stress. The proportion of non- workers who reported feeling stressed increases 0.8–3.9 percentage points more in Daegu than in other provinces after COVID-19. The amount of the increase is 7.7 percentage points greater for wage workers and 10.9 percentage points higher for self-employed people than non-workers. The estimation results consistently show that the psychological well-being of self-employed people in Daegu deteriorated the most after the COVID-19 outbreak. Discussion and Conclusion Although previous studies document that the COVID-19 pandemic has had psychological consequences worldwide (Rajkumar, 2020), less is known about whether the psychological impact of the pandemic has been even across sociodemographic groups. Because the pandemic has persisted for an extended period, examining the heterogeneous effects of the pandemic is critical to better understand the pandemic’s consequences. This intersection of understanding can also inform policymakers about how to address the psychological well-being problems brought on by the pandemic more effectively. In this study, we investigated how the COVID-19 outbreak impacted life satisfaction and stress in South Korea, and whether these effects differ depending on sociodemographic characteristics including gender, age, education, income, and employment type. This article found that the initial surge of COVID-19 cases negatively affected psychological well-being. We found that the proportion of people dissatisfied with life increased by 2.8–6.5 percentage points more in Daegu. Daegu is a place that experienced a more dramatic diffusion of COVID-19 compared with other provinces after the initial COVID-19outbreak. Similarly, the proportion of people with stress increased more in Daegu than in other provinces by 5.8–8.9 percentage points. The negative impact on psychological well-being is significantly greater for men, young adults, middle-aged adults, self-employed people, and middle-income people. On the other hand, the highest-educated (a master’s degree or higher) and high-income people reported feeling less stressed than other groups after the initial surge of the pandemic. In this study, we found interesting patterns about the heterogeneous effects of the pandemic on psychological well-being. First, this study found that men got more stressed and dissatisfied with their lives. This is inconsistent with previous research that show that women’s psychological well-being is more affected by the pandemic than men’s psychological well-being (Mooi-Reci & Risman, 2021). These results in this study may indicate that the psychological well-being consequences of the pandemic may be largely driven by economic shock and insecurity. Unlike other countries, men’s employment declined significantly more than women’s in South Korea in the early days of COVID-19 (Aum et al., 2020). Despite an increase in the number of dual-earner families in Korea, men are traditionally expected to take primary responsibility of financially supporting their family (Park & Chesla, 2007). Thus, compared to women, men may perceive economic uncertainty as a more serious threat, and this may lead to lower psychological health among men. We also found that pandemic most severely affected the psychological well-being of the youngest group (ages 19–34) in this study. Interestingly, while the pandemic’s effect on life satisfaction is greater in the younger population, its impact on stress is greater in middle-aged individuals, especially those between 35-49 years old. These contrasting results provide support for potentially heterogeneous pathways through which the pandemic shapes psychological well-being for different age groups. During the pandemic, young people may perceive the future more pessimistically, resulting in lower levels of life satisfaction. Given that life satisfaction captures how one feels about their future directions and options (rather than an assessment of current feelings), it is possible that concerns about the future such as concerns about romantic relationships or marriage as well as employment may negatively affect life satisfaction among young people (Kim & Kim, 2021; Pierce et al., 2020). Among middle aged persons, the increased burden of childcare during the pandemic, a family stressor, may be more salient for their mental wellbeing (Buecker et al., 2020). Interestingly, the pandemic has no or little discernable impact on the psychological well-being of older adults (aged 65+) in this study. This is consistent with a previous study that found that, compared to younger and middle-aged adults, older adults’ mental health is more resilient as they are less concerned with harm to their physical health and safety as well as emotional well-being due possibly to higher levels of coping efficacy (Klaiber et al., 2021). Our findings on the heterogeneous effects of COVID-19 across different SES groups suggest that the pandemic has the strongest impact on the psychological well-being of high school-/college-educated and middle-income individuals. While the pandemic appears to have a negative impact on life satisfaction for all SES groups, it negatively affects stress for low and middle-SES groups, but not the highest SES group (i.e., master’s degree or higher and high-income group). Since the onset of the COVID-19 pandemic, working from home has increased due to the risk of infection. Considering that the jobs in which working from home is possible are typically high-paying jobs (Dingel & Neiman, 2020), high-income individuals may have lower stress related to work. The highest-SES groups have a lower risk of losing jobs (Aum et al., 2020). They may also be able to utilize their social and financial resources to successfully mitigate stress, an acute threat to psychological well-being during the pandemic (Daly et al., 2020). That said, when it comes to life satisfaction, the pandemic appears to change one’s fundamental attitudes towards one’s life, regardless of SES. Findings of this study also indicate that the pandemic most severely negatively affects the psychological well-being of self-employed workers, followed by wage workers and then unemployed individuals. Results lend support to the claim that economic hardships and uncertainty led by the pandemic may be the major driver of heterogeneous effects of the pandemic by employment type. Although both unemployed individuals and wage workers have been affected by the pandemic-related economic downturn and are worried about future economic ramifications of the pandemic (Monitor, 2020), self-employed workers have been hardest hit by the pandemic (Fana et al., 2020; Wolfe & Patel, 2021). In particular, despite their effectiveness, social distancing measures that forced night curfews and business suspensions play a major role in business disruptions (Koren & Pető 2020). Their downstream consequences in turn negatively affect the psychological well-being of self-employed individuals. This study contributes to a growing body of literature about how the COVID-19 pandemic affects psychological wellbeing. This study estimates the causal effect of COVID-19 on psychological well-being by taking advantage of the fact that the transmission of the virus was concentrated only in one area at the beginning of the COVID-19 pandemic in South Korea. Our study provides a comprehensive account of the heterogeneous effects of the pandemic on life satisfaction and stress with a focus on differences by gender, age, education, income, and employment type. To the authors’ knowledge, this is the first study to investigate multiple sources of heterogeneous impacts in a single study. Our findings have important policy implications. Despite the creation and implementation of several interventions designed and implemented to improve people’s psychological well-being during the pandemic, policymakers did not consider potential heterogeneity in the psychological costs of the pandemic when developing such interventions. Our findings may guide policymakers and practitioners to tailor intervention programs to help relieve psychological distress for individuals in different sociodemographic groups. For example, given the prolonged pandemic and potential habituation of social distancing, long-term and practical policies are needed to promote the psychological well-being of self-employed individuals. Providing self-employed individuals with temporary financial support as a means to compensate for income loss may not be a long-term solution for their recovery from psychological downturns. Moreover, despite evidence of heterogeneous effects, our findings document that the pandemic has adversely affected the psychological well-being of everyone. Therefore, policymakers should continue to consider policy interventions that benefit the psychological well-being of all individuals, irrespective of their sociodemographic characteristics. This study has limitations. First, we only investigate the short-term effects of the COVID-19 pandemic in the early stages of it due to data limitation. It may be meaningful for subsequesnt research to analyze the longer-term effects of COVID-19 on life satisfaction and stress and how the effects dynamically change during the pandemic. Second, we did not quantitatively evaluate relative importantce of each mechanisum through which COVID-19 affects life satisfaction and stress, while we estimate the net effect of COVID-19. Future research that quantitatively evaluates the relative importance of each mechanism, such as mediation analysis, needs to be performed. Third, our measures of psychological well-being are limited. It may be necessary for future research to conduct a more detailed analysis using multidimensional measures of well-being.Fig. 1 Time trends of the number of confirmed cases per 1000 people by region: January 2020–May 2020 Fig. 2 Trend of psychological well-being by region: May 2010–May 2020 Fig. 3 Trend of the difference in psychological well-being between Daegu and Other Provinces by sociodemographic group: May 2010–May 2020 Fig. 4 Trend of the difference in psychological well-being between Daegu and Other Provinces by socio-economic status: May 2010–May 2020 Table 1 Summary statistics (1) (2) (3) (4) All Daegu (treatment) Other provinces (control) Difference Spread of COVID-19 The number of confirmed cases per 1000 people on May 31 0.22 2.86 0.09 2.77 Mental health Ratio of people with life 0.168 0.185 0.167 0.017* dissatisfaction before COVID-19 (0.374) (0.388) (0.373) (0.004) Ratio of people with life 0.123 0.162 0.121 0.041* dissatisfaction after COVID-19 (0.329) (0.369) (0.327) (0.009) Ratio of people with stress 0.619 0.626 0.618 0.008 before COVID-19 (0.486) (0.484) (0.486) (0.006) Ratio of people with stress 0.534 0.586 0.531 0.055* before COVID-19 (0.499) (0.493) (0.499) (0.014) Sociodemographics Gender(Male=1) 0.497 0.484 0.498 −0.014 (0.500) (0.500) (0.500) (0.005) Age 44.6 44.70 44.62 0.083 (13.84) (13.96) (13.84) (0.147) Marriage(Married=1) 0.652 0.647 0.652 -0.006 (0.476) (0.478) (0.476) (0.005) Education level Middle school or lower 0.155 0.162 0.154 0.007 (0.361) (0.368) (0.361) (0.004) High school 0.324 0.300 0.325 −0.025* (0.468) (0.458) (0.468) (0.005) College 0.469 0.489 0.467 0.022* (0.499) (0.500) (0.499) (0.005) Master or higher 0.053 0.049 0.054 −0.005 (0.225) (0.216) (0.225) (0.002) Household income Low income 0.260 0.283 0.258 0.025* (0.438) (0.451) (0.438) (0.005) Middle income 0.521 0.538 0.521 0.017 (0.500) (0.499) (0.500) (0.005) High income 0.219 0.179 0.221 −0.042* (0.414) (0.384) (0.415) (0.004) Employment type Wage worker 0.475 0.449 0.476 −0.027* (0.499) (0.497) (0.499) (0.005) Self-employed 0.177 0.154 0.178 −0.024* (0.382) (0.361) (0.383) (0.004) Not working 0.348 0.397 0.346 0.051* (0.476) (0.489) (0.476) (0.005) Observations 178,796 10,306 168,490 178,796 1. Standard deviations are in parenthesis. 2. *p<0.001; p<0.01, p<0.05 Table 2 The effects of COVID-19 on psychological well-being (1) (2) (3) Observations (a) Dichotomous variable Life dissatisfaction 0.028 0.040* 0.065* 178,796 (0.004) (0.006) (0.009) Stress 0.058* 0.085* 0.089* 178,796 (0.009) (0.008) (0.012) (b) Likert scale values Life dissatisfaction 0.080* 0.085* 0.144* 178,796 (0.019) (0.025) (0.027) Stress 0.084* 0.127* 0.154* 178,796 (0.010) (0.011) (0.019) Province-specific time trend None Linear Quadratic 1. *p<0.001; p<0.01; p<0.05. 2. Standard errors are clustered at the province-level. 3. All models commonly control age, a dummy of gender, dummies of education level, dummies of job status, dummies of marital status, dummies of household income, province fixed effects, and year fixed effects Table 3 The effects of COVID-19 on psychological well-being by gender and age (1) (2) (3) (4) (5) (6) Life dissatisfaction Stress (a) Gender COVID-19 0.020 0.032* 0.057* 0.047* 0.074* 0.078* (Base=female) (0.004) (0.005) (0.009) (0.008) (0.008) (0.013) COVID-19×Male 0.016* 0.016* 0.016* 0.021* 0.021* 0.021* (0.002) (0.002) (0.002) (0.004) (0.004) (0.004) (b) Age COVID-19 0.066* 0.078* 0.104* 0.052* 0.079* 0.085* (Base=ages 19–34) (0.005) (0.006) (0.009) (0.010) (0.009) (0.010) COVID-19×Ages 35–49 −0.038* −0.038* −0.038* 0.024* 0.024* 0.024* (0.001) (0.001) (0.001) (0.006) (0.006) (0.006) COVID-19×Ages 50–64 −0.061* −0.062* −0.062* 0.019 0.019 0.019 (0.004) (0.004) (0.004) (0.013) (0.013) (0.013) COVID-19×Ages 65+ −0.070* −0.070* −0.070* −0.092* −0.092* −0.092* (0.003) (0.003) (0.003) (0.013) (0.013) (0.013) Observations 178,796 178,796 178,796 178,796 178,796 178,796 Regional time trend None Linear Quadratic None Linear Quadratic 1. p<0.001; p<0.01; p<0.05. 2. Standard errors are clustered at the province-level. 3. All models commonly control age, a dummy of gender, dummies of education level, dummies of job status, dummies of marital status, dummies of household income, province fixed effects, and year fixed effects Table 4 The effects of COVID-19 on psychological well-being by socioeconomic status (1) (2) (3) (4) (5) (6) Life dissatisfaction Stress (a) Education COVID-19 0.003 0.015 0.040* 0.007 0.034 0.038 (Base=Middle school or lower) (0.007) (0.007) (0.013) (0.013) (0.012) (0.015) COVID-19×High school 0.027* 0.027* 0.027* 0.071* 0.071* 0.071* (0.008) (0.008) (0.008) (0.006) (0.006) (0.006) COVID-19×College 0.031* 0.031* 0.031* 0.072* 0.072* 0.072* (0.008) (0.008) (0.008) (0.009) (0.009) (0.009) COVID-19×Master or higher 0.008 0.008 0.008 −0.116* −0.116* −0.116* (0.013) (0.013) (0.013) (0.009) (0.009) (0.009) (b) Household income COVID-19 −0.002 0.010 0.036* 0.034 0.062* 0.067* (Base=low income) (0.004) (0.006) (0.008) (0.011) (0.011) (0.014) COVID-19×Middle income 0.055* 0.055* 0.055* 0.084* 0.084* 0.083* (0.005) (0.005) (0.005) (0.006) (0.006) (0.006) COVID-19×High income 0.002 0.002 0.002 −0.097* −0.097* −0.097* (0.005) (0.005) (0.005) (0.010) (0.010) (0.010) (c) Employment type COVID-19 0.004 0.016 0.042* 0.008 0.035* 0.039* (Base=Non-worker) (0.004) (0.006) (0.008) (0.009) (0.009) (0.013) COVID-19×Wage worker 0.019* 0.019* 0.019* 0.077* 0.077* 0.077* (0.002) (0.002) (0.002) (0.007) (0.007) (0.007) COVID-19×Self-employed 0.108* 0.108* 0.108* 0.109* 0.109* 0.109* (0.006) (0.005) (0.005) (0.007) (0.007) (0.007) Observations 178,796 178,796 178,796 178,796 178,796 178,796 Regional time trend None Linear Quadratic None Linear Quadratic 1. *p<0.001; p<0.01, *p<0.05. 2. Standard errors are clustered at the province-level. 3. All models commonly control age, a dummy of gender, dummies of education level, dummies of job status, dummies of marital status, dummies of household income, province fixed effects, and year fixed effects Declarations Conflicts of interest The authors declare that they have no confilict of interest. Informed Consent This research did not involve human participants. Human or Animal Rights This research did not involve human or participants or animals, and did not require research ethics approval. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Albanesi, S., & Kim, J. (2021). The gendered impact of the covid-19 recession on the us labor market (Tech. Rep.). National Bureau of Economic Research. Alon, T. , Coskun, S. , Doepke, M. , Koll, D., & Tertilt, M. (2021). 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==== Front Neurocrit Care Neurocrit Care Neurocritical Care 1541-6933 1556-0961 Springer US New York 36450970 1641 10.1007/s12028-022-01641-w Original Work Do Cerebral Cortex Perfusion, Oxygen Delivery, and Oxygen Saturation Responses Measured by Near-Infrared Spectroscopy Differ Between Patients Who Fail or Succeed in a Spontaneous Breathing Trial? A Prospective Observational Study http://orcid.org/0000-0001-8661-8546 Louvaris Zafeiris [email protected] 12 Van Hollebeke Marine 12 Poddighe Diego 12 Meersseman Philippe 12 Wauters Joost 34 Wilmer Alexander 34 Gosselink Rik 12 Langer Daniel 12 Hermans Greet 35 1 grid.5596.f 0000 0001 0668 7884 Research Group for Rehabilitation in Internal Disorders, Department of Rehabilitation Sciences, Faculty of Movement and Rehabilitation Sciences, Katholieke University Leuven, Campus Gasthuisberg O&N4, Herestraat 49, Box 1510, B-3000 Leuven, Belgium 2 grid.410569.f 0000 0004 0626 3338 Department of Intensive Care Medicine, University Hospitals Leuven, Leuven, Belgium 3 grid.410569.f 0000 0004 0626 3338 Medical Intensive Care Unit, Department of General Internal Medicine, University Hospitals Leuven, Leuven, Belgium 4 grid.410569.f 0000 0004 0626 3338 Laboratory for Clinical Infectious and Inflammatory Disorders, Department of Microbiology and Immunology, University Hospitals Leuven, Leuven, Belgium 5 grid.5596.f 0000 0001 0668 7884 Laboratory of Intensive Care Medicine, Division of Cellular and Molecular Medicine, Katholieke University Leuven, Leuven, Belgium 30 11 2022 113 20 2 2022 7 11 2022 © Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background Alterations in perfusion to the brain during the transition from mechanical ventilation (MV) to a spontaneous breathing trial (SBT) remain poorly understood. The aim of the study was to determine whether changes in cerebral cortex perfusion, oxygen delivery (DO2), and oxygen saturation (%StiO2) during the transition from MV to an SBT differ between patients who succeed or fail an SBT. Methods This was a single-center prospective observational study conducted in a 16-bed medical intensive care unit of the University Hospital Leuven, Belgium. Measurements were performed in 24 patients receiving MV immediately before and at the end of a 30-min SBT. Blood flow index (BFI), DO2, and %StiO2 in the prefrontal cortex, scalene, rectus abdominis, and thenar muscle were simultaneously assessed by near-infrared spectroscopy using the tracer indocyanine green dye. Cardiac output, arterial blood gases, and systemic oxygenation were also recorded. Results During the SBT, prefrontal cortex BFI and DO2 responses did not differ between SBT-failure and SBT-success groups (p > 0.05). However, prefrontal cortex %StiO2 decreased in six of eight patients (75%) in the SBT-failure group (median [interquartile range 25–75%]: MV = 57.2% [49.1–61.7] vs. SBT = 51.0% [41.5–62.5]) compared to 3 of 16 patients (19%) in the SBT-success group (median [interquartile range 25–75%]: MV = 65.0% [58.6–68.5] vs. SBT = 65.1% [59.5–71.1]), resulting in a significant differential %StiO2 response between groups (p = 0.031). Similarly, a significant differential response in thenar muscle %StiO2 (p = 0.018) was observed between groups. A receiver operating characteristic analysis identified a decrease in prefrontal cortex %StiO2 > 1.6% during the SBT as an optimal cutoff, with a sensitivity of 94% and a specificity of 75% to predict SBT failure and an area under the curve of 0.79 (95% CI: 0.55–1.00). Cardiac output, systemic oxygenation, scalene, and rectus abdominis BFI, DO2, and %StiO2 responses did not differ between groups (p > 0.05); however, during the SBT, a significant positive association in prefrontal cortex BFI and partial pressure of arterial carbon dioxide was observed only in the SBT-success group (SBT success: Spearman’s ρ = 0.728, p = 0.002 vs. SBT failure: ρ = 0.048, p = 0.934). Conclusions This study demonstrated a reduced differential response in prefrontal cortex %StiO2 in the SBT-failure group compared with the SBT-success group possibly due to the insufficient increase in prefrontal cortex perfusion in SBT-failure patients. A > 1.6% drop in prefrontal cortex %StiO2 during SBT was sensitive in predicting SBT failure. Further research is needed to validate these findings in a larger population and to evaluate whether cerebral cortex %StiO2 measurements by near-infrared spectroscopy can assist in the decision-making process on liberation from MV. Keywords Brain Blood flow index Near-infrared spectroscopy Respiratory muscles Weaning failure Ventilator Spontaneous breathing trial FWO12U5618N 180512N G053721N G053721N Louvaris Zafeiris Langer Daniel Hermans Greet ==== Body pmcIntroduction For the majority of patients receiving mechanical ventilation (MV), weaning can be accomplished during the first liberation attempt; however, this is not the case for 15–30% of MV patients [1, 2]. Reducing the length of time patients are on MV decreases the risk of ventilator-associated complications, including respiratory and locomotor muscle weakness, infections, and mortality [3–7]. On the contrary, failure rates between 10 and 30% have been described during early discontinuation from MV [8–12]. Reintubated patients have a higher incidence of hospital mortality, increased length of intensive care unit (ICU) and hospital stay, prolonged duration of MV, and an increased need for tracheostomy [13]. Studies to identify parameters to predict weaning outcomes have demonstrated that several parameters, and not only respiratory ones, should be taken into account [8]. Energy supply to the respiratory muscles plays a critical role during the weaning process [14]. Elegant studies in animal models have shown a redistribution of blood flow away from the brain during spontaneous breathing to maintain an adequate energy supply to the respiratory muscles [15, 16]. More recently, studies in humans demonstrated that patients who fail a spontaneous breathing trial (SBT) can exhibit a reduction in perfusion in their splanchnic area and peripheral nonworking muscles that may be redistributed to the respiratory muscles [17, 18]. Nevertheless, alterations in perfusion to the brain during the transition from MV to SBT are still poorly understood in weaning patients. Accordingly, this study aimed to assess changes in cerebral cortex perfusion, oxygen delivery (DO2), and tissue (i.e., cerebral cortex) oxygen saturation (%StiO2) during the transition from MV to SBT to determine whether these changes differ between patients who fail and patients who succeed an SBT. To address the aim of the study, we used near-infrared spectroscopy (NIRS) in conjunction with the injections of indocyanine green dye (ICG) [18–21] and simultaneously assessed local hemodynamic and tissue %StiO2 responses of the cerebral cortex (prefrontal cortex area), respiratory muscles, and nonworking muscles in consecutive weaning patients during MV and SBT. We hypothesize that SBT-failure patients would exhibit reduced cerebral cortex perfusion during the transition from MV to SBT compared with SBT-success patients. Methods Ethics All procedures were performed in accordance with the ethical standards of the institutional review board of Universitair Ziekenhuis/Katholieke University Leuven and with the 1964 Helsinki Declaration and its later amendments, which have been described in the published protocol [23]. Ethical approval was obtained from the responsible local ethical committee (Ethische Commissie Onderzoek UZ/KU Leuven protocol ID: S60516), and the study was registered with ClinicalTrials.gov (identifier NCT03240263). Written informed consent was obtained from all patients. On the day of measurement, oral consent was allowed (i.e., nodding or shaking the head) only for patients who were unable to sign written consent because of physical weakness. This procedure was always performed in the presence of a witness (i.e., a nurse or doctor). Written consent was obtained afterward as soon as possible. Study Design and Participants This single-center prospective observational study was conducted in a 16-bed medical ICU of the University Hospital Leuven, Belgium, in 24 weaning patients between January 2019 and March 2020. Unconscious patients and patients unable to comprehend the study information were excluded from the study [23]. The inclusion criterion was patients on MV for more than 48 h who underwent their first SBT. Exclusion criteria were as follows: patients with acute or past brain injury (given concerns for motor deficits and brain swelling because the latter could affect the NIRS signal), preexisting neuromuscular disease, spinal cord injury above T8, skeletal pathology, severe kyphoscoliosis, congenital deformities, and contractures (given concerns for severe impairments in chest wall movement). Furthermore, we excluded patients with liver cirrhosis and chronic renal failure or with allergic reactions to iodine (given concerns for contradictions to ICG injections); patients with edema, trauma, or hematoma skin lesions at the sites of NIRS measurements (could hinder the placement of NIRS sensor probes); and patients with poor general prognosis or anticipated fatal outcomes [23]. This study complies with the Strengthening the Reporting of Observational Studies in Epidemiology statement. Protocol Measurements were performed during two conditions on the day clinicians judged that the patients were ready to wean: (1) on MV, immediately preceding the planned SBT, for a period of 30 min and (2) during a 30-min SBT (Fig. 1). A readiness-to-wean assessment was performed according to a local protocol [23]. The SBT in all patients was performed with either low (≤ 8 cm H2O) or zero level pressure support ventilation and continuous positive airway pressure (≤ 5 cm H2O). The mode of MV that was used prior to the SBT was continuous positive airway pressure with pressure support for 22 patients and bilevel positive airway pressure for two patients.Fig. 1 The timing of the different measures during MV and SBT. Black arrows indicate data collected at specific time points during MV and SBT. Dotted arrows indicate data recorded continuously during MV and SBT. CO cardiac output, FiO2 inspiratory oxygen fraction, Hb hemoglobin, HR heart rate, MAP mean arterial pressure, MV mechanical ventilation, PaCO2 partial pressure of arterial carbon dioxide, PaO2 partial pressure of arterial oxygen, PEEP positive end-expiratory pressure, pH hydrogen ion concentration, PS pressure support, P0.1 occlusion pressure at 100 milliseconds as an indicator of neuromuscular activation index of the respiratory system, RR respiratory rate, SaO2 arterial oxygen saturation, SBT spontaneous breathing trial, SV stroke volume, VE minute ventilation, VT tidal volume, %StiO2 fractional oxygen saturation Readiness-to-Wean Criteria Readiness-to-wean criteria included the assessment of the following: (1) resolution of the acute phase of the disease for which the patient was intubated, (2) adequate oxygenation (partial pressure arterial oxygen/inspiratory oxygen fraction [PaO2/FIO2] of 150–200 (P/F Ratio) requiring positive end-expiratory pressure [PEEP] ≤ 8 cm H2O and FIO2 ≤ 0.5), (3) absence of fever (temperature < 38 °C), (4) hemodynamic stability (e.g., heart rate ≤ 140 bpm), (5) stable blood pressure and no or minimal vasopressors (dobutamine ≤ 5 μg/kg/minute, norepinephrine ≤ 0.1 µg/kg/minute), (6) absence of myocardial ischemia, (7) adequate hemoglobin (e.g., hemoglobin level > 7–8 g/dL), (8) adequate mentation, and (9) adequate cough [23]. Evaluation of SBT Outcomes Criteria assessed for evaluating the success or failure of the SBT included all of the following: (1) adequate gas exchange (Peripheral capillary oxygen saturation [SpO2] ≥ 85–90%, PaO2 ≥ 55–60 mm Hg, pH ≥ 7.32, and increase in partial pressure of arterial carbon dioxide [PaCO2] ≤ 10 mm Hg), (2) adequate ventilatory pattern (respiratory rate ≤ 30–35/minute, change in respiratory rate during SBT < 50%), (3) hemodynamically stable (heart rate < 120–140 bpm, changes in heart rate during SBT < 20%, systolic blood pressure < 180–200 mm Hg and > 90 mm Hg, and change in blood pressure during SBT < 20%), and (4) subjective clinical signs (no changes in mental well-being and comfortable, no sweating, no paradoxical breathing). The decision to extubate the patient was made by the treating clinician, independent of the investigators and blinded to measurements performed. Hemodynamic Status Assessment To assess whether any local changes observed might reflect changes in global hemodynamics, we assessed stroke volume, heart rate, and cardiac output continuously by pulse contour analysis using a specific sensor (Pulsioflex Monitor, Pulsion Medical Systems SE) [24, 25]. The device has been validated against clinical gold standard methods, and the results suggest that the pulse contour analysis method provides clinically acceptable cardiac output trend assessment, particularly in hemodynamically stable patients [26–29]. The calculation of cardiac output was performed five times per minute (every 12 s). Cardiac output data were exported in document file format and stored on disk for off-line analysis. Cardiac output and heart rate data were averaged over 60 s and were aligned with respiratory and tissues hemodynamic and oxygen saturation responses. Ventilator Settings and Respiratory Parameters Monitoring of the patients on MV before, during, and following the SBT was facilitated by using an electronic recording platform (MetaVision, iMD-Soft, Needham, MA) [30]. This platform can store information on patient demographics, anthropometric and clinical characteristics, and blood gas values measured by arterial blood gas analysis completed by the clinical team. The platform is also connected with the mechanical ventilator; thus, it continuously records and stores ventilatory settings data, such as pressure support and PEEP, as well as respiratory parameters, such as respiratory rate, tidal volume, minute ventilation, and FIO2 [30]. P0.1, which is the occlusion pressure at 100 milliseconds, was measured as an indicator of the neuromuscular activation index of the respiratory system [31]. P0.1 was measured in awake patients on MV immediately before the start of the SBT and at the end of the 30-min SBT. Tissue Blood Flow Index andDO2 Assessments by NIRS Cerebral cortex and respiratory and peripheral muscle blood flow indices (BFIs) were measured by a methodology validated in humans and various clinical populations [19–22, 32] using NIRS (Hamamatsu Photonics KK) in combination with injections of the tracer ICG (NIRS-ICG) [23]. Each injection included a dose of ICG of 5 mg diluted in 1 mL of saline, followed by a rapid 10-mL flush of isotonic saline [23]. The sets of NIRS optodes (light transmitter and emitter) were transcutaneously positioned as follows: for the brain over the prefrontal cortex area at an adequate distance to avoid interference with the midline sinus, for the scalene muscles on the left or right posterior triangle of the neck, and for the upper rectus abdominis on upper right third of rectus abdominis below the costal cartilage. The fourth set of NIRS optodes was placed on the thenar eminence muscle to represent a nonworking muscle site, as previously described [23]. Path lengths of 24.0 cm for the cerebral cortex and 18.0 cm for both respiratory and thenar muscles were set up according to the manufacturer’s recommendations. The separation distance between the NIRS light transmitter and receiver probes was 40 mm, thus allowing a maximum NIRS penetration depth of 20 mm. ICG concentration curve data were exported by NIRS to a document file format and stored on disk for off-line analysis. ICG concentration curves were analyzed by using the Chart5 version 5.4.2 (ADInstruments) program. A low-pass filter with a cutoff frequency of 0.5 Hz and smoothing window width (by using the triangular Bartlett window function) of nine points was used to smooth the ICG curves for BFI calculation. NIRS data were sampled at 6 Hz. Tissue %StiO2 Assessment by NIRS Continuous measurements of tissue %StiO2 were also performed by NIRS. NIRS-derived %StiO2 is a real-time and rapidly responsive absolute index of local tissue %StiO2 that is calculated by the ratio of microvascular oxygenated hemoglobin concentration (HbO2) to total hemoglobin concentration (tHb) and is expressed in percentage [(HbO2/tHb) × 100] [32]. %StiO2 reflects the dynamic balance between local tissue DO2 and consumption and therefore the capacity of tissue to match oxygen supplies relative to its metabolic demands [33]. Because of high ICG tissue concentrations, the passage of the dye bolus through the tissue may interfere with hemoglobin signals; thus, %StiO2 data (sampled at 6 Hz) were averaged over 15 s immediately before the ICG injection and were aligned with hemodynamic and respiratory responses. Calculations Systemic arterial O2 (SaO2) content was calculated using the following formula: [(1.34 × hemoglobin level × SaO2) + (0.003 × PaO2)]. Systemic DO2 was calculated by multiplying cardiac output and SaO2content. All blood gases were measured immediately before and after the SBT by arterial blood gas analysis. Tissue DO2 was calculated by multiplying tissue BFI and SaO2 content and was presented in arbitrary units (nanomole/second × milliliterO2). The primary outcome of the study was the difference in change in cerebral cortex BFI during the transition from MV to SBT between SBT-success and SBT-failure patients. Secondary outcomes include the differences in change in respiratory and thenar muscle BFI, change in cerebral cortex and respiratory and thenar muscle %StiO2, ventilator settings, and change in breathing pattern parameters, hemodynamic parameters, and blood gas parameters [23]. Statistical Analysis Categorical values are presented as numbers and proportions and compared using the χ2 test. Continuous data are expressed as median and interquartile range. The normality of the data was examined by the Shapiro–Wilk test. There were no missing data in this study. Within-group (before and after SBT) and between-group (SBT success and SBT failure) comparisons were performed by paired t-tests or Wilcoxon signed-rank tests and unpaired t-tests or Mann–Whitney U-tests, respectively, according to the distribution of the data. A mixed-model analysis of variance was applied to examine whether groups (i.e., SBT success and SBT failure) responded differently during MV versus at the end of the SBT for all the aforementioned physiological variables. Because PaCO2 serves as one of the fundamental regulators of cerebral blood flow, Spearman's rank correlation was employed to determine the strength and direction of this relationship between the SBT-success and SBT-failure groups. Furthermore, if any of the cerebral cortex NIRS variables revealed a differential response between the SBT-success and SBT-failure groups, further exploratory analyses would involve the determination of the optimal cutoff, sensitivity, and specificity with the use of a receiver operating characteristic (ROC) curve. Specifically, an ROC curve was used to examine the diagnostic accuracy of the prefrontal cerebral %StiO2 to discriminate SBT failure from SBT success [34]. The Youden index method (i.e., the maximum of vertical distance of the ROC curve from the point [x, y] on the diagonal line [chance line]) was used to determine the optimal cutoff value of the prefrontal cerebral %StiO2 [35]. Statistical significance was defined as p < 0.05 (two-sided). Data were analyzed using the GraphPad Prism software. Sample Size Calculation The initial sample size of the study was estimated to be 20 SBT-failure patients [23], which was based on an expected effect size (Cohen’s d) of 0.467. This effect size was calculated from the mean difference of cerebral cortex BFI (i.e., 6.70 nmol/second) and the corresponding pooled standard deviation (i.e., 14.0 nmol/second), from a previous study investigating interhemispheric differences in cerebral cortex BFI in critically ill patients [20]. However, because of the outbreak of the COVID-19 pandemic in Belgium in March 2020, research activities were put on hold. Because the medical ICU of University Hospital Leuven in Belgium hospitalized patients with COVID-19 during all waves of the pandemic and no guarantee could be provided on when research activities would resume, inclusion in the trial was stopped prematurely. Results Characteristics of Patients From January 2019 to March 2020, 287 consecutive patients from the medical ICU were screened for the study. Of those, 263 patients were excluded from the study because of poor general prognosis or anticipated fatal outcome (n = 99); chronic renal failure (n = 48); preexisting neuromuscular disease (n = 44); liver cirrhosis (n = 24); edema, trauma, or hematoma skin lesions at the sites of NIRS measurements (n = 22); acute or past brain injury (n = 8); spinal cord injury above T8 (n = 8); skeletal pathology that severely impaired chest wall movements (n = 7), and allergic reactions to iodine (n = 3). Therefore, the actual number of patients recruited for the study was 24 patients (i.e., n = 16 SBT-success patients and n = 8 SBT-failure patients). Demographics, anthropometrics, and clinical characteristics of all patients and the two subgroups separately (i.e., SBT success and SBT failure) are presented in Table 1. No differences in demographics and anthropometrics and clinical tools such as APACHE (Acute Physiology and Chronic Health Evaluation), SOFA (Sequential Organ Failure Assessment), GCS (Glasgow Coma Scale), and RASS (Richmond Agitation-Sedation Scale) scores were found between SBT-success and SBT-failure groups. Only patients in the SBT-failure group had the clinical diagnosis of heart failure (n = 2 of 8), and 4 of 8 (50%) patients had the clinical diagnosis of diabetes mellitus (vs. 13% in the SBT-success group). The SBT-failure group had a significantly longer duration of MV prior to the SBT compared with the SBT-success group (Table 1). All patients who failed the SBT failed at 30 min. The reasons recorded for the SBT-failure group were hypercapnia (n = 2 patients), hypoxemia (n = 2 patients), tachypnea (n = 2 patients), tachycardia (n = 1 patient), and changes in mental well-being and comfort (n = 1 patients). Extubation was successful for all patients in the SBT-success group.Table 1 Demographics, anthropometrics, and clinical characteristics All patients SBT success group SBT failure group Subjects, n 24 16 8 Sex, male/female (%) 14/10 (58/42) 8/8 (50/50) 6/2 (25/75) Age, years 64 (58–74) 61 (50–73) 71 (64–75) Weight, kg 70 (57–100) 67 (54–108) 74 (59–95) Height, cm 173 (163–180) 169 (161–179) 170 (165–180) BMI 24.6 (19.3–31.2) 24.3 (18.9–35.9) 25.4 (20.7–29.8) Body temperature, °C 37 (36.5–37.1) 36.9 (36.3–37.1) 37.0 (36.6–37.2) APACHE II score 22 (14.8–28.0) 21.0 (15.0–27.3) 25.0 (10.3–28.8) SOFA score 5.0 (4.0–8.0) 4.0 (4.0–6.0) 7.5 (5.0–8.8) GCS score 11 (10–11) 11 (10–11) 10 (7–11) RASS score 0.0 (− 1.8 to 0.0) 0.0 (− 1.0 to 0.0) − 0.5 (− 2.8 to 0.0) Duration on MV before SBT, days 5.5 (3.3–8.0) 4.0 (3.0–6.8) 7.5 (6.3–17.0)* Diagnosis, n (%) Septic shock 1 (4) 1 (6) 0 Heart failure 2 (8) 0 (0) 2 (25) Respiratory 16 (67) 11 (69) 5 (63) Other 5 (21) 4 (25) 1 (12) Preexistent comorbidities, n (%) Cardiovascular diseases 3 (13) 3 (19) 0 COPD 6 (25) 3 (19) 3 (38) Diabetes mellitus 6 (25) 2 (13) 4 (50) Obesity 2 (8) 2 (13) 0 All patients included in the study were White. Values are expressed as median and interquartile range (IQR 25–75%) or proportion and percentage. Level of significance, p < 0.05. APACHE II score was assessed on the day of intensive care unit admission. SOFA score was assessed on the day of SBT. The intubation causes for the SBT-failure group were acute respiratory failure due to pneumonia (n = 5), cardiac arrest (n = 2), and hemorrhagic shock (n = 1). The intubation causes for the SBT-success group were acute respiratory failure due to pneumonia (n = 11), septic shock (n = 1), acute pancreatitis (n = 2), and gastrointestinal hemorrhage (n = 1). SBT-failure and SBT-success groups were balanced for sex APACHE II Acute Physiology and Chronic Health Evaluation II, BMI body mass index, COPD chronic obstructive pulmonary disease, GCS Glasgow Coma Scale, MV mechanical ventilation, RASS Richmond Agitation-Sedation Scale, SBT spontaneous breathing trial, SOFA Sequential Organ Failure Assessment Significant differences between SBT success and SBT failure Cardiovascular Parameters, Blood Gas Values, and Ventilatory Responses During MV, cardiovascular parameters were similar between groups. Changes in cardiovascular responses when transitioning from MV to SBT were also similar for both groups (Table 2). During the transition from MV to SBT, percentage SaO2 was significantly reduced in the SBT-failure group. Differential responses of PaCO2 (p = 0.021), respiratory rate (p = 0.0006), and minute ventilation (p < 0.0001) were observed between SBT-failure and SBT-success patients (Table 2).Table 2 Hemodynamics, blood gas values, arterial oxygen content and delivery, ventilatory settings, and respiratory parameters on MV and during SBT SBT success SBT failure p value MV 30-min SBT MV 30-min SBT Cardiovascular parameters CO, L/min 7.1 (4.2–8.1) 7.4 (4.3–9.1) 6.9 (4.7–8.8) 7.3 (4.9–10.8) 0.580 HR, beats/min 82 (63–98) 91 (69–104) 84 (67–96) 92 (68–104) 0.780 SV, ml/beat 80 (64–99) 74 (60–100) 93 (62–105) 91 (57–122) 0.378 MAP, mmHg 80 (74–88) 86 (78–93) 83 (72–90) 84 (80–90) 0.605 Blood gas values PaO2, mm Hg 86.1 (73.4–97.7) 84.9 (73.4–90.5) 84.9 (77.9–95.7) 82.5 (70.2–95.8) 0.831 PaCO2, mm Hg 42.8 (36.9–47.9) 43.0 (35.1–49.5) 42.9 (39.5–47.2) 47.5 (40.4–57.1) 0.021 SaO2, % 96.4 (94.7–98.1) 96.4 (95.3–97.4) 96.5 (95.8–97.4) 94.4 (92.4–97.1) 0.081 Arterial pH 7.48 (7.42–7.49) 7.45 (7.42–7.47) 7.44 (7.37–7.50) 7.41 (7.33–7.47) 0.165 Hb, g/L 9.5 (8.5–10.4) 9.6 (8.5–10.4) 9.0 (7.7–9.5) 9.0 (7.8–9.6) 0.830 Systemic arterial oxygen content and delivery CaO2, mL O2/L 123 (111–133) 125 (111–136) 118 (100–125) 111 (96–122) 0.076 DO2, mL O2/min 844 (494–1,010) 908 (545–1,195) 808 (570–851) 849 (457–1,040) 0.195 Ventilatory settings and respiratory parameters PS, cm H2O 10.0 (10.0–10.0) 5.0 (5.0–5.0) 10.0 (10.0–11.5) 5.0 (0–5.0) 0.124 PEEP, cm H2O 5.0 (5.0–8.0) 5.0 (1.3–5.0) 6.0 (5.0–7.8) 2.5 (0–5.0) 0.348 RR, breaths/min 18.5 (16.5–20.0) 21.5 (16.3–24.5) 21.5 (16.5–22.8) 28.5 (21.5–32.3) 0.0006 VT, mL/min 443 (404–534) 388 (361–489) 385 (336–482) 391 (341–516) 0.052 VE, L/min 8.4 (7.0–10.2) 8.7 (6.7–10.9) 7.9 (5.9–9.5) 11.2 (9.6–13.3) < 0.0001 FiO2, % 30 (26–40) 30 (26–40) 33 (26–35) 33 (26–35) – P0.1, cm H2O 1.74 (0.87–3.47) 4.53 (1.00–6.07) 2.90 (0.70–3.33) 5.10 (3.5–6.9)** 0.091 Values are expressed as median and interquartile range CaO2, arterial oxygen content; CO, cardiac output; DO2, oxygen delivery; FiO2, inspiratory oxygen fraction; Hb, hemoglobin; HR, heart rate; MAP, mean arterial pressure; MV, mechanical ventilation; PaCO2, partial pressure of arterial carbon dioxide; PaO2, partial pressure of arterial oxygen; PEEP, positive end-expiratory pressure; pH, hydrogen ion concentration; PS, pressure support; P0.1, occlusion pressure at 100 milliseconds as an indicator of neuromuscular activation index of the respiratory system; RR, respiratory rate; SaO2, arterial oxygen saturation; SBT, spontaneous breathing trial; SV, stroke volume; VE, minute ventilation; VT, tidal volume p values indicate the comparison of the changes between patients with SBT success and SBT failure Within-groups significant differences. Level of significance: p < 0.05 (two-sided) Local Tissue BFI and %StiO2 Responses During the transition from MV to SBT, prefrontal cortex BFI significantly increased in the SBT-success group (p = 0.016) but not in the SBT-failure group (Table 3, Fig. 2a). We could not confirm a significant differential response in prefrontal cortex BFI between groups (Table 3). Similarly, prefrontal DO2 only significantly increased in the SBT-success group (p = 0.009; Table 3). In the SBT-success group, a strong and positive relationship was found between changes from MV to SBT in PaCO2 and prefrontal cortex BFI (ρ = 0.728, p = 0.002; Fig. 3) that was not observed in the SBT-failure group (ρ = 0.048, p = 0.934; Fig. 3). During the transition from MV to SBT, prefrontal cortex %StiO2 decreased in six of eight patients (75%) in the SBT-failure group, compared to 3 of 16 patients (19%) in the SBT-success group (p = 0.007), resulting in a significant differential %StiO2 response between groups (p = 0.031; Table 3, Fig. 2b). No differential responses for BFI, DO2, and %StiO2 were observed for scalene and abdominal muscles during the transition from MV to SBT between groups (Table 3). However, thenar muscle %StiO2 significantly decreased in the SBT-failure group, resulting in a differential response between groups (p = 0.018) (Table 3).Table 3 Local tissue hemodynamic and oxygenation responses on MV and during SBT SBT success SBT failure Interaction effect (group × time), p MV 30-min SBT MV 30-min SBT Local tissues BFI, nanomole/second Prefrontal cortex 7.28 (4.64–14.27) 10.31 (7.37–14.04) 8.10 (6.79–11.01) 10.10 (6.49–14.6) 0.535 Scalene 5.86 (3.15–6.99) 6.50 (5.11–8.75)* 5.70 (4.61–8.5) 8.13 (5.0–12.9)* 0.363 Rectus abdominis 4.10 (2.61–7.42) 6.23 (2.84–8.01)* 4.13 (2.58–7.01) 6.07 (3.04–8.62)* 0.849 Thenar muscle 4.08 (1.32–9.32) 3.46 (1.59–11.86) 4.12 (0.83–5.88) 2.21 (1.1–3.74) 0.776 Local tissues DO2, nanomole/second × mlO2 (arbitrary unit) Prefrontal cortex 889 (544–1641) 1324 (903–2,046)* 996 (661–1,405) 925 (768–1562) 0.110 Scalene 701 (427–862) 867 (610–1065)* 573 (511–989) 706 (548–1254)* 0.907 Rectus abdominis 481 (325–933) 811 (458–1029)* 369 (242–782) 630 (301–920)* 0.825 Thenar muscle 447 (194–1092) 426 (197–940) 375 (80–740) 218 (87–408) 0.372 Local tissues fractional %StiO2, % Prefrontal cortex 65.0 (58.6–68.5) 65.1 (59.5–71.1) 57.2 (49.1–61.7) 51.0 (41.5–62.5) 0.031 Scalene 65.2 (53.5–72.4) 68.0 (57.0–74.2)* 52.2 (45.9–58.0) 53.8 (42.9–63.8) 0.840 Rectus abdominis 80.7 (53.8–87.0) 81.2 (57.4–86.2) 65.0 (56.2–69.6) 66.5 (56.4–75.2) 0.454 Thenar muscle 62.3 (60.1–66.5) 64.0 (59.8–71.9) 54.8 (35.6–57.9) 45.4 (32.6–57.3)* 0.018 Values are expressed as median and interquartile range (IQR 25–75%) BFI, blood flow index; DO2, oxygen delivery; mlO2, ml (milliliter) O2; XXX; MV, mechanical ventilation; SBT, spontaneous breathing trial; %StiO2, oxygen saturation *Within-group significant differences. Level of significance: p < 0.05 (two-sided) Fig. 2 a Individual values of the prefrontal cortex blood flow index (BFI) during mechanical ventilation (MV) and spontaneous breathing trial (SBT) between SBT-success group (gray circles) and SBT-failure group (open circles). The reasons recorded for SBT failure were hypercapnia (patients #5 and #6), hypoxemia (patients #3 and #7), tachypnea (patients #1 and #8), tachycardia (patients #4), and changes in mental well-being and comfort (patients #2). b Individual values of the prefrontal cortex fractional oxygen saturation (%StiO2) during MV and SBT between SBT-success group (gray circles) and SBT failure group (open circles). The reasons recorded for SBT failure were hypercapnia (patients #3 and #6), hypoxemia (patients #2 and #7), tachypnea (patients #4 and #5), tachycardia (patients #1), and changes in mental well-being and comfort (patients #8) Fig. 3 Left panel: Scatter plot, Spearman’s rank correlation coefficients, and significance levels between changes from mechanical ventilation (MV) to spontaneous breathing trial (SBT) in cerebral cortex blood flow index (BFI) and partial pressure of arterial carbon dioxide (PaCO2) for the SBT-success group (gray circles) and SBT failure group (open circles). The reasons recorded for SBT failure were hypercapnia (patients #6 and #7), hypoxemia (patients #3 and #4), tachypnea (patients #2 and #5), tachycardia (patients #1), and changes in mental well-being and comfort (patients #8). ROC Curve Analysis A ROC curve showed that the optimal threshold of prefrontal cerebral cortex %StiO2 to predict SBT failure was > 1.6%, exhibiting 94% sensitivity, 75% specificity, and an area under the curve of 0.79 (95% confidence interval: 0.55–1.00; p = 0.024) (Fig. 4).Fig. 4 Receiver operating characteristic (ROC) curve for the cutoff point of the prefrontal cerebral oxygen saturation (%StiO2). The diagonal line represents a test that is expected a priori to have no discriminatory value. The Youden index method (i.e., the maximum of vertical distance of the ROC curve from the point [x, y] on the diagonal line [chance line] indicated by a black arrow) was used to determine the optimal cutoff value (red point) of the prefrontal cerebral %StiO2 (i.e., − 1.6%) Discussion To our knowledge, this is the first study that simultaneously and noninvasively assessed cerebral cortex and respiratory muscle perfusion, DO2, and %StiO2 responses on MV during an SBT. In contrast to our hypothesis, there was no differential response in perfusion and DO2 of the prefrontal cortex while transitioning from SBT between patients with SBT failure and patients with SBT success (Table 3). However, our findings did show a differential response in prefrontal cortex %StiO2 in the SBT-failure group compared with the SBT-success group (Table 3). The ROC curve indicated high sensitivity (i.e., 94%) and specificity (i.e., 75%) for a decrease of 1.6% in prefrontal cerebral cortex %StiO2 for the prediction of SBT failure (Fig. 4). The latter results suggest that prefrontal cortex %StiO2, in addition to the well-established broader physiological, respiratory, and psychological criteria, might be a useful tool to help predict SBT failure [36, 37]. NIRS-derived %StiO2 is the ratio of microvascular HbO2 to total hemoglobin (tHb), reflecting the balance between tissue DO2 and O2 consumption [32]. We postulate that the differential cerebral cortex %StiO2 response we found between SBT-success and SBT-failure groups may result from an insufficient increase in cerebral cortex perfusion in the latter group, necessary to meet cerebral cortex O2 requirements. Because NIRS mostly reflects venous %StiO2 [38] and therefore tissue oxygen extraction, the decrease in cerebral cortex %StiO2 values might indicate insufficient delivery to the cerebral cortex. Indeed, we observed a significant increase in prefrontal cortex perfusion and cerebral cortex DO2 in the SBT-success group, but not in the SBT-failure group (Table 3), during the transition from MV to SBT. This hypothesis is supported by the lack of association between PaCO2 and cerebral cortex BFI in the SBT-failure group, in contrast to the positive relationship we found in the SBT-success group (Fig. 3). Normal physiological response to hypercapnia includes a profound effect on cerebral blood flow because it causes marked dilation of cerebral arteries and arterioles and increased blood flow [39]. Therefore, the greater increase in PaCO2 in the SBT-failure group would be expected to act as a driver for a greater increase in cerebral BFI compared with the SBT-success group, which was not the case (Fig. 2). The transition from positive pressure ventilation to SBT requires an increase in the DO2 to the respiratory muscles [40]. In both groups, there were significant increases in the perfusion and DO2 of scalene and abdominal muscles during the SBT to maintain adequate respiratory muscle oxygen supply (Table 3). Previous studies have reported that patients who fail an SBT may exhibit increased sympathetically mediated peripheral vasoconstriction, leading to restrictions in the oxygen availability of peripheral nonworking muscles in an attempt to support oxygen supply to the respiratory muscles [17, 18]. Our findings are consistent with others, demonstrating a significant reduction in thenar muscle %StiO2 in the SBT failure group (Table 3). In addition, we did not observe any differential responses in systemic oxygenation and hemodynamic response between SBT-success and SBT-failure groups (Table 2). Therefore, the differential response in the cerebral cortex and thenar muscle %StiO2 between groups (Table 3) cannot be explained by systemic oxygenation or hemodynamic changes. The differential cerebral cortex %StiO2 response between SBT-success and SBT-failure groups could either be a marker profile or an epiphenomenon, or possibly this response could in part contribute to SBT failure. The prefrontal cortex receives input from multiple cortical regions and is involved in emotional, attention, motivational, executive function, and decision-making processes and may act as a relay station in the central nervous fatigue-related network, regulating central command through peripheral feedback [41]. The role of the prefrontal cortex functions in SBT failure was outside the scope of this study. Thus, studies to implement objective measures of neural respiratory drive by assessing esophageal pressure, transdiaphragmatic pressure, and respiratory muscles electromyography, together with cerebral %StiO2 measures, are necessary to confirm this hypothesis. Nevertheless, our data support previous observational studies showing that NIRS can be a valuable tool for the detection of cerebral ischemia [36]. Indeed, intraoperative and postoperative cerebral cortex deoxygenation, as determined by NIRS, can predict complications such as neurocognitive dysfunction, acute kidney failure, wound infection, and myocardial infarction following cardiac and noncardiac surgeries in adult and pediatric patients [42–49]. Our pilot study has important limitations. The study is limited by the single-center design and the small sample size. Therefore, the power to detect differences in baseline characteristics and certain outcomes, such as cerebral cortex BFI, may be limited. Although the study might be underpowered, we believe that the findings are novel and advance our understanding in this area of interest. This study focused on the prefrontal cortex; thus, the “true” global brain response might not necessarily mirror the local responses investigated here in the prefrontal region of the forehead. Indeed, the pattern of blood flow responses could be different among brain regions, as indicated by studies measuring blood flow in the internal carotid and vertebral arteries in humans [50]. We chose to assess the perfusion and %StiO2 responses in scalene (in the absence of technology to assess the diaphragm) and rectus abdominis muscles because both muscles have been documented to play a critical role in SBT outcomes [51, 52]. We also took into account previously published data from our group showing appropriate scalene and rectus abdominis NIRS signal responsiveness under progressive respiratory efforts in patients with chronic lung diseases and critically ill populations [53, 54]. We did not assess the perfusion and %StiO2 responses in the sternocleidomastoid muscle to avoid potential NIRS signal artifacts caused by movements of the patient’s head. In addition, although supply could be maintained to scalene and rectus abdominis muscles, these data might not be applicable to other primary respiratory muscles, such as the diaphragm. Another limitation of the study includes the nonstandardized setting of PEEP and pressure support ventilation during the SBT. Different levels of pressure support and PEEP may lead to variable responses in the work of breathing and respiratory muscle perfusion and %StiO2 responses in the two groups. Along these lines, we could also expect different behaviors of respiratory and cerebral cortex %StiO2 according to the specific SBT settings. This must be considered in future studies exploring SBT failure by NIRS. The SBT-failure group had a significantly longer duration of MV prior to the SBT compared with the SBT-success group. Furthermore, only patients in the SBT-failure group had the clinical diagnosis of heart failure (n = 2 of 8), and 4 of 8 patients (50%) had diabetes mellitus Table (1), with a potential mechanism of limited capability to augment systemic oxygen delivery. However, the central hemodynamic responses did not differ between SBT-failure and SBT-success groups (Table 2). Differences in the intubation cause, comorbidities, and days on MV between study groups must be considered as potential confounders in future studies exploring SBT failure by NIRS. In addition, the inclusion of patients with acute or chronic brain injuries should be considered in future studies exploring weaning failure by brain imaging techniques. In this study, the assessment of primary physiological parameters at only two points (i.e., during MV and at the end of the SBT) did not allow us to explore the evolution of NIRS and other relevant physiological changes during the SBT to the timing of the SBT failure. This study design is similar to that of a recent study that used noninvasive methodologies, such as electrocardiography, echocardiography, and lung ultrasound evaluation before and after an SBT, to describe whether weaning-induced pulmonary edema occurred in patients with or without diaphragm dysfunction [55]. Finally, this study did not examine the reproducibility of the BFI method for measuring cerebral blood flow to avoid exposing the patients to unnecessary ICG injections to address issues beyond the primary scope of this study. However, previous data revealed that the coefficient of variation of the prefrontal cerebral cortex BFI measured by the NIRS-ICG technique during repeated measures (reproducibility was evaluated during six ICG injections at 5-min intervals in 14 critically ill patients) was low (i.e., median coefficient of variation: 10%, first and third interquartile range: 4.9–18.5%) [22]. Conclusions To conclude, during the transition from MV to SBT, we identified a differential response in prefrontal cortex %StiO2 between SBT-failure and SBT-success patients, possibly due to the insufficient increase in prefrontal cortex perfusion in the SBT-failure group. During the SBT, a drop in prefrontal cerebral cortex %StiO2 > 1.6% was found to have the best association with SBT failure, with 94% sensitivity. This study is hypothesis generating, and a large multicenter study is now needed to confirm these findings to validate the role of NIRS-derived cerebral cortex %StiO2 responses in supporting decisions about the timing of liberation from MV. Acknowledgements We thank Ms Emily Hume (Northumbria University, Newcastle, UK) and Mr. Neeraj Shah, MD (Guy’s and St Thomas’ National Health Service, (NHS) Foundation Trust, London, UK), for language editing of the manuscript. Author Contributions All authors contributed to the conception and designed research. ZL, MVH, and DP performed experiments and analyzed and visualized the data. All authors interpreted the results of the experiments. ZL drafted the manuscript. All authors revised and edited the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors approved the final version of the manuscript. Source of Support Dr Zafeiris Louvaris received a postdoctoral fellowship of the Research Foundation—Flanders (FWO) (#12U5618N). Prof. Greet Hermans received an FWO Senior Clinical Researcher Fellowship (#180512 N). Prof. Greet Hermans and Prof. Daniel Langer received an FWO project grant (#G053721N). Conflict of interest The authors declare that they have no conflicts of interest. Ethical Approval/Informed Consent Ethics approval was obtained from the responsible local ethical committee (Ethische Commissie Onderzoek Universitair Ziekenhuis/Katholieke University Leuven protocol ID: S60516). Written informed consent was obtained from all patients. Clinical Trial Registration ClinicalTrials.gov identifier NCT03240263. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Daniel Langer and Greet Hermans have contributed equally to this work. ==== Refs References 1. Béduneau G Pham T Schortgen F Epidemiology of weaning outcome according to a new definition. The wind study Am J Respir Crit Care Med 2017 195 772 83 10.1164/rccm.201602-0320OC 27626706 2. McConville J Kress J Weaning patients from the ventilator N Engl J Med 2013 368 1068 9 23484845 3. 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==== Front Cluster Comput Cluster Comput Cluster Computing 1386-7857 1573-7543 Springer US New York 3813 10.1007/s10586-022-03813-x Article Security provisions in smart edge computing devices using blockchain and machine learning algorithms: a novel approach Mishra Kamta Nath [email protected] Kamta Nath Mishra was born on August 15, 1973, in Chandrauta Village of Kushinagar district of Uttar Pradesh, India. He received his Bachelor of Science (B.Sc., 3 years) degree from the University of Gorakhpur, India, in 1992, and Master of Computer Application (M.C.A., 3 years) degree from Madan Mohan Malviya Engineering College (Currently MMMUT), Gorakhpur, U. P., India in 1996. Dr. Mishra completed his M.Tech. (Software Systems) degree from Birla Institute of Technology and Science (BITS) Pilani, India in 2003 and Ph.D. (Engg.) from the C.S.E. department of B.I.T. Mesra—India, in May 2015. Dr. Mishra has more than twenty-one years of teaching and research experience. Currently, he is working as an Assistant Professor (Senior Grade) at Dept. of CS&E, B.I.T. Mesra, Ranchi, India since August 2009; He has worked as a faculty member in the Department of Computer Science, University of Sebha (A public University), Libya, from October 2006 to July 2009. He was a senior lecturer at B.I.T. Mesra, (Noida Campus) from July 2004 to September 2006. Dr. Mishra has worked as a senior project engineer from September 2003 to June 2004 and project engineer from September 2000 to August 2003, in the Centre for Development of Advanced Computing (Ministry of Communication & I.T., Govt. of India) Noida, Uttar Pradesh. Before joining CDAC, Dr. Mishra worked as a lecturer in the CS&E department at Krishna Institute of Engineering & Technology (KIET), Ghaziabad, India, from July 1998 to August 2000. Dr. Mishra has published two booksfifteen book chapters, and more than forty research papers in journals and conferences of international repute. His research interest includes Biometric Systems, Image Processing, Analysis of Algorithms, and Distributed Cloud Computing. Dr. Mishra is a professional member of IEEE Biometric Society U.S.A., and A.C.M., U.S.A. Bhattacharjee Vandana [email protected] Vandana Bhattacharjee is working as a Professor in the Department of Computer Science and Engineering, Birla Institute of Technology, Mesra, Ranchi. She completed her B. E. (C.S.E.) in 1989 from BIT Mesra and her M. Tech and Ph. D in Computer Science from Jawaharlal Nehru University New Delhi in 1991 and 1995 respectively. She has several National and International publications in Journal and Conference Proceedings. She has authored a book on Data Analysis. She is a Life Member of the Computer Society of India. Her research areas include Machine Learning and its applications. Currently, she is working on Deep Learning techniques applied to the domains of Software Fault prediction, Classification of Images, Disease prediction, analysis of Remote sensing images, and sentiment analysis. Saket Shashwat [email protected] Shashwat Saket hails from Ranchi, Jharkhand. He has completed his secondary education from D.A.V. Public School, Pundag, Ranchi, and his higher secondary education from Delhi Public School, Sail Township, Ranchi. Currently, he is pursuing his Bachelor of Technology from Birla Institute of Technology, Jharkhand, India, in Computer Science & Engineering with a specialization in Computational Intelligence. He is intrigued with Python, R language and has a further ambition to follow up his research in the fields such as Deep Learning, Machine Learning, Data Science, AI in Medical, Generative Adversarial Networks, Cryptanalysis, and Network Security. Mishra Shivam Prakash [email protected] Shivam Prakash Mishra was born in Ghaziabad, Uttar Pradesh, and completed his major part of schooling, including secondary and higher secondary education from Bethany Convent School Naini, Allahabad. Currently, he is pursuing his Bachelor of Technology from Birla Institute of Technology, Jharkhand, India, in Computer Science & Engineering. He aspires to follow his further research in Deep Learning, Machine Learning, Cryptography, and Cryptanalysis. grid.462084.c 0000 0001 2216 7125 Department of Computer Science & Engineering, Birla Institute of Technology, Ranchi, India 30 11 2022 126 16 7 2022 21 10 2022 5 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. It is difficult to manage massive amounts of data in an overlying environment with a single server. Therefore, it is necessary to comprehend the security provisions for erratic data in a dynamic environment. The authors are concerned about the security risk of vulnerable data in a Mobile Edge based distributive environment. As a result, edge computing appears to be an excellent perspective in which training can be done in an Edge-based environment. The combination of Edge computing and consensus approach of Blockchain in conjunction with machine learning techniques can further improve data security, mitigate the possibility of exposed data, and it reduces the risk of a data breach. As a result, the concept of federated learning provides a path for training the shared data. A dataset was collected that contained several vulnerable, exposed, recovered, and secured data and data security was precepted under the surveillance of two-factor authentication. This paper discusses the evolution of data and security flaws and their corresponding solutions in smart edge computing devices. The proposed model incorporates data security using consensus approach of Blockchain and machine learning techniques that include several classifiers and optimization techniques. Further, the authors applied the proposed algorithms in an edge computing environment by distributing several batches of data to different clients. As a result, the client privacy was maintained by using Blockchain servers. Furthermore, the authors segregated the client data into batches that were trained using the federated learning technique. The results obtained in this paper demonstrate the implementation of a Blockchain-based training model in an edge-based computing environment. Keywords Blockchain technology Edge computing Federated learning systems Machine learning techniques Voting classifier http://dx.doi.org/10.13039/501100007254 Birla Institute of Scientific Research 00071 00071 00071 00071 Mishra Kamta Nath Bhattacharjee Vandana Saket Shashwat Mishra Shivam Prakash ==== Body pmcIntroduction The fourth major paradigm shift in modern computing is edge computing. It is processing data closer to the point where this is generated. With the digital revolution, it is now possible to reduce the latency of client–server architecture, particularly on a large scale. Edge computing is a distributed information technology architecture that processes client statistics at the network's edge. It is based on the idea that instead of bringing data closer to the computer, we bring the computer closer to the data. Edge computing relocates some storage and compute resources away from the central data center and closer to the data source. The three pillars of edge computing, namely autonomy, edge device security, and data sovereignty, are among its most significant advantages [1, 2]. While the disadvantages of the edge computing could be summarized in its low redundancy, potential loss as well as data poisoning, longer outrage time. Mobile edge computing (MEC) devices are now widely used to solve artificial intelligence (AI) problems. Traditionally, AI requires a central server with powerful processing power for Machine Learning. This approach has several drawbacks, including communication overheads (CO) and data privacy issues (DPI) [3]. Federated learning is a machine learning technique that trains algorithms across multiple devices (edge/mobile) without exchanging or accumulating them in a single server [4]. Unlike traditional machine learning, which requires data sets to be brought to a single location, federated learning promotes trust in machine learning (ML) processes by keeping the data at the source and directing the model toward it [5]. Each device has its own set of local data ready to be trained on. In a single epoch, the weights are calculated and returned to the MEC. The MEC fine-tunes the model before sending it out for iteration [6, 7]. This process is repeated until the desired level of accuracy is obtained. In the current Federated Learning system, users must trust the MEC system for model aggregation. What started out as a benefit may turn out to be a disadvantage. Figure 1 depicts the classical federated learning architecture.Fig. 1 Classical Federated Learning Architecture The interconnection having Mobile Edge Computing (MEC) servers, aggregation and global data at one end and Internet of Things (IoT) devices, phones at other end are given in Fig. 1. It fosters the model training at local and global levels. While the model is being trained at some of the devices, it is simultaneously being tested at other devices. The model trained here is further improved at the global level with added noise and some improvements. This keeps the check and balance on the model accuracy. Further, it provides us with a faster and more accurate model. But along with the given advantages, it paves a way for high security risk of several attacks such as data poisoning attacks, susceptible data breaching attack, etc. Blockchain aptly is a decentralized database. The transactions being managed by chain are the backbone of the technology. The blocks of data distributed are secured using the crypto-graphical methods. After a new block is merged into the chain, it is believed that the new block can interact the all the other blocks in the chain. Proof of Work (PoW) is one of the methods used for merging blocks by adding the hash function in the current block and then mines it. PoW provides a simple algorithm with a complete centralization as well as it procures a node free access. But along with the advantages, it is lucid that there is a waste of energy. Transmission of learning parameters is highly vulnerable to attacks when travelling between nodes. The MEC server can be a single point of failure in an attack, and this model has scalability issues. Hence, a consensus approach of Blockchain based Federated Learning system may have following advantages over others [8, 9]:Advantage1: It is using decentralized approach. Hence, it has the advantages in terms of security and privacy. Advantage2: It does not allow a single point of failure attacks. Advantage3:It is highly scalable. The biggest reason for slowed progress is the unavailability of data. Differential privacy (DP) is a technique that protects user privacy by adding random noise to the data. It requires the output of a query to be the same when a user is removed from the original dataset [10]. The disadvantage with the differential privacy is that the picking of outliers such as max, sum can alter the meaning of the data and can provide random inferences. Along with this, the addition of the noise must be done at every level of queries leading towards the data breaches. In local differential privacy (LDP), the noise is added directly to the data by the individual data owners. In the global differential privacy (GDP), the noise is added by the complete dataset owner to the output of a query on the database [11]. Figure 2 defines a schematic representation of local differential privacy and global differential privacy in association to the mobile edge computing servers.Fig. 2 Types of differential privacies and fundamental solutions for Blockchain-based federated learning The contributions of this paper are as follows:i. Data security is established with susceptible data, recoverable data in an edge computing environment using two-level-architecture. ii. Proper security provisions for several breached data as well as susceptible data using differential privacy using data stored in blockchain based storage. iii. Addition of noise in the blocks of data securing anonymity of the data in the local differential privacy and further exaggerated in a global differential privacy. iv. Implementation of machine learning techniques for training the data from individual blocks with ensemble techniques procuring a locally asymptotic stability and further training the model in a federated edge-based environment for obtaining the global asymptotic stability of the data. v. Mitigate the risk of poisoning attack by segregating the data using consensus approach of blockchains. Further, differential privacy techniques are implemented in order to hinder the malicious user reaching out to the highly susceptible data. Literature review Throughout the spread of numerous IoT (Internet of Things) devices in the cloud environment, the authors’ encountered challenges in meeting the low latency and high bandwidth requirements, creating a void for decentralized computing, this later evolved into edge computing. Furthermore, edge computing depicts several security threat itineraries. Another issue arises when the central server goes offline for an extended period. In these circumstances, the best approach is to implement the consensus approach of Blockchain, which can be visualized as an actual chain of blocks that operates on the principle of decentralization. It is clear from this that machine learning models like deep learning, deep reinforcement learning, and reinforcement learning are difficult to adapt to IoT and Edge environments. This opens the door to a plethora of potential risks and is one of the most important areas for research enthusiasts. Edge Computing devices are vulnerable in terms of software and resources, allowing them to integrate with a variety of machine learning techniques. This section will concentrate on several works that have been completed in the respective domain. Nowadays, Blockchain has been homogenized into IoT and cloud environments; Sharma et al. [12] proposed Software-Defined Network (SDN) architecture blended with Blockchain, which is divided into three layers. The first layer is the device layer, which collects data from the devices. The raw data was processed by the second layer, the fog layer, with the help of the SDN controller. Furthermore, the third layer contains all of the received processed data. As a result, they collected, classified, and analyzed the streaming data obtained from IoT. They reduced the cost of end-to-end delay by bringing computational resources to the edge of IoT devices using Pan et al. [13] proposed a work demonstrating the EdgeChain framework for ensuring Blockchain collaboration in smart contracts. Edge devices collected data that IoT devices could access. Edge Chain's core concept is to combine Blockchains with a coin system, blend them under the IoT framework, and merge the resource pool of edge clouds. They proctored all transactions through Blockchains for security auditing and logging without overburdening the devices. Rahman et al. [14] proposed a Blockchain-based infrastructure that guarantees privacy and security-based spatial–temporal contract services. This infrastructure anchors several fog nodes at the host's edge and integrates them with computational intelligence methods. Further, they proposed a Mobile Edge Computing-based sharing economy system through the off-chain framework and Blockchain implementation to contain immutable ledgers. Xu et al. [15] discussed how edge computing could meet low latency, high security, and data privacy requirements. With the channel of the short distance between the edge computing devices and the power terminals, low delay is ensured while collecting the gigantic data. They implemented a deep learning mechanism to enchant the real-time data flow using examples such as load balancing in the intelligent energy system, power prices predictions, and many more. The network architecture used through the itineraries of edge computing following the peculiarities of security proved to be much more effective than the cloud computing frameworks implemented beforehand. Still, the security of each node that arises in the edge computing framework makes it strenuous to adept in general use. Thus, to overcome this, they proposed a security architecture named as the physical layer security that involved only end-to-end users constrained by the limits of resources and energy. This paper effectuated many artificial intelligence prototypes for achieving the required efficiency and security under the chain of intelligent grids associated with edge computing. It is significant to fortify the working of the critical infrastructural system in a society and its economy. The volume of data generated due to expanding IoT devices is turning out to be a hot topic in terms of scalability and security factors. Wu et al. [16] introduced the IoT/IIoT (Industrial Internet of Things) based critical infrastructure in industry 4.0. Moreover, it entered the Blockchain and edge computing paradigms in this reference. They also worked on a blend of these paradigms and analyzed how they could enter the scalable and secure environment era. They also surveyed state of the art for scalability and security purposes. One of the most arduous tasks is to provide security and privacy in the Internet of Things environment. Some of the limitations are solved with the introduction of Blockchain as a decentralized ledger. Pajooh et al. [17] presented the advancements of Hyper-ledger Fabric, which serves as a permission Blockchain for protecting edge computing devices in an environment of the locally employed process used in authentication. The proposed model also procured the traceability of data obtained from the IoT devices addressing scalability challenges. The paper encountered the execution of Hyper-ledger Fabric Blockchain framework desegregated with edge computing and the Internet of Things. Then they performed the tests on the VMware Virtual desktops and physical environmental setups such as Raspberry Pi devices. Apart from this, they secured the framework with multi-layered Blockchains with authentication protocols at IoT nodes of every cluster. The aftermath of this turned out to be a remarkable boost in the throughput of IoT applications. The obstacle behind the popularised usage of edge computing was data storage security, which played an indispensable role in the evolution of intelligent computing. The authors in [18] proposed a mechanism in which they combined Blockchain with the regeneration coding to improve the reliability and security of the stored data through edge computing. They also built global layers of Blockchains in a cloud service. They also formulated local Blockchain at IoT terminals for another pool of verification. The residual nodes then selected the terminal and repaired them with regeneration coding resulting in the development of resources under the umbrella of edge computing. Kuo et al. [19] further proposed ModelChain, which focuses on training medical health prediction framework. This allowed multiple institutions for the training in the environment of Blockchain using intelligent machine learning techniques and developed a framework for the prediction of medical health. Every site involved here fostered the estimation of model parameters. They applied transactional metadata and therefore integrated privacy-preserved models using intelligent computing. Rathore et al. [20] proposed BlockDeepNet, a Blockchain-based Deep Learning model that enlightened the collaborative paradigm of IoT and Deep Learning techniques. They verified the compatibility and feasibility of object detection under the IoT archetype through experimental analysis. However, the proposed model portrayed the need for higher computational resources, and the implementation would be impotent in the low computational machines. Ferran et al. [21] suggested a deep learning framework based upon BlockChain for the implementation under Smart Grid environment named DeepCoin. It achieved a very high throughput by applying a novel energy system associated with Byzantine fault tolerance. They used hash functions and short signatures using Blockchain technology. They prevented attacks on Smart Grids. Singh et al. [22] proposed a Deep Learning-based IoT-oriented framework under the store of a secure, secure smart city in the domain of Cyber-Physical systems and Software-Defined networking. Further, they compared their model with scalability and latency parameters and evaluated pre-existing security and privacy. But, it still suffers from hindrances occurring due to the centralization of the workspace. He et al. [23] studied the implications of edge computing with IoT and the security issues. They proposed a general framework for edge computing based upon a Blockchain environment. Further, they designed a smart contract in the private Blockchain network to fathom the hindrances caused by resource allocation in edge computing, focusing on multiple service subscribers providing good efficiency. They displayed the amalgamation of Artificial Intelligence with the Blockchain. Also, they simulated their model to achieve the accuracy of resource allocation in Edge Computing. Dai et al. [24] witnessed the implications of Edge empowered intelligent computing for medical prospects against COVID-19. They addressed concerns like the over-centralization of resources on the Internet of Medical Things and the hysteresis of digitalization in medical services, and hindrances in proper security over the medical data. They analyzed that the collaboration of the Internet of Medical things with Blockchain technologies can somehow elixir the severity of COVID-19. However, they had several challenges to overcome, such as the absence of context in this regard, latency, and privacy issues. They presented an architecture based upon the Edge Intelligence working on the sub-domain of Blockchain technologies at the Internet of Medical Things (IoMT) platform. After that, they monitored the initiation of the pandemic and traced the supply chain that must be broken down to hinder the spread of the pandemic virus. Li et al. [25] integrated the Blockchain and Federated learning system (BCFL) framework. They provided an in-depth survey of the framework and further discussed the insights of this new paradigm. They further studied the design of BCFL framework and discussed challenges and issues of this technology. Lastly, they discussed the applications of the framework. The researchers Nguyen et al. [26] presented the fundamental concept of Federated Learning and worked on the opportunities it opens for Mobile edge computing devices. They explored several topics such as incentive mechanisms, communication cost, resource distribution, security and privacy issues. They also surveyed the use of Blockchain based Federated Learning in the case of famous applications in an edge-based network environment which includes edge crowd sensing, edge content caching, and edge-based data sharing. Zhang et al. [27] proposed the architecture for sharing the data in autonomous vehicles in an edge-based environment. The architecture worked on the hybrid of permission-based-blockchain and federated learning systems using a local Directed Acyclic Graph (DAG). Moreover, they adopted Deep Reinforcement Learning (DRL) methods for selecting the significant nodes and further improving the effectiveness. Problems with existing federated learning systems The following are some of the fundamental problems with the existing federated learning system:Problem1: Users need to trust the MEC system for model aggregation. Problem2: Transmission of learning parameters is highly vulnerable to attacks. Problem3: The MEC server can be a single point of failure in an attack. Problem4: Organizations may not want to provide data for various reasons. Problem5: Malicious users may trace back the original owner of the data. Problem6: Scalability issues. Some of the existing solutions for above problems (Problem1 to Problem6) of federated learning system are as follow:Solution1: Poisoning attacks can be decreased by increasing the difficulty level of blockchain mining. Solution2: Using smart contracts to secure communication and message passing. Solution3: Differential Privacy approach to reduce the possibility of individual record identification. Proposed improvement in existing solutions Overview of the proposed approach The current issues for Blockchain-based federated learning system are attacks like model poisoning and data privacy threats that can make the network vulnerable to outside attacks; there can be attempts to train a model using designed falsie data; and individual record identification; attempt to reconstruct training set from the generated gradient. Therefore, the following steps are needed:Step 1: Increasing the difficulty level of consensus approach based Blockchain mining to decrease poisoning attacks. Step 2: Using smart contracts to secure communication and message passing. Step 3: Differential privacy approach to reduce the possibility of individual record identification. Figure 3 represents the addition of noise and the corresponding relation between the raw value, noise, secure data, and queries to retrieve the information. This model propels the idea of data security after the addition of noise. To receive further improvements in existing solutions, the following actions may be implemented:Action 1: Identification of individual records and private raw data, since it is one of the most severe threats that almost defeats the purpose of Federated Learning. Action 2: Study of current solutions using differential privacy concerning local devices. Action 3: Performing experiments to compare the effectiveness of the new consensus approach based Blockchain federated learning architecture with differential privacy-dependent machine learning algorithms. Fig. 3 Establishing different types of relationships amongst raw value, noise, secure datasets, and queries to retrieve information Proposed mathematical model The variables used in mathematical model are presented and described in Table 1. This section aims to formulate a mathematical model regarding the data breaching dynamics under an Edge environment. The authors have provided an approach for minimizing the risk of a data breach in an Edge Computing environment.Table 1 Nomenclature of variables used in research Symbol Description T Susceptible Data F Exposed Data R1 Susceptible Data after User Id, Password authentication J Already Breached Data and has the potential to breach other data R2 Vulnerable Data after OTP authentication S Recovered data, i.e., retrieved by applying several security measures under this paper B Influx rate of data η1 Rate of a general security breach, i.e., other than authentication η2 Rate of a security breach due to breach at Level 1, i.e., User Id/Password authentication η3 Rate of a security breach due to breach at Level 2, i.e., OTP authentication β1 Transfer rate of exposed data to susceptible data at Level 1, i.e., User ID/Password β2 Transfer rate of exposed data to breached data Γ Per ineffective attack rate δ1 Transfer rate of suspected data to already breached Data δ2 Transfer rate of data from suspected block to another block where suspected data was secured ω1 Rate of transfer of breached data to Vulnerable data after positive security risk ω2 Transfer rate of breached data to recovered data Ψ The recovery rate of breached data after OTP authentication security procedures W A matrix containing the Jacobians concerning the rate of other transitions between blocks of wi of and breached blocks, ∀i∈[1,5] G A matrix containing the Jacobians concerning the rate of appearance of new security attacks in the block of gi of and breached blocks ∀i∈1,5. JRFE Jacobian at Risk-Free Equilibrium τESP* τ at the Equilibrium for Security Provisions in Smart Edge Computing Devices using Block-chain and Machine Learning Algorithms The proposed mathematical model consists of following hypothesis:H1: We formulate an emphasis on a security model with a random influx rate (B > 0) and a general security breach rate (η1 > 0). H2: Exposed Data has undergone Username and Password-based authentication (β1 > 0). H3: Breaching of Username/Password authenticated data at a constant rate of (ω > 0). H4: The susceptible Data is breached through already breached data at a constant rate of (γ > 0). H5: Susceptible data are sent for the authentication at Username/Password-based authentication at a rate of δ1. H6:: Transfer rate of sensitive data after Username/Password authentication to secured data after implementing proper security protocols (δ2 > 0). H7: Already breached data are sent to OTP-based authentication at a constant rate of (ω1 > 0). H8: OTP authenticated data are secured at a constant rate of (ψ > 0). H9: Breached data at level 1, i.e., Username/Password authentication level unable to get recovered at a rate of (η2 > 0). H10: Data Breached at level 2, i.e., OTP authentication was unable to get recovered at a constant rate of (η3 > 0). H11: B > η2 > η3 > η1 > 0. Through the above hypothesis on data breaching at two-tier securities, the authors have observed the following equations to be apt as per Fig. 4: dTdt=B-γTJ-η1T+δ2R1 dFdt=γTJ-β1+β2+η1F dR1dt=β1F-(δ1+δ2+η1)R1 dJdt=δ1R1-ω1+ω2+η1+η2J+β2F dR2dt=ω1J-(η1+η3+ψ)R2 1 dSdt=ψR2+ω2J-η1S Positivity and boundedness of proposed model The data and parameters associated with it are assumed to be non-negative. Thus, the rate of alteration in the security factor of the data can be given by:2 dMdt=dTdt+dFdt+dR1dt+dJdt+dR2dt+dSdt 3 ⇒dMdt=B-η1M-η2J-η3R2 with M=T+F+R1+J+R2+S Now, it can be deduced that whenever there is no security risk, then (F = J = R2 = 0).Fig. 4 Schematic diagram for the flow to security breaches in a data block at two-tier authentications Hence, dMdT=B-η1M which defined the data size M having the storage of Bη1 as t→∞. It further defines the solution of (1) to lie in the range of τ={T,F,R1,J,R2,SϵR6+:Tt≥0,Ft≥0,R1≥0,Jt≥0,R2t≥0,St≥0,T+F+R1+J+R2+S≤Bη1}. Due to the positive invariant boundness of the solutions in the region defined by τ, the problem is well-formed. Security breach number (S0) We will implement the next-generation matrix method to define the security breach number at the Level 2 OTP-based authentication. The security breach number is designated as the average number of security breaches at Level 2 authentication, i.e., One Time Password authentication, when data from one breached block enters another block with totally susceptible data. We denote this number as S0. We can calculate this using the spectral radius of the GW−1 matrix, which is the most significant absolute value of their Eigen values. This is lucidly formed through the linearization of the Eq. (1). As per the principle of the next-generation matrix, we can deduce the security breach number as the spectral radius of the next generation matrix formed as GW−1 of the system of Eq. (1).4 gi-wi=dFdtdR1dtdJdtdR2dtdSdt=γTJ-β1+β2+η1Fβ1F-δ1+δ2+η1R1δ1R1-ω1+ω2+η1+η2J+β2Fω1J-η1+η3+ψR2ψR2+ω2J-η1S Accordingly,5 gi=γTJ0000,wi=β1+β2+η1Fδ1+δ2+η1R1-β1Fω1+ω2+η1+η2J-(δ1R1+β2F)η1+η3+ψR2-ω1Jη1S-(ψR2+ω2J) Here, in this equation, gi is the rate of appearance of new security attacks in the block and wi is the rate of other transitions between blocks of (i) of and breached blocks, ∀i∈[1,5]. The matrix for G and W can be formed as follows:6 G=∂g1∂F∂g1∂R1∂g1∂J∂g1∂R2∂g1∂S∂g2∂F∂g2∂R1∂g2∂J∂g2∂R2∂g2∂S∂g3∂F∂g3∂R1∂g3∂J∂g3∂R2∂g3∂S∂g4∂F∂g4∂R1∂g4∂J∂g4∂R2∂g4∂S∂g5∂F∂g5∂R1∂g5∂J∂g5∂R2∂g5∂S=00γT00000000000000000000000, where T0=Bη1, and,7 W=∂w1∂F∂w1∂R1∂w1∂J∂w1∂R2∂w1∂S∂w2∂F∂w2∂R1∂w2∂J∂w2∂R2∂w2∂S∂w3∂F∂w3∂R1∂w3∂J∂w3∂R2∂w3∂S∂w4∂F∂w4∂R1∂w4∂J∂w4∂R2∂w4∂S∂w5∂F∂w5∂R1∂w5∂J∂w5∂R2∂w5∂S 8 Hence,W=β1+β2+η10000-β1(δ1+δ2+η1)000-β2-δ1(ω1+ω2+η1+η2)0000-ω1(η1+η3+ψ)000-ω2-ψη1 Now, we know that W-1=AdjWW9 W-1=1β1+β2+η10000β1β1+β2+η1(δ1+δ2+η1)1(δ1+δ2+η1)000β1δ1+β2(δ1+δ2+η1)β1+β2+η1(δ1+δ2+η1)(ω1+ω2+η1+η2)δ1(δ1+δ2+η1)(ω1+ω2+η1+η2)1(ω1+ω2+η1+η2)00β2ω1(δ1+δ2+η1)+(ω1δ1β1)β1+β2+η1(δ1+δ2+η1)(ω1+ω2+η1+η2)(η1+η3+ψ)ω1δ1(δ1+δ2+η1)(ω1+ω2+η1+η2)(η1+η3+ψ)-ω1(ω1+ω2+η1+η2)(η1+η3+ψ)-ω2(ω1+ω2+η1+η2)(η1+η3+ψ)0(η1+η3+ψ)ω2β2(δ1+δ2+η1)+ψω1β1δ1-β1ω2δ1(η1+η3+ψ)+(δ1+δ2+η1)ψω1β2β1+β2+η1(δ1+δ2+η1)(ω1+ω2+η1+η2)(η1+η3+ψ)η1(η1+η3+ψ)δ1ω2+ω1ψδ1(δ1+δ2+η1)(ω1+ω2+η1+η2)(η1+η3+ψ)η1ω2(η1+η3+ψ)+ψω1(ω1+ω2+η1+η2)(η1+η3+ψ)η1ψη11η1 Therefore, we can say that our security breach number, as denoted by S0, is10 S0=κGW-1=γT0(β1δ1+β2(δ1+δ2+η1))β1+β2+η1(δ1+δ2+η1)(ω1+ω2+η1+η2) 11 S0=κGW-1=γB(β1δ1+β2(δ1+δ2+η1))η1β1+β2+η1(δ1+δ2+η1)(ω1+ω2+η1+η2) Also, we can say that for the steady-state condition, our system of Eq. (9) becomes: 12 B-γTJ-η1T+δ2R1=0γTJ-β1+β2+η1F=0β1F-δ1+δ2+η1R1=0δ1R1-ω1+ω2+η1+η2J+β2F=0ω1J-η1+η3+ψR2=0ψR2+ω2J-η1S=0 Here, we have discussed the local stability of a risk-free equilibrium and the equilibrium of Security Provisions in Smart Edge Computing Devices with system given in Eq. 12 by scrutinizing the following characteristics equations. Theorem 1 If S0 < 1, the risk-free equilibrium is locally asymptotically stable in the region τ0for Security Provisions in Smart Edge Computing Devices using consensus approach of Block-chain and Machine Learning Algorithms, otherwise it is unstable. Proof The Jacobian Matrix (12) formulates as τ0T=1,F=R1=J=R2=0, in a risk-free equilibrium condition.13 JRFEτ0=-η10-δ2-γ00-(β1+β2+η1)0γ00β1-(δ1+δ2+η1)0000δ1-(ω1+ω2+η1+η2)0000ω1-(ψ+η1+η3) This corresponding Eigen value of JDFE(τ0) are as follows:λ1=-η1 14 λ2=-ψ+η1+η3 Also, we can get the other three Eigen values through the equation of the cubic polynomial we have obtained.λ3+b1λ2+b2λ+b3=0, where b1=(β1+β2+3η1+δ1+δ2+ω1+ω2+η2), b2=β1+β2+η1δ1+δ2+η1+β1+β2+η1ω1+ω2+η1+η2+(ω1+ω2+η1+η2)(δ1+δ2+η1), b3=β1+β2+η1δ1+δ2+η1ω1+ω2+η1+η2-γ1β1δ1=(β1+β2+η1δ1+δ2+η1ω1+ω2+η1+η21-S0. If S0<1, then we can deduce that, b1>0;b2>0,b3>0andb1·b2>b3 [25, 26]. Thus, we can say that b1>0;b2>0,b3>0andb1·b2>b3. Therefore, by implementing the Routh-Hurwitz criterion, we can deduce that in the region if S0<1,JDFE(τ0), then it is locally asymptotically stable. Equilibrium for security provisions in smart edge computing devices using block-chain and ML algorithms We can obtain the Equilibrium for Security Provisions in Smart Edge Computing Devices using Block-chain and Machine Learning Algorithms with τT,F,R1,J,R2,S, after solving Eq. (12) simultaneously. Thus, we get the following equations:S=BS0δ1-(γδ1+S0ω1+ω2+η1+η2)JS0η1δ1 F=γJS0(β1+β2+η1) R1=(η1+η2+ω1+ω2)Jδ1 J=δ1R1ω1+ω2+η1+η2 R2=ω1J(ψ+η1+η3) 15 S=(ψω1+ω2ψ+η1+η3)Jη1ψ+η1+η3 Local stability of Equilibrium for Security Provisions in Smart Edge Computing Devices using consensus approach of Block-chain and Machine Learning Algorithms: Theorem 2 The Equilibrium for security breaches in a Smart Edge Computing Devices using consensus approach of Block-chain and Machine Learning Algorithms with τT,F,R1,J,R2,S, is locally asymptotically stable when S0>1; otherwise, it is unstable. Proof At the Equilibrium for Security Provisions in Smart Edge Computing Devices using Block-chain and Machine Learning Algorithms with τT,F,R1,J,R2,S, the variation matrix equation (12) becomes.16 τESPT,F,R1,J,R2,S=-(γJ+η1)0δ2-γT00γJ-(β1+β2+η1)0γT000β1-(δ1+δ2+η1)00000δ1-ω1+ω2+η1+η200000ω1-(ψ+η1+η2)0000ω2ψ-η1 The corresponding Eigen values are:λ1=-η1 λ2=-(ψ+η1+η2) After solving the fourth-degree polynomial, we can generate the other Eigen values as λ4+B1λ3+B2λ2+B3λ+B4=0, in which,B1=(γJ+η1+η2+δ1+δ2+ω1+ω2+4η1+η2) B2=γJ+η1β1+β2+η1+γJ+η1η1+η2+ω1+ω2+δ1+δ2+η1γJ+η1+β1+β2+η1ω1+ω2+η1+η2+β1+β2+η1δ1+δ2+η1+δ1+δ2+η1ω1+ω2+η1+η2 B3=(γJ+η1β1+β2+η1ω1+ω2+η1+η2+γJ+η1β1+β2+η1δ1+δ2+η1+γJ+η1ω1+ω2+η1+η2δ1+δ2+η1+ω1+ω2+η1+η2β1+β2+η1δ1+δ2+η1-γJβ1δ2-γTδ1β1) 17 B4=((γJ+η1β1+β2+η1ω1+ω2+η1+η2δ1+δ2+η1-γJ+η1γTδ1β1+γ2TJβ1δ1-γJ*δ2β1ω1+ω2+η1+η2) Through this, we get,18 B1>0,B3>0,B4>0B1B2B3>B32+B12B4⇒B1B2B3-B32+B12B4>0 Thus, through Routh–Hurwitz criterion, if S0>1, then the equation for Security Provisions in Smart Edge Computing Devices using Block-chain and Machine Learning Algorithms is locally asymptotically stable. Theorem 1 asserts the risk-free equilibrium if S0<1 and Theorem 2 gives an assertion for the equilibrium in the cases of security breaches if S0>1 in accordance with the Routh Hurwitz criterion. The locally asymptotically stable conditions determine the stability of equilibrium formed in the local block of data stored for mobile edge-based device. The risk-free equilibrium demonstrates that the security is established under the conditions that S0<1 and the data must be secured in local edge-based device if the security breach number (S0) is greater than 1. The globally asymptotic conditions determine the optimization of security criterion for several blocks forming a consensus approach based blockchain of secured data. The global asymptotic stability signifies that the device is secured under the data connection in a mobile edge based dynamic environment. Algorithmic representation of proposed steps In this paper, the algorithmic representation consists of two parts. The First algorithm will perform the classification using several classifiers and model them using metrics such as f1-score, accuracy. On the other hand, the second algorithm uses the federated learning techniques for implementing this model in an edge computing-based smart environment. Classification is a procedure for labelling the data into distinguished classes. There are several classification models in machine learning, such as Ridge Classifier, XGB Classifier, Logistic Regression, KNN classifier, Decision Tree Classifier, Random Forest Classifier, etc. [28, 29]. The authors implemented several standard classification models and found that the K-Neighbour Classification model is apt in terms of metrics such as accuracy and f1-score [30–32]. The K-Neighbour algorithm works by storing all the instances of the dataset that has been used in training using dimensional spaces. And using a simple majority approach from the neighbor nodes establishes the classification. Further, the authors considered several optimization algorithms such as S.G.D., Momentum S.G.D., Adam, and RMSprop for generating an optimized loss vs. epoch graph [33, 34]. Furthermore, the researchers used a voting classifier to ensemble multiple classifiers for the dataset of several blocks [35, 36].19 FpR=FPP 20 FNR=FNP 21 TpR=TPP 22 TNR=TNP 23 Accuracy=TN+TPTN+TP+FN+FP 24 Specificity=TNTN+FP 25 Precisionerror=TPFP+TP 26 Sensitivity=TPTP+FN In Eqs. (19) to (26), ‘FpR’ represents the false-positive ratio, ‘FNR’ represents the false-negative ratio, ‘TpR’ represents the true-positive ratio, and ‘TNR’ represents the true-negative Ratio whereas, ‘FP’ represents the number of false-positive cases, ‘FN’ represents the number of false-negative cases, ‘TP’ represents the number of true-positive cases, and ‘TN’ represents the number of true-negative cases [37, 38]. The accuracy, specificity, precision error, and sensitivity values for providing security provisions in smart edge computing devices using consensus approach of blockchain and machine learning algorithms can be obtained using Eqs. (23), (24), (25), and (26) respectively. The algorithmic representation for classification of data blocks using machine learning techniques for providing the security provisions in smart edge computing devices is given in Fig. 5.Fig. 5 Algorithmic representation for classification of data blocks using machine learning techniques Then they share the data with the clients and form the data for the runtime testing under a federated learning technique [38–40]. A federated learning technique implements the training of the algorithms over several decentralized edge computing enabled devices containing the blocks of data connected over the Blockchain [41, 42]. For the same, the authors implemented the steps given in Fig. 6 to obtain the aftermath in an edge computing-based environment for the security provisions.Fig. 6 Algorithmic representation for performing the data security in an edge-based environment The Algorithm 1 as shown in Fig. 5 implements several machine learning techniques on a single block of data and further approaches to determine a risk-free equilibrium for a locally asymptotically stabilized condition for a region τ0 defined by the block of data in an edge computing based mobile devices. The authors in this paper applied the simple training using naïve bayes classification and then improved the optimization using several models as per apt representation for the paper. The Algorithm 2 as shown in Fig. 6 henceforth processes the trained model using the methods proposed in Algorithm 1 for each block of data and hence performed a training using Multi-Layer Perceptron based model and obtained a risk-free equilibrium state in the total region defining the consensus approach based blockchain of data for globally asymptotically stabilized model. Experiments and result analysis In a dynamically changing non-linearity, the optimization of risk-free equilibrium can be achieved by solving the differential equations using machine learning in a supervised learning setup. In this paper, the authors considered the dataset having several features such as data vulnerabilities, data inconsistency for people of different ages, sex, location, etc. from various media portals namely press, print media, electronic media, television reports, radio coverage, and other social media such as Facebook, Twitter, LinkedIn, and Instagram. The dataset contains several vulnerable, exposed, recovered, and secured data. The labels were determined using the classification where the data displayed a risk-free equilibrium for locally asymptotic non-linear optimization when the security breach number S0 is less than 1 i.e., S0<1 the data portraying the risk of security breaches when security breach number S0 is greater than 1 i.e., S0>1 as per Theorems 1 and 2. Henceforth, minimization of S0 results in mitigating the security risk in an edge based locally asymptotic regions. Thus, the optimization goal is to minimize S0 with respect to increase in time. In this part the authors procured the dataset and classified them using the Naïve Bayes classification method to determine the class probability based upon the observed attributes [43, 44]. Further, the acquired data was normalized into the blocks of data with several chunks containing various severities. The re-sampling and approximation algorithms were applied on the blocks while keeping a check on the target class [45, 46]. Furthermore, the authors generated a heatmap using the dataset and observed the multiple features of the correlation matrix such as age, sex, cp, etc. Using the correlation matrix, the authors concluded that the Naïve Bayes classifier gave good results for the datasets. They observed aptly correct predictions for true labels tending towards 1.00, as presented in Fig. 7. The evaluation of a classification model is based upon the performance of metrics such as accuracy, precision, recall, F1 Score [47–49]. Thus, the authors find the accuracy and the F1 Score for the given classification. The accuracy score gives the fraction of correct predictions in the classification model [50, 51]. The F1 Score shows the harmonic mean of precision and recall. In our model, we obtained an accuracy of 86% and an F1 score of 87%, as per Appendix 1. Through these scores, the authors concluded that the model is classified correctly [52–54].Fig. 7 Generating Heat-map using Naïve Bayes Classification The authors appended several models given in Appendix 2, such as K-Neighbours Classifier, Decision Tree Classifier, Random Forest Classifier, MLP Classifier, etc. [55–57]. Further, they feed the data from several susceptible and non-susceptible blocks and resample them following the appended machine learning classification models [58–60]. The best Score for each model was calculated and compared. According to the dataset, K Neighbours Classifier procured the best results [61, 62]. The best Score for K Neighbours was found out to be 93.1% as mentioned in Fig. 8.Fig. 8 Result displaying the best Score achieved after using several models on our dataset Further, the authors generated a receiver operating characteristics curve (ROC curve) plotting the true positive rate vs. the false positive rate for the classification models [63–65]. The comparison of the classifiers can perform concerning area under the curve (AUC) [66, 67]. The performance of a classifier is directly proportional to the area under the curve in a ROC curve [68, 69]. In Fig. 9, the AUC for several classifiers has been depicted and it can be observed that the AUC values for K-Neighbours Classifier is highest, i.e., 0.873. According to the ROC AUC, the second-best classifier for the dataset is the MLP Classifier.Fig. 9 Generation of ROC curve for a different model used in our approach The classification report provides an insight into the behavior of the classifier around the global accuracy masking the multi-class datasets [70–72]. The authors analyzed and compared the classification report for several optimization algorithms used in training, such as Stochastic Gradient Descend Optimizer, Momentum Optimizer, Adam Optimizer, and RMSprop Optimizer [73–75]. The Stochastic Gradient Descent (SGD) optimizer generates the gradient of the cost function concerning the training dataset [76–78]. The authors developed the classification report for SGD optimizer in this paper. The classification report for Stochastic Gradient Descend Optimizer is presented in Fig. 10. The authors observed that class ‘0’ has a precision of 0.82 and recall of 0.76, further giving an F1 Score of 0.78 with a support of 41 observations. Class ‘1’ has a precision of 0.81 and a recall of 0.86, procuring an F1 Score of 0.83 with a support of 50 observations.Fig. 10 Classification report generated by using Stochastic Gradient Descend optimizer The momentum optimizer boosts the acceleration of the Stochastic Gradient descend in a particular direction and further stifle the oscillation of the gradient [79–81]. The classification report for Momentum Optimizer is presented in Fig. 11 [82, 83]. This optimizer suggests that class 0 has a precision of 0.76 and recall of 0.71, further giving an F1 Score of 0.73 with a support of 41 observations. Class 1 has a precision of 0.77 and a recall of 0.82, procuring an F1 Score of 0.80 with a support of 50 observations.Fig. 11 Classification report generated by using Momentum optimizer The Adaptive Moment Estimation implements the adaptive learning rates for every variable. It works as an amalgamation of AdaGrad and Momentum Optimizers [84–86]. The classification report for Adam Optimizer for data used in this paper is presented in Fig. 12 [87, 88]. This optimizer presents that class 0 has a precision of 0.74 and recall of 0.71, further giving an F1 Score of 0.72 with a support of 41 observations. Class 1 has a precision of 0.77 and a recall of 0.80, procuring an F1 Score of 0.78 with a support of 50 observations.Fig. 12 Classification report generated by using Adam optimizer The Root Mean Squared Propagation (RMSprop) portrays itself to use the decaying average of partial gradients implemented with the adaptation of step size measured for every parameter [89, 90]. The classification report for RMSprop Optimizer for data used in this paper is presented in Fig. 13 [91, 92]. This optimizer presents that class 0 has a precision of 0.74 and recall of 0.71, further giving an F1 Score of 0.72 with a support of 41 observations. Class 1 has a precision of 0.77 and a recall of 0.80, procuring the F1 Score of 0.78 with a support of 50 observations.Fig. 13 Classification report generated by using RMSprop optimizer The authors in this paper plot graph for loss which signifies the security breach, vs. Epoch. As per Theorem 1, if the security of the locally asymptotic region decreases significantly with increase in time, then the security of that specific region τ0 defined by the local block of data stored in an edge-based system increases. This stabilizes the equilibrium conditions of the risk-free state as mentioned in Theorem 1.This phenomenon illustrates time (in years) in x-axis and the minimization loss for the optimization function S0in y-axis for multiple optimizers as implemented in this paper [93, 94]. Figure 14 bestows a graph that gives the inference that SGD optimizer performs poorly in this model [95]. In the case of momentum of SGD, RMSprop performs significantly well in minimizing the data loss [96, 97]. The Adam Optimizer seems to be the best fit optimizer in this paper as we see that the time, the security breach is being minimized.Fig. 14 Generating a graph representing Training loss vs. Epoch using the different optimizers Then, the authors plot a graph in Fig. 15 between validation losses vs. Epoch, representing the possibility of security breach during the validation in terms of age (in years). The SGD optimizer gives us an insight that the model is under-fitting and needs some alteration in its parameters [98, 99]. The Adam Optimizer and RMSprop hint at over-fitting as there is a significant difference between the training and validation losses [100, 101]. The Momentum Optimizer gives better results for validation loss as the loss decreases concerning the time [102].The voting classifier implements the aggregation of different classifiers such as Random Forest Classifier, SVC (support vector classifier), Logistic Regression and predicts the class probability for the highest votes [103]. Thus, the authors implemented the ensemble classifier technique and aggregated the aftermaths procured from this model, as shown in Appendix 3 [104, 105]. In many cases, the ensemble classifier works better than the best classifier [106].Fig. 15 Generating a graph representing Validation loss vs. Epoch using the different optimizers The Area under the curve with true positive rate vs. false positive rate is plotted in Fig. 16 after the training of the voting classifier and found that the true positive rate is increasing better than individual classifiers [107]. This defined assertion for Theorem 1 which under the risk-free equilibrium of locally asymptotic stability confirms the secured atmosphere for batches of data. As per the optimization methods mentioned in Algorithm 1 depicted in Fig. 5, the ensemble classifier obtains a risk-free equilibrium condition in a locally asymptotic region. The aftermath for ensemble classifier is obtained with an area under curve (AUC) of 92%, which is better than K-Nearest Neighbour and all other classifiers implemented in this paper [108]. This result fostered the implementation of an Ensemble classifier in this model and minimized the security risk with several blocks of data [109].Fig. 16 Generation of area under curve for the model This paper determines the goal of securing the data over several blocks of the edge-based atmosphere each having locally stable regions τ0 and henceforth generating the combined stability of the data in a fully-fledged global server as per Algorithm 2 mentioned in Fig. 6. This serves the functionality of risk-free equilibrium in a globally asymptotic stabilized region. The results procured in this paper assert the security provisions for edge-based model using intelligent machine learning techniques. The authors in this paper secured an accuracy of 96% in the globally asymptotic regions. Table 2 describes the performance analysis of different edge-based algorithms synced with Blockchain and other technologies. Many algorithms worked for security in edge computing and Blockchain-based environment. These algorithms are compared on the metrics such as global accuracy, global loss, f1-score, and AUC curve. The proposed model using Edge Computing, consensus approach of Blockchain, and a voting classifier based on K-Neighbours, SVM, Random Forest, Logistic Regression lucidly performs better than the pre-existing algorithmic model. The authors shared the data blocks with several clients mapped with data shared amongst them. The data contained some vulnerable data, susceptible data as shown in Appendices 4 and 5. The influx of data with the clients is taken in the edge-based environment and henceforth trained using Multi-Layered Perceptron (Appendix 6). The global accuracy obtained using the following technique was observed as 96% with a worldwide loss of 1.51 as mentioned in Fig. 17, which gives a deduction that the model can efficiently minimize the risk of data leakage in an edge-based environment when the Data is shared amongst the clients in the form of blocks [110]. The consensus approach of Blockchain bolstered the security of the data in the edge-based environment.Table 2 Performance comparison of various security provision approaches for edge computing Algorithm name Classifier taken/technologies used Results/observation Global accuracy Global loss F1-score AUC 1 SDN Based Blockchain [12] S.D.N., Blockchain, Fog Nodes 87% 3% Not available Not available 2 Edge Chain [13] Edge Computing, Blockchain Not available Not available Not available Not available 3 Smart Grid [15] Smart Grids, Edge Computing, K-Nearest Neighbours 91% 8% Not available Not available 4 Hyper ledger Fabric Blockchain [17] Edge Computing, Hyper ledger Fabric, IoT Not available Not available Not available Not available 5 Hybrid Storage architecture [18] Blockchain, smart computing, Edge Computing Not available Not available Not available Not available 6 Model Chain [19] Blockchain, Machine Learning 78% 7% Not available Not available 7 Block Deep Net [20] Blockchain, Deep Learning 72% 5.4% Not available Not available 8 Deep Coin [21] Deep Learning, Blockchain, Smart Grid 92% 3.98 0.761 Not available 9 Deep Block Scheme [22] Blockchain, Deep Learning 89% 2.65 0.697 Not available 10 Asynchronous advantage actor-critic algorithm [23] Blockchain, Edge Computing, Deep Reinforcement Learning 76% 1.91% Not available Not available 11 Proposed approach Edge Computing, consensus approach of Blockchain, Voting Classifier based on K Neighbours, SVM, Random Forest, Logistics Regression 96% 1.5% 0.78 92% Fig. 17 Global accuracy and global loss as obtained after the training Applications of proposed work Edge Computing is a computing paradigm shift that paves the way for many modern computing applications. The theory associated with the proposed method can lead to several paths in the security of IoT, or widely used smart devices. As a result, we can say that the paper's applications are diverse and are given below:i. Banking is the first application that aptly defines the behaviour of mobile edge computing, because transactional data can be secured using consensus approach of blockchain and it allows for effective secured lines. ii. Medical wearables can be improved, and data poisoning can be reduced, allowing patient data to be secure and model training to improve in both local and global differential privacy. iii. The approaches used in this paper to build global and local model training frameworks can be obtained by an automated vehicle driving system. As a result, it will improve the driving experience in areas that have been trained for spatial models. iv. Gaming experiences based on cloud computing can be enhanced using the techniques provided, resulting in a secure and risk-free gaming environment. v. The advertisement sector can also benefit from having the noise parameters handy with the dataset in order to improve marketing strategies for specific products and secure their value in a global market. vi. The open networks provide several paths for security breaches. So, the anomaly detection can be performed while the network is sharing an application of the proposed model. Conclusions The authors used consensus approach of Blockchain and Machine Learning techniques to improve the security provisions in smart edge computing devices. The authors obtained the dataset, which contained susceptible data, exposed data, and recovered data etc. Further, the authors used several classification techniques, including Naïve Bayes, Random Forest, K-Neighbors, and SVM, and optimized with various optimizers, including SGD, Momentum SGD, RMSprop, and Adam. Furthermore, the authors combined these classifiers using ensemble classification techniques on several blocks of data and they obtained the best results using the ensemble classifier through these implementations. The AUC for the best classifier used in the model, the K-Neighbours Classifier, was 87% whereas the Ensemble classifier increased the accuracy to 92%. After training, the authors initiated the two-factor authentication process and assessed their vulnerability. Furthermore, the authors distributed the data in batches to several clients and carried out the federated learning in an intelligent Edge computing-based environment using a multi-layered perceptron model. Finally, the proposed model was trained with a global accuracy of 96% and a global loss of 1.51. The authors used consensus approach of Blockchain to maintain a data flow channel and provided a comparative analysis of machine learning and differential privacy approaches for obtaining secure data while including noise and other datasets in the actual information. The results obtained in this paper have room for improvement by performing the experiments from both the client and server sides using distributed and global differential privacy. Further research could be conducted by comparing state-of-the-art machine learning models with dynamic programming dataset-based algorithms. Experimenting with distributed and global differential privacy can be done on both the client and server sides. Appendix 1: Calculation of Accuracy score and F1 score using Naïve bayes approach Appendix 2: Appending models for the required classification of the dataset Appendix 3: Implementation of Ensemble classifier Appendix 4: Creation of clients for sharing the data using federated learning Appendix 5: Assigning data into the batches and then processing the train and test data for each client Appendix 6: Simple Multilayer perceptron model for the federated learning procedure Author contributions The third and fourth authors wrote the and conducted experiments in the supervision of first and second authors. Funding The fund for completing this work was given by Birla Institute of Technology, Mesra, Ranchi, India (Grant No. 00071). Data availability All data generated or analyzed during this study are included in this article (and its supplementary information files). Code availability The required software application or custom code is given in Appendices. Declarations Conflict of interest Being the corresponding author, I declare that there is no conflict of interest between the authors or with any organization. The information about relevant data (if any) has been provided by the authors in the manuscript. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Nguyen DC Ding M Pham Q-V Long L Seneviratne A Pathirana P Li J Niyato D Poor HV Federated learning meets blockchain in edge computing: opportunities and challenges IEEE Internet Things J. 2021 8 12806 12825 10.1109/JIOT.2021.3072611 2. Li Z RR-LADP: a privacy-enhanced federated learning scheme for internet of everything IEEE Consumer Electron. Magazine 2021 10 5 93 101 10.1109/MCE.2021.3059958 3. Nguyen DC Cheng P Ding M Lopez-Perez D Pathirana PN Li J Seneviratne A Li Y Poor HV Enabling AI in future wireless networks: a data life cycle perspective IEEE Commun. Surv. 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==== Front Drug Deliv Transl Res Drug Deliv Transl Res Drug Delivery and Translational Research 2190-393X 2190-3948 Springer US New York 36450964 1262 10.1007/s13346-022-01262-y Review Article Nanomedicines and nanocarriers in clinical trials: surfing through regulatory requirements and physico-chemical critical quality attributes http://orcid.org/0000-0002-5110-3582 Dri Diego Alejandro [email protected] Rinaldi Federica Carafa Maria Marianecci Carlotta grid.7841.a Department of Drug Chemistry and Technology, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy 30 11 2022 113 14 11 2022 © Controlled Release Society 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Elucidation of physical-chemical characteristics of investigational medicinal products should be established with suitable methodology. Characterization of nanomedicines and nanocarriers in clinical trials may require the definition of additional specific properties depending on the nature of the nanostructures or nanomaterials composing the investigational medicinal product. The availability of regulatory requirements and guidelines is investigated focusing on critical quality attributes for nanomedicines and nanocarriers, mapping them in a clinical trial setting. Current regulatory challenges and issues are highlighted. The increasing complexity of nanostructures, the innovative connotation of applied nanotechnology, and the lack in capillarity or misalignment of relevant guidelines and terminology may lead to a potential not standardized approach in the characterization of nanomedicines and nanocarriers in clinical trials and delays in the approval process. Further efforts and a proactive approach from a regulatory standpoint would be desirable to surf the wave of innovation that impact nanomedicines and nanocarriers in clinical trials, in order to support clinical drug development capitalizing on technological advances and still ensuring a strong regulatory framework. Graphical Abstract Keywords Characterization Clinical trials Critical quality attributes Nanocarriers Nanomedicines Regulatory ==== Body pmcIntroduction Requirements for a dossier to be submitted to the regulatory authorities (RAs) for a request of authorization of clinical trials (CTs) may differ from those required for the submission of a marketing authorization of a medicinal product. Information and data needed in the assessment of investigational medicinal products (IMPs) are mainly focused on the potential risks, inherent the specific nature of the product, and take into deep consideration, among others, the status of the drug development, the phase of the CT (phase I to IV) and its duration, the characteristics of the population in study (e.g., pediatric, vulnerable group of patients), the therapeutic area, and the specificity of the diseases, like in case of rare ones, or their pathology. The specifications set for the control of the drug substances used in the CTs, including the tests and their acceptance criteria foreseen for phase I or phase II CTs, may be reviewed and strengthened when the IMP is further tested in phase III CTs. Furthermore, additional parameters may need to be adjusted according to the clinical development stage. Detailed data on the IMPs manufacturing process may not be required unless critical new processes are implemented such as non-standard sterilization ones, which may not be reported in the Pharmacopoeia. However, when complex manufacturing processes are involved and the relationship between quality characteristics and in vivo performance is not perfectly demonstrated or even understood, such as with nanomedicines or nanocarriers, the manufacturing process and characterization of IMPs are critical information that is expected to be provided in the quality section of the investigational medicinal product dossier (IMPD) as part of the submission of an application for a request of a CT authorization [1]. On the other hand, in a marketing authorization, the expected use of the medicinal product in a wider number of patients implies that the state of the art of its quality must be ensured, illustrated in detail, including a consolidated and validated manufacturing process. Liposomal, micellar, and nanoparticulate preparations are considered specialized pharmaceutical dose forms deriving from non-standard processes and therefore requiring production scale validation data to be provided in the marketing authorization application dossier unless otherwise justified [2]. It is acknowledged that it may be challenging to set requirements able to cover into specific details every possible nature of a drug product. As an example, in the EU, general quality guidelines are available [3] and others also cover specific types of drug products such as IMPs [4], or multidisciplinary ones [5] including nanomedicines. During the benefit-risk assessment of CTs, dedicated guidelines for the evaluation of the quality of IMPs are taken into consideration; however, assessors at the RAs also refer to scientific guidelines on the quality of human medicinal products. This may be due to the lack of dedicated guideline intended to be applicable in a CT setting or due to the need of a more conservative approach because of the intrinsic characteristics of the IMP. It is not easy to identify CTs involving nanomedicines or nanocarriers in the public databases due to missing dedicated structured data fields in the clinical trial application (CTA) [6], able to code information on a nanomedicine, nanocarrier, or nanodevice and related characteristics. The sponsor of a CT is not prompted to declare if the IMP is a nanomedicine and to elucidate if the composition of the IMP includes nanocarriers, if due to the formulation of the IMP nanostructures or nanomaterials are involved, or if any nanotechnology is applied. Even now that the Clinical Trial Regulation (EU) No 536/2014 [7] is fully applicable in the EEA, the clinical trials information system [8] has no fields where the sponsor can state if the IMP is impacted by a nanotechnology. However, in the EMA pre-submission request form for a marketing authorization application [9], a checkbox is instead available to indicate if any nanotechnology applies. For CTs, the information can be only deduced by the description fields and by the text entered by sponsors in the databases, such as ClinicalTrials.gov [10], during the submission of a new application. This procedure has been used to retrieve the number of interventional CTs including the term “nano” and the trend in the number of submissions (Fig. 1) during the last 10 years.Fig. 1 Interventional clinical trials including the term “nano” with a study start date from 2010 to 2021. Searching criteria: other terms field, with interventional filter applied. Source: ClinicalTrials.gov (accessed 03 April 2022) It is not possible to distinguish how many CTs involve a nanomedicine rather than a nanodevice, if a nanocarrier is concerned, or if a specific nanotechnology is used; moreover, other databases [11] are even less informative. However, the growing trend in human drug product submissions to FDA containing nanomaterials [12] is confirmed, as also evidenced by the number of nanomedicines on the market [13, 14]. The increasing impact of nanotechnology in the manufacturing processes of nanomedicines and nanodevices is bringing along an increase in efficacy and accuracy and at the same time additional regulatory discussions and concerns on safety to human health and the environment [15]. Characterization of nanomedicines, their future perspectives, and a better understanding of the correlation between their physico-chemical properties and their pharmacokinetics need to be and are being widely investigated [16–19]. However, there is limited information on the regulatory framework and quality assessment associated to the clinical development phase of a nanomedicine or nanocarrier. Missing full description or not standardized characterization of nanomedicines tested in CTs may jeopardize the safety profile or represent weak development data, and may not considered sufficient to support the marketing authorization stage. We therefore investigate the current availability of regulatory requirements and guidelines on nanomedicines and nanocarriers and focus on potential critical quality attributes (CQAs), mapping them in a CT setting, where additional regulatory guidance and alignment across RAs of different regions is strongly needed so that during drug development phase, the safety of subjects in CTs is ensured and an early consistent approach to proper description and characterization is envisaged by sponsors limiting inconsistencies between early batches and commercial ones. The need of an improvement of the regulatory protocols is commonly recognized to be of fundamental importance to increase the industrial and clinical applications of nanomedicines. This is evidenced by a large number of papers addressing this issue in the recent years [20–22], including the Refine project [23], all attempting to support regulatory advances in the nanomedicine field. Regulatory requirements One of the most important regulatory steps in establishing the initial safety and efficacy profile of an IMP is the conduction of CTs. Such studies, carried out in humans, provide an opportunity to assess, for the first time, advances in pharmaceutical nanotechnology and the latest scientific innovations and advances in health care and prevention. During this crucial process, the quality of IMPs is assessed to confirm the physical-chemical characterization, the critical quality parameters, and function impacting the drug product performance and safety. The characteristics of nanotechnology-based products are challenging for regulatory approval processes and there are still many open questions in the regulation of nanomedicines and nanomaterials, starting from their assessment in a CT setting. Examples are related to not standard pharmacokinetics, environmental and accumulation issues, genotoxicity, representativeness of in vitro nanotoxicology tests, increased permeation, stability and manufacturing scale-up, nanomorphology and characterization, non-standardized terminology, and regulations [24]. In addition, limited dedicated guidelines are available to support quality, safety, and efficacy assessment of nanomedicines or nanocarriers in the specific context of CTs. A list of most relevant guidelines available to support medicinal product developers and CT sponsors in the preparation of the quality documentation presented in a request for authorization of CTs is reported in Fig. 2; the required information should be included in the chemistry manufacture and control (CMC) part of the IMPD for the evaluation of nanomedicines and nanocarriers. Looking at the incremental number of guidelines issued in the last 10 years, it is evident the attention and efforts that regulators dedicated to nanotechnology and its application to the pharmaceutical sector, even if it is also noted that RAs adopted different approaches and reacted with different timing. The trend in guidelines production reflects the impulse in sponsors’ submissions of CT applications containing nanotechnology-based products and highlights how the regulatory environment reacted to innovation when it has already reached the clinical trial stage.Fig. 2 Relevant guidelines on quality documentation and regulatory requirements for clinical trials, nanomedicines, and nanocarriers European Union (EU) Since 2012, the European Commission (EC) proposed in Europe a case-by-case approach to the assessment of nanomaterials [25]; however, in the document, there is no reference to CTs; a few opinions were also generated through the EC scientific committees on risk assessment of products of nanotechnologies and effects of nanosilver compounds [26, 27]. After the elaboration of a reflection paper on nanotechnology-based medicinal products for human use [28], EMA had a very productive period (2012–2016) in terms of reflection papers elaboration, on intravenous micellar systems [29], block copolymer micelle [30], intravenous liposomal products [31], coated nanomedicine products [32], and intravenous iron-based nano-colloidal products [33], followed by a break during the last 5 years. No general guideline on nanomedicines was ever developed, or a discussion launched on the need of a dedicated one in a CT context, which represents the front line where nanotechnology innovations applied to IMPs are facing for the first time a regulatory assessment process. The evaluation of innovation has been mandated to the innovation task force (ITF) in 2014 [34], and there are no signals that EMA intends to develop any further detailed guidelines or to review its own approach. Nevertheless, it is acknowledged that dedicated guidelines for CTs were developed and recently also updated, such as the guidelines on the requirements for the chemical and pharmaceutical or biological quality documentation concerning IMPs in CTs [35, 36]. The guideline for the chemical and pharmaceutical quality introduced in its scope, during one of the last updates, synthetic oligonucleotides, but the update did not incorporate any specific guidance on nanomedicines or nanocarriers, which are not cited. Even if huge efforts and progresses have been done, the regulatory framework for nanomedicines and nanocarriers in the EU remains fragmented and stratified [37] and would benefit from a harmonization process, including dedicated guidelines for early stages development and assessment in CTs. USA After the elaboration of a guidance to set the content and format of investigational new drug applications for phase I studies, the FDA elaborated in the first decade of the century also a guidance for industry to support with the use of current good manufacturing practice, for phase I investigational drugs [38, 39], and to support with the chemistry, manufacturing, and control information that would be submitted for phase II and phase III studies [40]. However, no reference to nanotechnology can be found in these documents. The increasing attention to nanotechnology products led to the publication in 2014 of an important guidance to discern whether an FDA-regulated product involves the application of nanotechnology [41] even if CTs were not specifically covered. There is no dedicated regulatory framework on nanomedicines, and the FDA assessment follows a product specific path, relying on consultation with sponsors to identify potential regulatory issues and on the assumption that the available requirements are sufficient to determine any potential toxicity profile. Consistent with other agencies, also, FDA privileges a case-by-case approach to the assessment of nanotechnology. However, it must be recognized that FDA reacted to the emerging application of nanotechnology with the publication of a fundamental guidance on drug products, including biological products that contain nanomaterials [42]. With this guidance, nanomaterial quality attributes and structural characterization of drug products containing nanomaterials were addressed, including in the scope eventually also CTs. A proportionate approach in the description and characterization of the IMP depending on the development stage is acknowledged, as far as it ensures safety during use in clinical trials. This represents a major step in the attempt to set a general framework for the identification of CQAs of the drug product, acknowledging also that the nanomaterial’s CQAs should be determined with regard to its function and potential impact on product performance. Recently, also, a guidance on liposome drug products was published [43]. Japan There is no evidence that a definition or a specific framework was ever adopted for nanomedicines in Japan that are regulated under the general framework of the pharmaceutical affairs law and seems to be evaluated on a case-by-case basis approach. However, a nanomedicine initiative working group is in place for discussions on regulatory requirements for nanomedicine development. The Pharmaceuticals and Medical Devices Agency (PMDA) and the Ministry of Heath Labour and Welfare (MHLW) collaborated in the preparation of the joint MHLW/EMA reflection paper on the development of block copolymer micelle medicinal products [30]. PMDA also assisted MHLW in the preparation of two management guidance on CT notifications containing the points to consider in case of some nanotechnology-based medicines [44]. A reflection paper on nucleic acids (siRNA)-loaded nanotechnology-based drug products [45] and a guideline for the development of liposome drug products [46] were issued in 2016 by MHLW. There is not a dedicated regulatory framework for the assessment of nanomedicines and nanocarriers in CTs. Canada Issues with nanomedicines in Canada are acknowledged since 2010 [47]. Benefit-risk evaluation and approval of nanomedicines are currently performed within the existing legislative and regulatory frameworks, there is in fact no explicit reference to nanomaterial in acts and regulations, and there is no dedicated guideline for the submission of nanotechnology products in CTs. However, Health Canada implemented a working definition of nanomaterials [48]. This is allowing to request specific information to improve the understanding of nanomaterials and for the assessment of potential risks and benefits or risk management purposes, of regulated product or substances, including therefore those in CT applications that may be or that may contain a nanomaterial. The types of information required are the intended use, function and purpose of the nanomaterial, and information regarding any end product in which it will be used; manufacturing methods; toxicological, eco-toxicological, metabolism, and environmental fate data that may be both generic and specific to the nanomaterial if applicable; and risk assessment and risk management strategies, if considered or implemented. The characteristics and physico-chemical properties that can be required on nanomaterials by Health Canada are available online [49]; however, there is no structured or dedicated approach for CTs. India An innovative and extremely interesting approach is the one adopted in India with the recent publication of the guidelines for evaluation of nanopharmaceuticals [50]. A definition of nanopharmaceutical and nanomaterial is provided (material having particle size in the range of 1 to 100 nm in at least one dimension), extending the range up to 1000 nm if the material exhibits physical, chemical, or biological phenomenon or activity, which are attributable to its dimension. An attempt to categorize nanopharmaceuticals either according to the degradability and nature of the nanomaterial or the nanoform of the ingredient is also presented. The guideline is listing a set of data that should be submitted to the RA in the submission of an application for a CT. In particular, data for the physico-chemical characterization of nanopharmaceuticals are defined, specifying that some of them need to be identified as CQAs, and that they should be listed along with the product specifications. In the specifications, moreover, apart from criteria for unique identification, identity and quantification of impurities, and stability data, it is explicitly required to provide in vitro/in vivo release kinetics of the drug/active ingredient (as applicable) and in vitro/in vivo degradation kinetics of the nanopharmaceutical in various simulated media. The added value with this approach resides in having a comprehensive list of general regulatory requirements for the evaluation of nanopharmaceutics and nanocarriers, applicable to CTs. However, and in alignment with the other RAs, it is recognized that information required for nanopharmaceuticals should be decided on case-by-case basis approach. Difficulties in the translation of nanotechnology health products into clinical application pass through several potential challenges such as the understanding of biological interaction, manufacturing complexity and costs, safety issues, and also facing regulatory standards [18], and dedicated efforts should be made in all these sectors. From a regulatory perspective, scientific challenges and regulatory needs for nanotechnology-enabled health products are well known, as discussed by the Joint Research Centre (JRC) [51]; however, additional guidelines particularly in a CT setting, where nanotechnology-impacted IMP would be assessed for the first time, would help in streamlining the overall process and in anticipating regulatory challenges that may be expected during the marketing authorization step. Developing a dedicated guidance for CTs would help to ensure that sponsors acknowledge the submission of any nanotechnology-impacted IMP, nanomaterial, or nanostructure associated with an IMP, such as innovative nanocarriers or supramolecular structures, that may affect the stability, the PK/PD properties, the size, the drug encapsulation efficiency, or the targeting properties, and that may carry along with innovation also unknown risks. In the case of non-ionic surfactant-based nanocarriers (e.g., niosomes, nanoemulsions, micelles), the elucidation of excipients and surfactant role in the IMP formulation should be explored and reported, focusing on in vivo stability or safety issues, and potential toxicity issues could be investigated in ad hoc designed CTs [52]. Coding in a guideline the minimum set of requirements to control the potential impact of nanotechnology on the safety profile of nanomedicines or nanocarriers would help to ensure that a sufficient and transparent level of data are submitted in a CT application and that chemistry manufacturing and control information meet regulatory requirements starting from an early-stage clinical development. The final desired scope would be to support the manufacturing process to achieve a desired quality drug product focusing on CQAs. Envisaging a risk-based approach or exploring other regulatory strategies could support a more proactive and dynamic regulatory framework, able to support innovation in nanotechnology, and drive efforts towards a personalized approach to medicine. But it still would need to ensure that a strong regulatory framework is in place. The question to address is if the current static regulatory framework is still able to support the emerging technology development or if a regulatory conceptual evolution is needed. Critical quality attributes A CQA is defined as a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality of a drug substance, excipients, intermediates (in-process materials), and drug product [53]. But how can we expect to define CQAs for nanomedicines or nanocarriers if there is still no consensus on the definition of the used terms? There is no standardization in the use of scientific terminology; many different terms are used as synonyms such as nanomedicines, nanoparticles, nanodrugs, or nanopharmaceuticals [54]. Even when the same term is used, in a regulatory environment, there are still misalignments in the adoption by different regulatory bodies. In 2011, the EC published a recommendation on the definition of a nanomaterial [55, 56]; however, EMA issued a definition of nanomedicines [28] and, in relation to quality aspects of nano-based medicines, adopted a working definition [57, 58]. The EC recently updated the definition of nanomaterial in a new recommendation [59] with the aim to support a coherent EU regulatory framework for nanomaterials and help to align legislation across all sectors. However, it is still not aligned with RAs in other regions, as it sets the size limits for the size range to 100 nm in contrast to the FDA guidance. The use of the term nanomaterial in FDA guidance documents does not represent a regulatory definition, and neither a definition is available for the term nanotechnology, nanoscale, or other related terms; in Canada, a working definition of nanomaterial is adopted [60]; a definition of nanomaterial can instead be found for India in the guidelines for evaluation of nanopharmaceuticals [50]. Even when a definition is provided, such as in the case of ISO standards [61], an approximate terminology is adopted when defining the nanoscale (approximately from 1 to 100 nm). Size limit adoption in the definitions is arbitrarily used and definitely not appropriate, at least in a CT setting and for medicinal products, where physical and chemical properties dependent on size continue to apply across regulatory imposed definitions [62]. Divergent descriptions or definitions of the term nanomaterial or nanomedicine are reported in Table 1.Table 1 The description or definition of nanomaterial or nanomedicine according to different regulatory bodies Regulatory body Description/definition of the term nanomaterial or nanomedicine EC Recommended definition “Nanomaterial” means a natural, incidental, or manufactured material consisting of solid particles that are present, either on their own or as identifiable constituent particles in aggregates or agglomerates, and where 50% or more of these particles in the number-based size distribution fulfill at least one of the following conditions: (a) One or more external dimensions of the particle are in the size range 1–100 nm (b) The particle has an elongated shape, such as a rod, fiber, or tube, where two external dimensions are smaller than 1 nm and the other dimension is larger than 100 nm (c) The particle has a plate-like shape, where one external dimension is smaller than 1 nm and the other dimensions are larger than 100 nm In the determination of the particle number-based size distribution, particles with at least two orthogonal external dimensions larger than 100 µm need not be considered. However, a material with a specific surface area by volume of < 6 m2/cm3 shall not be considered a nanomaterial EMA Definition The nanometric scale ranges from the atomic level at around 0.2 nm (2 Å) up to around 100 nm Nanomedicine is defined as the application of nanotechnology in view of making a medical diagnosis or treating or preventing diseases. It exploits the improved and often novel physical, chemical, and biological properties of materials at nanometric scale Working definition • Purposely designed systems for clinical applications • At least one component at nanoscale size that should not exceed 1000 nm • Resulting in definable specific properties and characteristics FDA Description Materials falling within either point 1 or 2 (1) A material or end product is engineered to have at least one external dimension, or an internal or surface structure, in the nanoscale range (approximately 1 to 100 nm) In addition, because materials or end products can also exhibit related properties or phenomena attributable to a dimension(s) outside the nanoscale range of approximately 1 to 100 nm that are relevant to evaluations of safety, effectiveness, performance, quality, public health impact, or regulatory status of products: (2) A material or end product is engineered to exhibit properties or phenomena including physical or chemical properties or biological effects that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to 1 µm (1000 nm) Health Canada Working definition It is at or within the nanoscale in at least one external dimension, or has internal or surface structure at the nanoscale It is smaller or larger than the nanoscale in all dimensions and exhibits one or more nanoscale properties/phenomena For the purposes of this definition: The term “nanoscale” means 1 to 100 nm, inclusive The term “nanoscale properties/phenomena” means properties which are attributable to size and their effects; these properties are distinguishable from the chemical or physical properties of individual atoms, individual molecules, and bulk material The term “manufactured” includes engineering processes and the control of matter India Definition The nanomaterial is generally defined as material having particle size in the range of 1 to 100 nm in at least one dimension. However, if a material exhibits physical, chemical, or biological phenomenon or activity which is attributable to its dimension beyond nanoscale range up to 1000 nm, the material should also be considered nanomaterial. Therefore, any pharmaceutical containing such material should also be considered nanopharmaceutical. ISO/TS 21623:2017 Definition Material with any external dimensions in the nanoscale or having internal structure or surface structure in the nanoscale (length range approximately from 1 to 100 nm) To identify regulatory needs and to enhance any static communication, sharing best practices and knowhow across regulatory agencies, a survey was recently conducted to identify regulatory experience with nanomedicines, information needs of regulators for the categorization and characterization of nanomaterials, and further steps that could support the acceptance of nanotechnology-based products in health care [63]. It was confirmed that regions have a different level of expertise in marketing nanomedicines, and that sharing experience and collaboration of regulatory bodies in the assessment of nanotechnology-based products would be an added value to face the foreseen increasing number of nanotechnology applications. However, to follow such a path, the prerequisite is a consistent terminology and categorization of nanomedicines, supporting communication and collaboration among regulatory bodies. When a list of nano-specific characteristics is proposed as relevant for the approval of CTs, even if not exhaustive, there is clear evidence that there is still not full alignment in the identification of crucial characteristics across regulatory bodies; this is not supporting sponsors in compiling quality data for the purpose of submitting international CTs in different regions. The consolidated takeaway message is that there is a strong need of an accurate characterization of physico-chemical properties by appropriate analytical methods of toxicity assessments including in vitro and in vivo testing, and that crucial to ensure the quality of nanomedicines is to focus on better understanding CQAs. The availability of a global list of CQAs and of a dedicated guideline would support the submission of quality data for nanomedicines and nanocarriers in CTs, and potential early access to nanotechnology health products would be streamlined, as detailed in Fig. 3.Fig. 3 Regulatory path forward for a CQA consolidation and nanotechnology health products potential early access Innovation in nanotechnology is a very fast evolving field, and regulatory frameworks are not always able to keep up with the speed of the ultimate nano-construct and potential application to the health sector, some are even creating new paradigms such as evolvable platforms for programmable nanoparticle-based therapies [64]. An evident contradiction is highlighted when these valuable projects are receiving public funding, but it is on the other hand clear that the final outcome could not be potentially supported by an equally dynamic and receptive regulatory framework. Even if many efforts have been spent by regulatory bodies so far in the field of nanomedicines and nanocarriers, the combination of speed of technology evolution and the increasing complexity of innovation seems to be an obstacle for regulators, unless a different approach and mindset are adopted by involving all potential stakeholders in the definition of a fit-for-purpose regulatory system [65]. Additional approaches are also proposed by stakeholders in the EU, including a centralized regulatory procedure, the harmonization of requirements to characterize nanomedicines, a scientific consensus on definitions, improved education, and a fostering of awareness on the complexity of nanomedicines [66]. However, regulatory acknowledge of innovation in the healthcare nanotechnology setting is definitely possible, as the Covid-19 emergency is showing, with the bursting introduction of the new technology based on lipid nanoparticles for mRNA vaccine delivery [67, 68]. There is a clear identification by EMA as a regulatory science research need, the one to develop an understanding of, and regulatory response to, nanotechnology and new materials in pharmaceuticals anticipated to be used in the coming 10 years [69]. Even if a fervent activity is noted across stakeholders as evidenced by the recent results of the Refine project in terms of future perspectives for advancing regulatory science of nanotechnology-enabled health products [70], it is still missing a dedicated discussion on the impact of nanotechnology innovation in the assessment of CTs. Nevertheless, there is a current activity carried out in the context of the EU innovation network horizon scanning with a topic proposal on nanotechnology, where hopefully this issue may emerge. To start supporting with such a process, stemming from the physical and chemical characterization of general nanomaterials-nanomedicines-nanopharmaceuticals and potential CQAs [71], we report in Table 2 the main critical parameters required in CTs for general types of nanomedicine as required by different regulatory bodies [42, 49, 50].Table 2 Cumulative CQAs for general types of nanomedicine according to different regulatory bodies Critical parameter Health Canada [49] (nanomaterial) FDA [42] (nanopharmaceuticals) DBT [50] (nanopharmaceuticals) Composition Entrapment efficacy Drug loading • Composition • Identity • Chemical composition • Assay of drug substance • Complete description of individual component(s) [e.g., API, nanocarrier material (single/multiphase) surface functional groups, contrast agents (theranostic), excipients, targeting ligands, etc.] • Encapsulation efficiency, loaded versus free drug content, with standard deviation • Percentage of drug loading with standard deviation Size • Particle size/size distribution • Average particle size • Particle size distribution (PSD) (description of d10, d50, d90, or polydispersity; modality) • Particle concentration • Nano-size range by number and/or intensity distribution, average size and polydispersion index, percentage of particle under each distribution with standard deviation • Molecular weight (drug and nanomaterials) Morphology • Morphology • General shape and morphology (aspect ratio) • Coating properties, including how coatings are bound to the nanomaterial • Porosity (if it relates to a function, e.g., capacity to load a drug) • Shape, surface texture information • State of drug (API) in nanomaterial (chemically conjugated/loaded/complexed with the nanocarrier) Stability • Agglomeration/aggregation (or other properties) • Chemical reactivity • Stability, both physical (e.g., aggregation and agglomeration or separation) and chemical • Particle concentration • Distribution of any drug substance associated with the nanomaterial and free in solution (e.g., surface bound or liposome encapsulated versus free drug substance) • Colloidal stability information for injections (06 batches) Structure Surface Nomenclature General properties • Structural integrity • Surface area/chemistry/charge/structure/shape • Surface-to-volume ratio • Water solubility/dispersibility • Electrical/mechanical/optical properties • Structural attributes that relate to function (e.g., lamellarity, core–shell structure) • Surface properties (e.g., surface area, surface charge, chemical reactivity, ligands, hydrophobicity, and roughness) • Crystal form • Empirical formula (drug and nanomaterials) • Chemical name, structure, crystal structure of drug and nanomaterial(s) • Surface charge with standard deviation (zeta potential) • Mechanical integrity/Properties (as applicable) • Viscosity (wherever applicable) • Average pH (wherever applicable) • Osmolality (wherever applicable) • Solubility/dispersion information (for injectable product) Impurities • Purity • Impurities • Residual solvent content as per ICH guidelines Sterility Apyrogenicity --- • Sterility, endotoxin levels, and pyrogenicity • Endotoxin/microbial load level for parental nanoformulations • Sterilization protocols/methods/stability post sterilization (as applicable) Drug release --- • In vitro release --- Other • Descriptions of the methods used to assign these determinations • Catalytic or photo-catalytic activity • Biodegradability of the nanomaterials and their constituents • Compatibility of the nanomaterial relevant to in-use conditions • Scalable GMP process description of the nanopharmaceutical preparation • Waste disposal method There is alignment in terms of most critical parameters: composition/entrapment efficacy drug loading, size, morphology, stability, structure/surface charge/nomenclature/general properties, and impurities. However, a different level of detail is provided by regulatory bodies in the requested information. This should not be read as a missing scrutiny of details in terms of characterization required by the RAs, as the assessor would require additional information if needed to guarantee the safety and positive benefit-risk balance. However, the approval process may be slowed down and not all required and updated information may be provided by the sponsor during the initial submission, particularly for multi-regional international CTs. Not all regulatory bodies instead are fully aligned with requests on sterility/apyrogenicity and especially drug release. Other requirements such as catalytic activity or GMP process description are also reported. CQAs are identified also for specific types of nanomaterials-nanomedicines-nanopharmaceuticals [29–33, 43, 45, 46], and even if parameters cannot be generalized, they should be taken into deep consideration as additional input on data and/or methods that can support or accelerate the process of identification of adequate characterization for new or forthcoming nanotechnologies. Among data to be considered and characteristics to be described also for general types of nanomedicines, particularly in terms of additional stability data and carrier functionality, the following not exhaustive list of features that are already coded for specific types of nanomedicines guidelines, such as liposomes or nucleic acid-loaded nanocarriers, could be in our opinion expanded in their scope and suggested as supportive also for general types of nanomedicines, as reported in Table 3.Table 3 Additional proposed parameters to be considered for nanomedicine characterization Critical parameter Description of additional parameters for consideration Morphology Bilayer characteristics, if a bilayer is present Stability studies over time at different storage temperature Stability studies in different media and at specific temperature to simulate biological environment Stability Stability studies taking into account in vivo administration (resistance to nebulization, or lyophilization resistance) Interaction and stability studies over time with biological environment (e.g., mucin interaction) Stability studies in terms of decomposition/degradation of drug loaded inside the nanocarrier in comparison with the free drug Surface Surface derivatization (PEG-targeting moieties) Polarity, microviscosity, rigidity and distribution of drug substance within nanocarriers, studies of nanocarriers in correlation with release capability Drug release In vitro drug release experiments in physiologically, clinically relevant media Selection of appropriate in vitro and in vivo models to assess nanomedicine efficacy Toxicity Toxicity evaluation of each nanocarrier’s component in comparison with components organized in the nanocarrier structure at the same concentration Conclusion There is no evidence of a unique rather than standardized approach in the development of guidelines and reflection papers for the evaluation of nanomedicines and nanocarriers, especially when focusing on IMPs in a CT setting. RAs are reluctant to adopt definitions of related terms such as nanomaterial, and prefer to rely on descriptions or working definitions that differ each other. The first step in removing obstacles through a potential standardization process would be the mutual recognition of terms used as synonyms in the same regulatory context. Regulatory bodies have been reacting with different timing and approaches to the emerging innovation of nanotechnology applied to the manufacture of medicinal products. Even if numerous guidelines have been recently developed and profound efforts have been made, it has not been possible to establish a consolidated and valid global platform to frame, from the point of view of the characterization in CTs, all the types of known nanomedicines and nanocarriers but especially those in development or yet to be discovered. Even if there is evidence of an attempt to provide a comprehensive list of requirements for the characterization of nanomedicines and nanocarriers in CTs, the common denominator across regulatory bodies is still a case-by-case basis approach. Available guidelines are fragmented and not aligned across different regulatory bodies. Additional efforts in the definition of the CQAs and the requirements for the characterization of nanomedicines and nanocarriers should be pursued and would be of benefit to encode them in the regulatory framework dedicated to CTs. CQAs should consider physico-chemical but also technological and biological attributes deriving from the potential transformations and fate of the product in the human body, including those that are function of their performance, or deriving from potential interactions and degradation processes in subjects. However, there will never be a convergence in CQAs if a semantic definition of what should be characterized is not achieved first. The high level of alignment actually available across regulatory bodies of different countries, in the use of a case-by-case basis approach during the evaluation process of a nanomedicine or nanocarrier, reflects the need to deal with continuous scientific, technological, and academic advances and increasing knowledge and expertise in manufacturing processes. Regulatory bodies should capitalize on the experience already acquired to envisage a more global and structured approach, potentially encompassing a renewed risk-based methodology or risk proportionate approaches in clinical trials [72, 73]. Current regulatory challenges and issues are highlighted, mainly due to the speed and complexity of innovation of nanotechnology applied to the health sector. The current lack in capillarity of relevant guidelines, particularly in a CT setting, can lead to a potential missing standardized approach in the characterization, and therefore not fully addressing CQAs, or in delays in the CT authorization process. Among major challenges for regulators in the coming years is the missing uniformity of regulations and guidelines and standardization of requirements for the characterization and control of nanomedicines and nanocarriers in CTs. To surf through the waves of the current and upcoming healthcare innovation and applied nanotechnology, a change of pace and approach to the evaluation of nanomedicines, nanocarriers, and nanotechnology health products is needed, starting from the CT setting. There is probably need to re-think how an already strong regulatory framework can increase efficiency by adapting itself, passing through its own innovation, encompassing for example a more inclusive strategy, with a higher level of involvement and early collaboration with stakeholders so that facing regulatory challenges would be part of the nanotechnology design and development phase. Author contribution All authors contributed to the study conception and design. Data collection and analysis were performed by Diego Alejandro Dri, Federica Rinaldi, Maria Carafa, and Carlotta Marianecci. The first draft of the manuscript was written by Diego Alejandro Dri and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of data and materials Data sharing is not applicable to this review article as no new data were created or analyzed in this study. Declarations Ethics approval and consent to participate Not applicable. 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==== Front J Anesth J Anesth Journal of Anesthesia 0913-8668 1438-8359 Springer Nature Singapore Singapore 36449089 3144 10.1007/s00540-022-03144-6 Original Article Efficacy of an aerosol suction device Free-100 M in removing aerosols produced by coughing to minimize COVID-19 infection http://orcid.org/0000-0002-4307-5032 Fujishiro Asuka [email protected] Asai Takashi Saito Tomoyuki Okuda Yasuhisa grid.416093.9 Department of Anesthesiology, Dokkyo Medical University Saitama Medical Center, 2-1-50 Minamikoshigaya, Koshigaya, Saitama 343-8555 Japan 30 11 2022 15 28 8 2022 20 11 2022 © The Author(s) under exclusive licence to Japanese Society of Anesthesiologists 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose The healthcare workers are at the greatest risk of being exposed to viral infection during airway management of a patient with coronavirus disease 2019 (COVID-19). An air extractor which suctions air around the patient's face would reduce the spread of viral aerosols during coughing, but no study has confirmed this. We assessed whether or not an air extractor reduces the amount of aerosols spreading toward the operator's face, during coughing of simulated patients. Methods After obtained approval of the study by a research ethics committee and written informed consent from 20 volunteers (and additional 20 volunteers), we asked each volunteer to lie supine on a table in a positive-pressure management operating room. As a cross-over design, we used an airborne particle counter (Handheld 3016, SGY company, Tokyo) to measure the aerosols approximately 30 cm above the participant's mouth, while the volunteer was coughing, with and without the use of an air extractor Free-100 M (Forest-one, Funabashi), facing the participant's mouth. In another 20 volunteers, the aerosols were measured, while each volunteer was lying supine, without coughing, and without the use of the air extractor. Results The aerosol count during coughing was significantly lower when the air extractor was used [median: 55 (interquartile range: 15–128)] than when it was not used [73 (44–201)] [p = 0.001, difference: 19 (95%CI: 4–70)]. Conclusions The Free-100 M air extractor would reduce, but do not remove all, aerosols produced by coughing of a patient, and thus may reduce the risk of infection of COVID-19. Keywords Airway management COVID-19 Aerosols Aerosol suction device ==== Body pmcIntroduction Severe acute respiratory syndrome-corona virus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is highly contagious, and is mainly transmitted through droplets and aerosols emitted from a patient with COVID-19, so that the healthcare workers are at the greatest risk of being exposed to viral infection during airway management [1–4]. Several methods have been proposed to protect healthcare workers from infection during airway management [1–3]. Standard personal protective equipment (PPE), such as P2/N95 masks, goggles, gloves, face-shields, and gowns, is recommended to wear during airway management, but it may not fully prevent viral infection. An "aerosol containment device", which covers the patient's head or the upper body, has been proposed to prevent spread of droplets and aerosols during airway management, but studies have shown that its use may not reduce, but may even increase, the risk of healthcare workers being exposed to a higher concentration of viral aerosols [3]. To prevent the problem of viral aerosols to escape out of an aerosol containment device, some have produced a negative air-flow environment by applying a suction mechanism [2]. One major problem with an aerosol containment device (with or without applying suction mechanism) is that airway management becomes more difficult [3, 5]. In addition, the spread of aerosols is marked when the patient is coughing, whereas it is minimal after rapid-sequence induction of anesthesia and neuromuscular blockade [6], so that the healthcare workers are at the greatest risk of being exposed to viral aerosols before or during placement of an aerosol containment device. We considered that an alternative method would be to place an air extractor to suction air around the patient's face (without placing an aerosol containment device). As a preliminary study, we carried out a simulation study [4], and have shown that an air extractor could successfully remove simulated viral aerosols, which were produced using a small-volume nebulizer placed in the mannequin’s mouth. Nevertheless, the velocity and the area of spreading droplets and aerosols during real coughing may be different from those of simulated aerosols, and thus the efficacy is not known of an air extractor in minimizing aerosol spread during coughing. The aim of the current study was to assess whether or not an air extractor reduces the amount of aerosols spreading toward the operator's face, during coughing of simulated patients. Methods We considered that it would be ethically not acceptable to study patients, as the efficacy of an air extractor has not been validated in humans, and thus we decided to carry out a study in 20 volunteers who had received vaccinations, whose body temperature had been within normal limits (< 37.5 degrees Celsius) for the last 4 weeks, and had not been close contacts with patients infected with SARS-CoV-2. Research ethics committee approved the study (approval number: 21002), and we obtained written informed consent from all the volunteered participants. Although this was an observational study, we have registered this study with JRCT (Japan Registry of Clinical Trials: jRCT1032210212). As a prospective crossover design, we asked each participant to lie supine on an operating table (with the height of approximately 1.0 m) which was placed in the center of an operating room with clean laminar airflow entering through the ceiling and exiting through wall vents, keeping the room at positive pressure. We used a handheld airborne particle counter (Airborne particle counter Handheld 3016, SGY company, Tokyo, Japan), which can simultaneously count particles of 6 different sizes (0.3, 0.5, 1.0, 2.5, 5.0 and 10.0 μm), and displays cumulative and differential data for particle counts. The measurement efficiency of the counter is 50% at 0.3 μm and 100% at 0.45 μm or more (ISO 21501–4 compliant). The particle counter was fixed to an intravenous stand. The monitor port was set approximately 30 cm above the participant's mouth. This distance was chosen, because an operator's face would be approximately 30 cm above the patient's mouth, when the operator performs airway management (manual ventilation using a facemask, insertion of a supraglottic airway, or tracheal intubation). To suction air around the patient's nose and the mouth, we used the Free-100 M (Forest-one, Funabashi, Japan) [4], which consists of a suction port, a length-variable swing arm, and directed high flow suction (with a 12-phase power vacuum), with ultra-low penetration air (ULPA) filter that removes 99.99% of 0.15 µm airborne pathogens (Fig. 1). The level of suction volume can be adjusted from 1 to 12, with a suction speed of 800 l.min−1 at the level 1 setting, and of 3,650 min−1 at the level 12 setting. For this study, we set the suction level of 12.Fig. 1 Free-100 M air extractor (Forest-one, Funabashi, Japan) The air extractor was placed by the operating table, and its suction port was placed approximately 20 cm above of the participant's chest, facing the participant's mouth. The amount of aerosols during coughing were measured, with and without the use of the air extractor, while the participant was coughing three times (of separate timings) in 30 s. The air extractor was used on three sessions of coughing, whereas the air extractor was not used on the other three sessions of coughing. Each experiment was conducted twice per person, and mean values were used for analysis. Timings of the use and non-use of the air extractor were made in a random order. The random order was made using a block randomization (in block of 6). For each measurement, the amount of aerosols were counted for 30 s. We counted the total number of aerosols of 0.3, 0.5, 1.0, 2.5 and 5.0 μm for each session. We also measured the amount of aerosols in the operating room, with the same setting, but without a volunteer on the operating table, and without running the air extractor. The primary outcome measure was aerosol counts, and the main hypothesis was that aerosol counts would be lower when the air extractor was used than when it was not used. Wilcoxon signed rank sum test was used to compare the aerosol counts with and without the air extractor, as the data were not normally distributed. P < 0.05 was judged significant. The 95% confidence intervals for the median difference in the aerosol counts were also calculated to estimate the difference. Power analysis was carried out for the primary outcome measure (the aerosol counts during coughing, with and without the use of the air extractor). The null hypothesis for this was that there was no higher or lower aerosol counts between the two circumstances, and thus the proportion of an event (either higher or lower) in the population (π) was 0.5. Cohen [7] defined the following effect size (g = π–0.5) conventions: small (0.05), medium (0.15), and large (0.25). We considered that the difference with and without the use of air extractor would be clinically meaningful, when the effect size was larger than the Cohen’s “large” (0.25), and thus we defined the effect size of 0.3 would be clinically meaningful. Twenty patients would be required to detect this size of the difference, with a power of 0.8, and P = 0.05. Responding to a reviewer’s comments to the initial draft, we carried out an additional study. Research ethics committee approved this additional study (approval number: 22088), and we obtained written informed consent from all the volunteer participants. Because this was an additional study, 20 volunteers in this part of the study were different from those for the main study. In this additional study, we asked each participant to lie supine on an operating table with the same settings for the main part of the study. The amount of aerosols was measured, while each volunteer was asked not to cough, and without running the air extractor. Mann–Whitney U test was used to compare the aerosol counts with and without the presence of a volunteer on an operating table (as the data were not paired). P < 0.05 was judged significant, as this was regarded as a subsidiary information. Results The amount of aerosols in the operating room, without a volunteer on the operating table, and without running the air extractor were 0 in 17 of 20 measurements, and 1 to 6 counts in the remaining 3 measurements, indicating that the amounts of aerosols in the operating room are approximately 0 (Fig. 2). An additional study has indicted that with a volunteer on the operating table, not coughing, and without running the air extractor, the amount of aerosols varied from 0 to 72, with the median of 3.5 (Fig. 2). The amount of aerosol measured was significantly higher when a volunteer was present than when a volunteer was absent on the table (P = 0.0043).Fig. 2 Aerosol count (30 cm above the participant's mouth) during coughing, with and without the use of an air extractor (Free-100 M, Forest-one, Funabashi, Japan), and while a volunteer was or was not lying on an operating table (without suctioning of air) (scatter plots with the medians and interquartile ranges). A dotted vertical line is drawn, as the data for “volunteer, not coughing, without suction” were taken from different volunteers from those in the main part of the study The aerosol count during coughing was significantly lower when the suction system was used [median: 55 (interquartile range: 15–128)] than when it was not used [73 (44–201)] [p = 0.001, difference: 19 (4–70)] (Fig. 2). Discussion We have confirmed that Free-100 M air extractor significantly decreased the amount of aerosols spread by coughing of simulated patients toward the operator's face, but a considerable amount of aerosols was sometimes detected even when the air extractor was being used. In a previous simulation study, we have visually confirmed that the air extractor could remove all the simulated viral aerosols, which were produced using a small-volume nebulizer placed in the manikin’s mouth [4]. Another study has also shown that an air extractor was effective in removing aerosols during simulated continuous breathing and a manikin-simulated cough [8]. Therefore, these simulation studies have indicated that an air extractor can prevent spread of viral aerosols emitted during coughing. Although we did not measure the volume of air emitted from each participant, the volume of air emitted by coughing should be less than the forced expiratory volume, and has been reported to be 0.5 l to 4.8 l, with the peak flow rate of up to approximately 930 l.min−1 (or 16 l.sec−1)[9]. The Free-100 M air extractor can suction up to 3,650 l.min−1 (60 l.sec−1) of air, so that suction volumes would be far much greater than the maximum volume of air emitted during coughing. Since the Free-100 M air extractor can suction 1,200 l.min−1 (20 l.sec−1) of air at the level 2 setting, the Free-100 M air extractor, in theory, can suction expired air during coughing by setting the suction level of 2 or greater. Despite the fact that these simulation studies and theoretical calculations indicate that the air extractor can effectively prevent spread of viral aerosols emitted by coughing, this study has shown that the Free-100 M air extractor reduces, but cannot remove all, the aerosols emitted by coughing. The reason is not clear for the considerably high aerosols were detected around the operator’s face during real coughing, while an air extractor was kept running. Nevertheless, one possible reason is that aerosols produced by coughing spread radially with a high velocity, so that a small suction port of the air extractor placed above a participant’s chest could not effectively suction the aerosols spread around the suction port. In our study, the suction port of the air extractor was placed approximately 20 cm above of the participant's chest, facing the participant's mouth. The efficacy of the air extractor may well have been affected by the location and the direction of the suction port, so that this positioning might not have been the optimal. Limitations of the study include that we did not study how effectively the use of an air extractor reduces the incidence of infection of the operator (true outcome measure). Nevertheless, it would be reasonable to deduce that the use of air extractor would reduce the spread of aerosols around an operator’s face, and that its use would reduce, but may not reliably prevent, the risk of infection of operator. Another limitation is that it is not clear whether or not all the amount of the aerosols detected by the counter were emitted from the participant’s airway during coughing. In a preliminary study, we noticed that the aerosol count in an “ordinary” room (even without anyone there) could be as high as 10,000, and this count did not change even when the air extractor was turned on. This indicates that in an “ordinary” room contains relatively high density of aerosols, and even if an air extractor effectively removes the air, the surrounding air (which would contain the same density of aerosols) would fill the space. Therefore, it would be technically difficult to determine how effectively the air extractor can remove the aerosols emitted from a patient. To minimize this difficulty, we chose to study in an operating room with positive-pressure management, where the density of aerosols in the air is much lower than the air in an “ordinary” room, and in fact, the count of aerosols in the operating room, where no volunteer was on the operating table was 0 or almost 0. An additional study has indicated that the amount of aerosols when a volunteer was lying on a table but not coughing ranged from 0 to 72, and although it was not a direct comparison, the amount was significantly higher than the amount when a volunteer was not lying on the table, but significantly lower than the amount when a volunteer was coughing. Therefore, aerosols detected during coughing would mainly from the person coughing, and that the air extractor could not remove aerosols produced by coughing. Clinical implications of this study include that, because the Free-100 M air extractor can reduce, but may not remove all, the aerosols emitted by coughing, the use of air extractor cannot make the standard PPE unnecessary, when the patient is known to be, or suspected to be, infected. Nevertheless, because PPE may not fully prevent viral infection, additional use of an air extractor to wearing PPE would reduce the risk further. It is known that viral aerosols may be coming out even from asymptomatic patients with negative test results for COVID-19 [10]. Therefore, an air extractor may be routinely used, to minimize a possible risk of infection to healthcare workers who are working by the patient. The Free-100 M is a transportable compact system, so that it is easy to transport it to the patient's side, and to start suctioning, even when an emergency airway management is required. In conclusion, the Free-100 M air extractor would reduce exposure of healthcare providers to infectious aerosols emitted by coughing, but a considerable amount of aerosols may still be exposed to healthcare providers. Author contributions All authors contributed to the study conception and design, material preparation, data collection and analysis. AF, TS and TA drafted the first manuscript, and all revising it critically for important intellectual content. All agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript. Funding AF received Research incentive grant from Dokkyo Medical University. Data availability Data are available on request. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Yamakage M Anesthesia in the times of COVID-19 J Anesth 2021 35 325 327 10.1007/s00540-020-02798-4 32451627 2. Hirose K Uchida K Umezu S Airtight, flexible, disposable barrier for extubation J Anesth 2020 34 798 799 10.1007/s00540-020-02804-9 32537706 3. Saito T Asai T Aerosol containment device for airway management of patients with COVID-19: narrative review J Anesth 2021 35 384 389 10.1007/s00540-020-02879-4 33226519 4. Saito T Fujishiro A Asai T Aerosol extractor for airway management of COVID-19 patients J Anesth 2021 35 323 10.1007/s00540-021-02916-w 33677713 5. Saito T Taguchi A Asai T Videolaryngoscopy for tracheal intubation in patients with COVID-19 Br J Anaesth 2020 125 e284 e286 10.1016/j.bja.2020.06.002 32571574 6. Simpson JP Wong DN Verco L Carter R Dzidowski M Chan PY Measurement of airborne particle exposure during simulated tracheal intubation using various proposed aerosol containment devices during the COVID-19 pandemic Anaesthesia 2020 75 1587 1595 10.1111/anae.15188 32559315 7. Cohen J The test that a proportion is 50 and the sign test In Statistical power analysis for the behavioral sciences 1988 2 New York Psychology press 8. Matava C Collard V Siegel J Denning S Li T Du B Fiadjoe J Fiset P Engelhardt T Use of a high-flow extractor to reduce aerosol exposure in tracheal intubation Br J Anaesth 2020 125 4 363 366 10.1016/j.bja.2020.07.014 9. 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==== Front Asia Pac J Manag Asia Pacific Journal of Management 0217-4561 1572-9958 Springer US New York 9853 10.1007/s10490-022-09853-6 Article Entrepreneurial passion and organizational innovation: The moderating effects of events and the competence to exploit events Li Megan Yuan [email protected] 1Megan Yuan Li Megan Yuan Li is an Assistant Professor in the College of Economics and Management at Nanjing University of Aeronautics and Astronautics. She received her PhD from the Department of Management at The Chinese University of Hong Kong. Her research focuses on the role of emotions in business strategy, entrepreneurship, and organizational innovation. E-mail: [email protected]. Makino Shige [email protected] 2Shige Makino Shige Makino is a Professor in the School of Economics at Kyoto University. He received his PhD from Ivey School of Business, the University of Western Ontario. His current research focuses on strategy and performance of multinational corporations. He is especially interested in exploring non-economic based motivations on economic activities and their performance consequences in international business. E-mail: [email protected]. http://orcid.org/0000-0001-6887-2532 Luo Lingli [email protected] 3Lingli Luo Lingli Luo is an Assistant Professor in the International Business School at Zhejiang University. She received her PhD in Management from UNSW Business School, University of New South Wales. Her research encompasses organizational aspirations, cluster status, reputation, entrepreneurship, and strategic management issues in turbulent environments. E-mail: [email protected]. Jiang Chunyan [email protected] 4Chunyan Jiang Chunyan Jiang is Professor in the School of Business at Nanjing University. She received her PhD from Department of Management, The Chinese University of Hong Kong. Her research interests include strategies for foreign market entry, entrepreneurship and research methods. E-mail: [email protected]. 1 grid.64938.30 0000 0000 9558 9911 College of Economics and Management, Nanjing University of Aeronautics and Astronautics, 29 Jiangjun Road, Jiangning District, Nanjing, Jiangsu Province China 2 grid.258799.8 0000 0004 0372 2033 School of Economics, Kyoto University, Yoshida-Honmachi, Sakyo-Ku, 606-8501 Kyoto, Japan 3 grid.13402.34 0000 0004 1759 700X International Business School, Zhejiang University, 718 East Haizhou Road, Haining, Zhejiang Province China 4 grid.41156.37 0000 0001 2314 964X School of Business, Nanjing University, Jinyinjie 16, Gulou District, Nanjing, China 1 12 2022 134 16 9 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. How do events, especially rare external events such as financial crises, wars, natural disasters, and the COVID-19 pandemic, affect the efficacy of entrepreneurial passion to drive organizational innovation? This study investigates the moderating role of events and entrepreneurs’ competence to exploit the events (opportunity competence) in the relationship between entrepreneurial passion and organizational innovation. Drawing insights from event system theory, we identified two critical event characteristics (i.e., event novelty and event criticality). Integrating the affect infusion model and the self-verification process in the identity literature, we argue that the two event characteristics and opportunity competence are crucial for entrepreneurs to exploit the benefits of entrepreneurial passion in promoting organizational innovation. After analyzing a survey sample of 435 entrepreneurs in Qinhuai Silicon Alley in China and an online survey of 202 entrepreneurs worldwide, we found that entrepreneurial passion exerts a stronger effect on organizational innovation when events are more novel and more critical to entrepreneurs, and when entrepreneurs have greater opportunity competence. We discuss these findings’ theoretical and practical implications later in this paper. Keywords Entrepreneurial passion Organizational innovation Event novelty Event criticality Opportunity competence the Scientific Research Foundation of Nanjing University of Aeronautics and Astronautics56XAB22024 Li Megan Yuan ==== Body pmcIntroduction Entrepreneurial passion is a prominent topic in both entrepreneurial research and practice. Passion can boost creativity in problem-solving (Bierly et al., 2000), persistence in the entrepreneurial process (Cardon & Kirk, 2015), employee engagement (Cardon, 2008), and venture growth (Baum & Locke, 2004; Baum et al., 2001), as well as attract potential investors (Hsu, Haynie, Simmons, & McKelvie, 2014; Mitteness et al., 2012). Recent studies have indicated that entrepreneurial passion is also effective in promoting organizational outcomes, particularly organizational innovation (Kiani et al., 2020; Ma, Gu, & Liu, 2017; Patel, Thorgren, & Wincent., 2015; Strese et al., 2018), yet entrepreneurial passion’s efficacy may vary under various circumstances. Contextual factors––such as entrepreneurs’ regulatory focus (Ma et al., 2017) and thinking style (Kiani et al., 2020), shared organizational vision (Strese et al., 2018), and environmental dynamism and complexity (Baron & Tang, 2011; Patel et al., 2015)––can moderate passion’s effects on organizational outcomes. However, these studies only considered feature-oriented factors (i.e., individual characteristics) or environmental characteristics (i.e., environmental dynamism or complexity) as major contextual factors. They overlooked the fact that entrepreneurs also experience various events that frequently force them to step out of their comfort zones. Events are a critical component of today’s dynamic environment; thus, they differ from feature-oriented factors that are static in nature. Moreover, events are discrete and bounded in space and time (Morgeson, 2005); thus, they largely differ from the environmental dynamism or complexity that captures the continuous and incremental changes in an industry over time (Boyd, 1990; Rescher, 1996). We argue that failing to incorporate events into an analysis of passion may elicit an incomplete understanding of the contextual theorizing of passion efficacy, as Johns (2017) points out: “If there has been a deficit in contextual theorizing, it is most apparent in a basic lack of theories that treat discrete events as contexts.” More specifically, it is unclear whether events’ characteristics and entrepreneurs’ exploitation of such events influence entrepreneurial passion’s efficacy in promoting organizational innovation. This research gap is critical because entrepreneurial passion’s efficacy may differ when entrepreneurs face different events and because passion’s role is highly salient in extreme conditions (i.e., novel or critical events in Morgeson 2005; threats in Staw et al., 1981; and punctuation in Tushman & Romanelli 1985). To address this research gap, we investigated how events moderate entrepreneurial passion’s efficacy in promoting organizational innovation. Scholars have reached the consensus that entrepreneurial passion comprises both affect (e.g., intensely positive feelings) and identity (the entrepreneurial identity’s salience) elements (Cardon et al., 2013). We drew on event system theory (EST) to capture core event characteristics’ implications on entrepreneurs’ information processing and behaviors. We borrowed further insights from the affect infusion model (AIM) (Forgas, 1995) and the self-verification process in the identity literature to explain how core event characteristics influence the effects of passion’s affect and identity elements on organizational innovation. For example, EST suggests that events induce substantive information processing when the events are more novel and critical to the entrepreneurs. Following AIM, we expected events to elicit greater passion infusion in entrepreneurs’ cognitive processing when the events are more novel and critical, thereby amplifying the relationship between passion and organizational innovation. Furthermore, EST suggests that novel or critical events inspire changes or creations, consistent with the entrepreneurial identity’s role expectations. According to the self-verification process in the identity literature (Heise, 1979; Stryker & Burke, 2000), we expect entrepreneurs to be motivated to engage in event-eliciting entrepreneurial activities (i.e., innovation) to verify their entrepreneurial identity. Therefore, when events are more novel and critical, passion’s identity element exerts a greater impact on organizational innovation. Aside from events’ characteristics, we also investigated the role of opportunity competence, which captures entrepreneurs’ competence to identify and exploit opportunities from events as a critical boundary condition of passion efficacy. Drawing from the AIM and the identity literature, we argue that greater opportunity competence facilitates the infusion of passion into entrepreneurial activities (Forgas, 1995) and enhances entrepreneurs’ confidence and motivation to confirm or enhance their entrepreneurial identity. We tested our theory using a field survey of 435 ventures located in Qinhuai Silicon Alley in China, with an emphasis on a series of government-initiated events. We gauged the findings’ robustness further by conducting an online survey of 202 entrepreneurs worldwide, with COVID-19 as the focal event. The findings generally demonstrated event characteristics’ moderating effects on the relationship between entrepreneurial passion and organizational innovation. Our study makes significant contributions to the entrepreneurial passion literature and the behavioral literature. First, we complemented extant research on entrepreneurial passion by delineating events as a critical boundary condition affecting passion’s efficacy in promoting organizational innovation. Recent studies have begun to examine passion’s varying effects under different individual and environmental contexts (Baron & Tang, 2011; Kiani et al., 2020; Ma et al., 2017; Patel et al., 2015; Strese et al., 2018). We built on this line of research by examining events as another highly different, yet critical context of passion efficacy. Furthermore, we contributed further insights into the entrepreneurial passion literature by investigating the interaction between passion and opportunity competence in promoting organizational innovation. Passion is treated as a key intangible asset in promoting organizational innovation (Makino et al., 2020). We built on this line of research by demonstrating that passion’s power cannot be fully exploited when entrepreneurs do not have enough competence to exploit opportunities from events, thereby highlighting the importance of integrating emotional and capability perspectives when studying passion’s efficacy. Finally, behavioral strategy scholars have suggested that to develop a more realistic theory of organizational decision-making, organizational studies should “merge cognitive and social psychology with strategic management theory and practice” (Powell et al., 2011: 1371). By examining passion and events’ interaction effects, which shape the cognitive processing strategies that entrepreneurs adopt, we acted on this call for an integrative approach by merging cognitive and social psychology in management research. Theoretical background and hypotheses Entrepreneurial passion Passion has been defined as “a strong inclination or desire toward an activity that one likes or loves, finds important, and in which one invests time and energy” (Vallerand et al., 2003: 757). Existing studies on entrepreneurial passion mainly have adopted two theoretical frameworks: the dualistic model of passion (Vallerand et al., 2003) and three types of entrepreneurial passion for specific role identities (Cardon et al., 2009). The former framework focuses on passion for the general identity of entrepreneur and identifies two types of passion based on the degree of internalization of the entrepreneurial identity (i.e., harmonious passion vs. obsessive passion). The latter framework focuses on passion for activities associated with different role identities (i.e., inventor, founder, and developer) and argues that passion for different role identities can elicit different effects (Cardon et al., 2009; Collewaert et al., 2016; Drnovsek et al., 2016). More importantly, entrepreneurs may experience different types of passion for activities associated with different role identities at different stages of the entrepreneurial process (Cardon et al., 2009). For example, passion for founding is most critical in the founding phase of a venture’s lifecycle (Collewaert et al., 2016), while passion for inventing is highly influential in generating product innovation, and passion for developing is most essential in ventures’ expansion decisions (Murnieks et al., 2020). Given our focus on innovation activities in the entrepreneurial process, we examined passion for inventing, which is linked to entrepreneurial efficacy in innovative idea development and opportunity exploration (Cardon et al., 2013; Katila & Ahuja, 2002). Thus, it is the type of passion most relevant to organizational innovation. Following Cardon et al. (2009: 517), we define entrepreneurial passion as “consciously accessible intense positive feelings experienced by engagement in entrepreneurial activities associated with roles that are meaningful and salient to the self-identity of the entrepreneur.” This definition indicates that entrepreneurial passion comprises two key elements. First, it has an affective aspect and involves experiencing intensely positive feelings. Second, it is identity-relevant, and the positive feelings are experienced for activities central to the individual’s self-identity. Further empirical studies have provided evidence that these two aspects are distinct from each other (e.g., Cardon et al., 2013). Entrepreneurial passion has been demonstrated to elicit several organizational benefits, e.g., venture growth (Baum & Locke, 2004; Baum et al., 2001) and attracting potential investors (Hsu et al., 2014; Mitteness et al., 2012). These effects mainly work through two mechanisms: Passion’s affect element broadens entrepreneurs’ cognitive repertoires (Cohn & Fredrickson, 2006), and passion’s identity element motivates entrepreneurs to be committed to entrepreneurial activities (Cardon et al., 2009). In the following sections on hypothesis development, we will discuss how these two elements of entrepreneurial passion provide direct and contingent values for promoting organizational innovation. Entrepreneurial passion and organizational innovation Passion for inventing (henceforth: passion) concerns entrepreneurs’ passion toward “activities associated with scanning the environment for new market opportunities, developing new products or services, and working with new prototypes” (Cardon et al., 2013: 379). We argue that passion’s affect and identity elements could facilitate organizational innovation for the following reasons. First, entrepreneurial passion, as a form of intense and positive emotion, can help improve the entrepreneurs’ perception on a wide range of information (Isen, 2002; Ashby & Isen, 1999), such as the market, technology and customer information (Delgado Carcía et al., 2015). Besides, passion also enhances the entrepreneurs’ abilities to conceive of new combinations and formulate creative ideas, which serve as precursors of organizational innovation (Cardon et al., 2009). Studies also have indicated that “passion, as an intangible, hard-to-measure quality of those asking for resources, can be powerful and critical in many endeavors that are aimed at creating something new in the society” (Chen et al., 2009: 119). Given these reasons, we argue that the new creations motivated by passion are expected to result in organizational innovation in the end. Second, entrepreneurs with positive emotions are likely to appraise risks positively and are willing to take risks. Multiple studies (e.g., Schwarz 1990; Schwarz & Clore, 1996) on individual-level phenomena have indicated that the risk-taking tendency is greater for individuals with positive emotions than those with negative emotions. At the organizational level, Huy (1999) also indicated that positive emotions created through playful activities could motivate organizational members’ innovative behaviors. Given that organizational innovation is often the consequence of risk-taking, entrepreneurs with higher passion levels are more likely to take risks and produce innovation outcomes. Furthermore, according to Cardon et al. (2013), passion emerges when the entrepreneurial identity is salient. Therefore, passion’s identity element is arguably a strong motivator for entrepreneurs to engage in entrepreneurial activities, e.g., identifying new opportunities, coming up with innovative ideas, and tinkering with creative solutions to meet important needs and solve problems (Cardon et al., 2009). These activities themselves are of particular importance in promoting organizational innovation (Ward, 2004). Finally, extant studies have demonstrated that passion can be transferred to other organizational members through both affect contagion and social identification processes (Barsade, 2002; Makino et al., 2020). When organizational members share their passion with each other, the shared passion will foster a “community of practice” that facilitates knowledge sharing and social exchange within the organization (Wenger & Snyder, 2000). This knowledge sharing and social exchange process will foster the emergence of new ideas, thereby promoting organizational innovation. Thus, we propose the following hypothesis: Hypothesis 1 Entrepreneurial passion for inventing is positively related to organizational innovation. Event characteristics as the context: event novelty and criticality As previous research suggests, entrepreneurial passion’s efficacy is subject to contextual factors, e.g., environmental complexity (Patel et al., 2015), organizational shared vision (Strese et al., 2018), entrepreneurs’ thinking style (Kiani et al., 2020), and regulatory focus (Ma et al., 2017). Building on this line of research, we expect event-related characteristics to affect entrepreneurial passion’s efficacy. Events may force people to step out of their routines (Morgeson, 2005), and these events usually function as the means by which people come to reevaluate their established behaviors. Several extant studies have investigated events’ influence on organizations (Hoffman & Ocasio, 2001) and on members within these organizations (Vuori & Huy, 2016). Events such as natural disasters, technological changes, or corporate scandals tend to exert varying degrees of influence on entrepreneurial decision making and behaviors (Salvato et al., 2020; Yiu et al., 2014). Event system theory (EST) is a comprehensive model that depicts the characteristics of and potential consequences elicited by an event (Morgeson et al., 2015). According to EST, an event’s strength is characterized by three aspects: novelty; disruption; and criticality. Event novelty reflects “the extent to which an event is different or varies from current and past behaviors, features, and events” (Morgeson et al., 2015: 520); thus, a novel event is usually a new or unexpected phenomenon (Morgeson, 2005). For example, introducing a new work procedure may be novel to the organization if it largely diverges from previous work processes. Event disruption reflects a discontinuity in the environment and breaks organizational routines (Morgeson et al., 2015). The term disruption usually is used interchangeably with intervention (Morgeson & DeRue, 2006) or upheaval (Gersick, 1991). Event criticality reflects “the degree to which an event is important, essential, or a priority” to an entity (Morgeson & DeRue, 2006: 273). The more critical the event, the more likely it is to attract unusual attention and responses (Morgeson et al., 2015). EST suggests that the three aspects of event strength are distinctive from each other, and that an event is likely to elicit the strongest emotional and behavioral reactions when it is novel, disruptive, and critical (Morgeson et al., 2015). As we will demonstrate in the next section, event novelty and criticality are related directly to the working mechanism of passion’s identity element, while event disruption is not. For example, entrepreneurs facing novel events are expected by key stakeholders to behave more creatively. These expectations under high event novelty are consistent with their entrepreneurial identity. Moreover, a critical event usually is deemed closely associated with the entrepreneurial identity, that is, the attribute of event criticality itself describes the association between the event and the entrepreneurial identity. Therefore, entrepreneurs may enhance their valued identity by engaging in dealing with critical events. However, a disruptive event does not carry clear implications on behavioral expectations associated with entrepreneurial identity. As suggested by EST (Morgeson et al., 2015), a disruptive event usually reflects the threat experienced from major disruptions. Existing studies suggest that individuals may respond differently to external threats. While some scholars argue that entrepreneurs tend to respond to perceived threats in the environment with risk-averse behavior because they perceive such actions to be associated with greater control (Staw et al., 1981; Sitkin & Pablo, 1992), others suggest that entrepreneurs are expected to seek risks in unfavorable circumstances because they feel that they have little to lose (Kahneman & Tversky, 1979). The above analysis suggests that a disruptive event may not necessarily lead to creative solutions; instead, entrepreneurs may resolve challenges by following existing protocols and procedures (Chen et al., 2021). Therefore, the behavioral expectations following disruptive events may or may not relate to the role expectations of an entrepreneur identity, i.e., innovation. Furthermore, as we show below, we drew from the AIM to argue for the moderating role of event characteristics between the affect aspect of passion and organizational innovation. While event novelty and criticality are core components included in the AIM, event disruption is not. Therefore, we did not include event disruption in our framework. In our analysis of events as the boundary condition, we drew insights from the AIM and identity literature (Burke & Reitze, 1991; Goffman 1959), corresponding to passion’s affect and identity elements. One of the main tenets of the AIM is that affect will be more likely to be infused in decision making when the events require a substantive processing strategy with a high degree of constructive processing, rather than a direct access strategy that adopts a preexisting evaluation (Forgas, 1995). A substantive processing strategy is most likely to be adopted when the event is novel and of high personal relevance, and when the subjects have a great need for cognition and adequate cognitive capacity (Morgeson et al., 2015; Petty & Cacipoppo, 1986). Therefore, we expect events to strengthen passion’s positive effects on organizational innovation by inspiring substantive processing. We also drew arguments from the identity literature to address events’ influence on the relationship between passion’s identity element and organizational innovation. Among the myriad theoretical approaches to identity, we focused on the self-verification process, which directs behaviors toward matching meanings associated with the situation (i.e., event) and the meanings associated with identity (Burke, 1991). We treated events as a context that raises various role expectations. Following Petriglieri (2011), an event’s relevance to entrepreneurs depends on the following primary appraisals: (1) whether it preserves or benefits the entrepreneur’s identity; (2) whether it harms the entrepreneur’s identity; (3) whether it holds potential for entrepreneurial identity growth; and (4) whether it holds potential to harm the entrepreneur’s identity. As we will demonstrate below, when an event is viewed as having high relevance, it is likely to enhance the entrepreneur’s tendency to confirm, enhance, or defend their valued identity (Heise, 1979; Stryker & Burke, 2000; Gecas & Burke, 1995), thereby amplifying the relationship between passion (identity element) and organizational innovation. Event novelty An organization may experience different events at different developmental stages, and different organizations likely will have divergent interpretations of the same event (Dutton & Jackson, 1987). When an event diverges from expectations, it is likely to be perceived as “novel.” Thus, a novel event usually represents a new or unfamiliar phenomenon. AIM (Forgas, 1995) suggests that novel events (the opposite of familiar events or targets in AIM) require substantive cognitive processing. Individuals may employ quick shortcuts or direct access strategies that rely on existing routines when faced with familiar events, but are less likely to do so when faced with novel events, as they are usually forced to step out of established routines (Morgeson et al., 2015). Thus, the more novel the event, the more likely people are to activate substantive cognitive processing and modify routine-based behavior. For example, the COVID-19 pandemic is an extremely rare and novel event for most firms, and a firm’s existing rules and procedures may not work effectively to deal with this event. Therefore, firms faced with a novel event need to seek new rules and procedures. Thus, our results suggest that entrepreneurs tend to adopt substantive processing strategies when faced with novel events. In addition, AIM has shown that affect tends to be infused into judgments when individuals are dealing with more atypical and novel events (Forgas, 1993, 1994). As mentioned earlier, novel events prompt substantive processing strategies, i.e., search. Considering that organizational innovation occurs through extensive search activities, we can conclude that the passion (specifically, the affect element) tends to have a greater impact on organizational innovation as the novelty of the event increases. Moreover, the occurrence of a novel event usually requires a creative response, such as the invention of a new product, and hence event novelty creates a social context that justifies entrepreneurial activity (Sine & David, 2003). Thus, novel events have the potential to reinforce entrepreneurial identity (Petriglieri, 2011). According to the self-verification process of the identity literature (Burke, 1991), if external role expectations (e.g., from stakeholders, etc.) in a given situation (i.e., event) enhance entrepreneurial identity, entrepreneurs will have a strong motivation to meet those role expectations. On the other hand, when external role expectations are inconsistent with the entrepreneurial identity, entrepreneurs will lose motivation to meet those role expectations (Shepherd & Haynie, 2009; Stryker & Burke, 2000). Following this logic, we argue that when entrepreneurial activity is consistent with the expectations of key stakeholders under novel events, entrepreneurs will have a strong motivation to confirm or reinforce their entrepreneurial identity. Thus, we propose: Hypothesis 2 The positive relationship between entrepreneurial passion for inventing and organizational innovation is stronger when entrepreneurs face events with greater novelty. Event criticality Different events may not attract entrepreneurs’ attention equally, and those of greater relevance are more likely to be viewed as more salient and attract a greater amount of attention and action. For example, Hoffman and Ocasio (2001) suggested that firms pay a great deal of attention to events that are crafted in a way that threatens their organizational identity. Entrepreneurship studies also have indicated that entrepreneurs tend to be emotionally and cognitively involved in certain activities (e.g., inventions) when they hold corresponding identities (e.g., an inventor identity; Breugst et al., 2012). These studies, when combined, suggest that entrepreneurs are more likely to respond proactively and intensively when an event is treated as more critical. The AIM suggests that, all things being equal, more critical events are likely to be processed substantively (Forgas, 1995), while less critical events will lead to direct access processing that adopts a preexisting evaluation. Ample evidence has indicated that even a slight variation in personal relevance, namely the level of criticality, may result in profound changes in information processing strategies (Brewer, 1988). For example, Mao et al. (2018) suggested that affect is more likely to influence individuals’ justice perceptions in high personal relevance conditions than in low ones. Therefore, we expect that when entrepreneurs treat an event as more critical to their entrepreneurial identity, they will be more likely to engage in substantive information processing, which invokes a high degree of constructive processing. As a result, their passion will be more likely to be infused into cognition and decision making, thereby exerting a greater influence on organizational innovation. Moreover, event criticality also provides a context in which entrepreneurs can enhance or defend their entrepreneurial identity, thereby promoting the conversion of passion into organizational innovation. As Morgeson et al. (2015) suggested, a critical event usually plays an important role in an entrepreneur’s long-term success. In our context, we argue that a critical event carries important implications for the survival and development of new ventures (Hermann, 1963). When a critical event is perceived as an opportunity, it may improve the likelihood of a venture’s success by providing entrepreneurs with critical resources and hence hold potential for entrepreneurial identity growth (Petriglieri, 2011). For example, meetings that the government organizes may provide entrepreneurs with potential collaborative opportunities. Government-initiated reward application events provide entrepreneurs with the possibility of attaining government support or preferential policy treatment. According to the self-verification process in the identity literature (Burke, 1991), when the meanings or role expectations associated with an event are consistent with the meanings of the entrepreneur identity, entrepreneurs will have stronger motivation to verify their identity by engaging in entrepreneurial activities, such as innovation. However, when an event is perceived as a threat, it may influence a firm’s survival and performance negatively. For example, external threats––e.g., technological transformation, financial crises, and the COVID-19 pandemic––likely “threaten the fundamental goals of an organization” (Weick, 1988: 305). We argue that these critical events may harm the entrepreneurial identity due to potential losses from such events (Petriglieri, 2011). As the self-verification process in the identity literature suggests, when external events change the situation such that “individuals perceive situated self-meanings and expectations of themselves as different from their identity standard, they act to counteract the disturbance” (Stets & Burke, 2000: 233). Indeed, extant studies have suggested that, faced with threats to their valued identity, entrepreneurs usually take proactive actions to defend their identities, rather than surrender (Powell & Baker, 2014; Jain et al., 2009). Therefore, these negative critical events also motivate entrepreneurs to engage in innovation as a way to defend their entrepreneurial identity. Thus, we propose: Hypothesis 3 The positive relationship between entrepreneurial passion for inventing and organizational innovation is stronger when entrepreneurs face events with higher criticality. Capability to exploit opportunities from events: opportunity competence According to the literature on appraisal of events (Lazarus, 1991), individuals’ response to events also depends on their appraisal of the competence to cope with events aside from the events’ relevance. Therefore, we also considered entrepreneurs’ competence to identify and exploit opportunities from events. In particular, we focused on opportunity competence, which is defined as entrepreneurs’ competence related to “recognizing and developing market opportunities through various means” (Man et al., 2002: 132). Following the AIM, we expected high opportunity competence to motivate exploratory learning, which, in turn, facilitates the use of a substantive processing strategy. Exploratory learning requires decision makers to attend to and learn from a broad set of alternative possibilities and usually is depicted as forward-looking actions that invoke substantial cognitive processes (Gavetti & Levinthal, 2000). However, when individuals do not have enough competence to process information from multiple sources, or when their processing competence is impaired, they tend to adopt a simplified heuristic processing strategy (Bodenhausen, 1993; Bodenhausen & Lichtenstein, 1987). Multiple studies have provided evidence that entrepreneurs may use simple heuristics to make decisions under high environmental complexity because of their limited cognitive capabilities to deal with information overload (Artinger et al., 2015; Busenitz & Barney, 1997). The above arguments based on the AIM suggest that a greater affect infusion occurs when entrepreneurs have greater opportunity competence. As discussed previously, this greater affect infusion will promote the conversion of entrepreneurial passion into organizational innovation. Furthermore, we argue that opportunity competence also enables entrepreneurs to express their entrepreneurial identity and, thus, promote organizational innovation. Entrepreneurs are better-equipped to deal with events when they have greater opportunity competence. Extant studies have found that individuals enjoy engaging in activities in which they have a strong belief in their ability to succeed (Baum & Locke, 2004), or when their interests align with their abilities (i.e., what they want to do is what they can do). Following this logic, entrepreneurs with greater competence to exploit opportunities are more likely to enhance their entrepreneurial identity by engaging in entrepreneurial activities, e.g., generating new ideas and promoting innovation. Thus, we propose: Hypothesis 4 The positive relationship between entrepreneurial passion for inventing and organizational innovation is stronger when entrepreneurs have greater opportunity competence. STUDY 1: Method Sample and data The data used in this study were collected in December 2019 via a survey of ventures in Qinhuai Silicon Alley, located in Jiangsu Province, China. Qinhuai Silicon Alley provides an ideal research setting in which to investigate passion for inventing and organizational innovation for three reasons. First, learning from Silicon Valley in Northern California, the Nanjing government designed Qinhuai Silicon Alley to promote firm innovation in Jiangsu Province’s Qinhuai District. The local government provides infrastructural support and facilitates collaborations between local firms and research institutes. Many ventures have moved to this area to take advantage of local government support and to build close business connections. Moreover, these ventures are relatively small in size,1 and entrepreneurs exert substantial influence on organizational decision-making and the innovation process. Therefore, entrepreneurs’ emotional aspects are highly relevant to organizational innovation. Three research assistants developed the survey in English, and then translated it into Chinese. We also commissioned back-translations from two independent translators to ensure conceptual equivalence. Considering that one of the authors engaged in a research collaboration project with government officials in Qinhuai District, we had a chance to involve local government officials in the data collection process. In this way, a high response rate to our survey was ensured. The survey was sent to 583 entrepreneurs involved with Qinhuai Silicon Alley ventures, and 545 responses were received, a response rate of 93.5%. After deleting surveys with missing information, we reached a final sample size of 435. Variables We adopted measurements of the variables in our study from the existing literature and measured each item using a five-point Likert scale. Appendix 1 provides the items for our key variables, along with the Cronbach’s alpha values and the percentage of variance explained for each variable. Dependent variable. Our dependent variable was organizational innovation. We derived our measurement scale for organizational innovation from existing research on corporate entrepreneurship (Guth & Ginsberg, 1990; Zahra, 1996), which comprises three aspects: innovation; venturing; and strategic renewal. Zahra (1996) developed 14 items to measure these three distinct aspects of corporate entrepreneurship, but innovation––i.e., introducing new products, services, and production processes––was the most relevant to the focus on organizational innovation in our study, while the other two factors,venturing and strategic renewal, was not relevant to organizational innovation. Therefore, we adopted Zahra’s (1996) scale. In particular, we extracted five items that cover the innovation aspects of Zahra’s measurement scale for corporate entrepreneurship (Zahra, 1996) to measure organizational innovation. The five innovation-related items explained 81.94% of the variance, with a Cronbach’s alpha of 0.91. Independent variables. We used the scale adapted from Cardon et al. (2013) to measure entrepreneurial passion. The scale incorporates two dimensions: (1) positive intense feelings for inventing activities and (2) the inventor identity’s centrality. The scale’s feeling dimension is a reflective measure comprising four items, whereas the identity dimension is a one-item measure of the inventor identity’s centrality. The feeling items are averaged, then multiplied by the identity item (Cardon et al., 2013). The four affect items explained 82.71% of the variance of the affect dimension of entrepreneurial passion, with a Cronbach’s alpha of 0.93. Event characteristics. Since its establishment, Qinhuai Silicon Alley has focused on stimulating regional innovation as its first priority. To facilitate regional innovation, the Qinhuai government initiated several events. We categorized these events under the overarching event of “building Qinhuai Silicon Alley,” given that these events were purported to facilitate regional innovation in Qinhuai Silicon Alley. We followed a two-step procedure by Morgeson (2005) and Morgeson and DeRue (2006) to collect and evaluate data regarding these events. First, we asked the entrepreneurs: “What are the major government-initiated events that have a great stake in the innovation of your firm since your entry into Qinhuai Silicon Alley?” The respondents were asked to write descriptions of the events. We also conducted interviews with two government officials to obtain a more comprehensive list of the events purported to facilitate regional innovation (event examples are presented in Table 1). We checked the consistency between the major events that the entrepreneurs proposed and those that the government proposed, and found that different entrepreneurs mentioned all the events that the two government officials proposed. Second, we asked the entrepreneurs to rate the novelty and criticality of the series of events based on established scales. To measure event novelty, we adopted a scale developed by Morgeson (2005). Considering that the measurement items reflect an event’s typicality (the opposite of novelty), we reversed the rating scores. The four items in the scale explained 89.28% of the variance, with a Cronbach’s alpha of 0.96. To measure event criticality, we used three items adapted from Morgeson and DeRue’s (2006) work. The three items are loaded on one factor and explain 80.25% of the variance, with a Cronbach’s alpha of 0.87. Table 1 Example events from the interviews Interpreting new policies The Qinhuai Government officials are very sensitive the new policies. They organized events to interpret these new policies, especially some preferential policies. For example, they made a brochure including all the recent new policies. Through their interpretation of these policies, I can get a clear understanding of which new policies most fit the current situation of our firm. Organizing training programs Sometimes the training is too much. For example, they are holding the Third Corporate Executive Training these days. However, as I have no financing needs, I am not interested in it. Despite of this, I know some other firms are in desperate need of this training. Product exhibition Silicon Alley frequently held product exhibitions. They asked each company to showcase their new products and invited the government officials at the city or provincial level to visit the exhibition. Although most of us are quite busy, we squeeze time to involve in this because we want to impress the municipal leaders. Last time, the leaders provided some help in our brand promotion. Organizing Conferences/meetings They will organize some international or national conferences on leading edge technologies. They recently organized one conference on artificial intelligence. I got to know my current business partner and got access to supply chain resources in this conference. Organizing talent projects They are promoting a “Zijinshan” talent project which aims to select promising innovation project. They selected me this time. This project requires a number of preparations. However, the final reward is attractive: besides the honor, they also provide financial support for the award winners, from 500,000 RMB to 1,000,000 RMB. We argue that these events provide a desirable context in which to test our model. First, considering that the government initiated these events, they are exogenous to the entrepreneurs. Moreover, extant studies have indicated that different entities may interpret the same event differently (Hoffman & Ocasio, 2001). Focusing on common events that entrepreneurs face enables us to control for possible extraneous variation without significantly losing variations among entrepreneurs’ perceptions of event characteristics. Opportunity competence. We used three items derived from the previous study to measure opportunity competence (Man et al., 2002). These items capture the entrepreneurs’ ability to recognize potential markets, as well as evaluate and utilize business opportunities. The items explained 92.27% of the variance, with a Cronbach’s alpha of 0.96. Control variables. We included several variables to control for possible confounding effects of entrepreneurs’ demographic characteristics (i.e., gender, age, education level, tenure at current firm, and tenure in current position), firm characteristics (i.e., firm type and firm size), and industry characteristics. We measured entrepreneurs’ gender using a dummy variable, coded 1 for males and 0 for females. We classified the entrepreneurs’ ages into four groups and assigned corresponding values to each one (1 = ages 21–30, 2 = ages 31–40, 3 = ages 41–50, and 4 = ages > 50). We categorized education level into three groups (1 = college graduate,2 2 = university graduate, and 3 = postgraduate or above). Entrepreneurs’ tenure at their current firms was measured by the number of months since they joined the firm, and tenure in their current positions was measured by the number of months they held their current positions. Firm type was measured using a dummy variable that indicates whether or not a firm is state-owned (1 indicates state-owned and 0 not state-owned). Given that Qinhuai Silicon Alley was established to “revitalize regional innovation,” many conventional state-owned firms set up new businesses as subsidiaries, and some top managers in the original state-owned firms were selected as entrepreneurs in the newly established subsidiaries. About 53% of the firms in our sample were state-owned. Firm size was measured based on total number of employees. Industry was measured using a binary variable with a value of 1 if the industry is high-tech and 0 otherwise, given our focus on organizational innovation. Analysis and results Table 2 presents the means, standard deviations, and correlations for the variables. We evaluated the variance inflation factor (VIF) values in our analysis, and the results indicated a maximum VIF of 2.59 across all regression models, far below the commonly accepted VIF threshold of 10 (Cohen et al., 2003). Therefore, we believe that multicollinearity did not significantly distort our analyses. Table 2 Descriptive statistics and correlation matrix in Study 1 Variables 1 2 3 4 5 6 7 8 9 10 11 12 13 1. Organizational innovation 0.91 2. Entrepreneurial passion for inventing 0.56* 0.93 3. Event novelty 0.61* 0.55* 0.96 4. Event criticality 0.61* 0.61* 0.67* 0.87 5. Opportunity competence 0.65* 0.64* 0.50* 0.62* 0.96 6. Gender -0.01 0.00 -0.08 -0.08 0.01 - 7. Age -0.17* -0.04 -0.21* -0.09 -0.13* 0.27* - 8. Education -0.18* -0.01 -0.07 -0.15* -0.08 0.14* 0.11* - 9. Tenure in current firm -0.29* -0.09* -0.29* -0.24* -0.21* 0.18* 0.61* 0.16* - 10. Tenure in current position -0.07 -0.03 -0.14* -0.16* -0.10* 0.16* 0.47* 0.06 0.59* - 11. Firm type 0.11* 0.00 0.12* -0.05 0.09* -0.01 -0.14* -0.21* -0.38* -0.12* - 12. Firm size -0.25* -0.02 -0.19* -0.06 -0.16* -0.01 0.22* 0.20* 0.54* 0.14* -0.55* - 13. Industry 0.12* 0.02 0.01 0.08 0.06 0.12* 0.01 0.15* 0.00 0.13* -0.07 -0.05 - Mean 3.65 3.86 3.83 3.30 3.57 0.53 1.96 1.95 80.13 44.56 0.53 1.61 0.33 SD 2.13 0.82 0.88 1.18 1.00 0.52 0.90 0.62 84.28 44.86 0.50 0.92 0.47 Notes: N = 435; * p < 0.05. The variables including organizational innovation, entrepreneurial passion for inventing, event novelty, event criticality, and opportunity competence are standardized with a mean of 0 and a standard deviation of 1. The means and standard deviations in this table show those of the items that compose each of main variables. Variables reliabilities are shown on the diagonal To reduce possible selection bias, we performed a t-test that compared the following characteristics between participating and non-participating firms: (1) Four items reflected the entrepreneurs’ gender, age, education level, tenure in current position, and tenure at current firm; and (2) three items reflected the ventures’ firm ownership type, size, and industry (high-tech or low-tech). The results indicated no statistically significant differences for any of these items, indicating that selection bias, if present, exerts no significant effect on our subsequent analyses’ generalizability. We also conducted Harman’s single-factor test to measure common method variance in our data (Podsakoff & Organ, 1986). A principal components factor analysis with an unrotated solution generated seven factors with eigenvalues greater than 1.0. The largest variance explained by a single factor was below 40% (less than 50%), suggesting that no single factor explained most of the covariance in all variables. To check further whether significant common variance exists among our key variables–– including organizational innovation, the feeing aspect of entrepreneurial passion for inventing, event novelty, event criticality, and opportunity competence––we followed a procedure that Breugst et al. (2012) suggested and ran multiple confirmatory factor analyses. In the first model, we loaded all indicators on their respective latent constructs. The fit indices indicated a generally acceptable model fit (χ2[180] = 1843.04; CFI = 0.95; RMSEA = 0.08; SRMR = 0.02). All the indicators loaded significantly (p < 0.001) on their respective constructs. To check whether the indicators could be subsumed under one construct, we loaded all the indicators on one latent factor in the second model, and the fit indices indicated a much worse model fit (χ2[180] = 5313.16; CFI = 0.45; RMSEA = 0.18; SRMR = 0.30). Comparing these two models suggests that no strong underlying component explains the variance in our main variables. To provide an additional check for any possible influence from common method variance (Podsakoff et al., 2003), we specified the third model by loading all the indicators on their respective latent factors, while also including an additional latent variable in the model. This latent variable represented the common variance extracted from all items and is allowed to influence all indicators. The model still did not converge after 3,000 iterations, providing further evidence that common method variance was not a major concern in our study. To test the hypotheses, we performed multiple ordinary least square (OLS) regression analyses, and the results were reported in Table 3. Model 1 is the baseline model and includes only control variables. Model 2 examines the main effect of passion for inventing on organizational innovation. In support of Hypothesis 1, the results indicated that passion for inventing is related positively to organizational innovation (b = 0.55, p < 0.001). Table 3 Regression models on entrepreneurial passion for inventing and organizational innovation in Study 1 Variables Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Model 7 Gender 0.09 (0.09) 0.06 (0.07) 0.07 (0.07) 0.09 (0.07) 0.03 (0.07) 0.05 (0.07) 0.08 (0.07) Age -0.04 (0.07) -0.03 (0.06) -0.00 (0.05) -0.08 (0.05) -0.03 (0.05) -0.01 (0.05) -0.03 (0.05) Education -0.27* (0.08) -0.23* (0.06) -0.23* (0.06) -0.15 (0.06) -0.16* (0.06) -0.14* (0.05) -0.18* (0.06) Tenure in current firm -0.00* (0.00) -0.00*** (0.00) -0.00* (0.00) -0.00 (0.00) -0.00 (0.00) -0.00† (0.00) -0.00* (0.00) Tenure in current position 0.00† (0.00) 0.00† (0.00) 0.00 (0.00) 0.00* (0.00) 0.00* (0.00) 0.00 (0.00) 0.00* (0.00) Firm type -0.03 (0.11) 0.01 (0.09) 0.00 (0.09) 0.05 (0.09) -0.04 (0.08) -0.03 (0.08) 0.04 (0.09) Firm size -0.12† (0.07) -0.13* (0.05) -0.11* (0.05) -0.16** (0.05) -0.10* (0.05) -0.12 (0.05) -0.14* (0.05) Industry 0.19† (0.10) 0.19* (0.08) 0.24*** (0.08) 0.16* (0.08) 0.18 (0.07) 0.21* (0.07) 0.21* (0.07) Entrepreneurial passion for inventing 0.55* (0.04) 0.37* (0.04) 0.36* (0.05) 0.28* (0.04) 0.11 (0.05) 0.27* (0.05) Event novelty 0.33* (0.05) 0.18* (0.05) 0.18* (0.05) Event criticality 0.32* (0.05) 0.12 (0.05) 0.21* (0.05) Opportunity competence 0.43* (0.05) 0.36* (0.05) Entrepreneurial passion for inventing × Event novelty 0.06† (0.03) -0.03 (0.04) 0.03 (0.04) Entrepreneurial passion for inventing × Event criticality 0.06† (0.04) -0.06 (0.05) 0.07† (0.04) Entrepreneurial passion for inventing × Opportunity competence 0.08* (0.03) 0.11* (0.04) Entrepreneurial passion for inventing × Event novelty × Event criticality 0.06 (0.03) Constant 0.87* (0.22) 0.70* (0.18) 0.53 (0.18) 0.50 (0.18) 0.45** (0.17) 0.30* (0.17) 0.41 (0.18) Adjusted R2 0.14 0.44 0.50 0.50 0.55 0.59 0.55 Notes: N = 435; * p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.1. We included the interaction term between Event novelty and Event criticality in the three-way interaction analysis, but did not report the result here as it is irrelevant to our hypothesis testing The moderating effects predicted in Hypotheses 2–4 were tested with Models 3–6. In Model 3, we added the interaction between passion for inventing and event novelty. The result (b = 0.06, p < 0.10) indicated marginal support for Hypothesis 2. We also found marginal support for Hypothesis 3 based on Model 4, as indicated by the weak significance of the positive coefficient of the interaction between passion for inventing and event criticality (b = 0.06, p < 0.10). In Model 5, we added the interaction between passion for inventing and opportunity competence. Consistent with Hypothesis 4, Model 5 indicated that opportunity competence strengthens the positive effect of passion for inventing on organizational innovation (b = 0.08, p < 0.001). To visualize four contextual factors’ moderating effects, we plotted the relationships between passion for inventing and organizational innovation under high (two standard deviations above the mean) and low (two standard deviations below the mean) levels of the four boundary conditions in Fig. 1. The plots clearly indicated that passion for inventing is more effective in promoting organizational innovation under higher levels of event novelty, event criticality, and opportunity competence. Fig. 1 The moderating effects Given the marginal moderating effects of event novelty and event criticality when they were analyzed separately, we examined and analyzed their combined moderating effect on the relationship between passion for inventing and organizational innovation. The results are reported in Model 7 of Table 3. In Model 7, we created a three-way interaction term comprising passion for inventing, event novelty, and event criticality. The results indicated that this three-way interaction term is statistically significant (b = 0.06, p < 0.01). This finding suggests that the positive effect of passion for inventing on organizational innovation is much stronger when event novelty and event criticality are higher simultaneously. Practically, this result suggests that the passion effect is strongest when the event is relevant to the entrepreneur and viewed as having a high degree of novelty. To further test this result, we divided our sample into four groups based on two dimensions (event novelty and event criticality) at two levels (low and high).3 We analyzed the effects of passion for inventing on organizational innovation in these four groups. The results indicated that the correlation between passion for inventing and organizational innovation (p < 0.001) was highest in the group of high event novelty and high event criticality, confirming our previous finding. Robustness check To gauge our findings’ robustness further, we conducted a few additional tests. First, one of the key premises in our arguments is that event novelty, event criticality, and opportunity competence motivate the use of a substantive processing strategy, thereby facilitating affect infusion into judgments and amplifying passion efficacy. To test this proposition, we examined these three factors’ direct influences on entrepreneurial cognitive processes. More specifically, we used exploratory learning as the dependent variable and included the three factors as the independent variables, as well as the control variables mentioned in the previous section. Exploratory learning was measured using four items based on existing research (He & Wong, 2004; Li et al., 2014). Our results indicated that all three factors exhibit a significant influence on exploratory learning (p < 0.001), suggesting that the three factors promote substantive processing. Second, we used exploratory innovation as an alternative measure of organizational innovation to test our hypotheses. Our measurement of exploratory innovation was derived from existing studies (Benner & Tushman, 2003; March, 1991). The results are provided in Table 4. Models 1–4 repeated our previous hypothesis testing, and Model 5 tested the combined moderating effect of event novelty and event criticality on the relationship between passion for inventing and exploratory innovation. The results were similar to those of the main test. Table 4 Regression models with exploratory innovation as an alternative dependent variable in Study 1 Variables Model 1 Model 2 Model 3 Model 4 Model 5 Gender -0.04 (0.08) -0.04 (0.08) -0.02 (0.08) -0.07 (0.07) -0.04 (0.08) Age -0.06 (0.06) -0.07 (0.06) -0.11† (0.06) -0.07 (0.06) -0.13* (0.06) Education -0.26* (0.07) -0.26* (0.07) -0.18 (0.07) -0.19* (0.06) -0.18*** (0.07) Tenure in current firm -0.00 (0.00) -0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Tenure in current position 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Firm type 0.13 (0.10) 0.17† (0.10) 0.19* (0.10) 0.08 (0.09) 0.24 (0.10) Firm size 0.04 (0.06) 0.04 (0.06) 0.01 (0.06) 0.07 (0.05) -0.01 (0.06) Industry 0.01 (0.09) 0.03 (0.09) -0.03 (0.09) 0.00 (0.08) -0.01 (0.08) Entrepreneurial passion for inventing 0.50* (0.04) 0.44* (0.05) 0.27* (0.05) 0.18* (0.05) 0.25* (0.06) Event novelty 0.14 (0.06) 0.10† (0.06) Event criticality 0.37* (0.06) 0.37* (0.06) Opportunity competence 0.49* (0.05) Entrepreneurial passion for inventing× Event novelty 0.09** (0.04) 0.09* (0.05) Entrepreneurial passion for inventing× Event criticality 0.05 (0.04) 0.06 (0.05) Entrepreneurial passion for inventing× Opportunity competence 0.12* (0.04) Entrepreneurial passion for inventing× Event novelty × Event criticality 0.09* (0.03) Constant 0.56* (0.20) 0.46 (0.20) 0.37* (0.20) 0.26 (0.19) 0.33† (0.20) Adjusted R2 0.31 0.33 0.38 0.45 0.40 Notes: N = 435; * p < 0.001, p < 0.01, * p < 0.05, † p < 0.1. We included the interaction term between Event novelty and Event criticality in the three-way interaction analysis, but did not report the result here as it is irrelevant to our hypothesis testing Third, as shown in Table 2, the correlations between passion for inventing and contextual factors (i.e., event novelty, event criticality, and opportunity competence) are relatively high. To determine whether passion for inventing may mediate the relationship between the contextual factors and organizational innovation, we estimated a structural equation model in which we treated the contextual factors as independent variables, passion for inventing as the mediator, and organizational innovation as the dependent variable. The results indicated poor model fit (χ2[204] = 1980.38; CFI = 0.84; RMSEA = 0.13; SRMR = 0.32). We also ran separate mediating models with each of these contextual factors as the independent variable, passion for inventing as the mediator, and organizational innovation as the dependent variable. All the models demonstrated poor model fit. The above evidence, to a certain degree, indicated that our data did not support the mediating effect of passion for inventing on the relationship between the contextual factors and organizational innovation. Finally, although existing literature treats passion as a driver of organizational innovation and performance (Baum & Locke, 2004; Drnovsek et al., 2016; Makino et al., 2020), recent research has indicated that venture progress exerts a positive influence on entrepreneurial passion (Gielnik et al., 2015). To attenuate the influence from the potential reverse causality problem, thereby mitigating passion’s possible endogeneity, we conducted a two-stage least squares (2SLS) regression. One critical issue in 2SLS was choosing valid instrument variables for passion (Wooldridge, 2012).4 Considering that existing studies have not suggested any instrument variables for passion explicitly, we drew on related research to create one instrument. According to a comprehensive review of literature on entrepreneurial passion, Cardon and Murnieks (2020) suggested that the roles of social environment and valued others should receive more attention in future research on the development of passion. Stenholm and Nielsen (2019) also empirically demonstrated that perceived social support from critical stakeholders, e.g., the government, is associated positively with entrepreneurial passion. First, perceived social support ignites positive emotions or affect, which is a critical element of entrepreneurial passion. Moreover, the experience of positive emotions will increase entrepreneurs’ engagement in entrepreneurial activities, which will promote their passion over time (Gielnik et al., 2015). Therefore, we chose social support, particularly perceived community support from the Silicon Alley government, as an instrument of passion. We derived this measure from Theodori (2001), which mainly encompasses the aspects of living environment, medical services, schools, shopping facilities, entertainment facilities, and Silicon Alley’s physical appearance. Conceptually, these aspects were not related directly to organizational innovation because they did not provide resources closely associated with organizational innovation, e.g., technology, finance et al. Moreover, they are unlikely to be affected by the government-initiated events purported to inspire regional innovation. In this way, using perceived community support as an instrument helps alleviate covariance among passion, organizational innovation, and events. Following Flammer (2018), we regressed entrepreneurial passion on the instrumental and control variables during the first stage and used passion’s fitted value as the predictor of organizational innovation during the second stage. The first-stage model’s results indicated that perceived community support significantly predicts entrepreneurial passion, and all the models’ minimum F value (33.10) exceeded the cutoff value of 9.08 suggested by Bascle (2008). The results provide empirical support for treating perceived community support as a valid instrument of entrepreneurial passion. As shown in Appendix 2, the 2SLS regression results generally were consistent with our previous findings, confirming our results’ reliability. Study 2: method Sample and data In Study 2, we tested our model using another sample, in which we focused on a different external event, the COVID-19 pandemic, to gauge our model’s generalizability. We believe that event novelty and criticality are representative characteristics of COVID-19 to most entrepreneurs, as it is highly novel and of great importance to the entrepreneurs. Following existing research using online surveys (e.g., Su et al., 2022), we collected data through Prolific, a web-based research institute that provides professional services for data collection, in April 2022. Our survey comprised full-time entrepreneurs of established firms. We distributed 281 questionnaires to entrepreneurs and excluded 79 from the analyses either because these entrepreneurs provided incorrect answers to the identifier questions or because of missing values, leaving us with 202 usable responses. To check for potential sampling bias, we performed a t-test to compare several entrepreneur- and venture-related characteristics between the remaining 202 responses and 79 rejected responses. The results indicated no significant differences in entrepreneurs’ gender, age, education level, tenure in current position, and tenure at current firm, as well as the ventures’ ownership type, firm size, firm age, and industry. We also conducted Harman’s single-factor test to measure common method variance in our data (Podsakoff & Organ, 1986). A principal component factor analysis with an unrotated solution generated six factors with eigenvalues greater than 1.0. The largest variance explained by a single factor was 20.41%; therefore, no single factor could explain most of the covariance in all the variables. Measures We adopted the same measurements and control variables used in Study 1. In measuring event characteristics, we changed the event from “building Qinhuai Silicon Alley” to “dealing with the COVID-19 pandemic since its outbreak in 2020.” Analysis and results Appendix 3 presents the variables’ descriptive statistics and correlations. The variables’ reliabilities can be found on the diagonal. We performed OLS analyses to test the hypotheses, and the results are provided in Table 5. Table 5 Regression models on entrepreneurial passion for inventing and organizational innovation in Study 2 Variables Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Gender -0.32 (0.13) -0.27 (0.12) -0.24† (0.12) -0.25 (0.12) -0.19 (0.12) -0.18 (0.11) Age -0.08 (0.07) -0.10 (0.07) -0.11† (0.07) -0.07 (0.07) -0.09 (0.07) -0.08 (0.06) Education -0.00 (0.09) -0.03 (0.09) -0.01 (0.08) -0.07 (0.08) -0.02 (0.08) -0.01 (0.08) Tenure in current firm 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) Tenure in current position -0.00 (0.00) -0.00 (0.00) -0.00 (0.00) -0.00 (0.00) -0.00 (0.00) -0.00 (0.00) Firm type 0. 30 (0.42) 0.31 (0.40) 0.36 (0.39) 0.31 (0.38) 0.27 (0.39) 0.35 (0.37) Firm size 0.42* (0.10) 0.42* (0.10) 0.38* (0.09) 0.38* (0.09) 0.41* (0.09) 0.34*** (0.09) Industry 0.33* (0.14) 0.24† (0.13) 0.18 (0.13) 0.23† (0.13) 0.20 (0.13) 0.17 (0.13) Entrepreneurial passion for inventing 0.05* (0.01) 0.05* (0.01) 0.04* (0.01) 0.03 (0.01) 0.02† (0.01) Event novelty -0.50 (0.17) -0.41 (0.16) Event criticality -0.07 (0.17) 0.01 (0.18) Opportunity competence -0.20 (0.17) -0.08 (0.18) Entrepreneurial passion for inventing × Event novelty 0.02* (0.01) 0.02† (0.01) Entrepreneurial passion for inventing × Event criticality 0.02* (0.01) 0.01 (0.01) Entrepreneurial passion for inventing × Opportunity competence 0.02** (0.01) 0.02* (0.01) Constant -0.33 (0.25) -1.19* (0.30) -1.15* (0.29) -0.97 (0.30) -0.91 (0.32) -0.67* (0.30) Adjusted R2 0.21 0.29 0.34 0.35 0.33 0.42 Notes: N = 202; * p < 0.001, > p < 0.01, * p < 0.05, † p < 0.1 As Model 2 revealed, entrepreneurial passion significantly influences organizational innovation (p < 0.001), thereby supporting Hypothesis 1. For Model 3, the interaction between entrepreneurial passion and event novelty was significant (p < 0.05), thereby supporting Hypothesis 2. Furthermore, Model 4’s results demonstrated a significant moderating effect from event criticality on the relationship between entrepreneurial passion and organizational innovation (p < 0.05), providing consistent support for Hypothesis 3. Finally, for Model 5, the interaction between entrepreneurial passion and opportunity competence was significant (p < 0.01), thereby supporting Hypothesis 4. To sum up, these findings provided consistent support for our hypotheses. Discussion In the present study, we discovered the moderating effects of events and entrepreneurs’ competence to exploit opportunities from events (opportunity competence) on the relationship between entrepreneurial passion and organizational innovation. Our findings indicated that entrepreneurial passion for inventing is likely to exert greater influence on organizational innovation when the events are both novel and critical, and when entrepreneurs have greater opportunity competence. These findings hold meaningful implications for theory and practice. Theoretical implications This study makes several contributions to the literature. First, we shed light on events’ role in examining the boundary conditions that affect entrepreneurial passion’s efficacy. Extant research suggests that events are at the heart of entrepreneurship and indicates that “the experienced event should be a principal focus in attempts to understand entrepreneurship” (Morris, 2015: 3). However, up until now, events’ significance in entrepreneurship has not been examined (Gartner, 1993; Morris, 2015). By examining the joint moderating role of event novelty and event criticality in the relationship between entrepreneurial passion and organizational innovation, we complement existing literature on entrepreneurial passion, which mainly has focused on the contingent influences of feature-based personal traits and environmental characteristics (Baron & Tang, 2011; Kiani et al., 2020; Ma, Gu, & Liu, 2017; Patel et al., 2015; Strese et al., 2018). Furthermore, by delineating events’ moderating effects on passion, we provide viable avenues for future research to investigate the interaction between events and passion. We encourage future researchers to adopt an event-oriented perspective to develop a more comprehensive understanding of entrepreneurial passion’s efficacy. Second, our study contributes to the research on the relationship between passion and innovation by integrating the emotional perspective with the cognitive and resource-based perspectives. Conventional research into the drivers of organizational innovation takes a (bounded) rational perspective by investigating the influences from external environment (Porter, 1981), and internal resources or capabilities (Barney, 1991). Different from these studies, research on passion takes an emotional perspective and treats passion as the critical driver of organizational innovation. Our findings showed that while passion is the strong internal force that facilitates innovation, its efficacy is bound by the external environment (i.e., events) and internal capabilities (opportunity competence). In this regard, we complement passion research by incorporating the cognitive and resource-based perspectives. We encourage future research to further integrate the emotional and rational perspectives when studying the efficacy of passion. For example, they may examine what entrepreneurs will do when the behavioral expectations from the external environment conflict with the entrepreneurial identity. Will they follow the behavioral expectations from key stakeholders, or insist on their identity, or will they choose a compromise? Third, our study contributes to the emotion literature by demonstrating the importance of incorporating both the affect infusion and self-verification mechanisms when studying the efficacy of passion. While the AIM focuses on how the attributes of the target, judge, or situation influence the selection of different information processing strategies and hence the degree of affect infusion (Forgas, 1995), the self-verification process in the identity literature focuses on how the behavioral expectations in a certain situation align with the identity (Burke, 1991). Our results demonstrate that both mechanisms work simultaneously when the focal emotion is identity-relevant like passion. Future research might explore the moderating role of other attributes in the AIM such as social desirability by also incorporating the self-verification process analysis. They may also make a nuanced analysis of the self-verification process by investigating entrepreneurs’ differential responses when behavioral expectations from the external environments provide opportunities or threats to their entrepreneurial identity. Fourth, our study also highlights the importance of integrating events’ differing characteristics. While existing event studies mainly have focused on investigating event characteristics’ main effects on individual or organizational consequences (e.g., Ilies et al., 2011; Zellmer-Bruhn, 2003), recent research has called for an interactionist perspective on various event characteristics (Chen et al., 2021). We complemented this new research stream by demonstrating the significant synergetic effect of event novelty and event criticality in enhancing entrepreneurial passion’s efficacy. While EST provides a comprehensive description of event characteristics, each dimension’s uniqueness and their interactions merit further investigation. For example, event criticality demonstrates an event’s relevance to the focal entity, i.e., it influences whether entrepreneurs will respond, while event novelty demonstrates the discontinuity in organizational routines that an event has elicited, i.e., it influences how entrepreneurs will respond. Furthermore, EST provides guidelines for what entrepreneurs are expected to do, while emotions determine what entrepreneurs are willing to do. By demonstrating the significant interaction effects of event characteristics and passion on organizational innovation, our work motivates future researchers to integrate EST with emotional theories to investigate their joint individual or organizational consequences. Furthermore, we provide further insights on the role of entrepreneurs’ cognitive capabilities in converting entrepreneurial passion into organizational innovation. Many studies have recognized passion’s importance in organizations, and recent research (e.g., Makino et al., 2020) has suggested that treating passion as a key intangible organizational asset holds parallel implications for knowledge-based assets (Nonaka, 1994). However, the power unleashed from passion cannot be converted fully into organizational benefits if entrepreneurs do not have enough opportunity-exploiting capabilities as part of opportunity competence. This finding indicates the importance of integrating the emotional and capability perspectives in studying passion’s effects. Finally, by drawing from the AIM and the identity literature, which were developed at the individual level to explain the relationship between entrepreneurial passion and innovation at the organizational level, this study provides a feasible avenue for investigating emotional elements’ effect on organizational innovation. Individuals have emotions just as they have cognitions. Behavioral scholars (e.g., Powell et al., 2011) have long called for a more “realistic” assumption about organizational decision-makers by incorporating psychological factors into the analysis of strategic issues. However, extant studies that aim to answer this call remain limited. One critical issue is the problem of “ecological fallacy” (Robinson, 1950) or “aggregation bias” (Rousseau, 1985), which is encountered when making parallel arguments across different levels of analysis. We believe that this problem should not hinder research progress in investigating cross-level phenomena. We advocate for more exploratory research to examine the effect of emotions, e.g., passion, on organizational outcomes by borrowing insights from theories developed at different levels. Practical implications Our studies also hold important implications for ventures that could increase their organizational innovation. First, a new venture’s founding teams usually comprise different individuals with varying characteristics and personalities. For example, entrepreneurs may have different identity orientations, i.e., some may emphasize the founder identity, while others may emphasize the inventor or developer identity. Considering that our findings indicated that passion for inventing plays a significant role in the innovation process, we suggest that when creating an entrepreneurial team, entrepreneurs’ passion for inventing should be considered. At the founding stage, entrepreneurs who view the founder identity as more important than other identities may play essential roles. However, as a venture moves to the development stage and pursues further improvement through innovation, entrepreneurs who emphasize the inventor identity may take greater responsibility than those with other identities. Second, new ventures also need to realize the boundary conditions that affect entrepreneurial passion’s efficacy. To better utilize entrepreneurs’ passion for inventing and improve organizational innovation, ventures could adapt events that may influence entrepreneurs’ perceptions of event novelty or event criticality. For example, facing novel events, entrepreneurs may realize that their passion for inventing will be extremely effective in improving their new ventures’ innovation performance. Although our study focuses on entrepreneurs’ passion, we argue that adapting these contextual factors also could improve other organizational members’ innovation performance when they share entrepreneurs’ passion. Third, we suggest that the government in Silicon Alley needs to consider the characteristics of the events they plan to initiate carefully. In particular, when the government plans to inspire innovation at regional firms, they should consider not only whether the events are critical to the entrepreneurs, but also whether the events are novel enough. In this regard, we encourage local governments to design events for regional ventures thoughtfully so that entrepreneurs can better exploit the benefits from these events. Limitations and future research Our study has several limitations that may provide opportunities for future research. First, we did not test the mediating mechanism that links passion for inventing and organizational innovation, although we performed robustness tests on cognitive information processing’s working mechanism, which undergirded the relationship. Considering that we focused mainly on examining boundary conditions of entrepreneurial passion’s effects, to maintain theoretical parsimony, we did not examine potential mediators. Further research might examine different mediating mechanisms and investigate how different moderators affect different mediating routes. Second, our survey data were cross-sectional, which may limit our ability to test causal relationships and track these relationships’ dynamics. We encourage future researchers to validate our findings using other samples. Third, we used a subjective measure of our dependent variable—organizational innovation—which may be subject to entrepreneurs’ individual bias. Although previous studies have validated this measure, we have tried to obtain more objective measurements of organizational innovation, e.g., the new product sales ratio or number of patents. However, as most ventures in our survey were unlisted firms, they were unwilling to report these data in the survey. We call for further research to test this model using more objective innovation measures. Fourth, entrepreneurs’ passion and organizational innovation are viewed frequently as existing on different levels. The mechanism that ties passion to organizational innovation may be more complex than we argued in our study. Future research could examine the more nuanced mechanisms between the key figures’ passion and organizational outcomes. For example, researchers could examine how key figures’ passion is shared among other organizational members, as well as how collective passion facilitates or impedes organizational outcomes. Fifth, following entrepreneurship research (Cardon et al., 2009), our model treats passion as the primary driver of innovation, and event characteristics as the moderators. However, interaction between passion and event characteristics may be more complicated than we proposed. Future researchers should conduct a more nuanced analysis of the different combinations of passion and event characteristics, e.g., divide combinations into four different types based on degree of entrepreneurial passion and event novelty (low vs. high), and compare the relative organizational innovation in these different scenarios. Finally, while our analysis of the moderating roles of event characteristics in the relationship between entrepreneurial passion and organizational innovation consists of two mechanisms, i.e., information processing and self-verification, we did not hypothesize these two mediating mechanisms for theoretical parsimony. Future research might select appropriate proxies for the information processing strategies and the self-verification processes, respectively, and examine a mediated moderation model to incorporate these two mechanisms. Appendix 1 Scale items Items Organizational innovation Over the past three years, this company… Has spent heavily (well above your industry average) on research and development. Has maintained world-class research and development. Has introduced a large number of new products to the market. Has acquired significantly more patents than its major competitors. Has pioneered the development of breakthrough innovations in its industry. Entrepreneurial passion for inventing It is exciting to figure out new ways to solve unmet market needs that can be commercialized. Searching for new ideas for products/services to offer is enjoyable to me. I am motivated to figure out how to make existing products/services better. Scanning the environment for new opportunities really excites me. Inventing new solutions to problems is an important part of who I am. Event novelty In building innovation in Qinhuai Silicon Alley, to what extent do you agree that: There is a clear, known way to respond to the event. There is an understandable sequence of steps that can be followed by the firm in responding to this event. The firm can rely on established procedures and practices in responding to this event. The firm had rules, procedures, or guidelines to follow when this event occurred. Event criticality In building innovation in Qinhuai Silicon Alley, to what extent do you agree that: This event was critical for my long-term success This event was important for me This event was a primary task for me Opportunity competence I can recognize potential markets; I can evaluate the advantages and disadvantages of potential business opportunities I can capture and implement the high-quality business opportunities Appendix 2 2SLS regression models on the relationship between entrepreneurial passion and organizational innovation Variables Model 1 Model 2 Model 3 Model 4 Gender 0.03 (0.08) 0.04 (0.07) 0.05 (0.07) 0.02 (0.07) Age -0.02 (0.06) 0.01 (0.06) -0.05 (0.06) -0.02 (0.05) Education -0.22* (0.06) -0.22* (0.06) -0.16 (0.06) -0.17* (0.06) Tenure in current firm -0.00*** (0.00) -0.00* (0.00) -0.00* (0.00) -0.00** (0.00) Tenure in current position 0.00 (0.00) 0.00 (0.00) 0.00* (0.00) 0.00* (0.00) Firm type 0.02 (0.09) 0.03 (0.09) 0.05 (0.09) -0.04 (0.08) Firm size -0.13* (0.05) -0.12 (0.05) -0.15 (0.05) -0.10* (0.05) Industry 0.20* (0.08) 0.24* (0.08) 0.19 (0.08) 0.20 (0.07) Instrumented passion for inventing 0.68* (0.05) 0.49* (0.07) 0.54* (0.08) 0.38* (0.08) Event novelty 0.25* (0.06) Event criticality 0.21* (0.06) Opportunity competence 0.36* (0.06) Instrumented passion for inventing× Event novelty 0.06† (0.04) Instrumented passion for inventing× Event criticality 0.08† (0.05) Instrumented passion for inventing× Opportunity competence 0.09* (0.03) Constant 0.63* (0.19) 0.51 (0.18) 0.47 (0.19) 0.45 (0.17) Adjusted R-squared 0.41 0.48 0.49 0.55 Note: N = 435; * p < 0.001, ** p < 0.01, * p < 0.05, † p < 0.1 Appendix 3 Descriptive statistics and correlation matrix in Study 2 Variables 1 2 3 4 5 6 7 8 9 10 11 12 13 1. Organizational innovation 0.91 2. Entrepreneurial passion for inventing 0.37* 0.83 3. Event novelty -0.27* -0.03 0.89 4. Event criticality 0.25* 0.20* -0.13* 0.78 5. Opportunity competence 0.41* 0.61* -0.08 0.11 0.81 6. Gender -0.01 -0.04 -0.06 0.03 -0.11 - 7. Age -0.24* -0.12 -0.03 -0.09 -0.16* 0.02 - 8. Education 0.08 0.08 0.05 0.11 0.01 0.06 -0.02 - 9. Tenure in current firm -0.20* -0.12 -0.05 -0.11 -0.15* 0.02 0.55* -0.03 - 10. Tenure in current position -0.24* -0.16* -0.03 -0.10 -0.17* 0.01 0.56* -0.06 0.94* - 11. Firm type 0.16* -0.01 -0.07 0.05 -0.04 0.10 0.01 0.10 -0.08* -0.12* - 12. Firm size 0.18* 0.08 0.08 -0.00 0.09 0.08 0.04 0.03 -0.08 0.14* 0.42* - 13. Industry 0.08 0.06 -0.12* 0.08 -0.02 0.25* 0.04 0.18* 0.03 0.13* 0.02 -0.02 - Mean 2.62 4.24 3.16 3.21 4.06 0.52 2.11 1.90 75.59 65.02 0.03 1.67 0.29 SD 1.11 0.68 1.19 1.02 0.68 0.50 1.02 0.67 67.89 62.04 0.15 5.07 0.45 Notes: N = 202; * p < 0.05. The variables including organizational innovation, entrepreneurial passion for inventing, event novelty, event criticality, and opportunity competence are standardized with a mean of 0 and a standard deviation of 1. The means and standard deviations in this table show those of the items that compose each of main variables. Variables reliabilities are shown on the diagonal Acknowledgements The authors thank the editor and two anonymous reviewers for their helpful comments. This study was supported by the Ministry of Education Project of Humanities and Social Science [grant number: 22YJC630073]; the Fundamental Research Funds for the Central Universities [grant number: 56XAB22024] and the National Natural Science Foundation of China [grant number: 72032002]. 1 Of the ventures in our sample, 60% have fewer than 100 employees, and 88% have fewer than 300 employees. 2 In China, colleges recruit students who did not meet the minimum admission score required for university admission on the National College Entrance Examination. 3 We used the means of event novelty and personal relevance as the cut-off points between high and low levels of event novelty and personal relevance, respectively,. The four groups include the following combinations: high event novelty and high event criticality; high event novelty and low event criticality; low event novelty and high event criticality; and low event novelty and low event criticality. 4 According to Wooldridge (2012), valid instruments should meet two requirements. First, the instrument variable z should correlate with the explanatory variable x, i.e., Cov (z, x) = 0. Second, the instrumental variable z should not correlate with the error term u, i.e., Cov (z, u) = 0. 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==== Front Nature Nature Nature 0028-0836 1476-4687 Nature Publishing Group UK London 36450978 5448 10.1038/s41586-022-05448-9 Article Common and rare variant associations with clonal haematopoiesis phenotypes Kessler Michael D. 1 Damask Amy 1 O’Keeffe Sean 1 Banerjee Nilanjana 1 http://orcid.org/0000-0003-0868-4884 Li Dadong 1 http://orcid.org/0000-0002-3303-8860 Watanabe Kyoko 1 Marketta Anthony 1 Van Meter Michael 2 Semrau Stefan 2 Horowitz Julie 1 Tang Jing 1 Kosmicki Jack A. 1 Rajagopal Veera M. 1 http://orcid.org/0000-0001-9816-5847 Zou Yuxin 1 Houvras Yariv 2 Ghosh Arkopravo 1 Gillies Christopher 1 Mbatchou Joelle 1 http://orcid.org/0000-0001-8344-2549 White Ryan R. 2 Verweij Niek 1 Bovijn Jonas 1 http://orcid.org/0000-0003-2027-1871 Parikshak Neelroop N. 1 LeBlanc Michelle G. 1 Jones Marcus 1 Regeneron Genetics Center GHS-RGC DiscovEHR Collaboration http://orcid.org/0000-0001-6187-4164 Glass David J. 2 Lotta Luca A. 1 http://orcid.org/0000-0002-1074-1203 Cantor Michael N. 1 Atwal Gurinder S. 2 http://orcid.org/0000-0001-6227-198X Locke Adam E. 1 http://orcid.org/0000-0001-9059-1825 Ferreira Manuel A. R. 1 Deering Raquel 2 http://orcid.org/0000-0002-7918-3561 Paulding Charles 1 Shuldiner Alan R. 1 Thurston Gavin 2 Ferrando Adolfo A. 1 Salerno Will 1 http://orcid.org/0000-0001-8645-4713 Reid Jeffrey G. 1 Overton John D. 1 http://orcid.org/0000-0003-0610-8322 Marchini Jonathan 1 Kang Hyun M. 1 http://orcid.org/0000-0002-6830-3396 Baras Aris 1 http://orcid.org/0000-0003-1509-1825 Abecasis Gonçalo R. 1 http://orcid.org/0000-0002-5829-8191 Jorgenson Eric [email protected] 1 1 grid.418961.3 0000 0004 0472 2713 Regeneron Genetics Center, Tarrytown, NY USA 2 grid.418961.3 0000 0004 0472 2713 Regeneron Pharmaceuticals, Tarrytown, NY USA 30 11 2022 2022 612 7939 301309 23 12 2021 14 10 2022 © The Author(s), under exclusive licence to Springer Nature Limited 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1–5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications. Exome sequence data from 628,388 individuals was used to identify 24 risk loci in 40,208 carriers of clonal haematopoiesis of indeterminate potential and link them to other conditions including COVID-19, cardiovascular disease and cancer. Subject terms Cancer genetics Genetics research Epidemiology Genome-wide association studies issue-copyright-statement© The Author(s), under exclusive licence to Springer Nature Limited 2022 ==== Body pmcMain As humans age, somatic alterations accrue in the DNA of haematopoietic stem cells (HSCs) due to mitotic errors and DNA damage. Alterations that confer a selective growth advantage can lead to the expansion of particular cell lineages, a phenomenon called clonal haematopoiesis. The presence of clonal haematopoiesis has been associated with an increased risk of haematological neoplasms, cytopaenias, cardiovascular disease (CVD), infection and all-cause mortality1–5. For this reason, identifying germline causes of clonal haematopoiesis has the potential to improve our understanding of initiating events in the development of these common diseases. Large-scale studies of the germline causes of clonal haematopoiesis have used samples from the UKB and other large cohorts, but those studies have been limited mostly to clonal haematopoiesis phenotypes that can be assessed using single nucleotide polymorphism (SNP) array genotype data, such as mosaic chromosomal alternations (mCA) and mosaic loss of sex chromosomes4,7,8 (mLOX and mLOY). Identifying individuals with CHIP, which is defined by somatic protein-altering mutations in genes that are recurrently mutated in clonal haematopoiesis, requires sequencing of blood1,2. Once a clone has expanded sufficiently, the somatic variants from this clone can be captured along with germline variants by exome sequencing. Since exome sequencing captures protein-altering variants, its large-scale application enables the detection of readily interpretable rare variant association signals, and can elucidate critical genes and pathways and potential therapeutic targeting9,10. So far, the largest genetic association study of CHIP has included 3,831 CHIP mutation carriers in a sample of 65,405 individuals and has identified four common variant loci11. Here, we use exome sequencing data to characterize CHIP status in 454,803 UKB10 and 173,585 Geisinger MyCode Community Health Initiative (GHS) participants. We then conduct a common variant genome-wide association study (GWAS) and rare variant and gene burden exome-wide association study (ExWAS) of CHIP by leveraging 27,331 CHIP mutation carriers from the UKB. We perform a replication analysis using 12,877 CHIP mutation carriers from the GHS cohort. To identify germline predictors of specific clonal haematopoiesis driver mutations, we also conduct GWAS and ExWAS in carriers of CHIP mutations from individual CHIP genes. We then compare genetic association findings for CHIP to those from analyses of other clonal haematopoiesis phenotypes determined from somatic alterations in the blood, including mCA, mLOX, mLOY and telomere length. Although GWAS of these non-CHIP clonal haematopoiesis phenotypes have been conducted4,7,12, none have evaluated the effect of rare variation. The ExWAS we perform here represents the first systematic large-scale exploration of the effect of rare variants on the genetic susceptibility of these phenotypes. Finally, we examine the clinical consequences of somatic CHIP mutations and germline predictors of CHIP in several ways. We first conduct a PheWAS13 of germline predictors of CHIP to understand their biological functions, and test cross-sectional phenotype associations of CHIP carrier status across 5,194 traits in the UKB. We then test the risk of incident cancer, CVD and all-cause mortality among specific CHIP gene mutation carriers and use Mendelian randomization to test for evidence of causal associations between CHIP and phenotypes of interest. Calling CHIP We used exome sequencing data from 454,803 and 173,585 individuals from the UKB and GHS cohorts, respectively, to generate large callsets of CHIP carrier status (Methods). In brief, we called somatic mutations using Mutect2 in a pipeline that included custom QC filtering (Extended Data Fig. 1a), and ultimately restricted our analysis to 23 well defined and recurrent CHIP-associated genes. This focused analysis identified 29,669 variants across 27,331 individuals in the UKB (6%), and 14,766 variants across 12,877 individuals in the GHS (7.4%). DNMT3A, TET2, ASXL1, PPM1D and TP53 were the most commonly mutated genes in both cohorts (Extended Data Fig. 2a). Although the GHS cohort had a wider age range, and therefore a larger number of older individuals, the prevalence by age was similar across cohorts, and reached approximately 15% by 75 years of age (Extended Data Fig. 1b,c). Prevalence of CHIP gene-specific mutations was consistent with recurrence patterns, with mutations in the most commonly mutated CHIP genes beginning to increase in prevalence at younger ages (Extended Data Fig. 1d,e and Supplementary Note 1). Somatic mutations within the IDH2 and SRSF2 genes co-occurred significantly more frequently than expected in both the UKB and GHS cohorts, whereas DNMT3A mutations co-occurred less frequently with other mutations than expected (Extended Data Fig. 2b,c and Supplementary Table 1). Among individuals with multiple CHIP mutations (Supplementary Note 2 and Supplementary Fig. 1), JAK2 mutations consistently had the highest variant allele fraction (VAF) (Supplementary Fig. 1b). CHIP demographics Compared with controls, CHIP carriers in both the UKB and GHS cohorts were older and more likely to be heavy smokers, consistent with previous studies11 (Table 1). Although our cohorts were predominantly comprised of European ancestry individuals, the prevalence of CHIP was similar across all ancestries (Supplementary Fig. 2). In multivariate logistic regression models, each additional year of age was strongly associated with an increased risk of CHIP in the UKB (odds ratio [range] = 1.08 [1.077–1.082], P < 10−300) and GHS (odds ratio = 1.06 [1.057–1.063], P < 10−300), and heavy smoking was strongly associated with CHIP carrier status in both UKB (odds ratio = 1.17 [1.14–1.21], P = 7.32 × 10−24) and GHS (odds ratio = 1.24 [1.10–1.41], P = 6.3 × 10−4). Overall, our results suggest that the prevalence of CHIP doubles every 9–12 years of life. These associations with age and smoking were stronger when restricting to high-VAF (≥0.1) CHIP carriers. In our multivariate modelling, women were significantly more likely to be CHIP mutation carriers than men in the UKB (odds ratio = 1.08 [1.05–1.11], P = 6.01 × 10−7), but not in the GHS (odds ratio = 1.01 [0.93–1.11, P = 0.77]). These associations were consistent when restricting to high-VAF CHIP carriers, although the risk of high-VAF CHIP was not significantly greater in women in the UKB (odds ratio = 1.035 [0.99–1.08], P = 0.126).Table 1 Descriptive statistics for CHIP mutation carriers Age (median) Sex (% female) Heavy smoking (%) Previous blood cancer (%) Any blood cancer (%) Any cancer (−NMSC, %) Severe COVID-19 (%) Ancestry (European, African, South Asian, other (%)) UKB CHIP 62 54.0 33.9 2.18 6.49 26.6 0.52 96 1.4 1.8 0.73 No CHIP 57 54.3 27.5 0.60 2.19 17.8 0.31 95 2.0 2.3 0.94 GHS CHIP 73 55.2 13.7 1.59 12.7 41.7 0.45 98 1.4 0.1 0.65 No CHIP 57 61.6 9.57 0.63 3.22 19.7 0.18 94 3.2 0.3 1.9 Genetic association with CHIP carrier status We first conducted genetic association analyses in the UKB cohort to identify germline loci associated with the risk of developing CHIP. In the common variant (minor allele frequency (MAF) > 0.5%) GWAS, which included 25,657 cases and 342,869 controls with European ancestry, we identified 24 loci (21 novel loci) harbouring 57 independently associated variants (Fig. 1 and Supplementary Table 2). To confirm these signals, we conducted a replication analysis in 9,523 CHIP cases and 105,502 controls of European ancestry from the GHS cohort. We estimated that we had sufficient statistical power in the GHS to detect 19.99 true and directionally consistent associations across lead SNPs from the 24 loci we identified in the UKB and achieved nominally significant (P < 0.05) replication for 15 SNPs (Supplementary Table 2). We used conditional analysis and statistical fine-mapping to further evaluate the independence of our genome-wide associations and found results to be consistent across methods (Extended Data Fig. 3, Supplementary Note 3, Supplementary Tables 3–6 and Supplementary Fig. 3).Fig. 1 GWAS of CHIP. Manhattan plot showing results from a genome-wide association analysis of CHIP. Twenty-four loci reach genome-wide significance (P ≤ 5 × 10−8, dashed line), and top-associated variants per locus are labelled with biologically relevant genes. Three of these loci have been previously identified (black), whereas 21 represent novel associations (red). Loci with suggestive signal (P ≤ 5 × 10−7) are labelled in grey. Association models were run with age, age2, sex and age × sex, and 10 ancestry-informative principal components as covariates. P-values are uncorrected and are from two-sided tests performed using approximate Firth logistic regression. We next sought to identify rare germline variants associated with CHIP. Since the CHIP phenotype is based on the presence of rare somatic variants in recurrently mutated genes, rare germline variants potentially misclassified as somatic can lead to false association signals. To address potential misclassification, we evaluated median VAF and association with age for each rare germline variant or gene burden associated with CHIP. We also conditioned these rare variant analyses on independent common variant signals to address confounding due to linkage disequilibrium (LD) (Supplementary Note 4). Ultimately, we identified a single rare germline frameshift variant in the CHEK2 gene that was significantly associated with CHIP (odds ratio = 2.22 [1.89–2.61], P = 8.04 × 10−22; Supplementary Table 7), remained so after conditioning on common variant signals (odds ratio = 2.90 [1.93–4.34], P = 2.40 × 10−7), and replicated in the GHS (odds ratio = 1.56 [1.19–2.04], P = 1.22 × 10−3). The two cancer-associated genes ATM and CHEK2 were associated with an increased risk of CHIP via rare variant gene burden testing (Supplementary Table 8), and we also found a significant gene burden association between rare loss of function (and missense) variants in the telomere maintenance and DNA replication associated gene CTC1 and an increased risk of CHIP (odds ratio = 1.55 [1.32–1.81], P = 5.24 × 10−8). Of these three gene burden associations, the ATM and CHEK2 signals were replicated in the GHS (P = 8.22 × 10−5 and P = 0.03, respectively), and VAF and age-association calculations suggested that all three of these gene burden signals were driven by germline variation. We also performed genome-wide association analyses in individuals of non-European ancestral background (Supplementary Note 5 and Supplementary Table 9). For each germline variant associated with CHIP and prioritized by clumping and thresholding, conditional analysis or fine-mapping (see Methods), we queried its associations across 937 binary and quantitative health traits from the UKB for which we have previously performed genetic association analysis10 (Supplementary Table 10). Overall, the traits with significant associations consisted predominantly of blood measures (that is, cells counts and biomarker levels), anthropometric measures related to body size, autoimmune phenotypes and respiratory measures. SNPs with the largest number of significant phenotypic associations included those at the HLA, TP53, ZFP36L2 and THADA, CD164 and MYB loci (Extended Data Fig. 4). Whereas associations with blood cell counts and biomarker levels are probably the direct result of expansion of individual cell lineages in blood, association with autoimmune phenotypes could reflect the consequences of disrupted immune system differentiation related to clonal haematopoiesis. Analyses of individual CHIP gene mutations To identify CHIP subtype-specific risk variants, we defined gene-specific CHIP phenotypes for each of the eight most commonly mutated CHIP genes. For each subtype, we selected individuals with mutations in one of the eight genes and no mutations in any of the other genes used to define CHIP. We then conducted genetic association analyses comparing these single CHIP gene carriers to CHIP-free controls, with replication in the GHS, and observed shared, unique, and opposing effects of associated loci on CHIP subtypes, including 8 genome-wide significant loci that were not significant in our overall analysis of CHIP (Fig. 2a, Extended Data Fig. 5 and Supplementary Tables 11–19).Fig. 2 Germline effect size comparisons across CHIP and Forest plots of PARP1 and LY75 missense variants. a, Using results from CHIP gene-specific association analyses, effect sizes of index SNPs are compared across CHIP subtypes. SNPs were chosen as those that were independent on the basis of clumping and thresholding (with some refinement based on our conditionally independent variant list) and genome-wide significant in at least one association with CHIP or a CHIP subtype. Certain loci showed notably different effects across CHIP subtypes, as seen at the CD164 locus, which was associated with DNMT3A CHIP and ASXL1 CHIP but not TET2 CHIP, and the TCL1A locus, which was associated with increased risk of DNMT3A CHIP but reduced risk of other CHIP subtypes (blue rectangles). b, Forest plots are shown reflecting the protective associations of a PARP1 missense variant (rs1136410-G) and two LY75 missense variants (rs78446341-A, rs147820690-T) with our DNMT3A CHIP phenotype in the UKB and GHS cohorts. Centre points represent odds ratios as estimated by approximate Firth logistic regression, with errors bars representing 95% confidence intervals. P-values are uncorrected and reflect two-sided tests. Numbers below the cases and controls columns represent counts of individuals with homozygote reference, heterozygote and homozygous alternative genotypes, respectively. DNMT3A, which was the most commonly mutated gene in the overall CHIP phenotype, had the largest number of significantly associated loci (n = 23), most of which overlapped with the overall CHIP association signals. Six loci achieved genome-wide significance in our DNMT3A CHIP analysis that were not significant in our overall analysis (RABIF, TSC22D2, ABCC5, MYB, FLT3 and TCL1A; Extended Data Fig. 5). Although most loci harboured variants that increased CHIP risk, two exceptions are noteworthy (Fig. 2b). At the PARP1 locus on chromosome 1, a tightly linked block of around 30 variants (29 in the 95% credible set from fine-mapping; Supplementary Table 6) with an alternate allele frequency (AAF) of 0.15 was associated with reduced risk of DNMT3A CHIP (odds ratio = 0.87 [0.84–0.90], P = 2.70 × 10−17). PARP1 has a role in DNA damage repair, and many variants in this block have been identified across multiple transcriptomic studies of blood as PARP1 expression quantitative trait loci (eQTLs) that associate with reduced PARP1 gene expression14–17. Furthermore, a missense variant (rs1136410-G, V762A) that is predicted as likely to be damaging (combined annotation dependent depletion (CADD) score = 27.9) is a part of this LD block, and has recently been reported to associate with improved prognosis and survival in myelodysplastic syndromes18 (MDS). At a locus on chromosome 2, rs78446341 (P1247L in LY75) was associated with reduced risk of DNMT3A CHIP (odds ratio = 0.78 [0.72–0.84], P = 3.70 × 10−10), and was prioritized by fine-mapping (Extended Data Fig. 3). LY75 features lymphocyte-specific expression (Supplementary Fig. 4a), and is thought to be involved in antigen presentation and lymphocyte proliferation19. We also identified a second rare (AAF = 0.002) missense variant (rs147820690-T, G525E) that associated with reduced risk of DNMT3A CHIP at close to genome-wide significance (odds ratio = 0.48 [0.36–0.63], P = 1.15 × 10−7). This variant was predicted as likely to be damaging (CADD = 23.6) and remains associated (odds ratio = 0.63 [0.51–0.77], P = 4.80 × 10−6) when conditioning on common variant signal in this locus (that is, this rare variant signal is independent of the common variant signal in this locus). This variant was also prioritized by fine-mapping (Extended Data Fig. 3 and Methods for jointly fine-mapping common and rare variants). Finally, these signals in PARP1 and LY75 replicated in the GHS (Fig. 2b). Among loci associated with multiple CHIP subtypes (Supplementary Note 6), we observed genome-wide significant association signals at the TCL1A locus that were not present in the overall CHIP analysis. This locus is notable because it exhibited genome-wide significant effects in opposing directions across CHIP subtypes (Extended Data Figs. 2a and 5 and Supplementary Table 20), with lead SNPs (for example, rs2887399-T, rs11846938-G and rs2296311-A) at the locus associated with an increased risk of DNMT3A CHIP (odds ratio = 1.14 [1.11–1.17], P = 2.13 × 10−20) but a reduced risk of TET2 CHIP (odds ratio = 0.75 [0.71–0.80], P = 9.14 × 10−22) and ASXL1 CHIP (odds ratio = 0.70 [0.65–0.76], P = 8.59 × 10−18). Effect estimates from the other five CHIP gene-specific association analyses were also consistent with protective effects. This is consistent with findings from a recent genetic association study of CHIP in the TOPMed cohort11, which identified a genome-wide significant positive association of the TCL1A locus and DNMT3A CHIP as well as a nominally significant opposing signal for TET2 CHIP. Additionally, the DNMT3A CHIP-increasing allele has been found to reduce the risk of mLOY in a recent GWAS7. This observation suggests that DNMT3A CHIP is distinct among clonal haematopoietic subtypes with regard to the genetic influence of the TCL1A locus, which may relate to the fact that TCL1A has been reported to directly interact with and inactivate DNMT3A20. CHIP and mosaic chromosomal alterations To evaluate the relationship between CHIP and other forms of somatic alterations of the blood, we used phenotype information on other types of clonal haematopoiesis that are available for UKB participants4,7,8,12. We first evaluated the phenotypic overlap between CHIP and mLOY, mLOX and autosomal mosaic chromosomal alterations (mCAaut). CHIP is distinct from mCA phenotypes (mCAaut, mLOX and mLOY), with more than 80% of CHIP carriers having no identified mCAs (Supplementary Fig. 4b). Furthermore, having an mCA is not significantly associated with being a CHIP carrier after adjusting for age, sex and smoking status (odds ratio = 1.02, P = 0.27). Carriers of only a single clonal haematopoiesis driver (that is, CHIP, mLOY, mLOX or mCAaut) were younger on average than those with multiple clonal haematopoiesis lesions, and mCAaut and CHIP carriers were youngest among single clonal haematopoiesis phenotype carriers (Supplementary Fig. 4c). We then conducted GWAS and ExWAS analyses of these somatic alteration phenotypes and evaluated the germline genetic contributions shared between CHIP and these traits (Supplementary Fig. 5 and Supplementary Tables 21–27). Genome-wide genetic correlation (rg)21,22 was nominally significant between CHIP and mLOY (rg = 0.27, P = 0.014 (uncorrected); Supplementary Table 21). Notably, variants at 4 loci (marked by the genes ATM, LY75, CD164 and GSDMC) showed similar associations with both CHIP and mLOY, whereas variants at the SETBP1 locus were negatively associated with CHIP and positively associated with mLOY. These comparisons suggest that despite being distinct clonal haematopoietic phenotypes, CHIP and mLOY share multiple germline genetic risk factors. Although the common variant association analyses of these other somatic alteration phenotypes were undertaken for the purpose of comparing to CHIP, and our results are consistent with recent published associations for these non-CHIP UKB somatic alteration phenotypes4,7,8, we also identified novel rare variant and gene burden associations via ExWAS analyses (Supplementary Note 7, Supplementary Tables 22–27 and Supplementary Fig. 6). We also extended our ExWAS analysis to telomere length and identified multiple novel rare variant associations (Supplementary Note 8 and Supplementary Tables 28–30). Phenotypic associations with CHIP Clonal haematopoiesis has been associated with an increased risk of haematologic malignancy and CVD, as well as other health outcomes including all-cause mortality and susceptibility to infection3,4,23,24. To test for expected as well as potentially novel associations, we performed cross-sectional association analyses across 5,041 traits (2,640 binary and 2,401 quantitative traits) from the UKB, curated as part of our efforts for the UKB Exome Sequencing Consortium. We performed Firth penalized logistic regression using CHIP gene mutation carrier status (that is, whether an individual had a mutation in our callset within a specific CHIP gene) as the binary outcome for 22 of the 23 CHIP genes in our callset (counts were too low for CSF3R; Methods), with age, sex and ten genetic principal components as covariates. Our results are consistent with previous findings, with the majority of associated phenotypes deriving from cardiovascular, haematologic, neoplastic, infectious, renal and/or smoking-related causes (Fig. 3, Supplementary Fig. 7 and Supplementary Table 31).Fig. 3 Phenome association profiles per CHIP subtype. Profiles are shown for each CHIP gene subtype reflecting phenome-wide association results. The y-axis (concentric circles) represents the proportion of phenotypes within a trait category that were nominally associated (P ≤ 0.05) with carrier status of the CHIP gene. A CHIP gene had to have at least one disease category with the proportion of associated phenotypes ≥ 0.2 to be included in the figure. As expected, haematological traits show the largest proportion of phenotypic trait associations overall. The largest number of cancer associations are seen for DNMT3A CHIP, whereas JAK2 CHIP shows the highest proportion of cardiovascular associations. Respiratory associations are most pronounced for ASXL1 CHIP. SUZ12 CHIP shows a unique profile across CHIP subtypes, with a higher proportion of ophthalmological and endocrine associations. Association models were run with age, age2, sex and age × sex, and ten ancestry-informative principal components as covariates. ASXL1 CHIP was associated with the largest number and widest range of traits, and many of these associations traced to correlates of smoking. SUZ12 CHIP showed a distinct association profile amongst CHIP genes, with a larger proportion of associations in endocrine and ophthalmologic traits than other CHIP genes. Many traits showed associations with DNMT3A CHIP and TET2 CHIP that were in opposing directions, including white blood cell count, platelet count and neutrophil count, which were all positively associated with DNMT3A CHIP and negatively associated with TET2 CHIP. These results are consistent with functional differences in the haematopoietic phenotypes of DNMT3A- and TET2-knockout mice25. Notably, body mass index (BMI) and fat percentage were negatively associated with DNMT3A CHIP and other leukaemogenic CHIP mutations (for example, JAK2, CALR and MPL), but are positively associated with other CHIP subtypes (for example, TET2 and ASXL1). We also observed significant associations between JAK2 mutations and gout, which may reflect the increased uric acid production that can accompany haematopoiesis26 and/or renal disease27, or even uric acid-independent associations identified between anaemia and gout28. Given recent reports that clonal haematopoiesis is associated with an increased risk of COVID-19 and other infections4,29, we also tested for an association between CHIP and COVID-19 infection in the UKB cohort30. When restricting to CHIP carriers with VAF ≥ 10% (Supplementary Note 9), we found that CHIP carrier status was significantly associated with COVID-19 hospitalization (odds ratio = 1.26 [1.07–1.47], P = 4.5 × 10−3) and severe COVID-19 infection (odds ratio = 1.55 [1.19–1.99], P = 8.5 × 10−4) in logistic regression models that excluded individuals with any previous blood cancers and that adjusted for age, sex, smoking, BMI, type 2 diabetes, active malignancy, and five genetic principal components. Analyses at the CHIP subtype level suggested that PPM1D carriers may be at elevated risk of severe COVID-19 (odds ratio = 5.42 [1.89–12.2], P = 2.8 × 10−4; Supplementary Note 9). Longitudinal disease risk among CHIP carriers Given the confounding that can bias cross-sectional association analyses, we performed survival analyses to evaluate whether individuals with CHIP at the time of enrolment and blood sampling in the UKB were at an increased risk of subsequent CVD, cancer and all-cause mortality. To do this, we generated aggregate longitudinal phenotypes of CVD, lymphoid cancer, myeloid cancer, lung cancer, breast cancer, prostate cancer, colon cancer and overall survival (that is, any death). Because prior longitudinal studies of CHIP and the risk of many of these outcomes have focused on high-VAF CHIP, we focused on CHIP carriers with VAF ≥ 0.10 for these analyses. To complement these longitudinal analyses, we used Mendelian randomization to evaluate the relationship between CHIP and subsequent disease (Extended Data Fig. 6a, Supplementary Note 10 and Supplementary Table 32). We observed a significantly increased risk of CVD in CHIP carriers (hazard ratio = 1.11 [1.03–1.19], P = 4.2 × 10−3), which was driven by TET2 CHIP (hazard ratio = 1.31 [1.14–1.51], P = 1.3 × 10−4; Supplementary Fig. 8a). However, this risk estimate is lower than the hazard ratio of 1.59 recently reported by Bick et al.6 in an analysis of CHIP from the first 50,000 UKB participants (hereafter referred to as the 50k UKB subset) with exome sequencing data available. Therefore, we restricted our analysis to the 50,000 individuals from the previous study and found that the estimated hazard ratio is indeed higher in this subset (hazard ratio = 1.30 [1.06–1.59], P = 0.013; Supplementary Fig. 8b). Bick et al. also observed a cardio-protective effect of IL6R rs2228145-C (a genetic proxy for IL-6 receptor inhibition) among CHIP carriers in the 50k UKB subset, so we repeated that analysis in both the 50k UKB subset and the full UKB cohort (n = 430,924 in these analyses). We observed the same CHIP-specific protective IL6R effect in the 50k UKB subset as previously reported (hazard ratio = 0.60 [0.40–0.89], P = 0.012), however we did not find any IL6R effect in the full cohort (hazard ratio = 0.99 [0.91–1.07], P = 0.784, n = 430,924; Extended Data Fig. 7a–d). These results were consistent when varying which CHIP mutations we used to define CHIP case status, as well as when using different VAF thresholds and a variety of CVD endpoint composites (Methods). We did not find any association between CHIP and CVD, nor a CHIP-specific protective IL6R effect, when repeating this analysis in the GHS cohort (Supplementary Figs. 8d and 9a, b). Furthermore, we did not find evidence for a casual association between CHIP and CVD when using a two-sample Mendelian randomization approach (Supplementary Note 10, Supplementary Fig. 10 and Supplementary Table 32). We next tested whether CHIP carriers are at an increased risk of haematologic and solid cancers, and whether risk differed by CHIP mutational subtype for the three most common CHIP genes (that is, DNMT3A, TET2 and ASXL1; Extended Data Figs. 7–9 and Supplementary Figs. 11–14). To control for the possibility that toxic chemotherapeutic treatment for previous cancers might drive the development of CHIP mutations31 and/or otherwise confound association analyses, we performed all analyses after excluding individuals with any diagnoses of cancer prior to DNA collection. As expected, we found CHIP carriers with VAF ≥ 0.10 to be at a significantly elevated risk of developing any blood cancer (hazard ratio = 3.88 [3.46–4.36], P = 9.10 × 10−117; Supplementary Fig. 11a), and we identified similarly elevated risk when replicating these analyses in the GHS (Supplementary Fig. 11d). We also estimated the risk of CHIP on neoplastic myeloid subtypes, including acute myeloid leukaemia (AML), MDS and myeloproliferative neoplasms (MPN), and found that high-VAF CHIP carriers have more than 23-fold increased risk of acquiring an MPN (hazard ratio = 23.11 [17.63–30.29], P = 1.60 × 10−114) (Extended Data Fig. 8). As expected, we identified a significant association between myeloid leukaemia and CHIP by Mendelian randomization (Supplementary Note 10, Supplementary Fig. 12 and Supplementary Table 32). We then tested whether CHIP carriers had an increased risk of developing solid tumours, and found that high-VAF carriers are at significantly increased risk of developing lung cancer (hazard ratio = 1.64 [1.42–1.90], P = 1.10 × 10−11), and more modest increased risk of developing prostate cancer (hazard ratio = 1.18 [1.05–1.32], P = 5.30 × 10−3) and non-melanoma skin cancer (hazard ratio = 1.14 [1.04–1.24], P = 4.7 × 10−3; Fig. 4 and Supplementary Fig. 13). We also observed a non-significant increased risk of developing breast cancer (hazard ratio = 1.14 [0.99–1.31], P = 0.062) and no increase in risk for the development of colon cancer (hazard ratio = 0.95 [0.78–1.15], P = 0.59; Supplementary Fig. 13). Models estimating event risk on the basis of CHIP mutational subtype (for example, DNMT3A CHIP) suggest that these associations with prostate and breast cancer are driven primarily by DNMT3A mutations. Only the association with lung cancer was replicated in the GHS (Fig. 13e), although sample sizes were limited for the analyses in the GHS owing to how the biobank data were ascertained (Methods).Fig. 4 Increased risk of lung cancer among CHIP carriers. a, Forest plot and table featuring hazard ratio estimates from Cox proportional hazard models of the risk lung cancer among CHIP carriers. Error bars represent a 95% confidence interval. Associations are similar across common CHIP subtypes, as well as among CHIP carriers with lower VAF (≥2%). Models are adjusted for sex, low density lipoprotein, high density lipoprotein, smoking status, pack years, BMI, essential primary hypertension, type 2 diabetes mellitus, and 10 genetic principal components specific to a European ancestral background. HR, hazard ratio. UKB 450K, the 450,00-participant full UKB dataset. DNMT3A+ represents subjects with DNMT3A CHIP and at least one other type of CHIP mutation. b, Estimated associations via four Mendelian randomization methods between CHIP and lung cancer. Each point represents one of 29 instrumental variables (that is, conditionally independent SNPs) that were identified in the UKB cohort as associated with CHIP. The x-axis shows the effect estimate (beta) of the SNP on CHIP in the UKB cohort, and the y-axis shows the effect estimate (beta) of the SNP on lung cancer in the GHS cohort. The slope of each regression line represents the effect size estimated by respective methods. IVW, inverse variance weighted. Given the strong associations between CHIP and both blood and lung cancers, and the associations between smoking and both CHIP and lung cancer, we performed additional analyses stratified by smoking status to test whether these associations were driven by smoking and merely marked by CHIP mutations. Although smoking status is difficult to ascertain, we used an inclusive ‘ever smoker’ definition to minimize the likelihood that individuals labelled as non-smokers had engaged in any smoking (Methods). High-VAF CHIP carriers had an increased risk of developing blood cancers in both smokers (hazard ratio = 3.95 [3.25–4.78], P = 2.80 × 10−44) and non-smokers (hazard ratio = 3.97 [3.43–4.58], P = 1.10 × 10−77; Supplementary Fig. 14a, b). Notably, lung cancer risk for high-VAF CHIP carriers was significantly elevated among both smokers (hazard ratio = 1.67 [1.41–1.97], P = 1.5 × 10−9) and non-smokers (hazard ratio = 2.02 [1.53–2.67], P = 8.30 × 10−7 ; Extended Data Fig. 9a,b). These associations were driven by DNMT3A and ASXL1 CHIP carriers, with both estimated to have elevated lung cancer risk in both smokers and non-smokers. We replicated the association between CHIP carrier status and lung cancer in both smokers and non-smokers in the GHS (Extended Data Fig. 9c,d). Overall, these models suggest that CHIP mutation carriers are at an elevated risk of both blood cancer and lung cancer, independent of smoking status. We also found support for a causal association between CHIP and lung cancer (inverse variance weighted odds ratio (ORIVW) = 1.55 [1.34–1.80], P = 8.90 × 10−9; Fig. 4 and Extended Data Table 1), as well as more modest support for causal associations between CHIP and melanoma (ORIVW = 1.39 [1.13–1.1.71], P = 0.0021), CHIP and non-melanoma skin cancer (ORIVW = 1.26 [1.13–1.41], P = 5.30 × 10−5), CHIP and prostate cancer (ORIVW = 1.20 [1.03–1.1.39], P = 0.017), and CHIP and breast cancer (1.17 [1.04–1.31], P = 0.01), when performing Mendelian randomization (Extended Data Fig. 6a, Supplementary Note 10 and Supplementary Table 32). Although there is a concern that variants predisposing to CHIP via cancer-associated pathways (for example, telomere biology, DNA damage repair and cell cycle regulation) may confound these associations via horizontal pleiotropy, Egger-based Mendelian randomization methods that account for this bias by fitting a non-zero intercept provided additional support for these associations. Finally, the risk of death from any cause was significantly elevated among high-VAF CHIP carriers (hazard ratio = 1.27 [1.18–1.36], P = 2.70 × 10−11), and was similar across DNMT3A, TET2 and ASXL1 CHIP subtypes (Extended Data Fig. 6b). In this study, we present the largest assessment to date of individuals with CHIP mutation carrier information, as well as the use of these calls to identify novel common and rare variant loci associated with CHIP and CHIP subtypes. These loci, which have shared, unique and opposing effects on the risk of developing different types of CHIP and other somatic alterations of the blood, highlight the fact that germline variants can predispose to clonal expansions, and that CHIP encapsulates a complex set of heterogeneous phenotypes. We further show that the genetic aetiology of CHIP is reflected in its clinical consequences, as the risk of various clinical conditions is differentially associated across CHIP gene mutations. The new loci identified in this study provide a foundation on which to investigate the biological mechanisms that lead to specific features of CHIP. For example, among CHIP-associated loci, variants in the TCL1A locus that are associated with an increase in the risk of DNMT3A CHIP have the opposite effect on the risk of all other CHIP and clonal haematopoiesis subtypes. Coupled with recent findings that link the role of TCL1A in mLOY to lymphocytes7 (for example, B cells), our results further suggest TCL1A as a critical mediator of clonal haematopoiesis as well as clonal haematopoiesis subtype-specific differences. Several novel loci associated with DNMT3A CHIP harbour genes that are potential targets for the development of new treatments to prevent or slow the expansion of CHIP clones. Both PARP1 and LY75 contain missense variants associated with reduced risk of CHIP and of DNMT3A CHIP specifically. The variants in the PARP1 locus are significantly associated with reduced PARP1 gene expression in whole blood32 (P ≤ 1 × 10−13), and the V762A missense variant (rs1136410-G) has been recently reported to associate with improved prognosis and survival in MDS18. Given the well-established role of PARP1 in DNA repair33, and that a recent CRISPR screen study in zebrafish identified PARP1 inhibition as a selective killer of TET2 mutant haematopoietic stem cells34, it seems plausible that a therapeutic strategy that inhibits PARP1 might be viable for the antagonization of CHIP clone expansion. Furthermore, PARP1-inhibiting drugs are already approved for use in the treatment of BRCA-mutant cancers35. Conversely, PARP1 inhibition is known to cause haematologic toxicity and to increase the risk of treatment related haematologic malignancy36. Therefore, further research is needed to test whether PARP1 inhibition may be appropriate for use in antagonizing the expansion of CHIP clones, and whether any effect is clonal haematopoiesis subtype-specific. The more common LY75 missense variant (rs78446341-A, P1247L) is located in the extracellular domain of lymphocytic antigen 75 (also known as DEC-205 or CD205), and has a role in antigenic capture, processing and presentation37. The rarer LY75 missense variant (rs147820690-T, G525E) is located in a C-type lectin domain and reported to interact directly with this receptor’s ligand. LY75 is expressed predominantly in haematopoietic-derived cells37,38 (and particularly dendritic cells), and its ablation impairs T cell proliferation and response to antigen challenge19. The protective associations with this variant that we identified appear to be most pronounced for DNMT3A CHIP and mLOY, and highlight LY75 as a potential therapeutic target for the antagonization of clonal haematopoiesis in general. Although most of the phenotypic associations we observe in our cross-sectional analyses are expected associations with haematologic and oncologic traits, the associations we identify with obesity and body mass traits are of particular interest. This relationship between body mass and CHIP may relate to inflammatory or hormonal signalling, and directions of effect that we estimate are consistent with recent findings that DNMT3A CHIP reduces bone mineral density via increases in macrophage-mediated IL-20 signalling39. The fact that the association we report between obesity and body mass and CHIP are in opposing directions across CHIP subtypes (for example, negative in DNMT3A CHIP and positive in TET2 CHIP and ASXL1 CHIP) suggests that the relationship between CHIP and adiposity is complex and requires further investigation. Perhaps most unexpectedly, we found associations between CHIP and CVD to be more modest than previously reported1–3. DNMT3A mutations do not associate with CVD, which is consistent with the absence of any association between CHIP and CVD when applying Mendelian randomization. However, this pattern is not seen across CHIP associations with solid tumours, which we found to be driven by DNMT3A, and to be supported by Mendelian randomization. Overall, our results further clarify the role of CHIP mutational subtypes in the development of cancer and CVD and emphasize the importance of viewing (and potentially treating) different CHIP subtypes as distinct haematologic preconditions. Whereas Bick et al. 6. found statistical support for reduced CVD incidence among CHIP carriers with an IL6R coding mutation (rs2228145-C) serving as a genetic proxy for IL-6 inhibition, we do not find any support for this association when extending their analysis from the first 50,000 exomes in the UKB to the full cohort of 450,000 exomes, nor when repeating this analysis in 175,000 exomes from the GHS cohort. The signal identified across the first 50,000 exomes may result from a chance ascertainment bias40. Alternatively, whereas the rs2228145-C variant is thought to mimic IL-6 inhibition, and therefore confer protection from heart disease41, neither our analysis nor Bick et al. found evidence that rs2228145 carriers are protected from CVD in subjects without CHIP. Therefore, it is possible that this mutation is a poor proxy for IL-6 inhibition, and that direct pharmacological inhibition of IL-6 may still antagonize the interplay between CHIP clone expansion and the onset of CVD. This study benefits from its biobank-scale size, which we leverage to further resolve clonal haematopoiesis subtypes and broadly assess clinical phenotypes associated with CHIP. However, limitations include the potential inclusion in our CHIP callset of a small number of germline variants, a lack of serial sampling, and a lack of experimental data to characterize the mechanisms underpinning the novel associations that we identify. Although we have taken many steps to ensure the quality of our callset and analysis (Supplementary Notes 11 and 12 and Supplementary Figs. 15–18), the misclassification of somatic variants with high VAF as germline variants, and/or the misclassification of true germline variants as somatic clonal haematopoiesis variants (for example, germline variants at genomic positions identified as clonal haematopoiesis hotspots) remain challenges inherent to calling and analysing CHIP and clonal haematopoiesis when using population scale genomic data. Serial sampling would enable the evaluation of changes to CHIP clones over time, and future studies that focus on such serial analysis at large scale will be able to better estimate CHIP subtype-specific clonal changes and clinical risk. Such increased data assets would also likely facilitate the identification of additional genes that show recurrent mutation during clonal haematopoiesis, as well as how such mutations relate to one another (that is, in dependency, mutual exclusivity and temporal order). Nonetheless, we identify many novel common and rare variant associations with CHIP and other clonal haematopoiesis phenotypes, which help to set the stage for future functional, mechanistic and therapeutic studies. On the whole, our analyses emphasize that CHIP is really a composite of somatic mutation-driven subtypes, with shared genetic aetiology and distinct risk profiles. Methods Study approval UKB study: ethical approval for the UKB study was previously obtained from the North West Centre for Research Ethics Committee (11/NW/0382). The work described herein was approved by UKB under application number 26041. GHS study: approval for DiscovEHR analyses was provided by the Geisinger Health System Institutional Review Board under project number 2006-0258. Exome sequencing and variant calling Sample preparation and sequencing were done at the Regeneron Genetics Center as previously described10,40. In brief, sequencing libraries were prepared using genomic DNA samples from the UKB, followed by multiplexed exome capture and sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform using S2 (first 50,000 samples) or S4 (all other samples) flow cells. Read mapping, variant calling and quality control were done according to the Seal Point Balinese (SPB) protocol40, which included the mapping of reads to the hg38 reference genome with BWA MEM, the identification of small variants with WeCall, and the use of GLnexus to aggregate these files into joint-genotyped, multi-sample VCF files. While certain UKB exome analysis efforts have used calls generated with the OQFE pipeline42, this pipeline has only been used to a limited degree for disease association analysis. Therefore, we chose to use calls from the SBP pipeline, which have been used very extensively for disease association analysis, including the largest set of association analyses done with UKB exome data10. Depth and allelic valance filters were then applied, and samples were filtered out if they showed disagreement between genetically determined and reported sex, high rates of heterozygosity or contamination (estimated with the VerifyBamId tool as a FREEMIX score > 5%), low sequence coverage, or genetically determined sample duplication. Calling CHIP To call CHIP carrier status, we first used the Mutect2 (GATK v4.1.4.0) somatic caller43 to generate a raw callset of somatic mutations across all individuals. This software aims to use mapping quality measures as well as allele frequency information to identify somatic mutations against a background of germline mutations and sequencing errors. We used data generated from gnomAD v2 as the reference source for germline allele frequency44. We generated a cohort-specific panel of normals, which Mutect2 uses to estimate per-site beta distribution parameters for use in refining somatic likelihood assignment. Since CHIP is strongly associated with age, we chose 100 random UKB samples from 40 year olds and 622 samples from individuals less than 18 years of age in GHS to build these cohort-specific panels of normals. By evaluating the degree to which default Mutect2 filtering excluded known CHIP hotspot mutations, we noted that the default Mutect2 pass/fail filters were too stringent. Therefore, we initially considered all Mutect2 variants (that is, even those that did not pass default Mutect2 filtering), and proceeded to perform our own QC and somatic mutation call refinement. As an initial refinement step, we selected variants occurring within genes that have been recurrently associated with CHIP according to recent reports from the Broad2, the TOPMed Consortium11, and the Integrative Cancer Genomics (IntOGen) project45. We then filtered putative somatic mutations using the outlined functional criteria2. Next, we performed additional QC steps, which consisted of (1) removing multi-allelic somatic calls, (2) applying sequencing depth filters (total depth (DP) ≥ 20; alternate allele depth (AD) ≥ 3, F1R2 and F2R1 read pair depth ≥ 1), (3) removing sites flagged as panel of normals by Mutect2 (unless previously reported), (4) removing indels flagged by the Mutect2 position filter, (5) removing sites within homopolymer runs (a sequence of ≥5 identical bases) if AD < 10 or VAF < 0.08, (6), removing missense mutations in CBL or TET2 inconsistent with somaticism (that is, P-value > 0.001 in a binomial test of VAF = 0.5), (7) removing novel (not previously reported) variants that exhibited characteristics consistent with germline variants or sequencing errors. That is, we excluded variants that had a median VAF ≥ 0.35, since approximately 97% of previously reported variants (that is, from a recent study of CHIP by the TOPMed consortium11) had a median VAF < 0.35. Beyond this, we evaluated the frequency distributions of known variants (stratified by effect—that is, missense or non-missense) to discern thresholds for newly identified variants (that is, AF (allele frequency) of novel variants ≤ AF of previously reported variants). Additionally, novel G>T or C>A SNV calls were evaluated for oxidation artifacts46. Specifically, variants with a maximum alternate allelic depth < 6 (across all samples) and < 2 supportive reads from F1R2 (C>A) or F2R1 (G>T) mate pairs were removed, respectively. Given that > 90% of mutations belonged to 23 recurrent CHIP-associated genes, we restricted to variants occurring within these genes as a final step to maximize the specificity of our callset. These genes consisted of the 8 most frequent mutated CHIP genes (DNMT3A, TET2, ASXL1, PPM1D, TP53, JAK2, SRSF2 and SF3B1), a collection of CHIP-associated genes containing SNV hotspots (BRAF, CSF3R, ETNK1, GNAS, KRAS, GNB1, IDH2, MPL, NRAS, PHF6 and PRPF8), and CHIP-associated genes of haematological interest (CBL, CALR, RUNX1 and SUZ12). Our final CHIP set of CHIP mutation carriers consisted of 29,669 CHIP mutations across 27,331 unique individuals from UKB, and 14,766 CHIP mutations across 12,877 unique individuals from GHS. Variant allele fraction (VAF) was calculated using AD/(reference allele depth (RD) + AD). Defining CHIP and mosaic phenotypes CHIP phenotypes were derived based on our mutation callset, whereas mosaic chromosomal alteration (mCA) phenotypes were derived based on previously published mCA calls from the UKB4,7,8. First, we used International Classification of Diseases (ICD) codes to exclude 3,596 samples from UKB and 1,222 samples from GHS that had a diagnosis of blood cancer prior to sample collection. We also excluded 13,004 individuals from GHS whose DNA samples were collected from saliva as opposed to blood. For all of the phenotypes we generated and analysed in this study, we used a combination of cancer registry data, hospital inpatient (HESIN) data, and data from general practitioner records to ascertain ICD10 codes. The majority of our cancer data came from the cancer registry, which we supplemented with the other sources. We then defined multiple CHIP and mosaic phenotypes based on whether carriers did (inclusive) or did not (exclusive) have other somatic phenotypes. For example, individuals with at least one CHIP mutation in our callset were defined as carriers for a CHIP_inclusive phenotype, whereas anyone with a CHIP mutation as well as an identified mCA was removed from this inclusive phenotype in order to define a CHIP_exclusive phenotype (20,606 cases and 342,869 controls). Our association analysis with CHIP used this CHIP_inclusive phenotype, which included 25,657 cases and 342,869 controls of European ancestry in UKB, and 11,821 cases and 135,106 controls of European ancestry in GHS. These counts reflect the samples with European ancestral origin that remain in each cohort after removing those with non-CHIP clonal haematopoiesis (60,991 in UKB and 0 in GHS, as we did not call mosaic chromosomal alterations in GHS), and those with missing meta data (348 in UKB and 4,893 in GHS). We defined mLOY carriers as male individuals with a Y chromosome mCA in the UKB mCA callset that had copy change status of loss or unknown, mLOX as individuals with an X chromosome mCA in the UKB mCA callset that had copy change status of loss or unknown, and mCAaut carriers as individuals with autosomal mCAs. We then refined these inclusive phenotypes to define exclusive versions, with mLOY_exclusive consisting of carriers with no X chromosome or autosomal mCAs (36,187 cases and 151,161 controls), mLOX_exclusive consisting of carriers with no Y chromosome or autosomal mCAs (10,743 cases and 364,072 controls), and mCAaut_exclusive consisting of carriers with no Y or X chromosomal alterations of any kind (11,154 cases and 364,072 controls). These exclusive phenotypes were used for all analyses comparing CHIP with mosaic phenotypes, as this approach facilitated the generation of four non-overlapping phenotypes (that is, CHIP, mLOY, mLOX, and mCAaut) that could be compared. We also defined CHIP gene-specific phenotypes by choosing carriers as those with mutations in our callset from a specific gene and no mutations in any other of the 23 CHIP genes defining our callset. For example, CHIP DNMT3A carriers were those with ≥ 1 somatic mutations in our callset within the DNMT3A gene, and no mutations in our callset in any of the other 23 CHIP genes we used for our final callset definition. The set of 364,072 controls used in UKB that had no evidence of any clonal haematopoiesis (that is, no CHIP or mCAs) was considered as our set of healthy controls, and was used across all association analyses in UKB. Genetic association analyses To perform genetic association analyses, we used the genome-wide regression approach implemented in REGENIE47, as described10. In brief, regressions were run separately for data derived from exome sequencing as well as data derived from genetic imputation using TOPMed48, and results were combined across these data sources for downstream analysis. Step 1 of REGENIE uses genetic data to predict individual values for the trait of interest (that is, a polygenic risk score), which is then used as a covariate in step 2 to adjust for population structure and other potential confounding. For step 1, we used variants from array data with a MAF > 1%, < 10% missingness, Hardy–Weinberg equilibrium test P-value > 10−15 and LD pruning (1,000 variant windows, 100 variant sliding windows and r2 < 0.9), and excluded any variants with high inter-chromosomal LD, in the major histocompatibility region, or in regions of low complexity. For association analyses in step 2 of REGENIE, we used age, age2, sex and age × sex, and 10 ancestry-informative principal components as covariates. For analyses involving exome data, we also included as covariates an indicator variable representing exome sequencing batch, and 20 principal components derived from the analysis of rare exomic variants (MAF between 2.6 × 10−5 and 0.01). Significance cutoffs and rare variant burden testing were set according to the power calculations and logic outlined by Backman et al.10. In brief, we used P ≤ 5 × 10−8, P ≤ 7.14 × 10−10, P ≤ 3.6 × 10−7, for common, rare and burden associations, respectively. Results were visualized and processed using an in-house version of the FUMA software49. Association analyses were performed separately for different continental ancestries defined based on the array data, as described10. Replication of associations signals in the GHS cohort To calculate the power to achieve replication in the GHS cohort, we first adjusted for the effects of ‘winner’s curse’, which are expected when choosing significant associations signals on the basis of a genome-wide threshold50. To do this, we used the conditional likelihood approach described by Ghosh et al.51 as implemented in the winnerscurse R package (version 0.1.1), which adjusts the estimated betas from genome-wide significant associations signals. These adjusted effect estimates are provided in Supplementary Table 2 (column Effect_adj). We then used these adjusted effect estimates to calculate the expected power to detect each lead signal in the GHS replication phase using the GHS sample size, allele frequencies, CHIP prevalence, and an alpha level of 0.05. To summarize our expected power across the replication phase, we summed the power across all lead variants and reported the number of SNPs that replicated at P < 0.05 as a proportion of the cumulative power to detect those variants. Identifying independent signals from association results We used three different approaches to identify independent signals across loci that associated with CHIP. First, we used a clumping and thresholding approach (C&T)52 in which index SNPs at each significantly associated locus were defined greedily as those with the lowest P-value. Clumping was then done by extending linkage blocks laterally to include all SNPs that have P < 1 × 10−5 and r2 > 0.1 with the index SNP. Any SNP within a clump was then removed from further analysis. This process was repeated as long as there was ≥ 1 additional SNP in the locus with P ≤ 5 × 10−8. After all clumps were made, we merged any clumps (that is, LD blocks) with overlapping genomic ranges. Since this approach did not feature any iterative conditioning nor model variant effects jointly, we also used conditional joint analysis as implemented in GCTA COJO53 and statistical fine-mapping as implemented in FINEMAP54 to identify independent/causal signals. COJO was run with a subset of 10,000 unrelated European ancestry samples from UKB as an LD references, and with a COJO adjusted P-value threshold of 5 × 10−6, an info score threshold of 0.3, and a MAF cutoff of 0.01. FINEMAP was run with the shotgun stochastic search algorithm using a maximum of 30 causal variants. We included variants in the FINEMAP analysis that had P < 0.1 in inverse variance weighted meta-analysis, and MAF > 0.001. The LD matrices used for the FINEMAP analysis were constructed as weighted meta LD matrices derived from the LD matrices from UKB and GHS. The LD matrices from UKB and GHS were computed independently using the same sets of samples included in each GWAS. Fine-mapping variants at the LY75 locus To further evaluate whether the rare variant association at the LY75 locus (rs147820690-T) was independent of other common and rare variant signals, we performed joint fine-mapping (with FINEMAP) on common and rare variants at this locus while including rarer variants then used in our genome-wide fine-mapping. In contrast to the genome-wide fine-mapping described above, this fine-mapping sensitivity analysis was done only in the UKB, was focused on the LY75 locus, and included all variants in our dataset. That is, the fine-mapping analysis was run as described above, but with a MAF > 0.0000000001. While FINEMAP suggests 3 credible sets are most parsimonious at this locus (posterior probability = 0.8), which is consistent with the results we report when preforming genome-wide fine-mapping, the fourth credible set (posterior probability = 0.11) identifies rs147820690-T as the top signal (PIP = 0.133) among 9,417 variants in the 95% credible set. This fine-mapping approach also prioritizes rs78446341-A (CPIP = 0.92, CS = 2). Furthermore, the median pairwise LD between SNPs in this fourth credible set is very low (6.7 × 10−4, compared with 0.995, 0.962, and 0.831 for the first three credible sets, respectively). Therefore, these fine-mapping results provide additional support for both LY75 missense variants, as well as the fact that the rs147820690-T rare variant signal is not driven by the tagging of other rare variants. PheWAS across CHIP-associated variants Using 937 traits from the UKB, we queried association results for 171 SNPs from our GWAS of CHIP. These SNPs represent the union of those identified by clumping and thresholding, conditional analysis with GCTA COJO, and fine-mapping with FINEMAP (fine-mapped SNPs were chosen if they had one of the highest two posterior inclusion probabilities—that is, PIPs—in any credible set). While this group of SNPs does include signals with P < 5 × 10−8 in our CHIP GWAS, these SNPs represent signals prioritized as conditionally independent and/or likely to be causal, and we therefore deemed them worthy of exploration via PheWAS. Some of these subthreshold signals featured many significant PheWAS associations (P < 5 × 10−8 in the PheWAS), and likely merit further evaluation (for example, ZFP36L2/THADA locus on chromosome 2, and THRB locus on chromosome 3). The traits used in this PheWAS represent the subset of the 5,041 traits used in our cross-sectional analyses of phenotypic association with CHIP mutations carrier status for which we have previously reported common variant associations10. In brief, for ICD10-based phenotypes, cases were required to have one or more records of diagnosis in the electronic health records, death registry data implicating the disease, or two or more diagnosis in outpatient data mapped to ICD10. For non-ICD10 phenotypes (quantitative measures, clinical outcomes, survey and touchscreen responses, and imaging derived phenotypes), data were derived from the UKB Showcase. Participants who did not meet the case definition for a given ICD10-based phenotype were removed from the analysis if they had one diagnosis code in the outpatient data, and included as controls if they had no diagnosis in the outpatient data. Supplementary Table 10 includes ICD10 codes as well as trait names and descriptions. Genetic comparisons between CHIP subtypes For pairwise comparisons between CHIP gene mutation subtypes, we used the union set of index SNPs (that is, independent signals in genome-wide significant loci) from all of our CHIP and CHIP gene subtype associations. This resulted in 93 variants, which we used to compare effect sizes estimates between CHIP subtype pairs. Genetic correlations were calculated using LDSC version 1.0.1 with annotation input version 2.222. Defining smoking phenotypes We derived smoking phenotypes from the lifestyle and environment questionnaire in the UKB and from the electronic health records in the GHS. Since smoking is difficult to ascertain and control for, we used a variety of data to code multiple smoking phenotypes for various analyses. These smoking phenotypes consisted of (1) pack years, (2) number of cigarettes smoked per day, (3) age started/stopped smoking (UKB only), (4) former/current smoker, (5) ever smoker and (6) heavy smoker (smoked ≥ 10 cigarettes a day). The ever smoker phenotype was maximally inclusive, and coded as cases all individuals with any evidence of prior smoking across the aforementioned phenotypes. For our longitudinal analyses in UKB, we used the ‘current smoker’ and ‘pack years’ (which captures the cumulative effect of smoking over one’s lifetime) as covariates in all models that did not stratify for smoking status. In the smoking stratified models, we stratified smokers based on the ‘ever smoker’ phenotype and further adjusted for pack years within the smokers subgroup. For our longitudinal analyses in GHS, we used the ‘ever smoker’ and ‘pack years’ phenotypes as covariates in all models that did not stratify for smoking status, and stratified smokers in the same manner as we did in the UKB analyses. For linear models that evaluated the overall relationship between age, sex, and smoking, we used the ‘heavy smoker’ coding. Otherwise, all other analyses used the aforementioned ‘ever smoker’ phenotype as a covariate. Phenotypic associations with CHIP To test for known as well as potentially novel associations, we used REGENIE47 to perform Firth-corrected tests for association between our CHIP gene-specific phenotypes and 5,041 traits (2,640 binary traits and 2,401 quantitative traits) from the UKB (version 5). To do this, we coded each CHIP gene-specific phenotype as 1 if an individual had any somatic CHIP mutation in the gene and 0 otherwise and formatted these binary codings as pseudo-genotypes to analyse with REGENIE. Regression models were run as described previously, with age, sex, and genetic principal components as covariates10. After filtering out association tests where the total number of somatic carriers was <5, we were left with 83,779 total association tests (Supplementary Table 31). Only 22 out of 23 CHIP gene subtypes were tested for association across phenotypes as we did not have enough carriers of CSF3R mutations to meet our minimum threshold of 5 somatic carriers that were also disease cases. Quantitative traits were transformed using a reverse inverse normalized transformation (RINT); effect size estimates from these associations are in units of standard deviation. Traits used in this analysis did not exclude any samples on the basis of having a diagnosed haematological disease or malignancy prior to sequencing date. To visualize high-level phenotypic patterns across these CHIP gene-specific phenotypes (Fig. 3), we categorized phenotypes by disease group10, and calculated the proportion of phenotypes per disease group per gene that were associated at a P ≤ 0.05 alpha level (uncorrected). To visualize the most significant of these associations, we plotted effect sizes (Supplementary Fig. 7) by disease category for all associations with P ≤ 1 × 10−5. Risk modelling among CHIP carriers We performed longitudinal survival analyses using cox proportional hazard models (coxph function) as implemented in the survival R package. Given that CHIP is strongly correlated with age, models used age as the time scale with interval censoring with age at first assessment and age at event or censoring. This allows for an implicit adjustment for age within the proportional hazard models. In UKB, individuals with follow-up time in excess of 13.5 years (3% of the dataset) were censored due to departures from the proportional hazards model. Analyses were performed on individuals of European ancestral background. All models included 10 genetically determined European-specific principal components as covariates, and all analyses excluded individuals genetically determined to be third-degree relatives or closer. In GHS, we had limited sample size with which to perform these longitudinal analyses. This was because GHS samples were collected at later ages (due to the nature of the biobank and the timing of our partnership) and fewer patients had disease onset dates subsequent to sample collection (that is, the time period where the onset of CHIP can be evaluated). Furthermore, in GHS, we could not derive an all-cause mortality phenotype due to the nature of the EHR data available to us. This incomplete ascertainment may also explain why our odds ratio estimates for risk of haematologic malignancy among CHIP carriers are lower in the GHS cohort. We used a variety of CHIP codings as variables in our models to test for potential differences between high/low VAF CHIP and/or CHIP subtypes. First, we subset CHIP carrier status by gene (DNMT3A, TET2, ASXL1, DNMT3A or TET2) and/or VAF (≥0.1) to test for potential differences between degree of clonal expansion (that is, high/low VAF CHIP) and/or CHIP subtypes. Additional analyses were run restricting CHIP mutation calls to previously reported variants (for example, Jaiswal et al.2), as well as restricting to carriers of DNMT3A mutations with at least one mutation in another CHIP gene. Controls were defined with two approaches: (1) any individual without CHIP mutations (the coding used in the results we report) and (2) those without any genetic evidence of clonal haematopoiesis (that is, healthy controls, as defined above, which did not change our results). The CHIP gene-specific coding described above varies from the phenotypic coding definitions used in our GWAS/ExWAS, which required carriers to have mutations only in the specified CHIP gene and no mutations in any other CHIP genes. Since mutational exclusivity becomes less common as VAF increases (that is, carrying a single mutation with VAF ≥ 0.1 and no other mutations), and substantially lowers sample size, we chose this adjusted definition for these longitudinal analyses of disease incidence. For the composite phenotypes described below, we relied heavily on ICD10 codes from cancer registry data, hospital records and general practitioner records, and supplemented these with self-reported data and procedure codes (OPCS4). We defined prevalent disease on the basis of event codes occurring before sample collection and used this definition to exclude samples from longitudinal analysis of incident disease. For these main analyses, we did not use any minimum number of days to diagnosis from sample collection as an additional filtering criterion (see Supplementary Note 12 for more details). In UKB, cardiovascular disease was defined with the following ICD10 codes obtained from primary care, HES (hospital episode statistics), or death registry data: I21, I22, I23, I252, I256, Z951, Z955, I248, I249, I241, I251, I255, I258, I259, I630, I631, I632, I633, I634, I635, I637, I638, I639, I651, ICD9 codes: 410, 412, and OPCS codes: K40, K41, K44, K45, K46, K49, K502, K75 and K471. ICD9/ICD10/OPCS diagnoses or procedures recorded prior to enrolment date and self-report codes 1075 (heart attack/myocardial infarction), 1095 (cabg), 1523 (heart bypass), 1070 (coronary angioplasty or stent), 1583 (ischaemic stroke), 1083 (stroke) were used to identify prevalent CVD cases. These were chosen to best reflect the coding use by Bick et al. in their study of CHIP6. In GHS, we used ICD10 codes I20–I25 and I60–I69, CPT codes from 33510–33523 (CABG, not continuous), 33533–33536, 35500, 35572, 35600, and 92920–92975 (PCI, not continuous). We also adjusted the CVD coding in GHS to exclude cerebrovascular events (that is, excluded I60–I69); association results were similar. The CVD coding we used for our Mendelian randomization analysis was comparable to these definitions but did not include ICD10 codes for cerebrovascular events. For the CVD models, we included sex, LDL, HDL, pack years, smoking status (current vs former, determined by self-reported data), BMI, essential primary hypertension, and type 2 diabetes mellitus as covariates. The results we reported used a composite of myocardial infarction (MI), coronary artery bypass graft (CABG), percutaneous coronary intervention (PCI), and coronary artery disease (CAD), based on the coding described above, and also included death from any of these events. Results were similar when our composite included ischaemic stroke (ISCH.TR), as well as when we repeated analyses with a subset of recurrent CHIP mutations derived from Jaiswal et al.2 or restricting carrier calls to variants in DNMT3A or TET2. We also excluded samples with any diagnosis of malignant blood cancer prior to sequencing (n = 3,596). Missing LDL and HDL values were median imputed, and individuals on cholesterol medication had their raw LDL values increased by a factor of 1/0.68, similar to Bick et al.6. IL6R missense variant (rs2228145-C) genotypes were modelled dominantly (coded as 1 for carriers of any allele and 0 otherwise), and we modelled the effect of this allele in CHIP -stratified proportional hazard models, and also tested for IL6R × CHIP interaction in a full (non-stratified) model. Models considering only the initial 50k UKB individuals restricted to intersection between our unrelated UKB sample set and the samples reported by Bick et al. 6. For visualization, Kaplan–Meier estimates were generated with the survfit function in the aforementioned survival package (version 3.2.13) and plotted using the ggsurvplot function from the survminer package (version 0.4.9). For models of cancers and overall survival risk tested using all CHIP carriers, high-VAF (VAF ≥ 0.1) CHIP carriers, and carriers of specific CHIP gene mutations, we used unrelated European samples that did not have any cancer diagnoses prior to sample collection (N = 360, 051 after the removal of 33,816 samples with a prior diagnosis of cancer). Results were qualitatively the same when repeating these analyses without excluding samples that had a diagnosis of any malignant cancer prior to sample collection date. Cancer phenotype definitions were derived from medical records indicating the following ICD10 codes: C81–C96, D46, D47.1, D47.3, D47.4 for blood cancers, C81–C86, C91 for lymphoid cancers, C92, C94.4, C94.6, D45, D46, D47.1, D47.3, D47.4 for myeloid cancers, C50 for breast cancers, C34 for lung cancers, C61 for prostate cancers, C44 for non-melanoma skin cancers (NMSC), and C18 for colon cancers (five total solid cancers). Myeloid subtypes were defined as follow: AML (C92), MDS (D46), MPN (D47.1, D47.3, D47.4). Given the rareness and/or non-specificity of myeloid codings C93–95, and that the majority of these codings overlapped with those that we used for the myeloid composite described above (that is, we already captured these samples using the previously described codings), we did not include these codings in our composite. However, we performed sensitivity analyses that used a myeloid definition that did include C93–C95, with findings equivalent to those described in our main results (Supplementary Note 12). For our lymphoid composite, we decided to combine lymphoma with lymphoid leukaemia for multiple reasons. First, in some clinical diagnostic situations (for example, T cell lymphoblastic lymphoma and T cell lymphoblastic leukaemia; Burkitt lymphoma and mature B cell ALL), the distinction between ‘leukaemia’ and ‘lymphoma’ is made on the basis of blast percentage in bone marrow (that is, > 20% blasts diagnosed as leukaemia), and may not reflect meaningful biological differences. Consistently, 22% of C91 codings are already captured in our C81–C86 codings. Moreover, the majority of cases across these codings correspond to tumours derived from mature B cells, namely chronic lymphocytic leukaemia (CLL) and mature non-Hodgkin lymphoma. Given data supporting that mature T cell lymphomas and also some mature non-Hodgkin B cell tumours may arise from hematopoietic stem and progenitor cells55–57, we considered the relationship between a composite of mature lymphoid tumours and CHIP. For blood cancers, we also included cases that self-reported leukaemia, lymphoma, or multiple myeloma. These models included the same covariates as described for CVD (with the exception that we did not adjust cholesterol level based on medication usage). Additionally, models estimating risk for sex-specific cancers (that is, prostate and breast) restricted to individuals of the relevant sex and did not adjust for sex as a covariate. For smoking stratified modelling of blood and lung cancer, we used our stricter definition of smoking (ever vs never) and included pack years as a covariate in models testing risk among smokers. To test a more conservative cutoff for excluding patients with a diagnosis of haematologic malignancy prior to sequencing (that is, exclude individuals with a diagnosis prior to 90 days after DNA collection date rather than prior to the DNA collection date itself), we conducted sensitivity analyses for the longitudinal modelling of the risk among CHIP carriers of acquiring blood cancers (for example, blood cancer, myeloid, lymphoid, AML, MDS and MPN). These results were the same as those reported in our main results (Supplementary Note 12). Polygenic risk scores Polygenic risk scores were calculated with Plink58 as a weighted sum of the effects across all conditionally independent variants we identified with GCTA COJO (74 variants, P ≤ 5 × 10−6) We performed association tests using logistic regression, with binary phenotypes of interest (that is, our CHIP subtype phenotypes—for example, TET2 CHIP, and so on) as the dependent variable, this polygenic risk score as the independent variable of interest, and age, sex, smoking status (ever vs never), and 10 genetic principal components as covariates. Software The code is publicly available and can be found at https://github.com/rgcgithub/regenie. The REGENIE software for whole-genome regression, which was used to perform all genetic association analysis, is available at https://github.com/rgcgithub/regenie. GCTA v1.91.7 was used for approximate conditional analysis. SHAPEIT4.2.0 was used for phasing of SNP array data. Imputation was completed with IMPUTE5. Somatic calling was done with Mutect2 (GATK v4.1.4.0). We use Plink1.9/2.0 for genotypic analysis as well as for constructing polygenic risk scores. FINEMAP was used for fine-mapping, and genetic correlations were calculated using LDSC version 1.0.1 with annotation input version 2.2. Beyond standard R packages, visualization tools, and data processing libraries (for example, dplyr, ggplot2 and data.table), we used the survival (version 3.2.13) and survminer (version 0.4.9) packages for survival analyses, the MendelianRandomization package for Mendelian randomization (version 0.6.0), and the winnerscurse package (version 0.1.1; https://amandaforde.github.io/winnerscurse/) to adjust GWAS effect size estimates for the effects of Winner’s Curse. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Online content Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-05448-9. Supplementary information Supplementary Information This file contains supplementary information for numerous analyses, and includes 12 supplementary notes, which are referenced throughout the Article. Reporting Summary Members lists for Regeneron Genetics Center and GHS-RGC DiscovEHR Collaboration. Supplementary Figures This file contains Supplementary Figs. 1–18. Supplementary Tables Supplementary Tables 1–33 - see `Table Descriptions’ worksheet within the file for details. Extended data figures and tables Extended Data Fig. 1 Workflow to Identify CHIP and Prevalence Estimates For Carriers of CHIP Mutations. A. Graphic depicting at a high-level the workflow used to collect and sequence the exomes of multiple large cohorts and to then identify CHIP mutations from this data. B-C. CHIP prevalence increases with age of donor at time of DNA collection in both the UKB (B, n = 484,629 individuals; one-sided F-test, P < 10−16) and GHS (C, n = 157,724 individuals; one-sided F-test, P < 10−16) cohorts, with the centre line representing the general additive model spline and the shaded region representing the 95% confidence interval. D-E. Similar to B-C, the prevalence of CHIP mutations per CHIP gene for each of the top 8 most common CHIP genes increase with age in the UKB (D, n = 484,629 individuals; one-sided F-test, P < 10−16) and in GHS (E, n = 157,724 individuals; one-sided F-test, P < 10−16). Extended Data Fig. 2 Count Distribution and Pairwise Enrichments of Clonal Hematopoiesis of Indeterminate Potential (CHIP) Gene Mutations. A. Total number of individuals with mutations (y axis, log10 scale) in each of the 23 genes that were used to determine CHIP status across the UKB (blue) and GHS (red) CHIP callsets. B-C. Pairwise mutation counts across the UKB (B) and DiscoverEHR (C) callsets across individuals with at least two identified CHIP mutations. The color scale reflects the significance of the p-value for association between mutated CHIP gene pairs as determined by logistic regression. Per CHIP gene pair, these models included CHIP gene 1 mutation carrier status as the outcome, CHIP gene 2 mutation carrier status as the predictor, and age, sex, and smoking status (ever vs never) as covariates. P values are log10 transformed (see Table S1 for complete enrichment results). Extended Data Fig. 3 Finemapping results at the LY75 locus on chromosome 2. A. Fine-mapping the summary statistics from our association analysis of CHIP prioritizes the P1247L missense variant (rs78446341-A, AAF = 0.02) as highly likely to be the causal variant driving one of three causal signals at this locus (CPIP = 0.913). At the top of the panel, a locus zoom plot shows marginal association results after inverse variance weighted meta analysis across UKB and GHS (p-values are uncorrected and derive from two-sided tests performed using approximate Firth logistic regression and subsequent meta analysis). Top common variants, including those prioritized by clumping and thresholding and COJO from UKB associations are highlighted with black circles. The rs78446341-A missense variant is highlighted as well and is in low linkage disequilibrium (LD) with the other SNPs. FINEMAP estimated 3 signals were most parsimonious here (PP = 0.55). B. Fine-mapping the summary statistics from our association analysis of DNMT3A-CHIP prioritizes the P1247L missense variant (rs78446341-A, MAF = 0.02, CPIP = 0.20, CS = 4) and the rarer G525E missense variant (rs147820690-T, AAF = 0.002 CPIP = 0.60, CS = 2) as likely to be the causal variants driving the signal at two out of four causal signals at this locus. Here, FINEMAP estimated 3 signals (PP = 0.57) or 4 signals (PP = 0.41) were likely; we report results for K = 3 in Table S6 and show results from K = 4 here. The other prioritized signals are those identified by clumping and thresholding and COJO: rs12472767-C (2-159925824-T-C, CPIP = 0.99, CS = 1) and rs12472767-C (2-159821048-C-T, CPIP = 0.28, CS = 3). CS: Credible Set, PP: Posterior Probability, PIP: Posterior Inclusions Probability, CPIP: Conditional Posterior Inclusion Probability. Extended Data Fig. 4 Results from a phenome-wide association analysis. Results from a phenome-wide association analysis are shown for the thirty SNPs from our GWAS that had the largest number of significant associations (P < 5 x 10−8). Associations are most common among hematological, body mass, and auto-immune traits (seen across the ‘dermatology’, ‘gastroenterology’, and ‘other’ phenotypic categories). For visualization, associations with –log10(P) < 50 were set to 50. Association models were run with age, age2, sex, and age-by-sex, and 10 ancestry-informative principal components (PCs) as covariates. P-values are uncorrected and derive from two-sided tests performed using approximate Firth logistic regression. See Table S10 for full associations results. Extended Data Fig. 5 GWAS of CHIP Subtypes. Manhattan plot showing results from a genome-wide association analysis of CHIP subtypes. While we ran CHIP subtype analysis for each of the 8 most recurrently mutated CHIP genes (Tables S11–S19), we show Manhattan plots for the 5 CHIP subtypes that had at least 1 genome-wide significant common variant association. These included DNMT3A-CHIP (23 significant loci), TET2-CHIP (6 significant loci), ASXL1-CHIP (2 significant loci), TP53-CHIP (1 significant locus), and JAK2-CHIP (1 significant locus). Novel biologically relevant genes are labeled at each locus, with red denoting novel loci, black identifying previously identified loci and grey identifying loci with suggestive signal (P < 5 x 10−7). Association models were run with age, age2, sex, and age-by-sex, and 10 ancestry-informative principal components (PCs) as covariates. P-values are uncorrected and are from two-sided tests performed using approximate Firth logistic regression. Extended Data Fig. 6 Results from Mendelian Randomization models and incident risk of death among CHIP carriers. A. Forest plot of results from Two Sample Mendelian Randomization (MR) modeling of the effect of CHIP on 20 traits of interest (including the two quantitative traits BMI and ALT). Reported p-values are uncorrected, and reflect two-sided Z-tests derived from an inverse variance weighted (IVW) MR procedure. Significant causal association between CHIP and breast cancer, prostate cancer, non-melanoma skin cancer, melanoma, myeloid leukemia, and lung cancer are supported by these models. As expected, estimates of germline effect on CHIP from UKB and GHS are strongly correlated (odds ratio = 1.94 [1.76–2.13], P = 3.2 x 10−42). B. CHIP and its most common subtypes are significantly associated with death from any cause across UKB. Hazard ratio (HR) estimates from cox-proportional hazard models are shown, with error bars that represent a 95% confidence interval. P-values are uncorrected and derive from two-sided Wald tests. Models are adjusted for sex, LDL, HDL, pack years, smoking status, BMI, essential primary hypertension, type 2 diabetes mellitus, and 10 European specific genetic PCs. Extended Data Fig. 7 CVD Incidence in IL6R Mutation Carriers with and without CHIP. A-B. Survival curves are drawn showing that IL6R p.Asp358Ala mutation carriers (green) are not an elevated risk of CVD incidence (y-axis) compared with individuals without CHIP (blue) in either the first 50K individuals from UKB (A) or the full 450K cohort (B). C-D. In contrast, IL6R p.Asp358Ala mutation carriers are estimated to be at a reduced risk of CVD events (C) (HR = 0.60), but only in the first 50K samples from UKB (D). Models are adjusted for sex, LDL, HDL, pack years, smoking status, BMI, essential primary hypertension, type 2 diabetes mellitus, and 10 European specific genetic PCs. Hazard ratios (HR) were estimated using cox-proportional hazard modeling, with p-values uncorrected and derived from two-sided Wald tests. Extended Data Fig. 8 Incident risk of myeloid cancer subtypes among CHIP carriers from the UKB. A-C. Forest plots and tables featuring hazard ratio (HR) estimates from cox-proportional hazard models are shown, with error bars that represent a 95% confidence interval. CHIP and its most common subtypes are significantly associated with acute myeloid leukemia (AML) (A), Myelodysplastic Syndromes (MDS) (B), and myeloproliferative neoplasm (MPN) (C). Here, results are depicted from analyses in which we removed samples that had a diagnosis of malignant cancer prior to sequencing collection. Models are adjusted for sex, LDL, HDL, pack years, smoking status, BMI, essential primary hypertension, type 2 diabetes mellitus, and 10 European specific genetic PCs. Hazard ratios (HR) were estimated using cox-proportional hazard modeling, with p-values uncorrected and derived from two-sided Wald tests. Extended Data Fig. 9 Incident risk of lung cancer among CHIP carriers from the UKB and GHS cohorts. A-D. Forest plots and tables featuring hazard ratio (HR) estimates from cox-proportional hazard models are shown, with error bars that represent a 95% confidence interval. CHIP and its most common subtypes are significantly associated with lung cancer in both smokers and non-smokers across UKB (A-B) and GHS (C-D). Here, results are depicted from analyses in which we removed samples that had a diagnosis of malignant cancer prior to DNA collection. Models are adjusted for sex, LDL, HDL, pack years, smoking status, BMI, essential primary hypertension, type 2 diabetes mellitus, and 10 European specific genetic PCs. Hazard ratios (HR) were estimated using cox-proportional hazard modeling, with p-values uncorrected and derived from two-sided Wald tests. Extended Data Table 1 Results from Mendelian Randomization analysis of CHIP exposure on lung cancer risk Results from Mendelian Randomization analysis of CHIP exposure on lung cancer risk Statistical results are shown from seven MR methods with differing sensitivities to outliers and/or violations of the MR assumptions. P-values are reported uncorrected. The estimated intercept values are shown for the two MR-Egger-based methods that estimate these terms. All models provided support for a casual association between CHIP and lung cancer. Models were significant when run without variants at the TERT locus as instrumental variables, which provides support for a causal association above and beyond any pleiotropic effects at the TERT locus (grey text). Extended data is available for this paper at 10.1038/s41586-022-05448-9. Supplementary information The online version contains supplementary material available at 10.1038/s41586-022-05448-9. Author contributions M.D.K., A.D., S.O., S.S., N.B., D.L., J.T. and H.M.K. performed bioinformatic and statistical genetic analysis. S.O., K.W., A.M., Y.Z., A.G., C.G., J. Mbatchou, W.S., J.G.R. and H.M.K. contributed to data engineering and bioinformatic pipeline development. J.D.O. developed and oversaw exome sequencing efforts. M.D.K., A.D., S.O., K.W., M.V.M., J.H., J.A.K., V.M.R., Y.H., C.G., R.R.W., N.V., J.B., N.N.P., M.G.L., M.J., D.J.G., L.A.L., M.N.C., G.S.A., A.E.L., M.A.R.F., R.D., G.T., C.P., A.R.S., A.A.F., W.S., J.G.R., J.D.O., J. Marchini, H.M.K., A.B., G.R.A. and E.J. contributed to experimental design, interpretation of results, and genetic programme development. M.D.K. and E.J. drafted the manuscript, with significant input from A.D., S.O., M.V.M., G.T., A.A.F., H.M.K., A.B. and G.R.A. All authors reviewed and approved the final version of the manuscript. Peer review Peer review information Nature thanks Po-Ru Loh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Data availability Individual-level sequence data, CHIP calls and polygenic scores have been deposited with UK Biobank and are freely available to approved researchers, as done with other genetic datasets to date10. Individual-level phenotype data are already available to approved researchers for the surveys and health record datasets from which all our traits are derived. Instructions for access to UK Biobank data is available at https://www.ukbiobank.ac.uk/enable-your-research. Summary statistics from UKB trait are available in the GWAS catalogue (accession IDs are listed in Supplementary Table 33). As described10, the HapMap3 reference panel was downloaded from ftp://ftp.ncbi.nlm.nih.gov/hapmap/, GnomAD v3.1 VCFs were obtained from https://gnomad.broadinstitute.org/downloads, and VCFs for TOPMED Freeze 8 were obtained from dbGaP as described in https://topmed.nhlbi.nih.gov/topmed-whole-genome-sequencing-methods-freeze-8. Data used for replication, such as DiscovEHR exome sequencing and genotyping data, and derived CHIP calls, can be made available to qualified, academic, non-commercial researchers upon request via a Data Transfer Agreement with Geisinger Health System (contact person: Lance Adams, [email protected]). Competing interests M.D.K., A.D., S.O., N.B., D.L., K.W., A.M., M.V.M., S.S., J.H., J.T., J.A.K., V.M.R., Y.Z., Y.H., A.G., C.G., J. Mbatchou, R.R.W., N.V., J.B., N.N.P., M.G.L., M.J., D.J.G., L.A.L., M.N.C., G.S.A., A.E.L., M.A.R.F., R.D., C.P., A.R.S., G.T., A.A.F., W.S., J.G.R., J.D.O., J. Marchini, H.M.K., A.B., G.R.A. and E.J. are current employees and/or stockholders of Regeneron Genetics Center or Regeneron Pharmaceuticals. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. A list of authors and their affiliations appears at the end of the paper A full list of members and their affiliations appears in the Supplementary Information ==== Refs References 1. Jaiswal S Age-related clonal hematopoiesis associated with adverse outcomes New Engl. J. Med. 2014 371 2488 2498 10.1056/NEJMoa1408617 25426837 2. Jaiswal S Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease New Engl. J. Med. 2017 377 111 121 10.1056/NEJMoa1701719 28636844 3. Jaiswal S Ebert BL Clonal hematopoiesis in human aging and disease Science 2019 366 eaan4673 10.1126/science.aan4673 31672865 4. Zekavat SM Hematopoietic mosaic chromosomal alterations increase the risk for diverse types of infection Nat. 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==== Front Indian J Thorac Cardiovasc Surg Indian J Thorac Cardiovasc Surg Indian Journal of Thoracic and Cardiovascular Surgery 0970-9134 0973-7723 Springer Nature Singapore Singapore 1432 10.1007/s12055-022-01432-0 Case Report Primary pleuro-pulmonary Ewing’s sarcoma: multimodal approach with uniportal thoracoscopic resection http://orcid.org/0000-0002-6670-5615 Chinthareddy Rohan Reddy 1 http://orcid.org/0000-0002-6142-2544 Chandran Suganya 1 http://orcid.org/0000-0002-8550-9441 Muthirevula Arvind 2 http://orcid.org/0000-0001-7747-902X Srikrishna Srirangapatna Varadaraj 1 http://orcid.org/0000-0002-5543-8240 Lingaraju Vijay Cholenahalli [email protected] 1 1 grid.429938.d Department of Thoracic Surgery, Mazumdar Shaw Medical Centre, Narayana Institute of Health, 258/A, Hosur Road, Bommasandra Industrial Area, Bengaluru, 560099 Karnataka India 2 grid.415967.8 0000 0000 9965 1030 Department of Thoracic Surgery, St. James University Hospitals, Leeds Teaching Hospitals Trust, Leeds, LS9 7TF UK 1 12 2022 14 21 7 2022 17 10 2022 20 10 2022 © Indian Association of Cardiovascular-Thoracic Surgeons 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Primitive neuroectodermal tumors (PNETs) and Ewing’s sarcoma (ES) belong to the same family of malignant, small, round cell neoplasms of soft tissue or bone origin. With just around 30 cases reported so far, primary pleuro-pulmonary PNETs/ES are extremely rare in adults. We herein describe a case of a hexagenerian male diagnosed with primary pleuro-pulmonary PNETs/ES after tissue biopsy. He underwent neoadjuvant chemotherapy, and after significant downstaging of the tumor, he underwent complete resection of the lesion through uniportal video-assisted thoracoscopic surgery (u-VATS) followed by adjuvant chemo-radiotherapy. He was disease-free while under close follow-up for the past 1 year. Keywords Pleuro-pulmonary Ewing’s sarcoma Uniportal VATS ==== Body pmcIntroduction The Ewing’s sarcoma family of tumors (ESFT; see Table 1 for a list of abbreviations) incorporate both the well-recognized primary bone and the extra-skeletal soft tissue sarcomas. The first case of primary ESFT of the lung was reported in 1989 by Hammar and colleagues [1]. With just about 30 cases reported in the literature, primary pulmonary Ewing’s sarcoma (ES) is evidently rare. In our report, we present such a case, diagnosed and treated at our institution, with an aim to highlight the importance of the multimodal team approach in the evaluation and management of such cases, along with the feasibility of minimal invasive curative surgery for the same.Table 1 List of abbreviations BCL-2 B-cell lymphoma 2 family (apoptosis regulator proteins) CD31 Cluster of differentiation 31 (protein) CD34 Cluster of differentiation 34 (protein) CD99 Cluster of differentiation 99 (protein) CECT Contrast-enhanced computed tomography COVID-19 Coronavirus disease-2019 ES Ewing’s sarcoma ESFT Ewing’s sarcoma family of tumors FLI-1 Friend Leukemia Integration 1 (transcription factor) pan-CK pan-Cytokeratin (antibody cocktail) PET-CT Positron emission tomography-computed tomography PPES Primary pulmonary Ewing’s sarcoma PNET Primitive neuroendocrine tumor TTF-1 Thyroid transcription factor-1 (protein) u-VATS Uniportal video-assisted thoracoscopic surgery VMAT Volumetric modulated arc therapy VAC/IE Chemotherapy regimen — vincristine + adriamycin + cyclophosphamide alternating with ifosfamide + etoposide WT-1 Wilms tumor (related protein) Case report A 64-year-old gentleman, never-smoker, presented with a history of dry cough and reduced air entry in the basal aspect of the right chest. Initial imaging by contrast-enhanced computed tomography (CECT) of the chest revealed a centrally located right lower lobar mass (yellow arrow) extending across the fissure into the middle lobe (Fig. 1A) and a nodular lesion (yellow arrow) at the interface of the right hemidiaphragm and right lower lobe (Fig. 1B). Positron emission tomography with computed tomography (PET-CT) revealed a hypermetabolic mass in the right lower lobe of the lung, crossing the major fissure and involving the middle lobe (6.7 × 7.1 × 6.1 cm, SUV (Standardized uptake value) max 14.6), with hypermetabolic right lower paratracheal lymph nodes (SUVmax 5.3), without any distant lesions (Fig. 1C). A similar mass was noted at the interface of the right lower lobe medial basal segment and adjacent diaphragm with indistinct fat planes (2.2 × 3.1 cm, SUVmax 10.4). A computed tomography (CT)–guided biopsy revealed malignant small round cell tumor, which on immunohistochemistry was positive for vimentin, CD99, and FLI-1. It was negative for pan-CK, TTF-1, chromogranin, synaptophysin, WT-1, S-100, CD31, CD34, and BCL-2. After a multidisciplinary tumor board discussion, a diagnosis of primary pulmonary ES was made and he received neoadjuvant chemotherapy (4 cycles of vincristine + adriamycin + cyclophosphamide alternating with ifosfamide + etoposide (VAC/IE)). A re-staging PET-CT after completion of therapy suggested interval reduction in both size and metabolic activity of both the masses (fissural aspect mass, 2.4 × 2.8 cm; SUVmax, 3.4; medial basal segmental mass, 2.1 × 2.8 cm; SUVmax, 7.4) and metabolic activity in the mediastinal lymph nodes (SUVmax, 3.5), yet with abutment of segmental pulmonary arterial branches.Fig. 1 A CECT chest demonstrating a heterogenous mass lesion (yellow arrow) in the right lower lobe extending into the middle lobe across the fissure and B a nodular lesion (yellow arrow) on the medial aspect of the right hemidiaphragm. C PET-CT image depicting the hypermetabolic nature of the right lower lobe mass (Green arrow). Intraoperative view of D the parenchymal lesion (green arrow) in the major fissure and E the ovoid “bilobed” nodule (green arrow) on the right hemidiaphragm He underwent uniportal video-assisted thoracoscopic surgery (u-VATS) exploration, wherein, a well-defined whitish mass (3.0 × 2.0 × 3.5 cm) was noted within the right lower lobe (Fig. 1D). It was crossing the oblique fissure, involving the medial segment of the right middle lobe and close to the origin of the middle lobar artery. A bilobed mass (4.0 × 4.0 × 2.0 cm) was noted on the right hemidiaphragmatic pleural surface, infiltrating the diaphragm but with no obvious hepatic infiltration (Fig. 1E). A sub-centimetric parietal pleural nodule was noted on the lateral costal surface. We completed a right inferior bi-lobectomy (middle and lower lobes) with wide excision of hemidiaphragmatic nodule and the rent was repaired primarily. Additionally, a systematic mediastinal lymph node dissection with resection of pleural nodule was done. Postoperative period was uneventful and histopathological examination revealed a malignant round cell tumor within the lung and diaphragmatic nodule (Fig. 2). Pleural based nodule revealed focal necrosis with no residual tumor suggestive of response to chemotherapy and lymph nodes were negative. He received adjuvant radiation volumetric modulated arc therapy (VMAT) of 45 Gray in 30 fractions with an additional 9-Gray boost, followed by adjuvant chemotherapy (6 cycles VAC/IE regimen), which he tolerated well. The patient was kept under close follow-up during which he showed significant clinical and overall functional recovery. He was re-evaluated 1 year after the surgery with a PET-CT which showed no residual or interval recurrence of disease. However, the patient unfortunately succumbed to coronavirus disease 2019 (COVID-19)–related pulmonary sepsis, which led to respiratory failure.Fig. 2 A Histo-micrographic image of malignant small round “blue” cells. Immunostain positive for B FLI-1 and C vimentin Discussion First described in 1921 by the American pathologist James Ewing, ES is a neuroectodermal tumor characterized by monotonous small round cells. It usually develops in the second decade of life as a primary osseous malignancy, with a median age of 13 years [2]. ES usually metastasizes through hematogenous spread, with the lung being the most common destination (38%), followed by bones (31%) and the bone marrow (11%) [2]. An extra-skeletal primary source of this condition is extremely rare; a review of English literature revealed just around 30 cases of primary ES of the lung reported so far, thus emphasizing the need to rule out a musculoskeletal primary [3]. According to past and recent studies, 15–40% of the patients already have metastasis at the time of diagnosis [3]. Thus, a detailed clinical and radiological examination is warranted to appropriately stage the disease prior to embarking upon therapy [4]. Definitive diagnosis is arrived at by biopsy and histological evaluation. ES should be differentiated from other pulmonary malignancies, viz. small cell carcinoma, adenocarcinoma, squamous cell carcinoma, and lymphoma. Recently, the use of CD99 immunostain has increased the diagnostic accuracy [5], and the same was found to be focal positive in our case. Multimodality therapy, through a formal multidisciplinary tumor board consensus, is indicated. The disease-free survival rate has been significantly increased by managing these tumors with aggressive surgical resection in combination with multi-agent chemotherapy, with or without radiotherapy [6]. The 5-year disease-free survival rate for ES of bone is estimated to be 60 to 70% for localised disease managed with multi-agent chemotherapy and surgical resection [7]. Surgery has been performed in 25 cases so far, either as a standalone therapy or in combination with chemotherapy or radiotherapy [8, 9], with none of them having undergone VATS. We performed a right inferior bi-lobectomy with diaphragmatic repair successfully through a uniportal VATS approach for our patient, with at least 12-month disease-free survival before losing him to COVID-19. Conclusion Extra-osseous ES affecting the lung and diaphragm primarily is extremely rare. It needs to be differentiated from the more common non-small cell and small cell cancers of the lung. The diagnosis can only be confirmed with immune-histochemical studies. An inter-disciplinary team management with multimodality treatment is necessary and hardly ever straightforward. Complete surgical resection with adjuvant therapy offers the best chance for cure. Given the availability of experience and expertise, the u-VATS approach was a feasible option in our patient and the surgical outcome was successful. Acknowledgements We extend our gratitude to the Chair of the Department of Anaesthesia, Mazumdar Shaw Medical Centre, Narayana Health City, and Dr Akhila Lakshmikantha, Consultant, Department of Pathology, Mazumdar Shaw Medical Centre, Narayana Health City. Author contribution All the authors have provided their intellectual contribution to the manuscript. The final draft was read and approved by all the authors. Funding No funding or other support was acquired from any external or institutional sources. Declarations Consent for publication The personal information of the patient was anonymized, and hence, a waiver of consent was provided by the Insti-tutional Review Board and Ethics Committee. Ethics approval The personal information of the patient was anonymized and procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Helsinki Declaration 1975, revised in 2000 and 2008). Accordingly, the Narayana Health Academics Ethics Committee (NHAEC) approved this case report for publication prior to submission (letter no. NHH/AEC-CL-2021–703). Conflict of interest The authors have no conflicts of interest to declare that are relevant to the content of this article. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Hammar S Bockus D Remington F Cooper L The unusual spectrum of neuroendocrine lung neoplasms Ultrastruct Pathol 1989 13 515 560 10.3109/01913128909074534 2552631 2. Bernstein M Kovar H Paulussen M Ewing’s sarcoma family of tumors: current management Oncologist 2006 11 503 519 10.1634/theoncologist.11-5-503 16720851 3. Horowitz ME, Malawer MM, Woo SY, Hicks MJ. “Ewing’s sarcoma family of tumors: Ewing’s sarcoma of bone and soft tissue and the peripheral primitive neuroectodermal tumors,” in Principles and practices of pediatric oncology, P. A.Pizzo and D. G. Poplack, Eds., pp. 831–888, Lippencott-Raven, Philadelphia, Pa, USA, 3rd edition, 1997. 4. Bacci G Ferrari S Comandone A Neoadjuvant chemotherapy for Ewing’s sarcoma of bone in patients older than thirty-nine years Acta Oncol 2000 39 111 116 10.1080/028418600431076 10752664 5. Gachechiladze M Skarda J Ibrahim M Primitive neuroectodermal tumor (PNET) of the lung in an adult woman World J Surg Oncol 2014 12 374 10.1186/1477-7819-12-374 25475214 6. Shet N Stanescu L Deutsch G Primary extraosseous Ewing sarcoma of the lung: case report and literature review Radiol Case Rep 2015 8 832 10.2484/rcr.v8i2.832 27330628 7. Hwang SK Kim DK Park S-I Kim Y-H Kim HR Primary Ewing’s sarcoma of the lung Korean J Thorac Cardiovasc Surg 2014 47 47 50 10.5090/kjtcs.2014.47.1.47 24570867 8. Sohn AJ Lang B McCarroll M Agarwal A Primary pulmonary Ewing sarcoma/peripheral primitive neuroectodermal tumor Proc (Bayl Univ Med Cent) 2020 33 646 648 33149376 9. Deokar KK Kunjir NG Ghorpade S Primary Ewings sarcoma of the lung J Clin Diagn Res 2015 9 XD01 XD03 25738070	36471861	PMC9713175	NO-CC CODE	2022-12-02 23:22:56	no	Indian J Thorac Cardiovasc Surg. 2022 Dec 1;:1-4	utf-8	Indian J Thorac Cardiovasc Surg	2,022	10.1007/s12055-022-01432-0	oa_other
==== Front Empir Econ Empir Econ Empirical Economics 0377-7332 1435-8921 Springer Berlin Heidelberg Berlin/Heidelberg 2334 10.1007/s00181-022-02334-1 Article The effect of infrastructure investment on tourism demand: a synthetic control approach for the case of Kuelap, Peru http://orcid.org/0000-0001-9199-4677 Lahura Erick [email protected] 123 Sabrera Rosario [email protected] 3 1 Economic Research Division, Central Reserve Bank of Peru (BCRP), Lima, Peru 2 grid.440592.e 0000 0001 2288 3308 Department of Economics, Pontifical Catholic University of Peru, Lima, Peru 3 grid.430666.1 0000 0000 9972 9272 Departamento de Ingeniería Económica y Contabilidad Corporativa, Universidad Científica del Sur, Lima, Perú 1 12 2022 136 4 2 2022 13 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. In this paper we estimate the effect of infrastructure investment on tourism demand. For this purpose, we analyse the case of the Kuelap Archaeological Complex (Peru), which became more attractive and accessible following the construction of Peru’s first cable car system and the redevelopment of a nearby airport located in the city of Jaen. In order to estimate the effect of this infrastructure investment package on the number of visitors to Kuelap, we construct a synthetic control using characteristics from several archaeological sites in Peru similar to Kuelap. This synthetic control allows us to estimate the potential number of visits to Kuelap had the infrastructure investment package not been made. Thus, the causal effect is the difference between the actual visits to Kuelap and the ones to the synthetic control. The results show that the number of visits to Kuelap increased by approximately 100% in the wake of the infrastructure investment package. Keywords Kuelap Infrastructure investment Tourism Synthetic control JEL Classification C21 H54 L83 R42 Z30 ==== Body pmcPrior to the COVID-19 pandemic, tourism generated about 10% of the world’s GDP and created 1 in 10 jobs worldwide (World Tourism Organization 2019). In Peru, tourism had also been an important economic activity, representing around 4% of GDP. However, since the beginning of the pandemic, tourism has been one of the most affected sectors in the majority of countries due to the closure of international borders and the restriction of non-essential activities. Thus, international tourist arrivals registered a historic contraction of 73% in 2020 and by the end of 2021 it still remains 73% below its prepandemic levels (World Tourism Organization 2022). Among others, a potential strategy to speed up the recovery of tourism is by improving accessibility to places of interest, which implies building and/or renovating airports and roads, among others. The main purpose of this paper is to estimate the causal effect that such public infrastructure investment may have on tourism demand. For this, we study the case of the Kuelap Archaeological Complex, a tourist site located in the Peruvian Amazon. Kuelap has received more visitors since the opening of the first cable car system in Peru (March 2017) and has benefited from the redevelopment of Jaen airport (one of the closest airports to the archaeological site). This has improved Kuelap’s accessibility by increasing the availability of flights to and from Lima, the capital of Peru. The hypothesis of this paper is that this investment package in tourism infrastructure (the construction of the cable car system and redevelopment of the airport of Jaen) has had a significant effect on the number of visits to Kuelap. To empirically evaluate this hypothesis, we use the infrastructure investment package (IIP) that benefited Kuelap as a case study and build a “synthetic control” unit, which is similar to Kuelap in terms of the number of visits before treatment but has not benefited from the IPP. Thus, the causal effect of the IIP (the treatment) on the number of visitors (variable of interest) to Kuelap (treated unit) is the difference in the number of visitors between Kuelap and the “syntectic control” unit after treatment. The candidates for the control unit are all tourist sites similar to Kuelap according to the classification of the Ministry of Foreign Trade and Tourism (MINCETUR), namely all those that are classified as “archaeological sites” (type) and “buildings” (subtype). However, given that none of these other tourist sites display a trajectory of visits similar to that of Kuelap, a synthetic control is constructed following the methodology originally proposed by Abadie and Gardeazabal (2003) and Abadie et al. (2010), and taking into account some recent developments and recommendations by Botosaru and Ferman (2019), Ferman and Pinto (2019), Ferman et al. (2020), Chernozhukov et al. (2021), Ferman and Pinto (2021), Chernozhukov et al. (2022), and Kaul et al. (2022). The baseline synthetic control is obtained by weighting the number of visits to each tourist site according to a set of relevant predictors that include only pre-treatment lags of the outcome variables and no covariates. The results show that investment in tourism infrastructure has significantly increased the number of visits to Kuelap since 2017. In particular, it is estimated that this investment package doubled the number of visits to Kuelap. Robustness exercises, related to the elimination of sites that were affected by a weather phenomenon known as El Niño and the inclusion of covariates related to tourism development (such as proportion of domestic tourists and guests per accommodation, among others), do not change the main results. The literature on tourism as an economic activity has focused on the analysis of its relationship with economic growth. The tourism-led growth hypothesis has been one of the most studied in this literature (for instance, Risso and Brida 2009; Rodríguez et al. 2014; Banerjee et al. 2015, 2016; Faber and Gaubert 2019) and has been empirically assessed using mainly time series econometric techniques (such as cointegration and Granger causality tests). Another branch of the literature has been devoted to the analysis of the relationship between public policies and tourism (Rodríguez et al. 2014; Imikan and Ekpo 2012, among others), and the study of the relationship between public investment in transport infrastructure and tourism (Álvarez-Díaz et al. 2019; Monterrubio et al. 2020; Kanwal et al. 2020, among others). However, there are few empirical studies that estimate the causal effect of public investment on tourism (for example Deng et al. 2019; Doerr et al. 2020; Chow et al. 2021; Albalate et al. 2022; Shu et al. 2022), and none that analyse the causal effect of the construction of a cable car system on tourism. As far as we know, this is the first paper that estimates the impact of public investment in tourism infrastructure on tourism demand for the Peruvian case. Also, our research contributes to the scarce literature that employs a comparative case study with a synthetic control to study the causal effect of public investment in infrastructure on tourism. Finally, it is the first paper that analyses the causal effect of the construction of a cable car system on tourism. The rest of this paper is organised into six sections. Section 1 presents a brief review of the economic literature on tourism. Section 2 describes the main stylized facts associated with the investment in tourism infrastructure that has led to an increase in visits to Kuelap. Section 3 presents the empirical methodology used to evaluate the hypothesis of interest. The data are described in Sect. 4. The results are presented and discussed in Sect. 5. Finally, the conclusions are presented in Sect. 6. A brief literature review The economic literature on tourism has focused on its long-run relationship with economic growth, the so-called tourism-led growth hypothesis. Most of this literature provides evidence of a positive impact of tourism on different economic variables, such as the accumulation of foreign reserves, investment and employment, among others. Empirically, this relationship has been analysed using time series econometric techniques such as cointegration and Granger causality.1 For instance, Risso and Brida (2009) analyse the contribution of tourism to Chilean economic growth and find that tourism is an important determinant of long-run economic growth; in particular, they estimate that the elasticity of tourism expenditure with respect to real GDP is 0.8 in the long run. Based on the existing literature on tourism and growth using time series, Brida et al. (2013) conclude that tourism Granger causes long-term economic growth; however, they warn that this result should be validated with regional and country-level studies. Along these lines, Faber and Gaubert (2019) study the case of Mexico and show that tourism has a positive economic effect in the long run, both at the local level (municipalities) and at the country level. Another branch of the literature focuses on the analysis of the relationship between public policies and tourism. Rodríguez et al. (2014) provide evidence of a long-run relationship between public investment in advertising and tourism demand in the Andalusia, Spain; furthermore, they show that advertising investment helps to predict tourist demand in the short run. Imikan and Ekpo (2012) study the relationship between investment in different types of infrastructure (water, electricity and transport, among others) and tourism development for the case of River State, Nigeria. Using multiple correlation and stepwise regression analysis, they find that investment in transport infrastructure is highly correlated with tourism development. Gulcan et al. (2009) study the relationship between public investment and value added created by hotels in the Aegean region of Turkey; using a panel data regression model they find that public investment in tourism has a positive effect on hotel activity. More recently, Deng et al. (2019) estimate the causal effect of a regional policy in western China (Western Development Strategy or WDS) on tourism using a quasi-experiment. Specifically, they use a sharp regression discontinuity design where the distance of each city from the geographical boundary of the WDS determines the point of discontinuity. The results of Deng et al. (2019) indicate that the WDS policy, which includes infrastructure projects, has had a positive effect on the development of tourism in China. The recent literature has shown that investment in tourism infrastructure plays an important role in the development of this sector, promoting sustainable investment policies (Paramati et al. 2018) and cutting through the entire value chain of the tourism sector: transport and communications, hotel industry, restaurants and recreational facilities (Nguyen 2021). One area of this research has focused specifically on the relationship between transport infrastructure and the development of the tourism industry based on investments in different types of transport links including by air (airports and cable cars), land, and rail. The relationship between air transport and tourism has received considerable attention in the academic literature and among policy makers. Papatheodorou (2021) identifies three types of relevant contributions to the study of tourist air transport: (a) airline business models and regional tourism development, (b) the link between aeropolitics and tourism, and (c) the airline–airport relationship and the development of tourist routes (for example, Benedetti et al. 2012; Jian et al. 2017; Ferníndez et al. 2018; Álvarez-Díaz et al. 2019; Monterrubio et al. 2020, among others). Likewise, land transport infrastructure constitutes an important driver for the dynamism of tourism, generating the development of new tourist sites and motivating the support of local residents towards this activity. For example, Kanwal et al. (2020) demonstrates that the road infrastructure of the China–Pakistan Economic Corridor (CPEC) is positively related to community support for tourism. On the other hand, investment in high-speed rails (HSR) and its relationship with the tourism industry have been extensively studied, demonstrating that improvements in accessibility as a consequence of HSR lead to increased demand for tourism (Yin et al. 2019; Delaplace et al. 2014; Bazin et al. 2011; Kurihara Wu 2016). Research suggests that investment in transport infrastructure is associated with increased visitor numbers and consequently tourism development. However, there are less empirical studies that estimate the causal effect of investment in infrastructure on tourism. Recent studies have used difference-in-differences, instrumental variable and synthetic control approaches to estimate the effect of airport subsidies and HSR on the number of visitors, the development of national tourism and the efficiency of the tourism industry (Chow et al. 2021; Albalate et al. 2022; Shu et al. 2022). For instance, Doerr et al. (2020) employing a synthetic control approach and using data from Germany conclude that the new airport infrastructure promotes regional tourism development, due to the increase in tourist arrivals and income generated from this activity. Regarding research on cable car systems and tourism, the literature is scarce. Zhang et al. (2009) reports on visitors’ impressions of using a cable car in Wulingyuan (China), but finds that tourists’ motivations are focused on landscape aspects and not on the transportation experience. In a paper not directly related to tourism, Martinez et al. (2018) use instrumental variables to identify the causal effect of the cable car in La Paz, Bolivia, on the choice of mode of transport, the use of time, and employment; the results show significant benefits for the users of this type of transportation. Our study uses a synthetic control approach to estimate the causal effect on tourism demand of the construction of the first cable car system in Peru and the reconstruction of a nearby airport - an investment package on tourism infrastructure. Thus, this paper contributes to the existing literature in three ways. As far as we know, this is the first paper that estimates the impact of public investment in tourism infrastructure on tourism demand for the Peruvian case. Second, our research contributes to the scarce literature that employs a comparative case study with a synthetic control to study the causal effect of public investment in infrastructure on tourism. Finally, it is the first paper that analyses the causal effect of the construction of a cable car system on tourism. Kuelap and tourism infrastructure investment The department of Amazonas is part of the tourist circuit of north-eastern Peru,2 which offers attractive archaeological and natural sites. One of its main tourist attractions is the Kuelap Archaeological Complex or simply Kuelap, located at a distance of approximately 107 kms from Chachapoyas, the capital of Amazonas. Kuelap is the most important archaeological site of the Chachapoyas culture (fifth–sixteenth century). It is located on a limestone mountain at 3000 ms above sea level, on the left bank of the Utcubamba River. The site occupies an area of approximately 6 hectares, surrounded by a perimeter wall of 15 ms height, and is built with heavy but finely edged limestone. Across the plateau there are more than 550 circular structures with different architectural functions (Ministry of Foreign Trade and Tourism 2017). Peru has 23 archaeological sites displaying similar characteristics to Kuelap, including Machu Picchu, Chavin de Huantar and Caral, among others (Ministry of Foreign Trade and Tourism 2019). The Kuelap cable car system The project to build a cable car system in Kuelap originated in December 2001, according to Supreme Resolution No. 536-2001-EF, which established the granting of concessions to the private sector for the execution of tourist infrastructure works for the Kuelap Archaeological Complex. On August 26, 2005, the public investment project (PIP) denominated “Implementation of Cable Cars between the town of Tingo Nuevo and the Kuelap-Amazonas Fortress” was registered. Its main goal was to reduce travel time and improve the quality of transport to reach Kuelap. In March 2013, under an inter-institutional collaboration agreement with MINCETUR, The Peruvian Finance Ministry’s Investment Agency (ProInversion) was put in charge of undertaking feasibility studies and developing a project proposal as a public–private partnership or PPP (Rodríguez et al. 2018). Table 1 details the main milestones in the process of development and construction of the first cable car system of Peru.Table 1 Milestones of the project “Kuelap cable car system”. Source: Own elaboration based on Rodríguez et al. (2018) Date Event 09-09-2013 Announcement and delivery of the Contest Rules 30-05-2014 Award of the good pro of the project 15-10-2014 Signing of the concession contract 13-08-2015 Beginning of construction 02-03-2017 Opening of the Kuelap cable car system The project was awarded to a French-Peruvian consortium in May 2014. Construction works started in August 2015 and led to a reduction in tourism activity, especially between August and December 2015, because traffic through the site’s access road was severely restricted. (During the day the road was open for just three hours.) In addition, maintenance work at the archaeological site meant that visits to some areas of the fortress were restricted. The opening ceremony of Peru’s first cable car line was in July 2016 and after a trial period full operations started in March 2017. According to Ministry of Economy and Finance (2017), the final cost of the construction of the cable car system was approximately US$ 27 million (S/ 89.2 millions). The project file projected a 50% increase in tourists during the cable car’s first ten years of operation; however, the actual number of visitors has been twice this forecast, increasing from 40,146 in 2014 to 102,905 in 2017. A simple cost-benefit analysis provided by Rodríguez et al. (2018) shows that the this outcome resulted in a reduction of costs of the cable car system of approximately US 370,000 during 2017. Furthermore, 350 workers were hired directly in order to operate the system. Airport infrastructure The average travel time from Lima to Kuelap by bus is approximately 24 h. However, it is possible to significantly reduce travel times by 18 h by taking a 1 h and 30 min flight to either Chachapoyas or Jaen, the closest cities to Kuelap. Travel times to Kuelap by car from Chachapoyas and Jaen take 3 h and 7 h, respectively. Overall, travel times by land can be an important factor that discourages an average tourist to travel to Kuelap, combined with very limited availability of flights which was the case until 2015. In recent years, investment has been made in these airports, which has allowed an increase in the number of passengers to Chachapoyas and Jaen. The general description of the airport investment programme, as well as the main milestones, is outlined in Table 2.Table 2 Airport investment in Chachapoyas and Jaen. Source: Own elaboration based on Private Investment Promotion Agency (2019), Regional Government of Amazonas (2016), Supervisory Board for Investment in Public Transport Infrastructure (2018), Andina (2017), Turismo News (2016), Turismo News (2017a) and Turismo News (2017b) Chachapoyas airport Jaen airport Description Project Airport modernisation Airport redevelopment Investment US$ 5.77 million (approx.) US$ 3 million (approx.) Infrastructure Remodelling of the passenger terminal Air terminal improvement New gantry construction Parking lot upgrade Parking lot paving Boarding area improvements Installation of basic services Operational technical equipment Distance to Kuélap 3 h approx. (without cable cars) 7 h approx. (without cable cars)) (By road) 2 h approx. (with cable cars) 6 h approx. (with cable cars) Milestones May 2016 Saeta airline started commercial flights between Tarapoto and Chachapoyas July 2016 Opening of the airport September 2016 LATAM airline started daily flights between Lima and Jaen March 2017 Air Majoro airline started direct flights between Lima and Chachapoyas July 2017 ATSA Airlines airline started regular direct flights between Lima and Chachapoyas Future project Master Plan for Development of Chachapoyas Third Group of Airports Airport In 2016, nearly US$ 9 million (30 million Soles) were invested in the redevelopment of the Jaen airport3 and in the refurbishment of the Chachapoyas airport, which allowed key airlines to start operating commercial flights to these airports. In the case of Chachapoyas airport, commercial flights from the city of Tarapoto restarted in May 2016 and in 2017 the airlines Air Majoro and ATSA Airlines started direct flights from the city of Lima. Jaen airport started receiving daily direct flights from Lima (LATAM airlines) from September 2016 onwards.Fig. 1 Monthly evolution of arrivals at airports and Kuelap. Note: The visits to Kuelap and arrivals at Jaen airport are measured on the left axis. Arrivals at Chachapoyas airport are measured on the right axis. The horizontal axis represents months. Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) Figure 1 shows the evolution of monthly visits to Kuelap and the arrival of passengers at the airports of Jaen and Chachapoyas between 2016 and 2018. It is evident that the evolution of passenger arrivals at these airports coincides with the evolution of visits to the Kuelap archaeological site. In particular, all three series increased during the high season for tourism to Amazonas (July–August). However, it can be observed that Jaen airport is more important than Chachapoyas in terms of the volume of passengers. For example, in the years 2017 and 2018 Jaen airport received 6.6 times more passengers than Chachapoyas.4Fig. 2 Evolution of visits to Kuelap: 2008–2018 Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) Evolution of the number of visits to Kuelap Figure 2 shows the evolution of the number of visits to the Kuelap Archaeological Complex between 2008 and 2018. A clear upward trend can be seen in the years leading up to 2015 when there is a drop in visitor numbers (from 40,146 to 36,385 visits) due to the construction works on the cable cars. In 2016 the number of visits recovered (56,010 visits), which coincided with the increase of commercial flights to Chachapoyas and Jaen due to the redeveloping and refurbishment of their airports. However, in 2017 a significant jump in the number of visits is observed (102,905 visits) which coincides with the inauguration of the cable car service and the availability of more flights. In addition, the number of visits in 2018 (110,068 visits) has remained well above the levels observed before 2017. The key question is to what extent the increase in visitor numbers to Kuelap from 2017 has been due to the infrastructure investments made. Our hypothesis posits that most of this increase is basically due to the construction of the cable car and the redevelopment of the Jaen airport. We assess this hypothesis in a comparative case study using a synthetic control. Empirical methodology: a synthetic control approach A comparative case study is an empirical approach used to measure the causal effect of an event, treatment or intervention (e.g. the enforcement of a policy or law) on a variable of interest when it is not possible to apply an experimental design. To implement a comparative case study, “units” (cities, regions, locations, etc.) exposed to an event (treatment, shock, etc.) and others not exposed to the event are required, and all units must be similar before treatment.5 We employ a comparative case study where Kuelap is the “treated” unit, investment in tourism infrastructure is the “treatment” and the number of visits to Kuelap is the variable of interest or the outcome variable. Let YtT represent the number of visitors to Kuelap in period t, for t=1,2,…,T,T+1,…,T∗, and assume that the treatment occurs in period t=T+1, so there are T preintervention or pre-treatment periods. To estimate the effect of the treatment on the treated unit, we need a control unit that replicates its counterfactual, YtC, i.e. the evolution of visits to Kuelap had the treatment not occurred. Then, the causal effect of the treatment is the difference between the number of visitor numbers to the treated unit, YtT, and its counterfactual, YtC, after treatment, i.e. YtT-YtC for all t>T. Natural candidates for a single control unit are any of the tourist sites that have the same Mincetur classification as Kuelap: archaeological sites/buildings. However, it is possible that none of those sites provides a close enough match to be a valid control.6 In this case, Abadie and Gardeazabal (2003) and Abadie et al. (2010) proposed the use of a “synthetic control” to approximate YtC, which is a weighted average of all J potential control units or donor pool. In order to be a valid estimate of the counterfactual, the synthetic control must satisfy two conditions. First, the synthetic control unit and Kuelap should display a similar number of visits before the intervention (for all t<T+1). Second, the synthetic control unit and Kuelap should be similar in terms of the preintervention values of the “predictors” of the number of visits. The predictors may include L lagged values of the visits to Kuelap (or linear combination of those values) and other M variables that can help predicting those visits, which are referred to as “covariates”, so that the total number of predictors is K=L+M. Estimation A synthetic control for Kuelap can be constructed by “optimally” weighting all the available sites. Formally, for each archaeological site j=1,2…,J that is part of the donor pool, we define a vector yj of dimension T×1 that contains the number of visits to archaeological site j along the preintervention periods (T periods). The synthetic control of Kuelap, y∗, is defined as the vector of visits that results from “optimally” weighting each yj using weights w1∗,w2∗,⋯,wJ∗:1 y∗=y1·w1∗+…+yj·wj∗+…+yJ·wJ∗=Y0w∗ where wj∗ represents the optimal weight of the archaeological site j in the synthetic control of Kuelap, Y0 is a matrix of order T×J whose columns are y1,y2,…,yJ, and w∗=[w1∗,w2∗,…,wJ∗]′ is a vector of order J×1 whose elements are weights that add up to one, w1∗+w2∗+…+wJ∗=1. The choice of optimal weights is made such that the resulting synthetic control replicates as close as possible the preintervention values of all the K predictors of visits to Kuelap. Kaul et al. (2022) call this procedure the “inner optimization” process and can be formalised as follows. Let x be a vector of order (K×1) containing the pre-treatment values of the K predictors and xj the corresponding vector for each archaeological site j, for j=1,2,3,…,J; let X0 be a (K×J) matrix whose columns are the vectors x1,x2,…,xJ. In addition, let V be a matrix whose values reflect the relative importance of each predictor (i.e. the weights of the predictors), which will be determined by a second procedure that Kaul et al. (2022) refer to as the “outer optimization”. Then, for a given V matrix, the vector of optimal weights w∗ is chosen to minimize the weighted distance between x and X0w, subject to the fact that the weights are nonnegative and they sum up to 1:2 Minw(x-X0w)′V(x-X0w)s.t.wj≥0,j=1,2,…,Jw1+w2+⋯+wJ=1 The solution to this problem is the vector w∗(V)=[w1∗,w2∗,…,wJ∗]′, which defines the synthetic control unit for Kuelap y∗=y1·w1∗+…+yj·wj∗+…+yJ·wJ∗, for a given matrix V. The next step consists of obtaining the optimal predictor weights, V∗, which is achieved by the so-called “outer optimization”. Abadie and Gardeazabal (2003) and Abadie et al. (2010) proposed to choose a positive definite and diagonal matrix V such that the synthetic control optimally replicates the trajectory of visits to Kuelap before treatment. Specifically, V∗ is the matrix that solves the following minimisation problem:3 MinV(z-Z0w∗(V))′(z-Z0w∗(V)) where z is a (m×1) vector containing all the preintervention values of the visits to Kuelap, Z0 is a (m×J) matrix with columns z1,z2,…,zJ, and zj is a (m×1) vector containing all the preintervention values of the visits to site j for j=1,2,…,J. An alternative way to obtain the predictor weights contained in V a regression-based method as in Bohn et al. (2014). Abadie et al. (2010) justify the use of the synthetic control method in a comparative case study under certain conditions. First, the intervention has no effect on the outcome variable before it is implemented; however, the anticipation of the intervention may affect preintervention outcomes, in which case the treatment period can be redefined and include the affected periods. Second, the outcomes of the untreated units are not affected by the intervention. However, if the pre-treatment fit of the synthetic control is poor (compared to the actual pre-treatment outcome variable), then Abadie et al. (2010) do not recommend the use of a synthetic control. A synthetic control allows the presence of unobserved confounders that vary over time. To see this formally, Abadie et al. (2010) assume that the outcome that would be observed for site i in period t in the absence of the treatment, YitN is described by the following factor model:4 YitN=δt+θtCi+λtμi+εit where δt is an unknown common factor that varies with constant factor loadings across units, Ci is a (K×1) vector of observed covariates (not affected by the intervention), θt is a (1×K) vector of unknown parameters, λt is a (1×F) vector of unobserved common factors that varies over time, μi is an (F×1) vector of unknown factor loadings, and εit represents unobserved idiosyncratic, transitory shocks with mean zero. Equation (4) is a generalization of the difference-in differences (DID) model with fixed effects: If λt is constant for all t, then Eq. (4) reduces to the DID fixed-effects model. Thus, the DID model allows for the presence of unobserved confounders μi but restrict their effect to be constant over time if λt=λ; thus, the confounders can be eliminated using the first difference transformation. In contrast, the synthetic control allows the effect of confounding unobserved characteristics to vary over time and, when the pre-treatment fit is very good, it provides an unbiased estimator of YitN for the treated unit i. However, in many applications, the pre-treatment fit is imperfect. Ferman and Pinto (2021) study the properties of the synthetic control, a demeaned version of the synthetic control, and the DID estimator when the pre-treatment fit is imperfect and provide some conditions under which those estimators can used with confidence. For instance, if the synthetic control unit recovers the levels of the treated unit and the preintervention fit is imperfect, then the synthetic control would be reliable if there is no important unobserved confounders that vary over time. In this case, if the DID and synthetic control provide similar results, then Ferman and Pinto (2021) suggest that the synthetic control is a good choice. As discussed by Ferman et al. (2020), there is lack of consensus about which predictors should be used in synthetic control applications. The standard practice is to use pre-treatment outcomes and covariates and to construct the synthetic control trying to balance at least on a long set of pre-treatment outcomes (Botosaru and Ferman 2019). Usually, linear combinations of preintervention outcomes are used as predictors, but there is no formal recommendation about it. If there are no good reasons to include covariates in the application, Ferman et al. (2020) recommend the use of the specification including all pre-treatment periods, given that it minimizes the root mean squared error (RMSE) in the pre-treatment period, and it is not subject to the choice of arbitrary pre-treatment outcome lags as predictors. On the other hand, the only reason not to use all pre-treatment periods is when there are good reasons to include covariates. In this case, it is recommended to use a specification that does not include all pre-treatment lags, otherwise all covariates would be irrelevant in the estimation of weights, as documented by Kaul et al. (2022): even though the inner optimization takes into account the covariates, the synthetic control obtained from the outer optimization will ignore all the covariates when all the pre-intervention values of the outcome variable are used as predictors. Based on the above discussion about the synthetic control method, our baseline synthetic control will be constructed using as predictors all the pre-treatment lags and no covariates, given we have limited economic information for each specific site. Then, the robustness of the results will be analysed by including covariates and taking into account the effect of the El Niño phenomenon in the donor pool and in the synthetic control unit. Inference One way to evaluate the significance and robustness of these estimates is to analyse whether the results could have been obtained by chance. In other words, how often would we have obtained an effect on visits of the estimated magnitude had we chosen an archaeological control site at random. To address these questions, Abadie and Gardeazabal (2003) and Abadie et al. (2010) proposed the use of “placebo tests”. It consists of applying the same methodology to tourist sites that did not experience any treatment (investment package) similar to Kuelap and analysing the resulting gap for each site, where the gap is defined as the difference between the actual number of visits and its synthetic counterpart. If any of the “placebos” create gaps of similar magnitude to those estimated for Kuelap, then we would conclude that there is no significant evidence of a positive effect of Kuelap’s infrastructure investment package. However, if none of the placebos show a positive gap as significant as the one associated with Kuelap, we can conclude that there is a positive and statistically significant effect of the infrastructure investment package. In order to evaluate Kuelap’s gap relative to the gaps obtained from the placebos obtained for each site, we follow Abadie et al. (2010) and analyse the distribution of the ratios of post/pre-intervention mean squared prediction errors (MSPE). The MSPE of site j is defined as the average of the squared difference between the number of visits to site j and its synthetic counterpart. If the synthetic control for Kuelap is such that the pre-treatment MSPE is zero and the post-treatment MSPE is large in magnitude, then the estimated gap reflects the effect of the investment package on the number of visitors to Kuelap. In particular, if the ratio “post-treatment MSPE / pre-treatment MSPE” (RMSPE) for Kuelap is the highest compared to the rest of sites, then the estimated effect is statistically significant: if a site was chosen at random, then the chances of obtaining a RMSPE as high as Kuelap’s would be approximately 1/J, which represents the p-value to test the null hypothesis of no significant effect of the treatment. Recently, some alternative inference methods for synthetic control analysis have been proposed, such as conformal inference (Chernozhukov et al. 2021) and t-test (Chernozhukov et al. 2022). In this paper our inference results of will be based on the approach proposed by Abadie et al. (2010). Data The information used has been obtained from MINCETUR and the National Institute of Statistics and Informatics (INEI). The data are annual and cover the period 2008–2018. Additionally, we collected monthly data on the number of visits from different tourist sites. MINCETUR has a database of the arrival of visitor numbers to 118 tourist sites in the country. It also maps and classifies tourist attractions by category, type and subtype, geographical location and other characteristics. Based on this classification, sites that are categorised as “cultural manifestations”, of the type “archaeological sites” and subtype “buildings” are considered as possible candidates for our control. Table 3 presents the list of tourist sites that fall into this classification by geographical location (department).7Table 3 Tourist sites: category “cultural manifestations”, type “archaeological sites” and subtype “buildings”. Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) Department Tourist site Number Amazonas Kuélap, Revash y Karajía. 3 Áncash Chavín. 1 Apurímac Saywite. 1 Ayacucho Intihuatana y Wari. 2 Cajamarca Otuzco y Cumbemayo. 2 Cusco Machu Picchu, Moray, Tipon, 6 Choquequirao, Pikillaqta y Raqchi. Huánuco Kotosh. 1 La Libertad Huaca Arco Iris, Huaca Sol, 4 Huaca Bruja y Nikán. Lima Caral. 1 Piura Narihualá. 1 Puno Sillustani. 1 Total 23 To build the synthetic control, we use as predictors the pre-treatment number of visits. Additionally, we also include variables related to key characteristics of tourism activity by region, such as average length of stay in accommodation facilities (length of stay), number of domestic tourists as a percentage of total number of tourists (domestic visitors), number of guests per accommodation facility (guests per accommodation), participation of tourism in GDP (tourism development), average level of education achieved by people over 15 years old (educational attainment), average annual temperature (regional temperature) and pre-treatment visits. The information is annual and corresponds to the period 2008–2018, where the sub-sample 2008–2014 is the pre-treatment period and the sub-sample 2015–2018 is the post-treatment period. Table 4 describes the variables and the sources. The descriptive statistics per region are detailed in “Section A of the appendix”.Table 4 Predictor variables employed to build the synthetic control Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) and National Institute of Statistics and Informatics (2019) Source Variable Definition MINCETUR Length of stay Average stay in accommodation facilities (in days) MINCETUR Domestic visitors Number of domestic tourists (% of total number of tourists) MINCETUR Guests per accommodation Number of guest arrivals per accommodation facility INEI Tourism development Gross value added at 2007 prices (% of GDP) INEI Educational attainment Average years of study achieved by the population over age 15. SENAMHI Regional temperature Average annual temperature, expressed in degree Celsius. MINCETUR Pre-treatment visits Total number of visits for each year between 2008 and 2014 Results Figures 3 and 4 show the evolution of the number of visits to Kuelap compared to the tourist sites located in Cusco (Peru’s main tourist destination) and to the rest of tourist sites outside Cusco, respectively, between 2008 and 2018.Fig. 3 Visits to Kuélap and other archaeological sites located in Cusco: 2008–2018 Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) Fig. 4 Visits to Kuélap and other archaeological sites: 2008–2018 Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) At first glance, none of the tourist sites display a trajectory of visits similar to Kuelap’s and thus would not qualify as a control unit. Therefore, a synthetic control was constructed following the methodology described in Sect. 3. The baseline synthetic control unit (SCA) is obtained using the pre-treatment values of the number of visits as the only predictors (no covariates included), and all the sites of the type “archaeological sites” and subtype “buildings” as the donor pool. As a robustness exercise, we present alternative specifications to estimate the synthetic control unit. First, given that in 2018 some of the tourist sites included in the donor pool were adversely affected by the “El Niño” weather phenomenon, we re-estimate the synthetic controls excluding those sites. Second, we estimate the synthetic control unit using two sets of potential covariates. As shown below, the results confirm the positive causal effect of the investment package on the number of visits to Kuelap. Baseline results The baseline synthetic control (Synthetic control A or SCA) is constructed using as predictors all the preintervention number of visits and no covariates. The donor pool consisted of all sites listed in Table 3 with the exception of “Revash” and “Karajia”, because the number of visitors to these sites has only been recorded since 2016; additionally, both sites are affected by the demand for Kuelap, since they are also located in the department of Amazonas. The weights of each site in the baseline synthetic Kuelap are displayed in the second column of Table 5. The synthetic Kuelap is a weighted average of six sites: Cumbemayo (weight of 0.17), Kotosh (weight of 0.20), Intihuatana (weight of 0.18), Huaca Brujo (weight of 0.31), Choquequirao (weight of 0.12), and Wari (weight of 0.31); the rest of the sites have zero weights in the synthetic control.Table 5 Synthetic control: Weights for each archaeological site Archaeological site Preintervention visits Synthetic control A Synthetic control B Choquequirao 0.12 0.08 Cumbemayo 0.31 0.23 Intihuatana 0.18 0.23 Kotosh 0.20 0.27 Machu Picchu 0.00 0.00 Moray 0.00 0.00 Otuzco 0.00 0.00 Pikillaqta 0.00 0.00 Raqchi 0.00 0.02 Saywite 0.00 0.00 Sillustani 0.00 0.00 Tipon 0.00 0.00 Wari 0.01 0.17 Caral∗ 0.00 Chavin∗ 0.00 Huaca Arco Iris∗ 0.00 Huaca Brujo∗ 0.17 Huaca Sol y Luna∗ 0.00 Narihuala∗ 0.00 Nikan∗ 0.00 Sites with (*) were affected by El Niño phenomenonn Columns (2), (3) and (4) of Table 6 display the pre-treatment visits to Kuelap, Cumbemayo (the site with the individual highest weight) and the baseline SCA, respectively. A simple comparison indicates that the SCA’s pre-treatment visits are more similar to Kuelap’s than Cumbemayo’s. Thus, the SCA replicates well the trajectory of preintervention visits.Table 6 Synthetic control units using all preintervention outcomes Source: Ministry of Foreign Trade and Tourism (2019) and authors’ estimates Predictors Kuelap Cumbemayo Synthetic control A Synthetic control B Visits in 2008 17,396 30,580 17,448 17,698 Visits in 2009 18,542 21,701 18,618 18,080 Visits in 2010 23,696 27,810 23,723 23,855 Visits in 2011 29,431 33,636 29,246 28,832 Visits in 2012 27,960 25,155 28,022 28,396 Visits in 2013 33,495 42,965 33,555 33,584 Visits in 2014 40,146 62,961 40,136 40,196 Fig. 5 Total visits: Kuelap, baseline synthetic control (SCA), and difference-in-differences (DID), 2008–2018 Source: Own elaboration Figure 5 shows the trajectory of visits to Kuelap and its synthetic control for the period 2008–2018. It is clear that the SCA replicates almost perfectly the preintervention visits to Kuelap, which makes the SCA a good counterfactual in order to estimate the causal effect of the investment package on the number of visits to Kuelap. Given that the difference between the visits to Kuelap and the synthetic control is positive in 2017 (when the cable car system started to operate) and 2018, we can conclude that the investment package had a positive effect on the number of visits to Kuelap. The top part of Table 7 displays the number of visits to Kuelap, Cumbemayo and SCA during the treatment period from which the causal effect of the infrastructure investment package can be calculated. It can be observed that the investment package had a negative impact during the first year of construction of the cable car system, due to the restriction in access to the Archaeological Complex related to the construction works; this is reflected by a gap (difference between actual and synthetic control visits) of -4668 visitors in 2015. However, the gap became positive in 2016 by 10,160 visitors and then was more than double in 2017 and 2018 (105% and 117%, respectively). Specifically, the estimated causal effect of the investment package on the number of visits to Kuelap is 52,610 visitors in 2017 and 59,612 visitors in 2018. On average, the estimated causal effect is 55,935 which is statistically significant at the 5% level (p-value = 0.048). For comparison purposes, the bottom part of Table 7 displays the estimated causal effect using the difference-in-differences (DID) estimator and robust standard errors as suggested by Ferman and Pinto (2019) when there is only one treated group. The DID estimator is positive (43,931 visitors) and statistically significant at the 10% level (p-value = 0.052). This effect is smaller than the estimated effect obtained with the synthetic control (A) but greater than the one obtained with Cumbemayo (34,889 visitors). However, the DID control unit does not replicate well the preintervention number of visits, as it is evident from Fig. 5.Table 7 Synthetic control and estimated treatment effects Kuelap Cumbemayo Synthetic control A Synthetic control B Visits during treatment period 2015 36,385 61,837 40,637 43,873 2016 56,010 66,611 45,850 49,225 2017 102,905 78,316 50,295 56,538 2018 110,068 64,879 50,807 56,425 Difference between Kuelap and controls 2017 24,589 52,610 46,367 2018 45,189 59,261 53,643 Average (2017–2018) 34,889 55,935 50,005 p-value 0.048 0.071 Difference-in-differences Effect 43,931 38,173 p-value 0.052 0.070 Using the t-test proposed by Chernozhukov et al. (2022), SCA is 53,880 with a t=13.26, and SCB is 49,423 with t=15.03; these results confirm that the causal effect is statistically significant. Own elaboration Fig. 6 Visit gaps for Kuelap and “placebo” gaps for other archaeological sites: 2008–2018 Source: Own elaboration Figure 6 illustrates the statistical significance of the causal effect using the “placebo tests” proposed by Abadie and Gardeazabal (2003) and Abadie et al. (2010) and discussed in Sect. 3. The figure displays the estimated gap for Kuelap and placebo gaps for other archaeological sites that did not experience a similar investment package in tourism infrastructure as Kuelap did during the period of analysis.8 It is clear that the positive gap for Kuelap is highly unusual and that its magnitude is the largest compared to the rest of the other sites. This is confirmed by the ratio of mean squared prediction error (MSPE) between post-treatment and pre-treatment for Kuelap and the sites that are part of the donor pool, which are displayed in Fig. 7. As can be observed, Kuelap’s ratio is larger than the rest, which means that if a site were chosen at random, then the chances of obtaining a ratio as high as Kuelap’s would be 1/21≈0.0476.Fig. 7 Ratio of post-treatment MSPE and pre-treatment MSPE: Kuelap and control sites Source: Own elaboration The effect of El Niño phenomenon on visits In 2017, several regions of the country were adversely affected by the El Niño phenomenon. According to the National Emergency Operations Center (COEN), Piura, Lambayeque, La Libertad, Ancash and Lima were the departments that registered the greatest damage ( Radio Programas del Perú (2017)). This event caused a decrease in visits to tourist sites within these regions which could reduce the number of visits to the synthetic control, especially if they have an important weight (for example, Huaca Brujo), and thus lead to a larger difference between actual visits and synthetic control and thus to an upward bias in the estimated causal effect. In order to avoid this potential bias, we re-estimated the synthetic control excluding the archaeological sites located in departments affected by the El Niño phenomenon in 2017. Figure 8, which displays monthly visits to these places between 2015 and 2017, shows a drop in visits during March, April and May 2017 compared to the same period in previous years.Fig. 8 Monthly visits to archaeological sites affected by the El Niño phenomenon: 2015, 2016 and 2017 Source: Own elaboration based on Ministry of Foreign Trade and Tourism (2019) The last column of Table 5 displays the weights of each site used to construct the alternative synthetic Kuelap excluding those site affected by El Niño phenomenon, which we call synthetic control (B) or SCB. The SCB is a weighted average of six sites: Kotosh (weight of 0.27), Cumbemayo (weight of 0.23), Intihuatana (weight of 0.23), Wari (weight of 0.17), Choquequirao (weight of 0.08) and Raqchi (weight of 0.02); the rest of the sites have zero weight in the synthetic control. It is worth noting that all sites not affected by El Niño that contribute to SCA remain in the SCB. The last column of Table 6 displays the pre-treatment visits to the SCB. A simple comparison indicates that the SCA’s pre-treatment visits are very similar to SCB’s. Figure 9 shows the trajectory of visits to Kuelap and the synthetic controls (A) and (B), for the period 2008–2018. It is clear that the SCB replicates almost perfectly the preintervention visits to Kuelap as SCA does. However, as expected, the SCB’s gap is positive but smaller than the SCA’s gap because we are excluding sites adversely affected by El Niño. Again, it is evident that the investment package had a positive effect on the number of visits to Kuelap.Fig. 9 Total visits: Kuelap, “Synthetic control”, and El Niño: 2008–2018 Source: Own elaboration The last column of the top part of Table 7 displays the number of visits to SCB during the treatment period. Similar to the case of SCA, it can be observed that the investment package had a negative impact during the first year of construction of the cable car system, which is reflected by a gap of -7488 visitors in 2015. However, the gap became positive in 2016 by 6785 visitors and then almost double in 2017 and 2018. Specifically, the estimated causal effect of the investment package on the number of visits to Kuelap is 46,367 visitors in 2017 and 53,643 visitors in 2018. On average, the estimated causal effect is 50,005 which is statistically significant at the 10% level (p-value = 0.071). As expected, in 2017 and 2018 the SCB’s gap is smaller than SCA’s gap because we are excluding sites adversely affected by El Niño. Again, the results indicate that the investment package in tourism infrastructure has had a positive and significant effect on the number of visitors to Kuelap. The effect of adding covariates The second robustness analysis we undertake is the estimation of the synthetic control using covariates. The results are displayed in Tables 8 and 9.Table 8 Synthetic control: Weights for each archaeological site Source: Own elaboration Archaeological site One control More controls Synthetic control A Synthetic control B Synthetic control A Synthetic control B Choquequirao 0.12 0.08 0.12 0.08 Cumbemayo 0.31 0.22 0.30 0.23 Intihuatana 0.18 0.22 0.19 0.23 Kotosh 0.20 0.27 0.20 0.27 Machu Picchu 0.00 0.00 0.00 0.00 Moray 0.00 0.00 0.00 0.00 Otuzco 0.00 0.00 0.00 0.00 Pikillaqta 0.00 0.00 0.00 0.00 Raqchi 0.00 0.02 0.00 0.02 Saywite 0.00 0.00 0.00 0.00 Sillustani 0.00 0.00 0.00 0.00 Tipon 0.00 0.00 0.00 0.00 Wari 0.01 0.17 0.01 0.17 Sites affected by the El Niño phenomenon Caral 0.00 0.00 Chavin 0.00 0.00 Huaca Arco Iris 0.00 0.00 Huaca Brujo 0.17 0.17 Huaca Sol y Luna 0.00 0.00 Narihuala 0.00 0.00 Nikan 0.00 0.00 Table 9 Synthetic control units Source: Own elaboration Predictors Kuelap One convariates More covariates Synthetic control A Synthetic control B Synthetic control A Synthetic control B Preintervention outcomes Visits in 2008 17,396 17,447 17,692 17,445 17,699 Visits in 2009 18,542 18,613 18,081 18,623 18,080 Visits in 2010 23,696 23,723 23,858 23,709 23,858 Visits in 2011 29,431 29,241 28,830 29,251 28,830 Visits in 2012 27,960 28,019 28,397 28,031 28,391 Visits in 2013 33,495 33,551 33,574 33,567 33,583 Visits in 2014 40,146 40,136 40,191 40,137 40,175 Controls Domestic visitors 76.52 85.5 88.3 85.7 88.3 Length of stay 1.14 1.39 1.42 Guests per accommodation 1380 1354 1255 Tourism development 1.34 2.23 2.09 Educational attainment 8.20 8.73 8.65 Regional temperature 14.58 17.1 17.3 Visits during treatment 2015 36,385 40,635 43,883 40,637 43,830 2016 56,010 45,827 49,238 45,873 49,172 2017 102,905 50,326 56,574 50,243 56,469 2018 110,068 50,818 56,468 50,795 56,351 Estimated effect 2017 52,579 46,331 52,662 46,436 2018 59,250 53,600 59,273 53,717 Average 2017–2018 55,914 49,966 55,967 50,077 p-value 0.048 0.071 0.048 0.071 As discussed in Sect. 3, when there exists uncertainty as to which covariates to include, then it is advisable to construct the synthetic control using the preintervention visits as the only predictors (Ferman et al. 2020; Kaul et al. 2022). In our case, the lack of reliable site-level data for potential predictors is the main limitation for not adding covariates in the baseline synthetic control. However, we estimate alternative synthetic controls in order to check if the results remain stable. First, we estimate a synthetic control adding as a covariate the number of domestic tourists as percentage of the total number of tourists (domestic visitors), which is the only covariate with information specific to each site. The second specification includes more covariates: average length of stay in accommodation facilities (length of stay), number of guests per accommodation facility (guests per accommodation), participation of tourism in GDP (tourism development), average level of education achieved by people above the age of 15 (educational attainment) and average annual temperature (regional temperature). We add covariates to both SCA and SCB. As seen in Table 9, the weights of the synthetic control (A) do not change when one or more covariates are added, as is formally shown by Kaul et al. (2022). This is also the case for SCB and the corresponding synthetic controls with one and more covariates. Also, the values of Kuelap’s covariates are very similar to those of the alternative synthetic controls, as displayed in Table 9. Finally, the estimated causal effects are very similar to those of the baseline SCA and SCB. Overall, the results show strong evidence that the investment package in tourism infrastructure has had a positive causal effect on the number of visitors to Kuelap. Conclusions This paper estimates the effect of public investment in tourism infrastructure on tourism demand for the Peruvian case. We analysed the case of the Kuelap Archaeological Complex, which has become more attractive and accessible after the construction of the first cable car system in Peru and the redevelopment of Jaen airport. For this purpose, we applied a comparative case study with a “synthetic control”, which was built using information from other similar archaeological sites in Peru. The results show that investment in tourism infrastructure generated an increase of approximately 100% in the number of visits to Kuelap, twice the initial forecast. Our findings provide evidence that promoting investment in infrastructure to facilitate access to archaeological sites with high tourism potential can be a useful tool to stimulate both tourism and by extension economic activity. Thus, investment in tourism infrastructure could contribute to overcoming the negative economic consequences of the Covid-19 pandemic. Choquequirao, located in the Cusco region and long considered the second Machu Picchu, is an archaeological site that has been attracting more tourists in recent years; however, the lack of accessibility to this place limits its potential for development and a significant increase in visitor numbers. An investment package similar to Kuelap’s could make it more accessible and attractive.9 Finally, this paper provides an example of how investment under a public–private partnership (PPP) scheme can be a useful tool to foster tourism. Appendix A: Descriptive statistics of the predictor variables Site Variable Mean SD Min. 25th p. 75th p. Max. Kuelap Pre-treatment visits 45,094.00 32,228.44 17,396.00 25,828.00 48,078.00 110,068.00 Length of stay 1.17 0.10 1.10 1.10 1.20 1.40 Domestic visits 78.43 4.67 69.00 76.18 81.75 83.28 Hotel demand 1365.69 111.87 1208.99 1286.64 1448.04 1534.82 Tourism development 1.37 0.05 1.29 1.35 1.41 1.46 Education 8.36 0.26 7.90 8.20 8.50 8.80 Regional temperature 14.88 0.53 14.00 14.75 15.18 15.60 Caral Pre-treatment visits 57,984.91 6335.22 45,095.00 55,650.50 61,534.00 66,425.00 Length of stay 1.35 0.05 1.30 1.30 1.40 1.40 Domestic visitors 93.17 2.10 90.49 91.11 95.04 96.00 Guests per accommodation 4637.93 712.35 3417.26 4344.45 5150.44 5392.01 Tourism development 4.57 0.19 4.28 4.40 4.74 4.76 Educational attainment 11.08 0.14 10.90 10.95 11.20 11.30 Regional temperature 19.51 0.71 18.70 19.00 20.03 20.80 Chavin Pre-treatment visits 69,929.73 16,738.07 46,156.00 58,453.00 79,672.00 96,642.00 Length of stay 1.25 0.05 1.20 1.20 1.30 1.30 Domestic visitors 87.77 3.50 82.58 84.96 90.71 91.85 Guests per accommodation 437.36 61.52 382.33 393.83 478.77 555.51 Tourism development 2.34 0.31 1.86 2.17 2.58 2.79 Educational attainment 9.46 0.19 9.10 9.40 9.55 9.70 Regional temperature 12.45 0.35 12.00 12.23 12.65 13.10 Choquequirao Pre-treatment visits 5768.82 1657.62 3334.00 4718.50 7341.50 8023.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 34.52 12.63 21.06 24.79 43.01 56.77 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Site Variable Mean SD Min. 25th p. 75th p. Max. Cumbemayo Pre-treatment visits 46,950.09 20,272.64 21,701.00 29,195.00 63,920.00 78,316.00 Length of stay 1.34 0.09 1.20 1.30 1.40 1.50 Domestic visitors 93.07 2.80 88.35 91.39 95.05 97.74 Guests per accommodation 1250.72 107.53 1096.11 1147.06 1337.54 1397.42 Tourism development 1.94 0.24 1.60 1.73 2.13 2.26 Educational attainment 8.16 0.17 7.80 8.15 8.30 8.30 Regional temperature 14.78 0.54 14.00 14.45 15.00 15.60 Huaca Pre-treatment visits 54,433.00 5674.30 45,833.00 50,224.00 58,497.50 63,405.00 Length of stay 1.25 0.05 1.20 1.20 1.30 1.30 Domestic visitors 78.04 1.57 75.06 76.94 78.92 80.62 Guests per accommodation 1780.69 174.56 1563.03 1662.68 1903.17 2127.01 Tourism development 2.28 0.16 2.07 2.13 2.44 2.48 Educational attainment 9.59 0.19 9.30 9.50 9.70 9.90 Regional temperature 20.55 0.89 19.30 19.93 21.15 22.20 Huaca Brujo Pre-treatment visits 42,607.73 13,056.07 16,474.00 37,579.00 48,212.50 67,262.00 Length of stay 1.25 0.05 1.20 1.20 1.30 1.30 Domestic visitors 82.83 3.05 78.28 81.18 84.53 87.75 Guests per accommodation 1780.69 174.56 1563.03 1662.68 1903.17 2127.01 Tourism development 2.28 0.16 2.07 2.13 2.44 2.48 Educational attainment 9.59 0.19 9.30 9.50 9.70 9.90 Regional temperature 20.55 0.89 19.30 19.93 21.15 22.20 Huaca Sol Pre-treatment visits 121,109.90 11,867.18 98,143.00 114,909.50 126,377.00 141,123.00 Length of stay 1.25 0.05 1.20 1.20 1.30 1.30 Domestic visitors 75.26 1.99 71.34 74.44 76.82 77.62 Guests per accommodation 1780.69 174.56 1563.03 1662.68 1903.17 2127.01 Tourism development 2.28 0.16 2.07 2.13 2.44 2.48 Educational attainment 9.59 0.19 9.30 9.50 9.70 9.90 Regional temperature 20.55 0.89 19.30 19.93 21.15 22.20 Site Variable Mean SD Min. 25th p. 75th p. Max. Intihuatana Pre-treatment visits 8099.91 4725.68 1129.00 4457.00 11,738.50 16,044.00 Length of stay 1.46 0.13 1.20 1.50 1.50 1.60 Domestic visitors 97.13 2.55 92.47 96.12 98.96 100.00 Guests per accommodation 1111.23 135.53 934.06 1024.77 1190.79 1343.56 Tourism development 1.26 0.04 1.20 1.23 1.28 1.32 Educational attainment 9.00 0.21 8.50 8.95 9.10 9.30 Regional temperature 18.36 0.38 18.00 18.03 18.70 19.00 Kotosh Pre-treatment visits 48,469.64 16,030.58 17,092.00 42,548.50 57,309.50 70,854.00 Length of stay 1.23 0.08 1.10 1.20 1.25 1.40 Domestic visitors 97.76 2.61 90.63 97.59 99.22 99.68 Guests per accommodation 1613.04 56.27 1531.91 1582.12 1636.85 1720.52 Tourism development 2.77 0.08 2.57 2.76 2.81 2.88 Educational attainment 8.44 0.22 8.10 8.30 8.60 8.70 Regional temperature 20.58 0.45 20.00 20.23 20.88 21.40 Machu Picchu Pre-treatment visits 1,026,919.00 300,337.60 583,480.00 774,216.00 1,277,352.00 1,491,840.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 26.87 2.82 22.79 24.44 28.28 31.29 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Moray Pre-treatment visits 186,340.60 173,026.30 39,957.00 63,487.00 309,383.00 517,909.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 36.94 4.35 29.85 34.11 39.64 44.06 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Narihuala Pre-treatment visits 23,746.09 5986.69 12,760.00 19,336.00 26,635.00 32,754.00 Length of stay 1.49 0.05 1.40 1.50 1.50 1.60 Site Variable Mean SD Min. 25th p. 75th p. Max. Domestic visitors 97.63 0.60 96.52 97.46 97.94 98.36 Guests per accommodation 1440.59 140.98 1189.95 1341.15 1546.53 1630.59 Tourism development 2.21 0.14 2.04 2.09 2.28 2.46 Educational attainment 9.26 0.10 9.10 9.20 9.30 9.40 Regional temperature 24.83 0.82 23.90 24.05 25.28 26.10 Nikan Pre-treatment visits 101,410.30 12,208.17 86,284.00 95,034.00 102,066.00 128,732.00 Length of stay 1.25 0.05 1.20 1.20 1.30 1.30 Domestic visitors 76.96 2.88 72.30 74.75 79.15 81.10 Guests per accommodation 1780.69 174.56 1563.03 1662.68 1903.17 2127.01 Tourism development 2.28 0.16 2.07 2.13 2.44 2.48 Educational attainment 9.59 0.19 9.30 9.50 9.70 9.90 Regional temperature 20.55 0.89 19.30 19.93 21.15 22.20 Otuzco Pre-treatment visits 59,680.91 29,702.54 26,361.00 37,409.00 87,188.00 103,872.00 Length of stay 1.34 0.09 1.20 1.30 1.40 1.50 Domestic visitors 95.58 2.16 92.22 94.19 97.42 98.19 Guests per accommodation 1250.72 107.53 1096.11 1147.06 1337.54 1397.42 Tourism development 1.94 0.24 1.60 1.73 2.13 2.26 Educational attainment 8.16 0.17 7.80 8.15 8.30 8.30 Regional temperature 14.78 0.54 14.00 14.45 15.00 15.60 Pikillaqta Pre-treatment visits 50,073.54 24,303.07 17,009.00 33,568.00 69,382.50 92,383.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 65.54 2.22 61.78 64.42 66.32 70.18 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Raqchi Pre-treatment visits 109,231.10 13,533.33 87,877.00 101,256.00 115,177.50 131,089.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 14.61 4.48 5.86 12.63 16.20 23.24 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Site Variable Mean SD Min. 25th p. 75th p. Max. Saywite Pre-treatment visits 2958.27 900.67 2131.00 2418.00 3046.50 5213.00 Length of stay 1.28 0.08 1.10 1.30 1.30 1.40 Domestic visitors 87.33 11.73 62.51 82.46 96.12 100.00 Guests per accommodation 1217.74 330.96 766.37 946.01 1446.66 1749.41 Tourism development 2.17 0.67 0.84 2.32 2.55 2.64 Educational attainment 9.02 0.24 8.70 8.80 9.20 9.40 Regional temperature 14.88 0.83 14.00 14.15 15.53 16.20 Sillustani Pre-treatment visits 81,472.46 13,132.82 51,068.00 80,149.50 90,344.00 95,008.00 Length of stay 1.29 0.07 1.20 1.25 1.30 1.40 Domestic visitors 29.11 8.37 13.23 25.17 34.59 40.01 Guests per accommodation 1438.97 80.31 1298.62 1402.60 1470.95 1612.51 Tourism development 2.11 0.09 1.96 2.06 2.17 2.23 Educational attainment 9.60 0.20 9.30 9.50 9.70 9.90 Regional temperature 10.43 0.65 9.00 10.23 10.75 11.50 Tipon Pre-treatment visits 64,434.64 33,410.48 24,497.00 40,522.50 98,256.50 119,598.00 Length of stay 1.63 0.07 1.50 1.60 1.70 1.70 Domestic visitors 65.92 5.48 53.24 64.58 67.19 76.50 Guests per accommodation 686.00 107.15 551.40 569.74 767.04 829.57 Tourism development 4.05 0.29 3.77 3.81 4.12 4.73 Educational attainment 9.55 0.20 9.20 9.40 9.70 9.80 Regional temperature 12.47 0.43 12.00 12.15 12.60 13.30 Wari Pre-treatment visits 42,216.00 23,683.14 16,105.00 25,125.50 58,922.00 81,033.00 Length of stay 1.46 0.13 1.20 1.50 1.50 1.60 Domestic visitors 94.21 3.03 90.08 92.01 96.84 98.96 Guests per accommodation 1111.23 135.53 934.06 1024.77 1190.79 1343.56 Tourism development 1.26 0.04 1.20 1.23 1.28 1.32 Educational attainment 9.00 0.21 8.50 8.95 9.10 9.30 Regional temperature 18.36 0.38 18.00 18.03 18.70 19.00 B: Archaeological sites Short name Full name Department Province District 1 Kulap Complejo Arqueológico de Kúelap Amazonas Luya Tingo 2 Revash Sitio Arqueológico Revash Amazonas Luya Santo Tomás 3 Karajia Sitio Arqueológico karagía Amazonas Luya Trita 4 Chavin Monumento Arqueológico Chavín de Huantar Áncash Huari Chavín De Huántar 5 Saywite Conjunto Arqueológico de Saywite Apúrimac Abancay Curahuasi 6 Intihuatana Centro Arqueológico Intihuatana Ayacucho Vilcashuaman Vischongo 7 Wari Complejo Arqueológico de Wari Ayacucho Huamanga Quinua 8 Otuzco Centro Arqueológico Ventanillas de Otuzco Cajamarca Cajamarca Los Baños Del Inca 9 Cumbemayo Monumento Arqueológico Cumbemayo Cajamarca Cajamarca Cajamarca 10 Machu Picchu Ciudad Inka de Machu Picchu Cusco Urubamba Machu Picchu 11 Moray Complejo Arqueológico de Moray Cusco Urubamba Maras 12 Tipón Complejo Arqueológico de Tipón Cusco Quispicanchi Oropesa 13 Choquequirao Parque Arqueológico de Choquequirao Cusco La Convención Vilcabamba 14 Pikillaqta Parque Arqueológico de Pikillaqta Cusco Quispicanchi Lucre 15 Raqchi Parque Arqueológico de Raqchi Cusco Canchis San Pedro Short name Full name Department Province District 16 Kotosh Complejo Arqueológico Kotosh Huánuco Huánuco Huánuco 17 Huaca Arco Iris Complejo Arqueológico Huaca Arco Iris La Libertad Trujillo La Esperanza 18 Huaca Sol Complejo Arqueológico Huaca del Sol y de la Luna La Libertad Trujillo Moche 19 Huaca Brujo Complejo Arqueológico Huaca el Brujo La Libertad Ascope Ascope 20 Nikan Palacio Nikán casa del centro La Libertad Trujillo Huanchaco 21 Caral Ciudad Sagrada de Caral Lima Barranca Supe 22 Narihualá Zona Arqueológica y Museo de Sitio Narihualá Piura Piura Catacaos 23 Sillustani Complejo Arqueológico de Sillustani Puno Puno Puno Source: Ministry of Foreign Trade and Tourism (2019) C: Variables Institution Source Indicator Variable Measure 1 MINCETUR Monthly Survey of lodging establishments Average length of stay in lodging establishments Length of stay Average number of days 2 MINCETUR Tourism Statistical Reports Number of domestic tourists (% of total number of tourists) Domestic visitors Percentage Institution Source Indicator Variable Measure 3 MINCETUR Monthly Survey of lodging establishments Guests arrivals per accommodation facility Guests per accommodation Number of guests per accommodation 4 INEI INEI Gross value added at 2007 prices (% of GDP) Tourism development Percentage 5 INEI National Household Survey Average years of schooling attained by the population over age 15 Educational attainment Years 6 SENAMHI SENAMHI Average annual temperature Regional temperature Degrees Celsius 7 MINCETUR Tourism Statistical Reports Total visits (2008 - 2014) pre-treatment number of visitors Source: Ministry of Foreign Trade and Tourism (2019), National Institute of Statistics and Informatics (2019) 1 See Brida et al. (2013) for a summary. 2 This tourist circuit also includes Cajamarca, La Libertad, Lambayeque and San Martin. 3 The reconstruction of this airport has not only benefited Kuelap but also other tourist destinations in the north-east of Peru. 4 In September 2019, the Ministry of Transport and Communications announced that it intends to update the “Master Plan for Development of Chachapoyas Airport” to expand the passenger terminal of the Chachapoyas airport, which could only receive small planes. On the other hand, the airport of Jaen receives planes with a capacity of 70 passengers or more and is part of the concession process of the “Third Group of Airports”. 5 If all units were exposed to the event, the level of exposure is required to be very different between the treated and the control group. 6 Our analysis showed that none of the other sites in Peru were a close enough match to use as a control. 7 More details can be found in “Table B of the Appendix”. 8 Moray and Nikan are excluded as they have the largest predictive errors in the placebo studies. 9 Non-official estimates indicate that with a system of cable cars providing access to Choquequirao from the nearest road could reduce travel times from a 2-day walk to less than 30 min. This paper is based on Lucely Puscan and Rosario Sabrera’s dissertation (Puscan and Sabrera 2019). Lahura et al. (2019) and Lahura and Sabrera (2020) are working paper versions of this paper. The authors are especially grateful to the anonymous referee, whose recommendations have allowed them to greatly improve the paper. The authors are also indebted to Magali Silva and Tanja Sturm for their valuable comments and suggestions. They also thank Iván Aldave, Iván Cosavalente, Marco Vega, and participants at the XXXVII Meeting of BCRP Economists (Lima, Perú, October 29th–30th, 2019). The opinions expressed in this research correspond to the authors and do not necessarily reflect the position of the institutions to which they are affiliated. 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==== Front Cybern Syst Anal Cybern Syst Anal Cybernetics and Systems Analysis 1060-0396 1573-8337 Springer US New York 504 10.1007/s10559-022-00504-8 Article Application of the Robust Methods for Estimation of Distribution Parameters with a Priori Constraints on Parameters in Economics and Engineering* Atoyev K. L. [email protected] Knopov P. S. [email protected] grid.467128.e 0000 0001 1090 4888 V. M. Glushkov Institute of Cybernetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine 1 12 2022 18 25 4 2022 © Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The approach to estimating distribution parameters with a priori constraints has been developed. The Lorentz six-sector model has been constructed. Based on this model, the authors have studied the relationships between food, energy, water resources, supply systems, epidemic, and social situations. They also have investigated the impact of the successive transitions of interconnected sectors to the deterministic chaos regime on the operation mode of the whole system. The method of risk assessment for food security and risk management has been elaborated. Keywords Lorentz model mathematical modeling economic development model optimal control deterministic chaos stochastic models ==== Body pmc *The work was partially supported by National Research Foundation of Ukraine (Grant # 2020.02/0121). Translated from Kibernetyka ta Systemnyi Analiz, No. 5, September–October, 2022, pp. 48–56. ==== Refs References 1. Sergienko IV Yanenko VM Atoev KL Optimal control of the immune response synchronizing the various regulatory compartments of the immune system. II. Identification of model parameters and missing data recovery Cybern. Syst. Analysis 1997 33 1 131 144 10.1007/BF02665951 2. K. L. Atoyev, L. B. Vovk, and S. P. Shpyga, “Studying the interconnection of food, energy and water resources using the three-sectoral Lorentz model,” The Intern. Sci. Techn. J. “Problems of Control and Informatics,” No. 3, 141–152 (2021). 10.34229/1028-0979-2021-3-12. 3. Atoyev K Knopov P Pepeliaev V Kisala P Romaniuk R Kalimoldayev M Wojcik W Sikora J The mathematical problems of complex systems investigation under uncertainties Recent Advances in Information Technology 2017 London CRC Press Taylor Francis Group 135 171 4. Kaplan JL Yorke JA Preturbulence: A regime observed in a fluid flow model of Lorenz Commun. Math. Phys. 1979 67 93 108 10.1007/BF01221359 5. The Global Risks Report 2021 (16th ed.), World Economic Forum (2021). URL: https://www3.weforum.org/docs/WEF_The_Global_Risks_Report_2021.pdf. 6. Magnitskii NA Sidorov SV New Methods for Chaotic Dynamics 2006 Singapore World Scientific 7. Kuramoto Y Chemical Oscillations, Waves, and Turbulence, Springer Series in Synergetics 1984 Berlin–Heidelberg–New York–Tokyo Springer-Verlag 8. Atoyev KL Golodnikov AN Gorbachuk VM Ermolieva TY Ermoliev YM Kiriljuk VS Knopov PS Pepeljaeva TV Zagorodny AG Ermoliev YM Bogdanov VL Ermolieva TY Food, energy and water nexus: Methodology of modeling and risk management Integrated Modeling and Management of FEW Nexus for Sustainable Development 2020 Kyiv Akademperiodyka 250 302 9. Global Financial Stability Report — COVID-19, Crypto, and Climate: Navigating Challenging Transitions, International Monetary Fund, October (2021). URL: https://www.imf.org/en/Publications/GFSR/Issues/2021/10/12/global-financial-stability-report-october-2021.	36471722	PMC9713179	NO-CC CODE	2022-12-08 23:16:02	no	Cybern Syst Anal. 2022 Dec 1; 58(5):713-720	utf-8	Cybern Syst Anal	2,022	10.1007/s10559-022-00504-8	oa_other
==== Front Z Gesundh Wiss Z Gesundh Wiss Zeitschrift Fur Gesundheitswissenschaften 2198-1833 1613-2238 Springer Berlin Heidelberg Berlin/Heidelberg 1786 10.1007/s10389-022-01786-0 Original Article COVID-19 policy analysis for 10 European countries http://orcid.org/0000-0002-1826-742X Takefuji Yoshiyasu [email protected] grid.411867.d 0000 0001 0356 8417 Faculty of Data Science, Musashino University, 3-3-3 Ariake Koto-Ku, Tokyo, 135-8181 Japan 1 12 2022 18 16 10 2022 20 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Aim The goal of this paper is to analyze the COVID-19 policies of 10 European countries, including Sweden, Finland, Norway, Italy, France, Germany, Poland, Belgium, the Netherlands, and Hungary, with a time-series policy analysis tool. Subject and methods The results of the COVID-19 policy analysis are based on a single time-series indicator, or daily population mortality rate: the number of COVID-19 daily cumulative deaths divided by the population in millions. The lower the score, the better the policy. Although many experts believe that the COVID-19 policy outcome analysis is premature, time series analysis is an excellent analysis that can provide information on the progress and transition of policy outcomes. In other words, the proposed time series analysis tool allows policymakers to identify and quantify when mistakes were made during the on-going COVID-19 pandemic. Results The COVID-19 policy analysis discovered many useful facts. Sweden failed due to the herd immunity approach. Hungary made a fundamental mistake in COVID-19 tactics. Countries such as Sweden, Hungary, Belgium, and Poland showed time-series changes that differed from the others. Conclusion Public health interventions can play a key role in mitigating the COVID-19 pandemic. The proposed policy analysis tool, hiscovid demonstrated the effectiveness of the time-series score behavior for discovering when policymakers made mistakes. Keywords COVID-19 policy Snapshot score Time-series score Policy analysis tool Hiscovid ==== Body pmcIntroduction COVID-19 policy is a matter of life and death and affects well-being. The more deaths there are, the more unhappy people become. The fewer the deaths, the fewer people are unhappy. Therefore, the impact from the well-being perspective plays an important role in evaluating COVID-19 policies. In other words, a single metric score is used throughout this study: dividing the number of COVID-19 deaths by the population in million, which is called the population mortality rate (Bauer et al. 2021; New York State 2022). A population mortality rate is the number of deaths due to a disease divided by the total population. In other words, the mortality rate can be computed by dividing the score by 1,000,000. The time-series mortality rate is equivalent to the single metric used in this paper. Time-series population mortality rates or time-series scores, rather than snapshot mortality rates, show the progress of the community and the outcome course of action on COVID-19. Thus, the calculated time-series scores can identify when policymakers made mistakes or when there was a change in community behavior and quantify those mistakes. In other words, snapshot mortality rates cannot identify when policymakers made mistakes because there is no progress to compare. The introduced time-series policy analysis in this paper is called, hiscovid (Takefuji 2022). The hiscovid tool is a Python Package Index application. Hiscovid allows policymakers to run on Windows, MacOS, and Linux operating systems as long as Python is installed on the system. The following command can install hiscovid after installing Python with the pip command. In the hiscovid analysis tool, the number of daily cumulative deaths by country is scraped over the Internet, and it is divided by the population in millions over time. In other words, score or daily population mortality rate by country is calculated by the number of daily cumulative deaths divided by the population in millions over time. The contribution of this paper is to introduce the time-series policy analysis tool, hiscovid, to be able to identify when policymakers made mistakes. This paper will examine the hiscovid results of 10 countries, including Sweden, Finland, Norway, Italy, France, Germany, Poland, Belgium, the Netherlands, and Hungary. They will be compared with New Zealand on time-series scores. The number of COVID-19 daily cumulative deaths monotonically increases so that the calculated scores cannot be corrected forever. The higher the score, the more COVID-19 deaths, which makes people unhappy. If policies to control COVID-19 were effective, there would be no COVID-19 deaths, and people would be happier. Methods The proposed time-series policy analysis, hiscovid allows policymakers to identify when they made mistakes or behavior changes in communities. The graphs generated have dates on the horizontal axis and scores or daily population mortality rates on the vertical axis. The calculated time series scores indicate the effectiveness of individual policies and their outcomes. The lower the score, the better the policy. The higher the score, the worse the policy. Instead of the snapshot policy analysis, time-series analysis plays a key role in identification of changes over time. A literature review was briefly conducted to explain the result of hiscovid. Scoring is based on the daily population mortality rate: the number of COVID-19 daily cumulative deaths divided by the population in millions. The number of COVID-19 daily cumulative deaths by country is scraped over the Internet. The COVID-19 policy analysis will discover many useful facts. Sweden failed due to the herd immunity approach. Hungary made a fundamental mistake in COVID-19 tactics. Countries such as Sweden, Hungary, Belgium, and Poland showed time-series changes that differed from the others. Results Figure 1 shows three graphs of Sweden, Finland, and Norway of time-series scores, respectively. Figure 1 can be generated by the following command.Fig. 1 Results of hiscovid for Sweden, Finland, and Norway as of Oct. 14, 2022 Sweden Sweden is the only country that implemented the concept of herd immunity (Ludvigsson et al. 2021). However, in Sweden, the herd immunity approach failed due to the large number of COVID-19 elderly death. Then, the COVID-19 policy in Sweden was updated after the herd immunity failure. In the debate on herd immunity, the most important policy indicator, the number of COVID-19 deaths was presented (Takefuji 2021). The more deaths, the worse the COVID-19 policy. The fewer deaths, the better the policy. Therefore, a score is introduced and normalized by the population in millions for scoring individual COVID-19 policies. Figure 1 shows that Sweden made three mistakes in March 2020, November 2020, and January 2022. This result indicates that the Swedish policy is leaky. A leaky policy means that a flat graph is not observed. A flat graph indicates that COVID-19 is successfully suppressed. Hungary To observe the progress of four countries, namely Italy, Germany, France, and Hungary, the following command can generate the progress scores results of four countries, as shown in Fig. 2.Fig. 2 Results of hiscovid for Italy, Germany, France, and Hungary There is a significant difference on scores between Hungary and Italy, Germany, and France. There are two reasons the worst outcome of COVID-19 occurred in Hungary (Karáth 2020). The first reason is due to the tactics used in Hungary. The tactics in Hungary shunned increased testing and installed lockdown limits, allowing people to go to work, go to the hairdresser, shop at the market, and attend funerals and weddings (Karáth 2020). The second reason is due to “the military dictatorship in Hungary” cited by (Karáth 2020), not by the author. The government appointed more than 100 hospital directors, who did not require medical or hospital management experience, to provide hands-on management, in order to monitor and control the use of medical resources during the pandemic. The hospital directors were not trusted, and thus soldiers and police officers were assigned to them. Figure 2 shows that Hungary made two big mistakes in October 2020 and November 2021. Italy Italy has been hit by an unprecedented crisis and was the first European country to be heavily swept by the COVID-19 pandemic (Bosa et al. 2022). The SARS-CoV-2 outbreak and related COVID-19 pandemic are the worst public health challenges Italy has endured in recent years. To observe distinguished mistakes in Italy, it was compared with the best policy of New Zealand. Figure 3 shows that Italy made two big mistakes in March 2020 and November 2020 and a medium mistake in January 2022.Fig. 3 Result of hiscovid for New Zealand and Italy Germany Germany was one of the first countries to initiate so-called social distancing measures (Schartau and Kirby 2020). Germany is said to be one of the most successful countries in the world in responding to COVID-19 (Okina, et al. 2020). Is this true? From November 2020, the score of Germany has been increasing. In other words, the snapshot policy analysis is not sufficient to state the robust analysis. Two time-series scores of New Zealand and Germany are compared, as shown in Fig. 4. Figure 4 shows that New Zealand successfully suppressed the COVID-19 pandemic until March 2022. New Zealand lifted border regulations for economics from March 2022 (New Zealand government 2022). The COVID-19 policy in Germany was successful until November 2020, with a flat graph in Fig. 4. The flat graph indicates suppressing the COVID-19 pandemic successfully. The diagonal graph with steeper slope shows that the policy is not successful because of the outcome of their policy. Figure 4 shows Germany made three mistakes in March 2020, November 2020, and November 2021.Fig. 4 Results of hiscovid for New Zealand and Germany France Or et al. summarized France’s response to COVID-19 (Or et al. 2022). France was one of the European countries hardest hit by the Covid-19 pandemic. The pandemic brought to light structural weaknesses in the health care system, including governance and decision-making processes, but also triggered changes to improve its resilience. In conclusion, France needs to strengthen the capacity of its health care system and improve cooperation between central and local level actors through participatory decision-making that takes into account the diversity of realities and needs at the local level. Figure 5 shows that France made three mistakes in March 2020, October 2020, and November 2021.Fig. 5 Results of hiscovid for New Zealand and France Poland The progress of the Belgium, Netherlands, and Poland scores, respectively, can be observed in Fig. 6. In the beginning of the COVID-19 pandemic, Poland was a successful country at suppressing COVID-19, but after October 2020, the score in Poland dramatically increased. The flat graph shows the success of the COVID-19 policy, while the diagonal graph shows the failure of the COVID-19 policy. The steeper the slope of the diagonal graph, the worse the policy.Fig. 6 Results of hiscovid for Belgium, the Netherlands, and Poland Polish covid advisers quit over the lack of science influence on policy (Dyer 2022). The Polish government had not introduced a shutdown because Poles have a “genetic resistance to regulations” developed over centuries. Figure 6 shows that Poland made two mistakes in October 2020 and November 2021. Belgium Policymakers failed to act adequately due to the complex political situation in Belgium without a strong centralized command from the government (Luyten and Schokkaert 2022). There are two reasons (Luyten and Schokkaert 2022). In the first wave, care home protection was not given sufficient priority. The second wave was larger than necessary because restrictive measures were not implemented in a timely manner. The difference between New Zealand and Belgium can be observed in Fig. 7. Belgium made two big mistakes in the beginning of March 2020 and in November 2021. The score of Belgium shows the COVID-19 policy is leaky and did not suppress the COVID-19 pandemic. However, during the period of May 2020 to October 2020, there is a flat graph in Fig. 7. Figure 7 shows that Belgium made two big mistakes in March 2020 and October 2020, and a medium mistake in October 2021.Fig. 7 Results of hiscovid for New Zealand and Belgium The Netherlands Hoekman et al. reported the Dutch COVID-19 approach (Hoekman et al. 2020). They concluded that compared to other countries, the Netherlands was slow and relatively mild in its response to the COVID-19 pandemic. This was consistent with the initial policy choice to consider COVID-19 as a severe influenza and to aim for herd immunity. The Dutch quickly abandoned containment in favor of mitigation with a focus on herd immunity. Contrary to the national policy, the three most northern provinces continued their containment strategy and were able to prevent local transmission of the virus. Figure 8 compares New Zealand and the Netherlands.Fig. 8 Results of hiscovid for New Zealand and the Netherlands Figure 8 shows that the Netherlands made three mistakes in March 2020, October 2020, and November 2021. Discussion A time-series policy analysis of 10 countries, including Sweden, Finland, Norway, Italy, France, Germany, Poland, Belgium, the Netherlands, and Hungary, was conducted using the hiscovid tool. The hiscovid tool is based on a single metric scoring: dividing the number of COVID-19 deaths by the population in millions. As a policy analysis tool, hiscovid analyzes time-series data to easily visualize changes in scores. In other words, hiscovid allows policymakers to identify when they made mistakes. If the COVID-19 policy is successful, a flat graph can be observed. If the policy is not successful, a diagonal graph can be seen. The steeper the slope of the diagonal graph, the worse the policy. The hiscovid results of 10 countries can be summarized as follows.Sweden made three mistakes in March 2020, November 2020, and January 2022. This result indicates that the Swedish policy is leaky. A leaky policy means that a flat graph is not observed. A flat graph indicates that COVID-19 is successfully suppressed. Hungary made two big mistakes in October 2020 and November 2021. Italy made two big mistakes in March 2020 and November 2020, and a medium mistake in January 2022. Germany made three mistakes in March 2020, November 2020, and November 2021. France made three mistakes in March 2020, October 2020, and November 2021. Poland made two mistakes in October 2020 and November 2021. Belgium made two big mistakes in March 2020 and October 2020, and a medium mistake in October 2021. The Netherlands made three mistakes in March 2020, October 2020, and November 2021. Sweden, Hungary, Italy, Germany, France, Poland, Belgium, and the Netherlands should compare their policy outcomes with the policy outcome of New Zealand. A literature review of countries’ responses to COVID-19 found that results were ambiguous and individual policies could not be quantified. Conclusion This paper shows how to quantify and score individual COVID-19 policy outcomes. The proposed time-series policy analysis tool, hiscovid, based on the population mortality rate – the number of COVID-19 daily cumulative deaths divided by the population in millions – can play a key role in mitigating the COVID-19 pandemic by observing the outcomes of policies to correct COVID-19 policies in the future. Authors’ contributions YT completed this research, wrote the program and the manuscript. Data availability Data is available at the following site: https://covid.ourworldindata.org/data/owid-covid-data.csv. Declarations Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Conflicts of interest The author has no conflict of interest. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Bauer P Brugger J König F (2021) An international comparison of age and sex dependency of COVID-19 deaths in 2020: a descriptive analysis Sci Rep 2021 11 19143 10.1038/s41598-021-97711-8 34580322 Bosa I Castelli A Castelli M Ciani O Compagni A Galizzi MM Garofano M Ghislandi S Giannoni M Marini G Vainieri M Response to COVID-19: was Italy (un)prepared? Health Econ Policy Law 2022 17 1 1 13 10.1017/S1744133121000141 33663622 Dyer O Covid-19: Poland’s medical council sees mass resignations over government inaction on pandemic BMJ (Clin Res Ed) 2022 376 o137 10.1136/bmj.o137 Hoekman LM Smits M Koolman X The Dutch COVID-19 approach: regional differences in a small country Health Policy Technol 2020 9 4 613 622 10.1016/j.hlpt.2020.08.008 32874861 Karáth K Covid-19: Hungary’s pandemic response may have been worse than the virus BMJ (Clin Res Ed) 2020 371 m4153 10.1136/bmj.m4153 Ludvigsson JF Engerström L Nordenhäll C Larsson E Open schools, Covid-19, and child and teacher morbidity in Sweden N Engl J Med 2021 384 7 669 671 10.1056/NEJMc2026670 33406327 Luyten J Schokkaert E Belgium’s response to the COVID-19 pandemic Health Econ Policy Law 2022 17 1 37 47 10.1017/S1744133121000232 34219632 New York State (2022) Basic statistics: about incidence, prevalence, morbidity, and mortality - statistics teaching tools. https://www.health.ny.gov/diseases/chronic/basicstat.htm New Zealand government (2022) Border to reopen in stages from 27 February. https://www.beehive.govt.nz/release/border-reopen-stages-27-february Okina Y et al (2020) What can we learn from Germany’s response to COVID-19?. NIRA opinion paper No.54. Oct. 2020. https://www.nira.or.jp/paper/e_opinion54.pdf Or Z Gandré C Durand Zaleski I Steffen M France’s response to the Covid-19 pandemic: between a rock and a hard place Health Econ Policy Law 2022 17 1 14 26 10.1017/S1744133121000165 33662232 Schartau P Kirby M Male mortality and the German response: lessons from COVID-19 Trends Urol Men’s Health 2020 11 3 26 28 10.1002/tre.752 Takefuji Y Open schools, Covid-19, and child and teacher morbidity in Sweden N Engl J Med 2021 384 17 e66 10.1056/NEJMc2101280 33646647 Takefuji Y (2022) Sustainable policy: don’t get infected and don’t infect others. J Hazard Mater Adv 8. 10.1016/j.hazadv.2022.100165	36471810	PMC9713180	NO-CC CODE	2022-12-02 23:22:56	no	Z Gesundh Wiss. 2022 Dec 1;:1-8	utf-8	Z Gesundh Wiss	2,022	10.1007/s10389-022-01786-0	oa_other
==== Front Drugs Drugs Drugs 0012-6667 1179-1950 Springer International Publishing Cham 1811 10.1007/s40265-022-01811-2 Letter to the Editor Comment on: “Global Consumption Trend of Antifungal Agents in Humans from 2008 to 2018: Data from 65 Middle‐ and High‐Income Countries” http://orcid.org/0000-0001-5626-2251 Denning David W. [email protected] [email protected] 12 1 grid.5379.8 0000000121662407 Manchester Fungal Infection Group, The University of Manchester, Manchester, UK 2 Global Action For Fungal Infections, Geneva, Switzerland 1 12 2022 13 14 11 2022 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcDear Editors, I read with interest the article by Pathadka et al. [1], and found the increased use of oral itraconazole in 27 middle-income countries intriguing. I was privileged to treat some of the first patients in the world with this new antifungal agent from 1987 onwards, contribute to the studies that led to its licensure in 1991 [2], and promulgate its use for non-approved indications such as allergic bronchopulmonary aspergillosis (ABPA). Our team at Global Action For Fungal Infections (GAFFI) successfully submitted itraconazole for inclusion on the World Health Organization (WHO)’s Essential Medicine List (EML) in 2017 [3, 4]. The authors show a big uptake in usage in 2015/6 preceding the WHO’s listing in 2017, and my speculations about the reasons for this is are as follows. Chronic pulmonary aspergillosis (CPA) requires long-term, usually oral, antifungal therapy in most patients. The European Society of Clinical Microbiology and Infectious Diseases together with the European Respiratory Society developed guidelines for the management of CPA that were published in early 2016 [5]. In August 2014, I presented these draft guidelines to the pulmonary society in China (with a large audience). Subsequently we estimated that there were ~ 488,000 CPA patients in China [6]. Locally manufactured Aspergillus antigen and antibody tests were then available in China. Do the authors know if the increase seen in itraconazole was particularly marked in China? Itraconazole is also useful for ABPA and what is now colloquially called ‘fungal asthma’. We published a slightly under-powered randomised, placebo-controlled study of itraconazole for severe asthma with fungal sensitisation showing significant benefit in quality of life in 2009 [7]. Other randomised studies of acute stage ABPA itraconazole versus corticosteroids from India [8] and all severe asthma also comparing itraconazole with (lower dose) corticosteroid from Iran [9] probably came too late to influence the consumption of itraconazole that the authors documented to 2018, and Iran is not included amongst the 38 middle-income countries. In low- and low-middle income countries, itraconazole remains a viable alternative to oral corticosteroid for severe asthma and ABPA in those who cannot afford biologics. Both acute but also recurrent vulvovaginal candidiasis (RVVC) are very frequent, with an estimated 138 million women suffering from the latter globally [10]. RVVC responds to long-term antifungal suppression with fluconazole, but replacement of Candida albicans with C. glabrata and fluconazole resistance in C. albicans reduce its effectiveness. It is likely itraconazole has been more frequently used for this indication. There has been increasing awareness of disseminated histoplasmosis in AIDS, thanks to the pioneering work of Mathieu Nacher, Antoine Adenis and colleagues in French Guiana [11]. Itraconazole is an important step-down and maintenance therapy for histoplasmosis, and some increased usage may have followed awareness and improved diagnostic capacity, with reduced deaths. The discovery of large numbers of such cases in Africa [12–15] should substantially increase usage further, although only three North African countries and South Africa were included in the survey. A long-term program of estimating the incidence and prevalence of serious fungal diseases (including CPA, ABPA, fungal asthma, RVVC and histoplasmosis) has now encompassed over 80 countries [16]. Engagement of national experts in these countries has alerted them to the relatively high prevalence of these conditions, and this may have contributed to local awareness. The first cases of terbinafine-resistant dermatophytosis (ringworm) were reported in India in 2016 [17]. Itraconazole for 3 weeks at a dose of 400 mg daily (more usually used for skin fungal infections) is about 75% effective in treating these inflammatory and disfiguring lesions. Do the authors know if there was a marked increase in consumption in India in 2017 and 2018 in response to this ongoing epidemic? The authors reference GAFFI’s 2016 summary of antifungal drug availability across the world [18]. This has been regularly updated since and fortunately many more countries do now have itraconazole approved for use (Fig. 1), but not Bolivia, Morocco, Senegal, The Gambia, Mauritania, Benin, Niger, Democratic Republic of Congo, Ethiopia, Ukraine Kyrgyzstan and Tajikistan [19]. However, the price of itraconazole remains unaffordable for many, and in most low- and middle-income countries it is not included with universal health coverage or government-funded medications.Fig. 1 Countries in which itraconazole is licenced for use 2021/22. Source: https://gaffi.org/antifungal-drug-maps/. Dark red = approved for use. Pink = not approved for use. Grey = no data available. Reproduced with permission The authors’ survey was until 2018. The echinocandin antifungal agents were added to the WHO’s EML in 2021 [20]. It will be of interest to observe whether this has an appreciable impact on usage over the coming years, especially in middle-income countries. Sincerely David W. Denning Professor of Infectious Diseases in Global Health, Manchester Fungal Infection Group, The University of Manchester, UK Chief Executive, Global Action for Fungal Infections, Geneva, Switzerland Declarations Funding No funding to declare. Conflicts of interest None pertain to this letter. Dr Denning and family hold Founder shares in F2G Ltd, a University of Manchester spin-out antifungal discovery company, and share options in TFF Pharma. He acts or has recently acted as a consultant to Pulmatrix, Pulmocide, Biosergen, TFF Pharmaceuticals, Pfizer, Omega, Novacyt and Cipla. He sat on the DSMB for a SARS CoV2 vaccine trial. In the last 3 years, he has been paid for talks on behalf of Hikma, Gilead, BioRad, Basilea, Mylan and Pfizer. He is a longstanding member of the Infectious Disease Society of America Aspergillosis Guidelines group, the European Society for Clinical Microbiology and Infectious Diseases Aspergillosis Guidelines group and recently joined the One World Guideline for Aspergillosis. Author contributions Not applicable. ==== Refs References 1. Pathadka S Yan VKC Neoh CF Global consumption trend of antifungal agents in humans from 2008 to 2018: data from 65 middle- and high-income countries Drugs 2022 82 1193 1205 10.1007/s40265-022-01751-x 35960433 2. Denning DW Lee JY Hostetler JS Pappas P NIAID Mycoses Study Group multicenter trial of oral itraconazole therapy of invasive aspergillosis Am J Med 1994 97 135 144 10.1016/0002-9343(94)90023-X 8059779 3. https://gaffi.org/itraconazole-voriconazole-and-natamycin-5-ophthalmic-preparation-proposed-as-essential-antifungal-medicines/. Accessed 19 Sept 2022. 4. https://gaffi.org/ruling-by-world-health-organisation-delights-doctors/. Accessed 19 Sept 2022. 5. Denning DW Cadranel J Beigelman-Aubry C Chronic pulmonary aspergillosis—rationale and clinical guidelines for diagnosis and management Eur Resp J 2016 47 45 68 10.1183/13993003.00583-2015 6. Zhou LH Li RY Denning DW Zhu LP Risk-based estimate of human fungal disease burden, China: old pathogen, new host, and new patterns Emerg Infect Dis 2020 26 2137 2147 10.3201/eid2609.200016 32818410 7. Denning DW O’Driscoll BR Powell G Randomized controlled trial of oral antifungal treatment for severe asthma with fungal sensitisation (SAFS), the FAST study Am J Resp Crit Care Med 2009 179 11 18 10.1164/rccm.200805-737OC 18948425 8. Agarwal R Dhooria S Singh Sehgal I A randomized trial of itraconazole vs prednisolone in acute-stage allergic bronchopulmonary aspergillosis complicating asthma Chest 2018 153 3 656 664 10.1016/j.chest.2018.01.005 29331473 9. Mirsadraee M Dehghan S Ghaffari S Mirsadraee N Long-term effect of antifungal therapy for the treatment of severe resistant asthma: an active comparator clinical trial Curr Med Mycol. 2019 5 4 1 7 32104737 10. Denning DW Kneale M Sobel JD Rautemaa-Richardson R Global burden of recurrent vulvovaginal candidiasis Lancet Infect Dis 2018 18 e339 e347 10.1016/S1473-3099(18)30103-8 30078662 11. Adenis A Nacher M Hanf M HIV-associated histoplasmosis early mortality and incidence trends: from neglect to priority PLoS Negl Trop Dis 2014 8 8 e3100 10.1371/journal.pntd.0003100 25144374 12. Mandengue CE Ngandjio A Atangana PJ Histoplasmosis in HIV-infected persons, Yaoundé, Cameroon Emerg Infect Dis 2015 21 11 2094 2096 10.3201/eid2111.150278 26488076 13. Oladele R Ayanlowo OO Richardson MD Denning DW Histoplasmosis in Africa: an emerging or a neglected disease? PLoS Negl Trop Dis 2018 12 e0006046 10.1371/journal.pntd.0006046 29346384 14. Ocansey BK Otoo B Asamoah I Cryptococcal and histoplasma antigen screening among people with HIV in Ghana and comparative analysis of OIDx histoplasma lateral flow assay and IMMY histoplasma enzyme immunoassay Open Forum Infect Dis 2022 9 7 ofac277 10.1093/ofid/ofac277 35854987 15. Oladele RO, Osaigbovo II, Akanmu AS, Adekanmbi O, Ekeng B, Yahaya M, Alex-Wele M, Okolo M, Ayanbeku T, Unigwe U, Akase I, Dan-Jumbo A, Israelski D, Denning DW, Pasqualotto AC, Chiller T. Ascertaining the current prevalence of Histoplasmosis in Nigeria’s Advanced HIV disease population. Emerg Infect Dis. 2022;28:2261–9. 16. https://gaffi.org/media/country-fungal-disease-burdens/. Accessed 19 Sept 2022. 17. Majid I Sheikh G Kanth F Hakak R Relapse after oral terbinafine therapy in dermatophytosis: a clinical and mycological study Indian J Dermatol 2016 61 5 529 533 10.4103/0019-5154.190120 27688443 18. Kneale M Bartholomew JS Davies E Denning DW Global access to antifungal therapy and its variable cost J Antimicrob Chemother 2016 71 3599 3606 10.1093/jac/dkw325 27516477 19. https://gaffi.org/antifungal-drug-maps/. Accessed 19 Sept 2022. 20. https://gaffi.org/antifungal-echinocandins-added-to-the-whos-essential-medicines-list/. Accessed 19 Sept 2022.	0	PMC9713181	NO-CC CODE	2022-12-11 23:16:18	no	Drugs. 2022 Dec 1; 82(18):1751-1753	utf-8	Drugs	2,022	10.1007/s40265-022-01811-2	oa_other
==== Front Drugs Drugs Drugs 0012-6667 1179-1950 Springer International Publishing Cham 1811 10.1007/s40265-022-01811-2 Letter to the Editor Comment on: “Global Consumption Trend of Antifungal Agents in Humans from 2008 to 2018: Data from 65 Middle‐ and High‐Income Countries” http://orcid.org/0000-0001-5626-2251 Denning David W. [email protected] [email protected] 12 1 grid.5379.8 0000000121662407 Manchester Fungal Infection Group, The University of Manchester, Manchester, UK 2 Global Action For Fungal Infections, Geneva, Switzerland 1 12 2022 13 14 11 2022 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcDear Editors, I read with interest the article by Pathadka et al. [1], and found the increased use of oral itraconazole in 27 middle-income countries intriguing. I was privileged to treat some of the first patients in the world with this new antifungal agent from 1987 onwards, contribute to the studies that led to its licensure in 1991 [2], and promulgate its use for non-approved indications such as allergic bronchopulmonary aspergillosis (ABPA). Our team at Global Action For Fungal Infections (GAFFI) successfully submitted itraconazole for inclusion on the World Health Organization (WHO)’s Essential Medicine List (EML) in 2017 [3, 4]. The authors show a big uptake in usage in 2015/6 preceding the WHO’s listing in 2017, and my speculations about the reasons for this is are as follows. Chronic pulmonary aspergillosis (CPA) requires long-term, usually oral, antifungal therapy in most patients. The European Society of Clinical Microbiology and Infectious Diseases together with the European Respiratory Society developed guidelines for the management of CPA that were published in early 2016 [5]. In August 2014, I presented these draft guidelines to the pulmonary society in China (with a large audience). Subsequently we estimated that there were ~ 488,000 CPA patients in China [6]. Locally manufactured Aspergillus antigen and antibody tests were then available in China. Do the authors know if the increase seen in itraconazole was particularly marked in China? Itraconazole is also useful for ABPA and what is now colloquially called ‘fungal asthma’. We published a slightly under-powered randomised, placebo-controlled study of itraconazole for severe asthma with fungal sensitisation showing significant benefit in quality of life in 2009 [7]. Other randomised studies of acute stage ABPA itraconazole versus corticosteroids from India [8] and all severe asthma also comparing itraconazole with (lower dose) corticosteroid from Iran [9] probably came too late to influence the consumption of itraconazole that the authors documented to 2018, and Iran is not included amongst the 38 middle-income countries. In low- and low-middle income countries, itraconazole remains a viable alternative to oral corticosteroid for severe asthma and ABPA in those who cannot afford biologics. Both acute but also recurrent vulvovaginal candidiasis (RVVC) are very frequent, with an estimated 138 million women suffering from the latter globally [10]. RVVC responds to long-term antifungal suppression with fluconazole, but replacement of Candida albicans with C. glabrata and fluconazole resistance in C. albicans reduce its effectiveness. It is likely itraconazole has been more frequently used for this indication. There has been increasing awareness of disseminated histoplasmosis in AIDS, thanks to the pioneering work of Mathieu Nacher, Antoine Adenis and colleagues in French Guiana [11]. Itraconazole is an important step-down and maintenance therapy for histoplasmosis, and some increased usage may have followed awareness and improved diagnostic capacity, with reduced deaths. The discovery of large numbers of such cases in Africa [12–15] should substantially increase usage further, although only three North African countries and South Africa were included in the survey. A long-term program of estimating the incidence and prevalence of serious fungal diseases (including CPA, ABPA, fungal asthma, RVVC and histoplasmosis) has now encompassed over 80 countries [16]. Engagement of national experts in these countries has alerted them to the relatively high prevalence of these conditions, and this may have contributed to local awareness. The first cases of terbinafine-resistant dermatophytosis (ringworm) were reported in India in 2016 [17]. Itraconazole for 3 weeks at a dose of 400 mg daily (more usually used for skin fungal infections) is about 75% effective in treating these inflammatory and disfiguring lesions. Do the authors know if there was a marked increase in consumption in India in 2017 and 2018 in response to this ongoing epidemic? The authors reference GAFFI’s 2016 summary of antifungal drug availability across the world [18]. This has been regularly updated since and fortunately many more countries do now have itraconazole approved for use (Fig. 1), but not Bolivia, Morocco, Senegal, The Gambia, Mauritania, Benin, Niger, Democratic Republic of Congo, Ethiopia, Ukraine Kyrgyzstan and Tajikistan [19]. However, the price of itraconazole remains unaffordable for many, and in most low- and middle-income countries it is not included with universal health coverage or government-funded medications.Fig. 1 Countries in which itraconazole is licenced for use 2021/22. Source: https://gaffi.org/antifungal-drug-maps/. Dark red = approved for use. Pink = not approved for use. Grey = no data available. Reproduced with permission The authors’ survey was until 2018. The echinocandin antifungal agents were added to the WHO’s EML in 2021 [20]. It will be of interest to observe whether this has an appreciable impact on usage over the coming years, especially in middle-income countries. Sincerely David W. Denning Professor of Infectious Diseases in Global Health, Manchester Fungal Infection Group, The University of Manchester, UK Chief Executive, Global Action for Fungal Infections, Geneva, Switzerland Declarations Funding No funding to declare. Conflicts of interest None pertain to this letter. Dr Denning and family hold Founder shares in F2G Ltd, a University of Manchester spin-out antifungal discovery company, and share options in TFF Pharma. He acts or has recently acted as a consultant to Pulmatrix, Pulmocide, Biosergen, TFF Pharmaceuticals, Pfizer, Omega, Novacyt and Cipla. He sat on the DSMB for a SARS CoV2 vaccine trial. In the last 3 years, he has been paid for talks on behalf of Hikma, Gilead, BioRad, Basilea, Mylan and Pfizer. He is a longstanding member of the Infectious Disease Society of America Aspergillosis Guidelines group, the European Society for Clinical Microbiology and Infectious Diseases Aspergillosis Guidelines group and recently joined the One World Guideline for Aspergillosis. Author contributions Not applicable. ==== Refs References 1. Pathadka S Yan VKC Neoh CF Global consumption trend of antifungal agents in humans from 2008 to 2018: data from 65 middle- and high-income countries Drugs 2022 82 1193 1205 10.1007/s40265-022-01751-x 35960433 2. Denning DW Lee JY Hostetler JS Pappas P NIAID Mycoses Study Group multicenter trial of oral itraconazole therapy of invasive aspergillosis Am J Med 1994 97 135 144 10.1016/0002-9343(94)90023-X 8059779 3. https://gaffi.org/itraconazole-voriconazole-and-natamycin-5-ophthalmic-preparation-proposed-as-essential-antifungal-medicines/. Accessed 19 Sept 2022. 4. https://gaffi.org/ruling-by-world-health-organisation-delights-doctors/. Accessed 19 Sept 2022. 5. Denning DW Cadranel J Beigelman-Aubry C Chronic pulmonary aspergillosis—rationale and clinical guidelines for diagnosis and management Eur Resp J 2016 47 45 68 10.1183/13993003.00583-2015 6. Zhou LH Li RY Denning DW Zhu LP Risk-based estimate of human fungal disease burden, China: old pathogen, new host, and new patterns Emerg Infect Dis 2020 26 2137 2147 10.3201/eid2609.200016 32818410 7. Denning DW O’Driscoll BR Powell G Randomized controlled trial of oral antifungal treatment for severe asthma with fungal sensitisation (SAFS), the FAST study Am J Resp Crit Care Med 2009 179 11 18 10.1164/rccm.200805-737OC 18948425 8. Agarwal R Dhooria S Singh Sehgal I A randomized trial of itraconazole vs prednisolone in acute-stage allergic bronchopulmonary aspergillosis complicating asthma Chest 2018 153 3 656 664 10.1016/j.chest.2018.01.005 29331473 9. Mirsadraee M Dehghan S Ghaffari S Mirsadraee N Long-term effect of antifungal therapy for the treatment of severe resistant asthma: an active comparator clinical trial Curr Med Mycol. 2019 5 4 1 7 32104737 10. Denning DW Kneale M Sobel JD Rautemaa-Richardson R Global burden of recurrent vulvovaginal candidiasis Lancet Infect Dis 2018 18 e339 e347 10.1016/S1473-3099(18)30103-8 30078662 11. Adenis A Nacher M Hanf M HIV-associated histoplasmosis early mortality and incidence trends: from neglect to priority PLoS Negl Trop Dis 2014 8 8 e3100 10.1371/journal.pntd.0003100 25144374 12. Mandengue CE Ngandjio A Atangana PJ Histoplasmosis in HIV-infected persons, Yaoundé, Cameroon Emerg Infect Dis 2015 21 11 2094 2096 10.3201/eid2111.150278 26488076 13. Oladele R Ayanlowo OO Richardson MD Denning DW Histoplasmosis in Africa: an emerging or a neglected disease? PLoS Negl Trop Dis 2018 12 e0006046 10.1371/journal.pntd.0006046 29346384 14. Ocansey BK Otoo B Asamoah I Cryptococcal and histoplasma antigen screening among people with HIV in Ghana and comparative analysis of OIDx histoplasma lateral flow assay and IMMY histoplasma enzyme immunoassay Open Forum Infect Dis 2022 9 7 ofac277 10.1093/ofid/ofac277 35854987 15. Oladele RO, Osaigbovo II, Akanmu AS, Adekanmbi O, Ekeng B, Yahaya M, Alex-Wele M, Okolo M, Ayanbeku T, Unigwe U, Akase I, Dan-Jumbo A, Israelski D, Denning DW, Pasqualotto AC, Chiller T. Ascertaining the current prevalence of Histoplasmosis in Nigeria’s Advanced HIV disease population. Emerg Infect Dis. 2022;28:2261–9. 16. https://gaffi.org/media/country-fungal-disease-burdens/. Accessed 19 Sept 2022. 17. Majid I Sheikh G Kanth F Hakak R Relapse after oral terbinafine therapy in dermatophytosis: a clinical and mycological study Indian J Dermatol 2016 61 5 529 533 10.4103/0019-5154.190120 27688443 18. Kneale M Bartholomew JS Davies E Denning DW Global access to antifungal therapy and its variable cost J Antimicrob Chemother 2016 71 3599 3606 10.1093/jac/dkw325 27516477 19. https://gaffi.org/antifungal-drug-maps/. Accessed 19 Sept 2022. 20. https://gaffi.org/antifungal-echinocandins-added-to-the-whos-essential-medicines-list/. Accessed 19 Sept 2022.	0	PMC9713184	NO-CC CODE	2022-12-02 23:22:56	no	DNP. 2022 Dec 1; 23(6):34-39	latin-1	null	null	null	oa_other
==== Front Ann Oper Res Ann Oper Res Annals of Operations Research 0254-5330 1572-9338 Springer US New York 5079 10.1007/s10479-022-05079-3 Original Research Perceived customer journey innovativeness and customer satisfaction: a mixed-method approach http://orcid.org/0000-0001-9964-3724 Vo-Thanh Tan [email protected] 1 http://orcid.org/0000-0002-8582-9445 Zaman Mustafeed [email protected] 2 http://orcid.org/0000-0001-9287-1150 Thai Trung Dam-Huy [email protected] 3 http://orcid.org/0000-0002-1290-4627 Hasan Rajibul [email protected] 4 http://orcid.org/0000-0002-8743-3348 Senbeto Dagnachew Leta [email protected] 5 1 Department of Marketing, Excelia Business School, CEREGE (UR 13564), 102 Rue de Coureilles, 17024 La Rochelle, France 2 Department of Marketing, EM Normandie Business School, Métis Lab, 20 Quai Frissard, 76600 Le Havre, France 3 grid.444827.9 0000 0000 9009 5680 International School of Business, University of Economics Ho Chi Minh City, Ho Chi Minh City, Vietnam 4 grid.95004.38 0000 0000 9331 9029 School of Business, Maynooth University, Maynooth, Ireland 5 grid.16890.36 0000 0004 1764 6123 School of Hotel and Tourism Management, The Hong Kong Polytechnic University, 17 Science Museum Rd, Tsim Sha Tsui, Kowloon, Hong Kong 30 11 2022 126 8 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. This research aims to understand the link between perceived innovativeness and customer satisfaction in the fine-dining catering segment. By employing a mixed-method approach, this paper proposes a multidimensional framework for measuring the perceived innovativeness of restaurants throughout the entire customer journey. Customer satisfaction was measured by considering online customer-generated data from TripAdvisor. The study not only finds a strong correlation between perceived innovativeness and customer satisfaction but also presents how fine-dining restaurants can employ user-generated data to co-innovate entire customer journeys and restaurant experiences. The results highlight menu-, service-, and customer experience-related innovativeness as the three most important criteria for fine-dining restaurant customers. Additionally, the results of the qualitative study indicate that in the context of fine-dining catering, the quality of the dishes, the service, and the customers’ experience with the service staff and chefs are the main elements of satisfaction that restaurants should consider in promoting innovation. Keywords Perceived innovativeness Customer journey innovation Customer satisfaction Fine-dining restaurants Customer-generated data AHP Fuzzy-TOPSIS ==== Body pmcIntroduction Data-driven innovation (DDI) consists of procuring, analyzing, and making decisions based on customer data (Babu et al., 2021; Bhatti et al., 2022; Rindfleisch et al., 2017). A huge amount of data obtained from using emerging technologies, such as blockchain, cloud computing, artificial intelligence, data analytics, and the Internet-of-things enable firms to create DDI in developing new products or improving existing ones (Akter et al., 2016, 2022; Babu et al., 2021; Ciampi et al., 2021; Duan et al., 2020; Wamba et al., 2017). According to Akter and Wamba (2016), digital giants, such as Amazon, Tencent, Google, Alibaba, Apple, and Facebook are experiencing stronger competitive advantages from DDI. Notably, in the unprecedented context of the COVID-19 outbreak, these digital giants witnessed an extreme increase in their turnover. For example, since the COVID-19 pandemic spread to Europe and the United States in the spring of 2020, Amazon has recruited more than 400,000 employees and posted a record profit for the period July to September for a second consecutive quarter at USD 6.3 billion (Greene, 2020). From an academic research standpoint, data and analytics are considered the core elements of the innovation and development of services and products (Akter et al., 2016, 2019; Babu et al., 2021; Ciampi et al., 2021; Ho-Dac, 2020; Lee, 2018; Wamba et al., 2017) or business models (Saura et al., 2022; Sorescu, 2017). The literature has also acknowledged that an organization’s innovativeness is critical in maintaining its competitive advantage (Acar et al., 2019; Akter & Wamba, 2016; Hollebeek & Rather, 2019; Kazadi et al., 2016; Kim et al., 2018; Yang et al., 2022; Zhan et al., 2018). Hence, to ensure the success of innovations in the marketplace, a consumer-centric view is crucial, highlighting the notion that the end consumer ultimately determines the success of an innovation (Cukier, 2021; Kunz et al., 2011; Tan & Staats, 2020). Customer satisfaction is recognized as a significant indicator of a product or service innovation (El Ouardighi et al., 2018; Giannakis et al., 2022; Hollebeek & Rather, 2019; Mahmoud et al., 2018). However, many studies on the relationship between innovativeness and customer satisfaction (e.g., Mahmoud et al., 2018) primarily focused on a single product or service (Hoeffler, 2003; Kunz et al., 2011), leaving a gap in terms of understanding the impacts of customer journey innovativeness on customer satisfaction and how firms can integrate customer-generated data into innovating. Customer journey mapping is an essential strategic management tool widely hailed by practitioners and academics for its usefulness in understanding an organization’s customer satisfaction. This can help an organization’s strategic management team identify which touchpoints are critical to customer satisfaction. It can also reveal key strategic initiatives at each touchpoint, resulting in cross-functional inputs to advance an organization’s service and product innovation (Hamilton et al., 2021; Klein et al., 2020; Rosenbaum et al., 2017). Moreover, Akter et al. (2019) claimed that gaining value from DDI is an integrative process, from idea generation to commercialization. Thus, we propose that customer journey innovativeness must be considered in determining whether a firm is successful in its innovation because customers review a range of firm activities to form an overall evaluation of its innovativeness (Hamilton et al., 2021; Kunz et al., 2011). Since 2010, “the gastronomic meal of the French” has been included in UNESCO’s list of intangible heritage. In 2016, the catering sector represented a turnover of 66 billion euros, making it the 5th sector in terms of job creation in France. Given the importance of the catering industry in French culture and economy, this research aims to examine the link between the customer journey innovativeness of French fine-dining restaurants and customer satisfaction, as well as to explore how these restaurants use customer-generated data to innovate. As highlighted by Vo-Thanh et al., (2022a, 2022b), fine-dining restaurants are different from fast-food or classic restaurants, as their customer journeys are much more complex. Fine-dining restaurants are defined based on their tangible and intangible attributes (Harr, 2008; Vo-Thanh et al., 2022a, 2022b). In addition to the excellent food choices, the visual presentation, refined decoration, use of high-quality tableware, interactions with the renowned chef, and faultless and precise services from the frontline employees also help provide customers with memorable and unique dining experiences, which induce customer satisfaction (Vo-Thanh et al., 2022a, 2022b). Meanwhile, innovativeness is described as the ability of an individual, organization, business, or even an economy to innovate, adopt, and develop a new product or service, or implement a new idea (Crawford & Di Benedetto, 2011; Rubera & Kirca, 2012). Although the importance of innovativeness is recognized in all sectors, studies on innovativeness have rarely dealt with the service industry (Ettlie & Rosenthal, 2011). Furthermore, the few studies on how customers perceive restaurant innovativeness (Kim et al., 2018; Sean Hyun & Han, 2012) have partially dealt with innovativeness and have not considered the entire customer journey. Notably, no study has been carried out on the perceived innovativeness of restaurants in France. Finally, studies that explore the use of DDI in fine-dining restaurants to create added value for customers and enhance competitive advantages are scant. At the same time, the development of information and communication technologies has radically changed consumer behaviors (Kirova & Vo-Thanh, 2019; Lamberton & Stephen, 2016; Xiang et al., 2015). In particular, the hospitality sector (e.g., hotel, restaurant, or leisure) has been greatly impacted by online review sites, mobile devices, and home food delivery (Clauzel et al., 2019; Kirova & Vo-Thanh, 2019; Vo-Thanh & Kirova, 2018; Vo-Thanh et al., 2021; Zaman et al., 2016a, 2016b, 2021). These review sites not only play a vital role in customers’ decision-making processes but also allow firms to capture and analyze data that can help them innovate their products or services (Vo-Thanh & Kirova, 2018; Zaman et al., 2016a, 2016b). To address the above research gaps, this study introduces the following research questions:Is there a relationship between perceived customer journey innovativeness and customer satisfaction in the context of fine-dining restaurants in France? How can fine-dining restaurants use customer-generated data to identify the main elements of customer satisfaction, innovate the customer journey, and co-create restaurant experiences? To answer these questions, this research established an evaluation grid of the perceived customer journey innovativeness of fine-dining restaurants, comprising six criteria and 32 subcriteria. First, the evaluation grid was developed through an extensive literature review and then refined with 37 MBA Hospitality Management (Executive Program) students who had at least three years of experience in the hotel and restaurant industries and held management positions at the time of the study. Second, the relationship between perceived customer journey innovativeness and customer satisfaction, as well as the elements of satisfaction, were examined. In this regard, the analytic hierarchy process (AHP), fuzzy-technique of order preference by similarity to ideal solution (TOPSIS) methods, and content analysis of customer online reviews were employed. By doing so, we explored the data of customer journey innovativeness of fine-dining restaurants to create DDI that can add more value for ensuring customer satisfaction and competitive advantage. Data-driven innovation DDI refers to the process, techniques, and technologies that allow firms to analyze big data (Babu et al., 2021; Bhatti et al., 2022) related to customers, business partners, and competitors based on innovation and value creation, thus helping firms generate competitive advantages (Kumar et al., 2016). In recent years, big data have gained particular attention from researchers in the tourism and hospitality field (Li et al., 2018; Mariani, 2020; Shamim et al., 2021). For example, Amatulli et al. (2019) demonstrated how Italian hotels use customer-generated online reviews to predict the sharing of negative emotional content. Zaman et al. (2022) highlighted the utilization of internal and external data in creating values for all the stakeholders of a destination. In addition, firms may use co-creation to include their customers in DDI (Bresciani et al., 2021). Meanwhile, customers’ experiences also play a vital role in product or service innovation (Bresciani et al., 2021; Vo-Thanh & Kirova, 2018). Kim et al. (2018) emphasized that a product or service could be considered innovative if end users (customers) find it innovative and if their perceptions of it are primordial. Therefore, we argue that fine-dining restaurants should include their customers in the innovation process. They should know their customers’ perception vis-à-vis the firm’s innovativeness and use these customer DDIs to co-innovate customer journeys and their dining experiences. In addition, customer satisfaction is one of the key performance indicators for firms in which a higher level of customer satisfaction improves a firm’s competitive advantage (Zaman et al., 2016b). Hence, if their innovations generate higher satisfaction rates, the competitive advantages of fine-dining restaurants are strengthened. Thus, based on customer-generated review data, fine-dining restaurants should consider what should be innovated to obtain the highest customer satisfaction levels from DDI sources. Perceived innovativeness of firms Measurement of innovativeness The innovativeness of firms is often measured by their investments in research and development (R&D) activities (Raymond et al., 2013) or through the number of patents they registered for a given period of time (Kleis et al., 2012). However, Deltour and Lethiais (2014) pointed out that in the context of small and medium-sized enterprises (SMEs), the measurement of innovativeness through investments in R&D and patents is not suitable because SMEs do not have enough budget to invest in such activities. Still, they are capable of developing a real capacity to implement other types of innovation (Forsman, 2011). Subramanian (1996) emphasized that the measurement of innovativeness is a multidimensional process that must be done over time. Hurley and Hult (1998) considered the speed of firms’ innovation adoption a criterion for measuring innovativeness and partly agreed with Subramanian’s (1996) view. Later, Wang and Ahmed (2009) state that firm innovativeness concerns their ability to innovate both in products and in processes. Therefore, some authors (e.g., Danneels & Kleinschmidtb, 2001; Kamins et al., 2007; Kunz et al., 2011) highlighted the necessity of taking a more holistic view of the perceived innovativeness of a firm. These authors recommend that a firm’s perceived innovativeness should be measured through its various activities and not just its new products. According to Kim et al. (2018), the perceived innovativeness of a firm can be measured from both managers’ (Binder et al., 2016; Sandvik et al., 2014) and customers’ (Jin et al., 2016; Kamins et al., 2007; Kunz et al., 2011) perspectives. In the current research, we focus on consumer-perceived innovativeness because, on the one hand, we adopt the conceptualization of the overall perceived innovativeness of the firm proposed by Kunz et al. (2011). On the other hand, a product or service is considered innovative if its users consider it to be such. Therefore, studying customers’ or users’ perceived innovativeness is crucial for firms (Kim et al., 2018; Kunz et al., 2011). Perceived innovativeness of fine-dining restaurants Restaurants are part of the service industry. Thus, we need to consider its specificities, such as intangibility, perishability, inseparability, and heterogeneity (Kim et al., 2018; Parasuraman et al., 1985). Kim et al. (2018) presented four dimensions of innovativeness for restaurants:Menu-related innovativeness This is measured by customers’ perceptions of the novelties and unique characteristics of a restaurant’s menu (Hubert et al., 2017; Kim et al., 2018). Kim et al. (2018) proposed measuring this through four subdimensions: quality, current food-related trends, menu uniqueness and variety, and personalization. Technology-based service-related innovativeness Service is intangible and can only be measured when delivered (Ding & Keh, 2017). The use of technology allows restaurants to better anticipate customer behaviors, gain competitive advantages, strengthen bonds with their customers, and build loyalty among them (Kim et al., 2018). This dimension is not only limited to the front of house (dining area), but also concerns the delivery of meals at home—a service that has proliferated with the development of digital platforms, such as Deliveroo, Uber Eats, Just Eat, Resto In, Frichti, or Nestor (Bordeaux, 2019). Home delivery has increased by about 20% per year for the last three years (e.g., 160 million euros in 2018) (Bordeaux, 2019). Kim et al. (2018) suggested measuring technology-based service-related innovativeness through four subdimensions: differentiation, technologies, utility, and advanced service. Experience-related innovativeness This refers to the creation of an atmosphere that allows customers to interact with employees and fellow customers (Ding & Keh, 2017; Kim et al., 2018). In an analysis of “The restaurant of the future: Engaging the next-generation customer,” Chick (2021) demonstrated how restaurants can engage their customers throughout the customer journey. To measure experience-related innovativeness, Kim et al. (2018) used three subdimensions: atmosphere, interaction (with employees and customers), and customer satisfaction. Promotional innovativeness This refers to the ability of a restaurant to introduce creative promotional activities using different channels, be they traditional and/or digital (Shankar et al., 2010). For example, in the summer of 2014, five Parisian hotels launched a highly successful innovative campaign with their “Pay What You Want” program (Zaman et al., 2016b). To measure promotion-related innovativeness, Kim et al. (2018) used five subdimensions: loyalty program, good deals, advertising, marketing manner, and communication. Notably, Kim et al. (2018) did not consider innovativeness related to a restaurant’s website, even though it is crucial in the digital era (Daries et al., 2018) due to changes in consumer behaviors (Lamberton & Stephen, 2016; Xiang et al., 2015) and the strong impacts of online review sites and home delivery tools on the restaurant sector (Clauzel et al., 2019; Vo-Thanh & Kirova, 2018; Vo-Thanh et al., 2021). In 2018, eight out of ten French people made purchases online, of whom 12% bought meals online (Laine-Devroede, 2019). In addition, consumers can now view online reviews and/or rate restaurants on review sites. These reviews are vital in shaping the choices of future customers (Zaman et al., 2016a). Moreover, the quality of a website can enhance both an organization’s online visibility and customer satisfaction (Bai et al., 2008). Therefore, in a context in which digital content influences consumption, restaurants should understand the entire customer journey and bring innovations to their websites. Measurement of the perceived innovativeness of the entire customer journey According to Bolden et al. (2017), the restaurant customers’ journey in the digital age consists of five stages. The first stage is about demand generation, where customers seek inspiration. At this stage, a company uses online and/or offline communication tools, such as social networks, online review sites, promotions, online booking platforms, or loyalty programs, to generate demand from potential consumers. Kim et al. (2018) considered this to be the dimension of promotional innovativeness. The next stage is ordering, which includes online menus, e-commerce, and payment. These aspects are also highlighted by Daries et al. (2018). The third stage is preparation, which involves inventory management, process management, and staff training. Kim et al. (2018) considered these aspects in studying menu-related innovativeness and technology-based service-related innovativeness. The fourth stage lies in service and delivery, which falls under experience-related innovativeness (Kim et al., 2018). The last stage refers to engagement (e.g., blogs and social networks), which, in turn, can generate demand (Daries et al., 2018). Considering the entire customer journey, we constructed the evaluation grid of the perceived innovativeness restaurants as follows:Step 1 A total of 37 MBA Hospitality Management (Executive Program) students were recruited. These students already had at least three years of experience in hotel and restaurant management. During the first three-hour session, we explained the restaurant customer journey in the digital age, as described by Bolden et al. (2017), along with the different criteria and subcriteria of restaurant innovativeness developed by Kim et al. (2018). Subsequently, we asked participants to work in groups (11 groups of three members and 1 group of four members) and to propose a restaurant innovativeness measurement framework (RIMF) in the digital age that they considered most relevant. Step 2 The participants were given three weeks to work on and refine the RIMF by interviewing at least three restaurant managers and three consumers of these restaurants based on the innovativeness criteria and subcriteria. Step 3 We organized a brainstorming session during which we discussed the proposed criteria and subcriteria and retained the final RIMF (Table 1). Table 1 Restaurant innovativeness measurement framework Criteria (C) Subcriteria (SC) References C1: Innovativeness of demand generation SC1.1: The restaurant has a website on which general information (about the restaurant, its chef, its dishes and prices, opening hours, geographical location, etc.) can be found SC1.2: The website is available in several languages with adapted expressions SC1.3: The website has good-quality photos of the dishes and presents them thematically in an innovative way SC1.4: The website gives information about the partners (including local partners, if any), the origin of the products, and the labels (ISO 9000, ISO 14000, and other ecological labels, if any) SC1.5: Online reviews are accessible from the website, which displays photos and comments from social networks in an innovative way SC1.6: The website offers different promotional offers and good deals SC1.7: The website has a responsive design, and the mobile site is also well-designed SC1.8: The website offers the possibility to communicate with the restaurant in real time (via Messenger, for example) SC1.9: The website offers the possibility of ordering online and offers secure payment channels with several choices SC1.10: The restaurant carries out advertising campaigns in an innovative way SC1.11: The restaurant offers innovative communication platforms (e.g., virtual communities) to gather ideas from customers SC1.12: The restaurant responds to customer feedback in an innovative way SC1.13: The restaurant partners with influencers and communicates in innovative ways Daries et al. (2018), Kim et al. (2018) C2: Innovativeness of preparation SC2.1: The restaurant uses digital tools to manage its inventory, orders, etc. (e.g., staff are equipped with digital tools) SC2.2: Waiting time is short thanks to the implementation of process innovation Bolden et al. (2017), Hurley and Hult (1998), Kim et al. (2018) C3: Innovativeness of menu SC3.1: The restaurant offers new flavors according to market trends SC3.2: The restaurant has unique menus/dishes compared with its competitors SC3.3: The restaurant often innovates its menus and presents them attractively SC3.4: The restaurant offers customers the opportunity to create their own menus SC3.5: The restaurant gives priority to local products Kim et al. (2018) C4: Innovativeness of service SC4.1: The restaurant provides exceptional service compared with its competitors SC4.2: Employees communicate in an innovative manner SC4.3: The restaurant uses technology to better serve customers (contactless payment, interactive tables, etc.) Kim et al. (2018) C5: Innovativeness of customer experience SC5.1: The restaurant has a unique atmosphere SC5.2: The restaurant has unique decorations compared with its competitors SC5.3: The restaurant organizes its activities in an innovative way SC5.4: The chef is approachable and often comes out to greet customers SC5.5: The restaurant uses technology to enhance the customer experience (e.g., the ability to see the chef’s preparation from the table) Bolden et al. (2017, Kim et al. (2018) C6: Innovativeness of customer engagement SC6.1: The restaurant is featured on online review sites and solicits customers to leave reviews SC6.2: The restaurant has a blog on which it regularly publishes information to maintain ties with its customers SC6.3: The restaurant communicates on social networks in an innovative way SC6.4: The restaurant has an innovative loyalty program Bolden et al. (2017) Application of AHP and fuzzy-TOPSIS methods By using the RIMF (Table 1), we evaluated 13 fine-dining restaurants in Paris (Restaurant Aux Lyonnais—R1, Restaurant Spoon—R2, Restaurant Kei—R3, Restaurant Champeaux—R4, Restaurant Le Pharamond—R5, Restaurant Huguette—R6, Restaurant Cucina—R7, IDA—R8, Anicia—R9, Plume Restaurant—R10, Bistrot Belhara—R11, 32 by Hervé—R12, et Brasserie Baroche—R13). These are voluntary members of a chain of restaurants called Les Collectionneurs1 under the famous chef Alain Ducasse (3-star Michelin chef). The study by Kim et al. (2018) did not distinguish among restaurant categories, which was identified as a limitation of their study. Our study addresses this limitation by studying fine-dining restaurants only. In addition, according to Gira Conseil (2021), superior, upscale, and luxury restaurants represent 23% of the overall restaurant revenue in France. Using the fuzzy-TOPSIS method (Chan et al., 2016; Hwang & Yoon, 1981; Lai et al., 1994) allowed us to rank restaurants according to their relative performances in terms of perceived innovativeness. In this regard, a questionnaire was developed according to the RIMF (Table 2).Table 2 Excerpt of the questionnaire used for evaluating each restaurant’s innovativeness Criterion 1: Innovativeness of demand generation Subcriterion 1.1: Does the restaurant’s website offer general information in an innovative way? Poor (Not at all or very little innovative) Good (Averagely innovative) Excellent (Very innovative) Subcriterion 1.2: Is the website available in several foreign languages with adapted expressions? Poor (Not at all or very little innovative) Good (Averagely innovative) Excellent (Very innovative) Subcriterion 1.3: Are the photos of the dishes of good quality and presented thematically? Poor (Not at all or very little innovative) Good (Averagely innovative) Excellent (Very innovative) The questionnaire consisted of linguistic terms (Table 3) based on fuzzy set theory (Zadeh, 1965). Zimmermann (1996) emphasized that natural representations are more efficient in measuring service performance. Li (2013) argues that using a fuzzy scale is more appropriate than a traditional Likert scale. Each linguistic term corresponds to a fuzzy number situated on a scale between 0 and 100. The membership function of each linguistic term has three values known as triangular fuzzy number (TFN; Li, 2013; Tsaur et al., 2002; Zadeh, 1965). For example, if we consider three linguistic terms, including Poor (Not at all or very little innovative), Good (Averagely innovative), and Excellent (Very innovative), and we assign 25, 50, and 100 for the Poor, Good, and Excellent terms, respectively, we can have the TFN for each linguistic term as follows:Table 3 Linguistic terms and their TFN Linguistic terms TFN for each linguistic term Poor (Not at all or very little innovative) (0, 25, 50) Good (Averagely innovative) (25, 50, 75) Excellent (Very innovative) (50, 75, 100) Table 3 only illustrates the TFN for the three linguistic terms. It should be noted that linguistic terms were adapted (from two to five linguistic terms) according to the question (Ma et al., 2007). As for the consumers’ evaluation, we had to find evaluators who had already eaten in the 13 selected restaurants and were willing to give their evaluations. To that end, we requested the help of Mr. Aldric D., who was, at the time of the study, owner and general director of the ZE Hotel Paris Opéra (a 4-star establishment, www.zehotel.fr) and president of the hotel owners of Alain Ducasse’s voluntary chain, Les Collectionneurs. Thanks to him, we were able to find seven people working in hotels in Paris who met the abovementioned conditions. Indeed, all seven evaluators were invited by the 13 chosen restaurants as part of the partnership, so they already knew those restaurants and their services very well. Notably, the hotels where these seven evaluators have been working are also part of Les Collectionneurs, and each of these evaluators was asked to evaluate the 13 selected restaurants. Table 4 summarizes their profiles.Table 4 Profiles of the selected evaluators Evaluator Position Age range E1 General Manager (Hotel 4) 40–49 years E2 Deputy Director (Hotel 4) 50–59 years E3 Front Office Manager (Hotel 4) 40–49 years E4 Receptionist (Hotel 4) 40–49 years E5 Receptionist (Hotel 4) 40–49 years E6 Marketing Director (Hotel 4) 40–49 years E7 Receptionist (Hotel 4*) 40–49 years If we consider n number of restaurants, R=(Rj,j=1,⋯,n); m number of criteria, C=(Cj,j=1,⋯,m) and t number of subcriteria,SC=SCj,h,h=1,⋯,t; and k number of evaluators, E=ep,p=1,⋯,k, we will obtain the result for restaurant i on criterion j from the following equation:1 RRiCj=1t∑t=1RRiSCj,t. By employing Eq. (1), we obtain the performance matrix–fuzzy as illustrated in Table 5.Table 5 Performance matrix–fuzzy C1 C2 … Cm R1 RR1C1 RR1C2 … RR1Cm R2 RR2C1 RR2C2 … RR2Cm … … … … … Rn RRnC1 RRnC2 … RRnCm The next step consists of defuzzification, which transforms the fuzzy numbers into non-fuzzy numbers (Zimmermann, 1996). To that end, we opted for the approach of Tsaur et al. (2002) by using the following equation:2 BNPij=UVij-LVij+MVij-LVij3+LVij. In the literature, the non-fuzzy number is called BNP = Best Non-Fuzzy Performance. Here, UVij stands for the upper value, MVij for the medium value, and LVij for the lower value of restaurant i on the criterion j. Therefore, we obtained the performance matrix–non-fuzzy in Table 6.Table 6 Performance matrix–non-fuzzy C1 C2 … Cm R1 BNPR1C1 BNPR1C2 … BNPR1Cm R2 BNPR2C1 BNPR2C2 … BNPR2Cm … … … … … Rn BNPRnC1 BNPRnC2 … BNPRnCm In multicriteria decision analysis, the weights of each criterion are critical. In this regard, AHP was used to determine the weights of the selected criteria. Based on Saaty’s (1977) scale (Table 7), a questionnaire was developed to make a pairwise comparison of the criteria. If we compare a set of criteria n, where the criteria are designated as a1,a2,⋯an and the weights of these criteria as w1,w2,⋯wn, respectively, the pairwise comparison can be presented in the questionnaire as follows:1-AHP A=a11⋯a1j⋯a1n⋮⋮⋮⋮⋮ai1⋯aij⋯ain⋮⋮⋮⋮⋮an1⋯anj⋯anm where aij≅1/aji (reciprocal value and always positive) and aij=aik/ajk. Note that in a real situation, wi/wj is unknown. In this respect, the problem for the AHP method is to find aij, for example, aij≅wi/wj. The weight matrix is presented as follows:2-AHP w1⋯wj⋯wnW=w1⋮wi⋮wnw1/w1⋯w1/wj⋯w1/wn⋮⋮⋮⋮⋮wi/w1⋯wi/wj⋯wi/wn⋮⋮⋮⋮⋮wn/w1⋯wn/wj⋯wn/wn Table 7 Saaty’s (1977) scale Verbal appreciation Intensity Reciprocal value Equally 1 1 Moderately 3 1/3 Strongly 5 1/5 Very strongly 7 1/7 Extremely 9 1/9 Intermediate values 2, 4, 6, 8 1/2, 1/4, 1/6, 1/8 If we multiply W by w, we will have:3-AHP w1⋯wj⋯wnW×w=w1⋮wi⋮wnw1/w1⋯w1/wj⋯w1/wn⋮⋮⋮⋮⋮wi/w1⋯wi/wj⋯wi/wn⋮⋮⋮⋮⋮wn/w1⋯wn/wj⋯wn/wnw1⋮wj⋮wn=nw1⋮wj⋮wn 4-AHP OtherwiseW-nIw=0, To determine the weights of the selected criteria, the respondents (i.e., the seven hospitality professionals who had eaten in the 13 selected restaurants) gave a relative verbal appreciation between the two criteria rather than a numerical judgment, using Saaty’s (1977) scale presented in Table 7. Saaty’s (1977) scale (Table 7) ranges from 1 to 9, where 1 indicates equal importance between two criteria, and 9 indicates extreme importance. On the one hand, given that solving Eq. (4-AHP) is an eigenvalue problem, we can obtain the comparative weights by finding the eigenvector w with the respective λmax that satisfies Aw = λmax w, where λmax is the largest eigenvalue of the matrix A (1-AHP). In other words, we have to find the eigenvector w with λmax for A-λmaxIw=0. On the other hand, one of the disadvantages of the AHP method is the inconsistency of judgments. Thus, to ensure consistency, we considered the consistency index (CI) and the consistency ratio (CR). According to Saaty (1980), the CR score should not exceed 0.1 to yield a relevant result. In the current study, the CR is obtained by using the following equation:5-AHP CI=λmax-n/n-1 where λmax is the largest eigenvalue, and n is the number of criteria. In addition, the following equation is also used:6-AHP CR=CIRI where RI (Table 8) refers to the random coherence indices constructed by Saaty (1977) using a large sample of random reciprocal matrices with the help of Saaty’s (1977) scale (Table 7).Table 8 Random indices (Saaty, 1977) Number of elements 3 4 5 6 7 8 9 10 11 12 13 RI 0.52 0.89 1.11 1.25 1.35 1.40 1.45 1.49 1.51 1.54 1.56 After obtaining the weights of the selected criteria (Wj) via the AHP method, we applied the formula (3) = BNPij×Wj to get the weighted-sum performance matrix–non-fuzzy. Finally, using the normalization method, we obtained the normalized weighted-sum performance matrix–no-fuzzy (Table 9).Table 9 Normalized weighted-sum performance matrix–non-fuzzy C1 C2 … Cm R1 BNPR1C1×w1 BNPR1C2×w2 … BNPR1Cm×wm R2 BNPR2C1×w1 BNPR2C2×w2 … BNPR2Cm×wm … … … … … Rn BNPRnC1×w1 BNPRnC2×w2 … BNPRnCm×wm Given that all the criteria should be maximized to be considered as high performance, the ideal solution (I∗) and the anti-ideal solution (I-) for each criterion were thus obtained from Eqs. (4) and (5).4 I∗=maxRRiCj\|j∈C,i=1,…,n 5 I-=minRRiCj\|j∈C,i=1,…,n The distances (D) with the ideal situation (Ii∗) and the anti-ideal solution (Ii-) were obtained from Eqs. (6) and (7).6 Di∗=∑j=1mRRiCj-maxRRiCj2i=1,…,n 7 Dii=1,…,n-=∑j=1mRRiCj-minRRiCj2i=1,…,n Finally, the relative proximity to the ideal solution was calculated using Eq. (8).8 Pi=Di-Di∗+Di-,i=1,…,n here 0≤Pi≤1. If Pi is close to 1, it shows that restaurant i is close to the ideal solution (Ii∗), thereby allowing us to rank the restaurants according to their performance in terms of perceived innovativeness. Customer satisfaction assessment In the hospitality industry, competition is intense, and customers are very demanding (Zaman et al., 2016b). Thus, customer satisfaction is crucial for a hospitality firm’s performance and competitive advantage (Anderson & Mittal, 2000; Zaman et al., 2016b). “Customer satisfaction” is defined as the positive attitude of consumers toward a product or service (Albayrak & Caber, 2015). However, customer satisfaction is a complex concept that depends on several criteria (Robinot & Giannelloni, 2010; Zaman et al., 2016b). In recent years, customers’ online reviews have been regarded as an essential source for assessing customer experience and satisfaction (Clauzel et al., 2019; Vo-Thanh & Kirova, 2018; Zaman et al., 2016a, 2016b). In line with these studies, we used customers’ online reviews and ratings on TripAdvisor to capture customer satisfaction with the 13 selected fine-dining restaurants. In this way, we can enrich our understanding of the link between perceived innovativeness and customer satisfaction. For the last point, after identifying the restaurant that obtained the best overall rating in terms of customer satisfaction, we captured only reviews related to that restaurant to examine the elements contributing to customer satisfaction. Operationally, to evaluate customer satisfaction, we first took customer ratings related to four criteria (food, service, value for money, and atmosphere) from the TripAdvisor review site (Fig. 1). Second, to obtain the weights of the four abovementioned criteria, we employed the AHP method, as explained in Sect. 3. Finally, the customer ratings were multiplied by the weights of these criteria to gain the weighted sum scores for customer satisfaction.Fig. 1 Illustration of customer ratings from TripAdvisor (Screenshot taken on 15/10/2021) Next, to identify the elements of perceived innovativeness that ensure customer satisfaction, a content analysis of customer reviews was performed on the best restaurant in terms of customer satisfaction from TripAdvisor. In particular, we analyzed 217 positive online reviews of that restaurant from October 1, 2018, to September 1, 2021. To ensure internal validity, positive reviews were selected when they achieved ratings of 5 (excellent) or 4 (very good) stars on a scale of 5 stars (Sann et al., 2020). After collecting the data, these were transferred to NVivo 12 software and analyzed by two of the researchers. In the initial processing step, we ran the text frequency to determine the most frequently appearing keywords. Then, a thematic content analysis was performed based on the six criteria obtained from the RIMF as key themes. First, following previous studies (e.g., Kirova & Vo-Thanh, 2019; Vo-Thanh & Kirova, 2018; Vo-Thanh et al., 2022a, 2022b), the two researchers coded the first 10 reviews together to reach agreement on the coding process. Second, each researcher independently analyzed the remaining corpus, strictly respecting the double encoding procedure (Kirova & Vo-Thanh, 2019; Miles et al., 2020; Vo-Thanh & Kirova, 2018; Vo-Thanh et al., 2022a, 2022b). Then, the qualitative results from the two researchers were compared. Coding consistency was achieved by comparing the coding results of both researchers. As indicated by Vo-Thanh et al., (2022a, 2022b), the two researchers utilized the Coding Comparison Query function in NVivo 12 software to accomplish this comparison. The agreement rate between the two researchers was 91.7%. Finally, the remaining inconsistencies were examined to obtain a consensus. The overall methodology is summarized in Fig. 2.Fig. 2 Illustration of the overall methodology used in the study Results First, thanks to the AHP method, we obtained the weights of six criteria presented in Table 10. The results show that Criteria 3 (menu-related innovativeness), 4 (service-related innovativeness), and 5 (experience-related innovativeness) are considered the most important criteria by the seven evaluators.Table 10 Weights of each criterion related to perceived innovativeness Criteria Weight Innovativeness of demand generation 0.08 (W1) Innovativeness of preparation 0.06 (W2) Innovativeness of menu 0.27 (W3) Innovativeness of service 0.22 (W4) Innovativeness of customer experience 0.26 (W5) Innovativeness of customer engagement 0.11 (W6) By applying the fuzzy-TOPSIS method, we obtained the ranking of the 13 fine-dining restaurants according to their perceived innovativeness (Table 11). The results in Table 11 reveal that none of the 13 restaurants reached the ideal solution. The most innovative restaurant is R9, with a score of 0.85, indicating that R9 has 85% similarity to the ideal solution. The least innovative restaurant is R5, with a low score of 0.19.Table 11 Ranking of restaurants according to their perceived innovativeness Rank Restaurants Performance related to perceived innovativeness (Pi) 1 R9 0.85 2 R12 0.84 3 R3 0.75 4 R2 0.73 5 R13 0.57 6 R11 0.54 7 R13 0.52 8 R8 0.43 9 R6 0.42 10 R1 0.39 11 R4 0.32 12 R7 0.23 13 R5 0.19 Second, regarding customer satisfaction, by using the AHP method, we obtained the weights of the four customer satisfaction criteria: food (0.39), service (0.28), value for money (0.18), and atmosphere (0.15). To calculate the weighted sum scores for customer satisfaction, we multiplied the customer ratings extracted from TripAdvisor (accessed on September 1, 2021) by the weights of each of the four customer satisfaction criteria (i.e., food, service, value for money, and atmosphere). Table 12 illustrates the weighted sum scores for customer satisfaction. The results indicate that R3 has the highest overall score (4.62 out of 5) and is ranked first in customer satisfaction.Table 12 Weighted sum scores for customer satisfaction Restaurants Food Service Value for money Atmosphere Weighted sum scores for customer satisfaction (out of 5) Rank R1 1.560 1.120 0.630 0.600 3.910 8 or 9 or 10 R2 1.560 1.260 0.720 0.525 4.065 7 R3 1.950 1.260 0.810 0.600 4.620 1 R4 1.365 0.980 0.540 0.450 3.335 13 R5 1.365 1.120 0.630 0.600 3.715 11 R6 1.560 1.120 0.630 0.600 3.910 8 or 9 or 10 R7 1.365 1.120 0.630 0.450 3.565 12 R8 1.560 1.120 0.630 0.600 3.910 8 or 9 or 10 R9 1.755 1.260 0.720 0.675 4.410 2 or 3 R10 1.755 1.260 0.720 0.525 4.260 4 R11 1.755 1.120 0.720 0.600 4.195 6 R12 1.755 1.260 0.720 0.675 4.410 2 or 3 R13 1.560 1.260 0.720 0.675 4.215 5 Next, we compared this customer satisfaction ranking with the perceived innovativeness ranking. The results indicate a fairly strong correlation between perceived innovativeness and customer satisfaction of the selected restaurants (R2 = 0.7324), as shown in Fig. 3 (Evans, 1996).Fig. 3 Correlation between perceived innovativeness and customer satisfaction of the selected restaurants The thematic content analysis of 217 positive online reviews of the restaurant ranked first in customer satisfaction (i.e., R3, Restaurant Kei) reveals that the most cited words are “dish” (171 recurrences), “chef” (162), “service” (125), “cuisine” (103), “menu” (99), and “flavor” (82). Customers expressed themselves in a very positive way vis-à-vis three criteria: innovativeness of menu, service, and customer experience, respectively. With 198 references, these three criteria account for 79% of all the references. A significant number of references (31%) refer to menu-related innovativeness. Customers were delighted with the flavor, precision, and presentation of the dishes offered by Restaurant Kei. They also greatly appreciated the priorities given by the restaurant to local products and its ability to respond to market trends.The dishes (asparagus, signature salad, duck and Wagyu beef, and dessert with strawberries and basil) are refined, tasty, and delicate. These dishes have been prepared with love and, above all, using local products. It is truly haute cuisine. (Comment 37) [...] All the dishes are beautiful, with absolutely incredible marriage of flavors. In each dish, we managed to separate, in the mouth, the announced flavors. Everything is light, smooth, [and] creamy as it should be, with an exceptional beauty of presentation. […] Each dish is a treat […]. Everything is a highlight. Moreover, in that lunch, from the caviar as a starter to the final desserts, everything was beautiful, original, tasty, and smooth. (Comment 103) The dishes followed one another with, always, the same precision: the signature salad was exceptional and never seen before, the bloody mary shrimp and caviar, as well as the Wagyu marked us forever! (Comment 49) I much appreciate the fact that the restaurant often changes menus. As a vegetarian, I love the vegetarian dishes that the restaurant offers, which is its strong point and perfectly responds to market trends. (Comment 18) With 25% of the references, service-related innovativeness was also highlighted positively by guests. They greatly enjoyed and recognized the professionalism, responsiveness, patience, and culinary knowledge of the staff.[…] and the room staff’s service was perfect, never intrusive. We have always liked the passionate explanations of the composition of the dishes, which is systematic in the Restaurant Kei. We will come back great with pleasure! [...] (Comment 89) […] We will also retain the performance of the restaurant supervisor with whom we discussed at length our common passion for the gastronomy [...] (Comment 162) A very good and atypical restaurant. The staff is perfect, friendly, smiling, and not snobbish, like in other places. […] (Comment 5) Customer experience-related innovativeness represents 23% of the references. In particular, customers experienced delightful moments when they could watch the chef prepare the dishes while waiting to be served. They also appreciated the unique decoration and atmosphere of the Restaurant Kei. Moreover, the friendliness and benevolence provided by the chef also seem to trigger a memorable and positive culinary experience, which in turn shapes a fundamental element of satisfaction.I really enjoy watching the chef prepare the dishes from my table. It reassures me about the dishes that I am going to experience. On the other hand, it allows me to psychologically reduce the waiting time. I don’t like to wait lengthily without doing anything at all. (Comment 55) […] When we left, Chef Kei simply introduced himself and we were able to exchange a few words about gastronomy. It was an unforgettable moment! (Comment 124) Faithfully accustomed to Gérard Besson, we were overwhelmed by his departure from the premises. Participants in the “four-handed” dinner for the handover of powers between Mr. Besson and Chef Kei, promises of very beautiful things were emerging without having the irrepressible desire to come and discover them. It took several years to say to ourselves: “Chick, let’s go! And there, Wow! The sublime! Amazing! and one could endlessly enumerate all the superlatives contained in the dictionary. Everything matches: the staff, the place (discreet and chic), the service—nothing is to be thrown away. In short, of a very high gastronomic standard in a very pleasant atmosphere. (Comment 13) Fourth visit and fourth extraordinary experience. All superlative merit. [...] The welcome and the location (quiet, beautiful, sober) deserve congratulations. Principle of the unique and surprise menu. [...] (Comment 23) Figure 4 presents the fine-dining restaurant innovativeness based on customer-generated online review data. It shows the most important elements of a customer’s fine-dining experience and which aspects restaurant managers should invest in to introduce innovations.Fig. 4 DDI based on customer-generated online review data Contributions, limitations, and future research Theoretical contributions Theoretically, this research contributes to extending the body of literature on operations management and strategic management by demonstrating how fine-dining restaurants should manage the perceived customer journey innovativeness to achieve the dual goals of satisfying their guests and achieving the sustainable development of their activities. First, this study proposes the RIMF that restaurant managers can use to assess restaurants’ perceived innovativeness throughout the entire customer journey. The study by Kim et al. (2018) did not take into consideration the entire customer journey; hence, our study filled this research gap by proposing the RIMF. Second, in the context of fine-dining restaurants, this research mobilized a mixed-method approach (i.e., the multicriteria decision-making method and the qualitative one), revealing a positive correlation between perceived customer journey innovativeness and customer satisfaction, with a more substantial explanatory power compared with the existing literature. This contribution is significant because prior research on this issue has, on the one hand, investigated a single product or service innovativeness (Hoeffler, 2003; Kunz et al., 2011). On the other hand, there has been a shortcoming in qualitatively explaining the correlation between perceived innovativeness and customer satisfaction in the existing literature (Xu et al., 2016). Third, our study enriches the literature and the implications of DDI by including customer data. Martin-Rios and Ciobanu (2019) presented different innovation strategies for hospitality firms to optimize their financial performance. However, no research has considered customer satisfaction as a key indicator of firm performance. In addition, DDI allows firms to include customers and their experiences in the innovation process as part of co-innovation (Bresciani et al., 2021). Thus, our study deepens the existing literature on co-innovation by putting customers of fine-dining restaurants at the center of the innovation process. In particular, customers were integrated into the evaluation process, and customer-generated online review data were included to better understand the customer journey and their dining experience and find ways to innovate them. Fourth, according to Kim et al. (2018) and Sean Hyun and Han (2012), few studies have examined the correlation between restaurants’ perceived innovativeness and satisfaction from a customer’s perspective. In addition, no study on this correlation has been conducted in the context of fine-dining restaurants in France. By addressing this gap, our study contributes to enriching the service innovativeness literature and fills the void of research on the French restaurant industry. This is especially significant because this industry plays a vital role in the French economy (5th sector in terms of job creation) and worldwide cultural heritage (UNESCO heritage). Managerial implications In this research, we employed both multicriteria decision-making methods (e.g., AHP and fuzzy-TOPSIS) and thematic content analysis of customer online reviews and ratings from TripAdvisor. This process enabled us to examine the link between perceived customer journey innovativeness and customer satisfaction and obtain—based on the actual lived experiences of consumers—the explanatory elements of the quantitative results. From a managerial point of view, this work proposes an evaluation grid of the perceived customer journey innovativeness of fine-dining restaurants and sheds light on the main elements of customer satisfaction, which can support restaurant managers in choosing effective and operational innovation strategies. To the best of our knowledge, this study is the first to propose a benchmarking tool for fine-dining restaurants. Benchmarking has been regarded as an effective tool for optimizing organizational performance (Aumüller et al., 2020; Yaseen et al., 2018). Therefore, our proposed framework (i.e., RIMF) will allow restaurant managers to compare their performance with that of their competitors and identify the best practices they should apply thereafter. Second, there have been profound changes in consumer behaviors, especially in the hospitality industry, due to the rapid development of online review sites, mobile devices, and home food delivery applications (Clauzel et al., 2019; Kirova & Vo-Thanh, 2019; Vo-Thanh & Kirova, 2018; Vo-Thanh et al., 2021; Zaman et al., 2016a, 2016b, 2021). Related to this, our RIMF considers all possible online and offline touchpoints of a restaurant customer’s journey, thus allowing restaurant managers to understand which of these require innovations. In other words, it provides a detailed overview of their performance in terms of innovativeness. For example, our analysis of online reviews shows that in the context of fine-dining catering, the quality of the dishes, room service, and experience with the room staff and the chef (all falling under three criteria, namely, menu-, service-, and experience-related innovativeness, respectively) are the main elements of customer satisfaction. Hence, restaurant managers should take these elements into consideration when implementing their innovation projects. Third, this study investigates the weights of four TripAdvisor criteria and underlines which of these are considered most important by fine-dining restaurant customers. This information will help restaurant managers determine which criteria they should work on as a priority and what types of information they should communicate to customers. Such information may also help managers optimize their operations. Concretely, from an expert standpoint, the weights of four fine-dining restaurant customer satisfaction criteria are as follows: food (0.39), service (0.28), value for money (0.18), and atmosphere (0.15). Therefore, food and service appear to be the two key criteria that should be prioritized by fine-dining restaurant managers. Furthermore, restaurant managers can use customer online reviews as a tool for co-creation and service innovation (Vo-Thanh & Kirova, 2018; Zhang et al., 2018). Finally, this study’s findings indicate that firms should employ DDI from big customer data to arrive at correct business decisions, implement sound strategies, and achieve efficient operations. Having access to the strategic resources of big data is not enough; firms must develop management capabilities to create value from such strategic resources and explore them for better business decisions. However, creating value from big data to achieve innovative outcomes is not an easy process (Saura et al., 2022; Shamim et al., 2021). Fortunately, DDI provides the right approach for procuring, analyzing, and making the right decisions (Babu et al., 2021; Bhatti et al., 2022). Furthermore, DDI enables businesses to derive value from data, suggest innovation, and advise on competitive advantages. From an operational perspective, DDI is not only confined to fine-dining restaurants but can also be applied to other industries, such as banking and retail. Limitations and future research directions Despite its significant contributions, this research calls for caution and has limitations that may lead to new research avenues. First, by using the suggested multicriteria decision-making methods, the robustness and relevance of the findings widely depend on the experts’ judgment. Therefore, the selection of the right experts involved in the decision-making process is crucial. Second, this study deals with fine-dining restaurants, a specific segment of the catering industry. Thus, it would be interesting to conduct further research on other segments of the industry to obtain a more global view of the relative weights of the identified criteria related to both customer satisfaction and restaurants’ perceived innovativeness throughout the entire customer journey. Wall and Berry (2007) suggested that customers of quick-service restaurants tend to have different service quality perceptions compared with customers patronizing other types of restaurants. Therefore, future studies should consider other types of restaurants (e.g., fast food). Third, our study focused only on the French context. 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==== Front Cell Tissue Res Cell Tissue Res Cell and Tissue Research 0302-766X 1432-0878 Springer Berlin Heidelberg Berlin/Heidelberg 3712 10.1007/s00441-022-03712-y Regular Article Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells Coatti Giuliana C. Paranjapye Alekh http://orcid.org/0000-0002-6541-2546 Harris Ann [email protected] grid.67105.35 0000 0001 2164 3847 Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106 USA 1 12 2022 19 31 5 2022 10 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-β signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies. Supplementary Information The online version contains supplementary material available at 10.1007/s00441-022-03712-y. Keywords Primary human epididymis epithelial cells Epididymis SMAD http://dx.doi.org/10.13039/100000071 National Institute of Child Health and Human Development HD0068901 Harris Ann ==== Body pmcIntroduction The epididymis is an important part of the male genital ducts, where it has a critical role in the maturation of spermatozoa. The human epididymis has three anatomical regions: the proximal segment, distal to the efferent ducts (head or caput), the elongated segment (body or corpus), and the distal segment (tail or cauda) (Sullivan and Mieusset 2016). Though, unlike in some other species, there are no septa demarcating these regions in the human organ, there is clear functional specialization between them. Processes of ion transport and response to hormone stimulus are enriched in the caput, suggesting a role in the maintenance of the luminal environment that is required for normal sperm maturation. In contrast, defense response processes were predominant in the transcriptome of corpus/cauda regions (Browne et al. 2015). Earlier, we described the establishment and characterization of primary cell cultures from the three segments of the human epididymis (Leir et al. 2015) using tissue samples from orchiectomy procedures. Orchiectomy is usually the first intervention in patients with testicular cancer and epididymal involvement is rare (Browne et al. 2016; Yang et al. 2018). Epididymis biopsies performed during epididymovasostomy procedures have also been used (Dubé et al. 2008). However, these successful primary cultures are dependent on a regular supply of surgical tissue samples, which became limiting during the COVID-19 pandemic. Moreover, the primary epididymal epithelial (HEE) cells have a limited proliferative capacity in culture. In general, though these cells can be passaged up to 8 times, they start to lose differentiation markers and replicative potential after 4–6 passages, in a donor-dependent manner. For both these reasons, our goal was to modify the HEE culture conditions in order to enhance their differentiated longevity and thus expand the primary epididymis cell cultures available for molecular and cell biological experiments. Replicative senescence and de-differentiation is a common feature of primary epithelial cells in culture. Others have shown that the transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) signaling activity is repressed in basal progenitor cells, but is highly active in differentiated epithelial cells (Mou et al. 2016). SMADs are transcriptional activators that transmit signals from cell surface-bound TGFβ family proteins (TGFβs, activins, and bone morphogenetic proteins (BMPs) to the nucleus). Thus, SMAD inhibition impairs the TGF-β /BMP signaling pathway. SMAD repression, in combination with WNT agonist and Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi) was shown to enhance the expansion and longevity of basal stem cells and other cell types (Levardon et al. 2018; Mou et al. 2016, 2021). Here, we use a similar protocol to attempt to extend the differentiated lifespan of primary HEE cells containing multiple cell types including basal progenitor cells (Browne et al. 2015; Leir et al. 2020a, b). We document the transcriptomic changes that accompany senescence of HEE cells and the reversal of some of these alterations by dual SMAD inhibition. Extended culture of HEE cells coincided with upregulation of genes in the TGF-β/BMP signaling cascade. TGF-β/BMP signaling is pivotal in determining cell fate and diverse cellular programs. Moreover, both TGF-β and BMP pathways are associated with both the control of reversible cell-cycle exit and with the maintenance of tissue stem cell niche populations (Guasch et al. 2007; He et al. 2004). When dysregulated, activation of the TGF-β/BMP pathways cause cell senescence and death (Schuster and Krieglstein 2002; Tominaga and Suzuki 2019). Exposure of HEE cells to the TGF-β/BMP antagonists, WNT agonist and ROCKi cocktail, restored the expression of TGF-β pathway members to pre-senescence levels. In addition, dual SMAD (DSI) treated HEE cells showed enhanced replicative potential and prolonged expression of differentiation markers. Material and methods Cell culture Human epididymis tissue was obtained with Institutional Review Board approval (donor 1, 24 years old; donor 2, 25 years old). Human epididymis epithelial (HEE) cells were isolated from the caput epididymis and cultured as described previously (Leir et al. 2015). P1 HEEs were cultured in Primaria flasks coated with type I collagen (1:60; Purecol 5005-B), either as described previously (CMRL1066 medium with 15% FBS, 2 mmol/L L-glutamine, 1 µg/mL hydrocortisone, 0.2 U/mL insulin, and 10−10 mol/L cholera toxin (NT)) or in the same media to which small molecule cocktail (dual SMAD inhibited, 1 µM A-8301, 1 µM DMH-1, 1 µM CHIR99021, and 5 µM Y27632) was added as per (Mou et al. 2016). For passaging, a fixed density of 2 × 104 cells/cm2 was maintained. RNA sequencing RNA was extracted from triplicate wells of p3 and p6 NT or DSI-treated HEE cells (from donor 1) using TRIzol, according to the manufacturer's protocol. cDNA was synthesized with oligo-dT, and libraries amplified and sequenced on a NovaSeq 6000 (SR 50 bp). Raw reads were aligned using STAR 2.7 (https://github.com/alexdobin/STAR) (Dobin et al. 2013). Aligned reads were assigned to genomic features with featureCounts version 1.6.1 (http://subread.sourceforge.net/) (Liao et al. 2014), and differential gene expression was analyzed using DEseq2 version 1.22.1. (https://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) (Love et al. 2014). Differentially expressed genes were filtered using log2 fold change ≥ + 1.5 and ≤ -1.5 and Benjamini–Hochberg adjusted p value ≤ 0.01. Dot plots for clustered results of each GO term were generated using the genome-wide annotation for human database and mapped using Entrez gene identifiers. Reverse transcription and quantitative PCR (RT-qPCR) RNA from p3 and p6 HEE cells (donor 2) was extracted using TRIzol, as above. cDNA was generated using TaqMan Reverse Transcription Reagents kit and oligo (dT)16. RT-qPCR was performed using primer pairs specific to each gene (supplementary Table 1) and SYBR green. β-2-Microglobulin expression was used to normalize gene expression levels. One-way ANOVA followed by Dunnett’s post-test was used to analyze the results. Western blots Whole cell lysates were obtained after lysing HEE cells in NET buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1X Sigma Protease Inhibitor). NT and DSI-treated cells at p3 and p6 from both donors were used. Nuclear and cytosolic fractions were obtained from p6 DSI-treated cells using NE-PER Nuclear and Cytoplasmic Extraction (Pierce Biotechnology) Reagents, following treatment of HEEs with 10 nM R1881 for 16 h. Proteins were analyzed by standard methods. Antibodies used for the androgen receptor (1:500, ab72742), SERPINE1 (1:1000, sc-5297), GAPDH (1:10,000, sc-47724), and Lamin A/C (1:1000, sc-7292) were used. Immunocytochemistry Primary adult HEEs were grown on 12-mm circular glass coverslips and fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized with 0.25% Triton-X100 in PBS for 15 min, followed by blocking for 1 h in 1% BSA in PBS. Antibodies used were specific to Androgen Receptor (1:100; sc-816), p63 (1:100, ab735), and KRT5 (1:200, ab52635). For detection, Alexa fluor-488 donkey anti-rabbit and Alexa fluor-647 goat anti-mouse secondary antibodies were used (1:2000). Proliferation assay and cell yield P6 HEEs (donor 1 and donor 2) were seeded in 96-well plates in triplicate. At 24, 48, 72, 96, 120, and 144 h post seeding, cells were fixed using methanol: acetic acid (3:1), and the number of cells in each well was counted after DAPI staining, using the BioTek Lionheart FX automated imager and Gen 5 image + analysis software (BioTek). Two-way Repeated Measure ANOVA comparing DSI with NT for each donor was used to analyze the data. To calculate the total yield, cells were grown to confluence in T25 culture flasks, trypsinized and counted. Then, 2 × 104 cells/cm2 were seeded into a new T25 flask at each passage. This procedure was repeated until the cell growth was arrested. The total yield is the addition of the number of cells counted at confluence at each passage. Results TGF-β/BMP signaling is activated in HEE cells with time in culture Primary HEE cells have a donor-dependent lifespan in culture, which may range from 3 to 8 passages (Leir et al. 2015). To evaluate the genome-wide impact of prolonged HEE culture, we performed RNA-seq on triplicate samples of passage 3 and passage 6 primary HEE cells from donor 1. Using a log2 fold change ≥ + 1.5 and ≤ -1.5 and adjusted p value ≤ 0.01, we identified 496 upregulated genes in passage 6 HEE cells in comparison to passage 3 cells (Fig. 1A). Among the most significantly upregulated transcripts in passage 6, HEE cells were those encoding members of the TGF-β signaling pathway including fibrillin 1 (FBN1), serpin family E member 1 (SERPINE1), transforming growth factor beta receptor 1 (TGFBR1), and thrombospondin 1 (THBS1).Fig. 1 The HEE cells transcriptome changes with time in culture. A Volcano plot of RNA-seq data showing up and down regulated genes in pre-senescent p6 NT HEE cells compared to proliferating p3 NT HEE cells. Volcano plot shows -log10 adjusted p value against log2 fold change. B Genes with log2 fold change ≥ + 1.5 and ≤ − 1.5 passing an adjusted p value threshold ≤ 0.01 were used for gene ontology process enrichment analysis. The dot plots show the top ten biological process (BP), cellular compartment (CC), and molecular function (MF) gene ontology terms that are enriched in DEGs either upregulated (top) or downregulated (bottom) in p6 compared to p3 NT HEE cells The proteins encoded by these genes act at different stages of the TGFβ signaling pathway. Fibrilin-1 forms extracellular microfibrils capable of regulating the bioavailability of TGFβ (Chaudhry et al. 2007). Thrombospondin-1 activates the TGFβ signaling cascade by controlling the conversion of latent to active TGFβ (Murphy-Ullrich and Poczatek 2000). TGFBR1 encodes a serine/threonine kinase responsible for transducing the TGFβ signal from the cell surface to the cytoplasm and SERPINE1 encodes one of the downstream targets of the TGFβ signaling (Sato et al. 1990). GO process enrichment analysis (Fig. 1B) performed for all the 496 upregulated differentially expressed genes (DEGs) revealed recurrent biological processes (BP) related to extracellular matrix organization and cell adhesion/locomotion. The prediction of these BPs was due to the overrepresentation of genes encoding extracellular matrix (ECM) structural constituents and binding proteins, such as ADAM metallopeptidase with thrombospondin type 1 Motif 5 (ADAMTS5), collagen type IV alpha 1 chain (COL4A1), and matrix metalloproteinase-14 (MMP14). Genes encoding integrins including integrin subunit alpha V (ITGAV) and integrin subunit beta 6 (ITGB6) were also amongst the top significant upregulated and highly expressed genes in passage 6 HEE cells. Several of these protein which participate in ECM remodeling are also part of the TGF-β pathway (Annes et al. 2004; Dong et al. 2017). We also identified 515 downregulated genes in passage 6 HEE cells in comparison to HEE cells at passage 3 (Fig. 1A). Among the significant downregulated genes are some with important functions in HEE cells (Browne et al. 2015). These include Defensin Beta 1 (DEFB1) and Claudin 2 (CLDN2), which encode proteins with roles in sperm motility (Diao et al. 2014) and tight junction permeability, respectively. In addition, Solute carrier family 6 member 6 (SLC6A6) and Solute carrier family 2 member 2 (SLC2A2) were also among the downregulated transcripts. These genes encode a sodium and chloride-ion dependent transporter and a glucose transporter, respectively. Recurrent GO terms (Fig. 1B) for the downregulated genes include categories related to cell cycle control and cell division, as HEE cells lost expression of several genes involved in chromosomal segregation, microtubule, and spindle organization and other related functions after prolonged (p6) time in culture. DSI-treated HEE cells show increased cell proliferation and extended in vitro lifespan Since the replicative senescence that occurred with time in HEE cultures was accompanied by activation of TGF-β signaling activation, we next tested the effect of a small molecule cocktail including TGF-β/BMP antagonists, a WNT agonist and ROCKi on HEE cells. This cocktail was shown elsewhere to enable the long-term culturing of basal cells from different epithelial tissues (Mou et al. 2016, 2021). Passage 1 HEE cells from two donors (donors 1 and 2) were cultured routinely for at least 6 passages either in CMRL1066 medium with supplements (non-treated, NT) (Leir et al. 2015) or the same media with the addition of DMH-1 and A8301 (TGF-β/BMP inhibitors), CHIR99021 (WNT activator) and Y-27632 (ROCK inhibitor), dual SMAD inhibited (DSI) (Mou et al. 2016). Examination of cell growth and morphology with increasing passage number (p) identified enlarged cells (white arrows) indicative of senescence in NT HEE cells at p4 (donor 1, Fig. 2A) and p3 (donor 2, Fig. S1). Proliferation of NT HEE cells at passage 6 was significantly lower than that of DSI-treated cells, as shown by counting fixed cells at serial time points after seeding (Fig. 2B). The growth of NT HEE cells was arrested by p7 and p8 for donors 1 and 2, respectively, while DSI-treated HEE cells exhibited growth arrest at p11 (donor 1) and p13 (donor 2) (Fig. 2C). Upon seeding one T25 flask per passaging event, the total yield of DSI over NT cells ranged between 1.6 and 1.9-fold (Fig. 2C).Fig. 2 DSI treatment impacts the morphology, proliferation and total yield of HEE cells. A Phase contrast images of serial passages of NT and DSI-treated HEE cells (donor 1). White arrows denote enlarged cells in the mixed population. B Graph denotes counts of DAPI-stained nuclei, fixed every 24 h for 6 days post-seeding. Data from p6 cells (donors 1 and 2) NT and DSI-treated HEEs are shown. The.p < 0.05 using a two-way Repeated Measure ANOVA comparing DSI with NT for each donor. C Table showing longevity and total yield of NT and DSI-treated cells (donors 1 and 2) (see methods for protocol) The DSI-treated HEE cells grew somewhat differently from NT cells, growing mostly as individual islands before confluence. Early passage DSI-treated HEE cells maintained a more homogeneous morphology up to p6, where enlarged cells started to appear within the population. In contrast, p6 NT cells had a very different (senescent) morphology compared to p3 NT cells. DSI treatment impacts TGF-β and BMP signaling pathways in HEE cells To determine the effect of dual SMAD inhibition on the HEE cell transcriptome, we performed RNA-seq on triplicate samples of p3 and p6 DSI-treated cells from donor 1 and compared these datasets to the transcriptomes of NT HEEs at the same passage number (Fig. 3A, B). Using a log2 fold change ≥ + 1.5 and ≤ -1.5 and adjusted p value ≤ 0.01, we identified 126 upregulated genes and 132 downregulated genes in DSI-treated HEE cells at p3 compared to control (Fig. 3A). Among the most downregulated genes are SERPINE1 and other members of the TGF-β pathway, such as SMAD family member 7 (SMAD7) and prostate transmembrane protein androgen induced 1 (PMEPA1). These genes encode respectively one of the signal transducers of the TFG-β/BMP cascade (SMAD7) and a negative regulator of TGF-β (PMEPA1) (Watanabe et al. 2010).Fig. 3 Impact of DSI treatment on HEE cell transcriptomes. A, B Volcano plot of RNA-seq data showing up- and downregulated genes comparing DSI -treated and NT HEE cells at p3 (A) and p6 (B). Plots show -log10 adjusted p value and log2 fold change. C For the same comparisons as in (A) and (B): RNA-seq DEseq2 normalized counts for TGF-β family members SERPINE1, FBN1, THBS1, and TGFBR1 (top); RT-qPCR data for SERPINE1 and TGFBR1 (middle, donor 1; bottom, donor 2). Data were normalized against NT p3. p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001 using a one-way ANOVA followed by Šídák's multiple comparisons test. D Representative western blot of total cell lysates from NT and DSI-treated cells shows lack of SERPINE1 in DSI-treated cells (donor 1); GAPDH is the loading control Using the statistical parameters described above, we identified 531 upregulated genes and 634 downregulated genes in DSI-treated HEE cells at p6 compared to the NT HEE p6 control cells (Fig. 3B). As shown in Fig. 3B and C, FBN1, SERPINE1, TGFBR1, and THBS1 were among the most downregulated targets in p6 DSI-treated HEE cells, consistent with these genes being upregulated in p6 HEE cells (Fig. 1A). SMAD7, PMEPA1, and other members of the TGF-β signaling cascade were also evident among genes downregulated in DSI-treated HEEs at p6. These transcriptome data were also confirmed at the protein level by western blot using an antibody specific for SERPINE1 (Fig. 3D), which showed elevated levels of the protein in p6 NT HEE cells compared to p3 NT HEE cells. No SERPINE1 was detected for DSI HEE cells, consistent with the RNA-seq data. Upregulation of several senescence-related markers were also detected in RNA-seq data from p6 NT cells but not in p6 DSI cells, which often showed the reduced expression (Fig. S2A). Consistent with this observation, NT cells but not DSI cells show enlarged nuclei at p6, one of the hallmarks of senescence (Davan-Wetton et al. 2021) (Fig. S2B). Upon the intersection of the DEG lists from the passage-matched comparisons of DSI-treated and NT cells and their interrogation on gene ontology analysis (Fig. S3), TGF-β-related and extracellular matrix-associated terms appears among the downregulated categories in DSI-treated cells. Although the RNA-seq data were generated on cells from donor 1, the results were validated using RT-qPCR on RNA extracted from both donors (Fig. S4). Based on our earlier description of the transcriptome of HEE cells (Browne et al. 2015), we chose to validate a set of caput cell-relevant genes. DSI-treated HEE cells maintain expression of markers of epididymal differentiation, including the androgen receptor To investigate the impact of the DSI treatment on cell population identity, we first used immunofluorescence to monitor the co-expression of p63 and KRT5, as markers of basal progenitor cells. The basal stem cell population was depleted rapidly in culture of both DSI-treated and NT HEE cells (Fig. S5). We next asked whether DSI-treated HEE cells maintain key markers of differentiation in HEE cells, again using our earlier data (Browne et al. 2015). To address this question, the RNA-seq normalized read counts were used to generate a heatmap of caput HEE cell identity genes. Hierarchical clustering performed on these targets showed that p6 DSI HEE cells maintained a transcriptome that was more similar to p3 NT HEE cells than to p6 NT cells (Fig. 4). Principal component analysis (PCA) of the total transcriptome shows closer association of passage 3 NT HEE cells to passage 6 DSI HEE cells than to passage 6 NT HEE cells (Fig. S6).Fig. 4 Heatmap and dendrogram of key markers of epididymal differentiation in NT and DSI-treated cells. DEseq2 normalized counts of each transcript are shown using RNA-seq data from p3 and p6 NT and DSI-treated cells (donor 1) Lastly, we examined the expression of the androgen receptor (AR) in DSI-treated HEE cells, since this is a pivotal transcription factor governing the differentiation and function of these cells. DSI treatment did not alter AR protein levels (Fig. 5A) or R1881-mediated AR activation (Fig. 5B). Nuclear localization of AR was also observed both in NT and DSI-treated HEEs (Fig. 5C).Fig. 5 DSI-treated HEE cells maintain expression of the androgen receptor. A, B Western blots probed with an antibody specific to AR. A Total protein from p3 and p6 NT and DSI-treated HEE cells, GAPDH is the loading control. B Nuclear and cytoplasmic fractions from R1881- treated HEE cells. GAPDH is the cytoplasmic marker and lamin A/C the nuclear marker. C Immunofluorescence images of p6 DSI-treated cells probed with AR antibody (green) and counterstained with DAPI (blue). (A, C donor 1; B donor 2) Discussion The primary cultures of human epididymis epithelial (HEE) cells that we described previously (Leir et al. 2015) have provided an extremely valuable tool to investigate the molecular basis of human epididymis function (Browne et al. 2016, 2018, 2019; Leir et al. 2020a, b; Yang et al. 2018). However, as for other primary epithelial cells, their restricted replicative potential limits their experimental applications. For example, they cannot be used for longer term gene editing. Our goals here were to examine the transcriptomic changes associated with loss of replicative potential and also determine whether we could prolong the life of primary HEE cells. We showed previously (Leir et al. 2015) that early passage caput-derived HEE cells are relatively small and fast-growing cells. With increased passage number, enlarged and irregularly shaped cells start to appear in the cultures, which ultimately reach replicative senescence and growth arrest. This ageing process is highly donor-dependent, but generally occurs by p8. To assess the molecular impact of extended culturing on HEEs, we compared the transcriptome of proliferating passage 3 cells with pre-senescent passage 6 cells. Major alterations in gene expression were observed, including down regulation of genes encoding proteins involved in cell division and upregulation of several members of the TGF-β signaling pathway. Of particular note is the profound upregulation of SERPINE-1 in passage 6 NT cells. SERPINE1 is a downstream effector of the TGF-β pathway and was found previously to be a critical target of p53 in the induction of replicative senescence (Kortlever et al. 2006). In addition to the loss of proliferative potential observed in NT cultures (Fig. 2C), a key feature that distinguishes senescence from other growth arrested cellular states is the “senescence-associated secretory phenotype” (SASP), comprising proinflammatory cytokines, chemokines, and proteases that are commonly secreted by senescent cells. Supporting our hypothesis, the expression of several SASP-associated genes was upregulated in NT cells compared to DSI -treated cells. The inhibition of the TGF-β/BMP signaling pathway using DMH-1 and A8301 was shown earlier to facilitate the expansion of primary airway cells and keratinocytes (Mou et al. 2016). Using the same strategy, basal cells isolated from mouse esophagus, epididymis, larynx/vocal fold, forestomach, and mammary gland could also be expanded to high passage numbers while still maintaining replicative potential (Mou et al. 2016). Building upon these results, we tested the effect of dual SMAD inhibition of the TGF-β/BMP signaling pathway on HEE cells. In comparison to NT HEE cells, DSI-treated HEE cells showed an extended lifespan in culture and produced more cells from the same starting number, across this time course. DSI-treated HEE cells were also actively proliferating at p6, when NT cells from this donor were already pre-senescent. Basal progenitor (KRT5 + /p63 +) cells were detected both in NT and DSI-treated HEE cells at p2, but no basal cells were evident in DSI-treated HEEs at p6. RNAseq analysis of p6 DSI-treated HEE cells revealed that DSI prevented the upregulation of TGF-β signaling pathway components that was evident in NT HEE p6 cells. PCA analysis showed that the transcriptome of DSI-treated p6 HEE cells was more similar to p3 NT HEE cells than NT HEE p6 cells. In addition, p6 DSI-treated HEE cells retained a molecular signature of important epididymal differentiation markers that was more similar to p3 than p6 NT HEE cells. Also relevant to the utility of this extended culture HEE model, DSI-treated HEE cells maintained normal expression of AR and its response to androgens. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file 1 (PDF 18220 KB) Acknowledgements We thank the Cleveland Clinic Central Biorepository for epididymis tissues, Dr. Shih-Hsing Leir for establishing primary HEE cell cultures, Dr. Jenny L. Kerschner for her contributions, and Dr. Pieter Faber at the University of Chicago Genomics Core for RNA-seq. Funding This work was supported by the National Institutes of Health (R01 HD068901) [A.H.]. Data Availability RNAseq data are deposited at NCBI GEO:GSE203535. Declarations Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of Case Western Reserve University research committee (CWRU IRB Protocol #2017–2099) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent (written) was obtained from all patients included in the study. Conflict of interest The authors declare no competing interests. Disclaimer The funders had no role in the design of the study, in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Annes JP Chen Y Munger JS Rifkin DB Integrin αvβ6-mediated activation of latent TGF-β requires the latent TGF-β binding protein-1 J Cell Biol 2004 165 5 723 734 10.1083/jcb.200312172 15184403 Browne JA Leir S-H Yin S Harris A Transcriptional Networks in the Human Epididymis HHS Public Access 2019 7 5 741 747 10.1111/andr.12629 Browne JA Leir SH Eggener SE Harris A Region-specific innate antiviral responses of the human epididymis Mol Cell Endocrinol 2018 473 72 78 10.1016/j.mce.2018.01.004 29339104 Browne JA Yang R Eggener SE Leir SH Harris A HNF1 regulates critical processes in the human epididymis epithelium Mol Cell Endocrinol 2016 425 94 102 10.1016/j.mce.2016.01.021 26808453 Browne JA Yang R Leir SH Eggener SE Harris A Expression profiles of human epididymis epithelial cells reveal the functional diversity of caput, corpus and cauda regions Mol Hum Reprod 2015 22 2 69 82 10.1093/molehr/gav066 26612782 Chaudhry SS Cain SA Morgan A Dallas SL Shuttleworth CA Kielty CM Fibrillin-1 regulates the bioavailability of TGFβ1 J Cell Biol 2007 176 3 355 367 10.1083/jcb.200608167 17242066 Davan-Wetton CSA, Pessolano E, Perretti M, Montero-Melendez T (2021) Senescence under appraisal: hopes and challenges revisited. 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==== Front Monatsschr Kinderheilkd Monatsschr Kinderheilkd Monatsschrift Kinderheilkunde 0026-9298 1433-0474 Springer Medizin Heidelberg 1657 10.1007/s00112-022-01657-8 Konsensuspapiere Wann sind Antikörperbestimmungen im Serum vor oder nach Impfungen sinnvoll und wann nicht? Stellungnahme der Kommission für Infektionskrankheiten und Impffragen des Bündnis Kinder- und Jugendgesundheit e. V. When are antibody determinations in serum before or after vaccination indicated and when not?Statement of the Committee for Infectious Diseases and Vaccinations of the Alliance Child and Adolescent Health Heininger Ulrich [email protected] Kommission für Infektionskrankheiten und Impffragen des Bündnis Kinder- und Jugendgesundheit e. V.von Both U. Grundhewer H. Heininger U. Huppertz H.-I. Iseke A. Knuf M. Korenke G. Ch. Müller A. [email protected] http://www.buendnis-kjg.de Kommission für Infektionskrankheiten und Impffragen, Bündnis Kinder- und Jugendgesundheit e. V., Chausseestr. 128/129, 10115 Berlin, Deutschland Redaktion Arndt Borkhardt, Düsseldorf Stefan Wirth, Wuppertal 30 11 2022 15 28 10 2022 © The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. In der Praxis stellt sich häufig die Frage nach der Sinnhaftigkeit der Bestimmung von Antikörperwerten bzw. Titern zur Überprüfung des Impfschutzes vor bzw. nach Impfungen. Dies kann in Ausnahmesituationen sinnvoll sein, z. B. bei Personen unter immunsuppressiver Therapie bzw. bei Verdacht auf Immundefizienz, bei Patienten mit unbekanntem Impfstatus sowie in definierten Risikosituationen (z. B. Überprüfung des Hepatitis-B-Impfschutzes nach Nadelstichverletzungen bzw. des COVID-19-Impfschutzes bei bestimmten Formen einer Immundefizienz). Im Gegensatz dazu sind für keine allgemein empfohlenen Standardimpfungen individuelle serologische Kontrollen empfohlen oder sinnvoll. Zum einen deswegen, weil aus den Zulassungsstudien oder langjähriger Anwendung bekannt ist, dass bei Einhalten der Impfempfehlungen eine ausreichend hohe individuelle Schutzwahrscheinlichkeit besteht (z. B. > 99 % bei Tetanus), zum anderen, weil die Impfstrategie primär auf indirekten Populationsschutz und nicht auf Schutz jeder einzelnen Person abzielt. Die wenigen Personen, die trotz Impfung nicht direkt geschützt sind („Impfversager“), profitieren somit indirekt von dem reduzierten Expositionsrisiko. Dies wurde an den Beispielen der oralen Poliomyelitis-Impfung in den 1960er-, der MMR-Impfung seit den 1970er- und der Hib-Impfung seit den 1990er-Jahren gezeigt. Diese Stellungnahme unserer Kommission zeigt die Evidenz zum sinnvollen Vorgehen auf, wann welche Antikörperbestimmungen nach bzw. vor einer Impfung hilfreich und aussagekräftig sind, und wann man sie besser unterlassen sollte. Sie lehnt sich dabei eng an die STIKO-Impfempfehlungen bzw. deren Anwendungshinweise an. Wir raten davon ab, dem Wunsch mancher Eltern nach medizinisch nicht begründeten Titerbestimmungen bei ihrem Kind nachzukommen. Questions about the usefulness of determining antibody values or titers to demonstrate protection often arise before or after vaccinations in immunization clinics. Such measurements may be useful in exceptional situations, e.g., in immunocompromised patients or those with suspected immunodeficiency, in patients with unknown vaccination status, and in certain defined risk situations (e.g., hepatitis B serology after needlestick injuries or after COVID-19 vaccination in patients with certain forms of immunodeficiency). In contrast, individual serological antibody measurements are neither recommended nor useful after generally recommended vaccinations. This is because either it is known from licensure studies or long-term use that there is a sufficiently high individual probability of protection (e.g., > 99% for tetanus) when the recommended vaccination schedules are adhered to or because the vaccination strategy is primarily aimed at indirect protection of the population and not at protection of each individual person. The few individuals who are not sufficiently protected after vaccination (vaccination failures) will still benefit indirectly from the reduced risk of exposure. This has been demonstrated with oral poliomyelitis vaccination in the 1960s, MMR vaccination since the 1970s, and Hib vaccination since the 1990s. This statement of our committee shows the evidence for a sensible approach, when and which antibody determinations are helpful and meaningful after or before vaccination and which are not. The statement is based on Germany’s Standing Committee on Vaccination (STIKO) recommendations and their administration instructions. We advise not to comply with the wish of some parents for titer determinations in their child that are not medically justified. Schlüsselwörter Antikörper Titer Schutzkorrelat Impfung Serologie Keywords Antibody determination Titer Correlate of protection Vaccination Serology ==== Body pmcIn der Praxis stellt sich immer wieder die Frage nach der Sinnhaftigkeit einer spezifischen, erregerbezogenen Bestimmung von Antikörperwerten (d. h. Konzentrationen im Serum) bzw. Titern (d. h. Verdünnungsstufen von Serum). Für manche, insbesondere diagnostische Fragestellungen kann das durchaus sinnvoll sein: z. B., ob es sich bei einem hoch fiebernden, nichtgeimpften Patienten mit Husten und makulopapulösem Exanthem um Masern handeln könnte (Test: IgM-Bestimmung im Serum). Auch zur Bestimmung des Impfschutzes vor bzw. nach immunsuppressiver Therapie bzw. bei bekannter oder Verdacht auf Immundefizienz, bei Patienten mit unbekanntem Impfstatus sowie in definierten Risikosituationen (wie z. B. Überprüfung des Hepatitis-B-Impfschutzes nach Nadelstichverletzungen) ergeben sich sinnvolle Fragestellungen. Dagegen lässt sich grundsätzlich festhalten, dass für keine der von der STIKO allgemein empfohlenen Standardimpfungen individuelle serologische Kontrollen empfohlen sind. Entweder,weil aus den Zulassungsstudien oder langjähriger Anwendung bekannt ist, dass bei Einhalten der Impfempfehlungen, inkl. eventueller Auffrischungen, eine ausreichend hohe individuelle Schutzwahrscheinlichkeit besteht (z. B. > 99 % bei Tetanus) oder weil die Impfstrategie primär auf indirekten Populationsschutz und nicht auf direkten Schutz einer jeden einzelnen Person abzielt (alle anderen Standardimpfungen). Das heißt, Personen, die trotz Impfung nicht direkt geschützt sein sollten, profitieren indirekt von dem reduzierten Expositionsrisiko. Das gilt für alle Standardimpfungen außer Tetanus. Dieses Konzept ist eindrucksvoll an den Beispielen der oralen Poliomyelitis-Impfung in den 1960er-, der MMR-Impfung seit den 1970er- und der Hib-Impfung in den 1990er-Jahren gezeigt worden. Diese Stellungnahme soll die verfügbare Evidenz zum sinnvollen Vorgehen darstellen, wann welche Antikörperbestimmungen nach bzw. vor einer Impfung hilfreich und aussagekräftig sind, und wann man sie besser unterlassen sollte. Sie lehnt sich eng an die STIKO-Impfempfehlungen bzw. deren Anwendungshinweise an [1–5]. Für welche Impfungen gibt es serologische Schutzkorrelate? Man muss sich über die fehlende oder eingeschränkte Aussagekraft vieler Antikörperbestimmungen zur Feststellung eines Impfschutzes bewusst sein. Oftmals sind die Testverfahren nicht geeignet, die Frage „Besteht Impfschutz vor Krankheit x?“ zu beantworten. Dementsprechend schwierig bis unmöglich ist dann die Interpretation des Ergebnisses. Nur, wenn ein sogenanntes serologisches Schutzkorrelat bekannt und zuverlässig ist, d. h. im Rahmen von Studien ermittelt und definiert wurde, kann die Bestimmung sinnvoll sein – aber selbst dann nur in klar definierten Situationen. Im Rahmen der Erstellung von Anwendungshinweisen zum Impfen bei Immundefizienz hat die STIKO im Grundlagenpapier 2017 die akzeptierten Schutzkorrelate in einer Tabelle dargestellt (Tab. 1 in [2]). In der Tabelle sind auch die Impfungen aufgeführt, für die kein zuverlässiges Schutzkorrelat bekannt ist. Die Zuverlässigkeit der Korrelate ist sehr heterogen. Im Falle von Masern heißt es: „bei Nachweis von Masern-IgG kann von einem Schutz ausgegangen werden“, was für neutralisierende Antikörper (die i. d. R. nur in Referenzlaboratorien verfügbar sind) eher zutrifft als für die im ELISA-Verfahren nachgewiesenen Antikörper. Neben der Prätestwahrscheinlichkeit ist hier auch die Güte des jeweils verwendeten Testverfahrens von entscheidender Bedeutung. Diese ist leider gemäß Ringversuchen – z. B. zur Bestimmung von Röteln-IgG [6] – sehr variabel. Dennoch bietet die Tabelle (Tab. 1) einen Anhalt zur Interpretation von Laborwerten bei individuellen Fragestellungen.Impfung Testverfahren Antikörperkonzentration/Titer Anmerkungen Diphtherie ELISA; Toxin NT 0,1 IU/ml – Hepatitis A ELISA 10 mIU/ml – Hepatitis B ELISA Anti-HBsAG: 10 IE/l Korrelat für Langzeitschutz: 100 IE/l Hib ELISA 0,15 μg/ml Anti-PRP Auch bei nichtnachweisbaren Antikörpern können Gedächtniszellen induziert worden sein HPV ELISA, Multiplex-Serologie n. d. – Masern ELISA, NT n. d. Bei Nachweis von Masern-IgG kann von einem Schutz ausgegangen werden Meningokokken hSBA ≥ 1:4 – Mumps ELISA, NT n. d. Bei Nachweis von Mumps-IgG kann von einem Schutz ausgegangen werden. Reinfektionen sind möglich Pertussis – n. d. Kein serologisches Schutzkorrelat bekannt Pneumokokken Unterschiedliche Serotypen in einem Impfstoff; Grenzwerte beruhen auf Studien bei Kindern, Übertragbarkeit auf Erwachsene fraglich – PCV ELISA PCV13: ≥ 0,35 μg/ml – PPSV ELISA; Opsonophagozytose PPSV23: 0,20–0,35 μg/ml; 1:8 Dilution Poliomyelitis NT > 1:4 Neutralisationstest weist definitionsgemäß schützende Antikörper nach Röteln ELISA 10–15 IU/ml Unterschiedliche Testverfahren international nicht standardisiert; zelluläre Immunität Tetanus ELISA 0,1 IU/ml – Varizellen ELISA, FAMA ELISA: n. d. FAMA: 1:2 bis 1:4 ELISA: je nach Labor und Test-Kit unterschiedliche Grenzwerte NT Neutralisationstest, n. d. nicht definiert, ELISA „enzyme-linked immunosorbent assay“, FAMA Fluoreszenz-Antikörper-Membran-Antigen-Test (nur im VZV-Referenzlabor Jena), PRP Hib Polyribosylribitol-Phosphat, hSBA „serum bactericidal activity with human complement“ Vorgehen in besonderen Risikosituationen Patienten mit Immundefizienz Für immunsupprimierte Patienten haben Antikörperbestimmungen eine andere Bedeutung als für immunkompetente Patienten. Wir empfehlen ein standardisiertes Vorgehen nach Vorgaben der STIKO in ihren Anwendungshinweisen zum Impfen bei Immundefizienz: Grundlagen [2], Impfen bei primären Immundefekterkrankungen und HIV-Infektion [3], Impfen bei Autoimmunkrankheiten, bei anderen chronisch-entzündlichen Erkrankungen und unter immunmodulatorischer Therapie [4] sowie Impfen bei hämatologischen und onkologischen Erkrankungen (antineoplastische Therapie, Stammzelltransplantation), Organtransplantation und Asplenie [5]. Beruflich indizierte Impfungen (Hepatitis B, Tollwut) Hepatitis B Von einer erfolgreichen Hepatitis-B-Impfung kann bei einem Anti-HBs-Antikörperwert ≥ 100 IE/l ausgegangen werden. Dieser Wert korreliert mit Langzeitschutz vor klinisch manifester Hepatitis B. Die Antikörperkontrolle sollte idealerweise 4 bis 8 Wochen nach Abschluss der Grundimmunisierung vorgenommen werden. Eine Kontrolle soll bei beruflicher Indikation erfolgen, welche die STIKO wie folgt definiert: „Personen mit erhöhtem arbeitsbedingten Expositionsrisiko, einschließlich Auszubildender, PraktikantInnen, Studierender und ehrenamtlich Tätiger mit vergleichbarem Expositionsrisiko, z. B. Personal in medizinischen Einrichtungen (einschließlich Labor- und Reinigungspersonal), Sanitäts- und Rettungsdienst, betriebliche ErsthelferInnen, PolizistInnen, Personal von Einrichtungen, in denen eine erhöhte Prävalenz von Hepatitis-B-Infizierten zu erwarten ist (z. B. Gefängnisse, Asylbewerberheime, Einrichtungen für Menschen mit Behinderungen).“ Ferner weist die STIKO darauf hin, dass die angeführten Personengruppen exemplarischen Charakter haben und keine abschließende Indikationsliste darstellen. Vielmehr soll man das tatsächliche Expositionsrisikos einschätzen [1].Bei Anti-HBs-Werten von 10–99 IE/l („Low Responder“) wird sofort eine weitere Impfstoffdosis empfohlen, wiederum gefolgt von einer Anti-HBs-Kontrolle nach 4 bis 8 Wochen. Falls der Wert wieder < 100 IE/l beträgt, empfiehlt die STIKO bis zu 2 weitere Impfstoffdosen, jeweils mit anschließender Anti-HBs-Kontrolle nach 4 bis 8 Wochen. Nach 6 erfolglosen Impfstoffdosen (Anti-HBs 10–99 IE/l) muss das weitere Vorgehen individuell besprochen werden (Erläuterungen in [7]). Erfolgreiche Impfserien mit deutlich mehr als 6 Impfstoffdosen sind dokumentiert. Bei einem Anti-HBs < 10 IE/l (sogenannte „Nonresponder“) sollen HBsAg und Anti-HBc zum Ausschluss einer chronischen HBV-Infektion im Serum bestimmt werden. Falls beide Parameter negativ sind, ist das weitere Vorgehen, wie oben bei „Low-Respondern“ beschrieben. Bei früher (z. B. im Säuglingsalter) gegen Hepatitis B geimpften Personen mit neu aufgetretenem Infektionsrisiko und unbekanntem Anti-HBs sollte erst eine weitere Hepatitis-B-Impfstoffdosis verabreicht werden, ehe die serologische Erfolgskontrolle erfolgt. Das Vorgehen ist dann, wie oben beschrieben. Ist ein Anti-HBs-Wert ≥ 100 IE/l dokumentiert, besteht Langzeitschutz, und Auffrischimpfungen sind nicht indiziert. CAVE: Nach Standardimpfungen im Kindes- und Jugendalter ist eine Anti-HBs-Titerkontrolle nicht sinnvoll. Tollwut Die STIKO empfiehlt für Laborpersonal, welches mit dem Tollwutvirus arbeitet, nach der Grundimmunisierung alle 6 Monate die Bestimmung von neutralisierenden Antikörpern gegen Tollwutviren im Serum. Eine Auffrischimpfung ist bei einem Wert < 0,5 IE/ml Serum indiziert. Vorgehen bei unbekanntem Impfstatus Bei fehlender oder unvollständiger Dokumentation von Impfungen sollte im Interesse der betroffenen Person von fehlenden Impfungen ausgegangen werden und altersentsprechend Nachholimpfungen empfohlen werden (Tabellen 10A–E der jeweils aktuellen Impfempfehlungen der STIKO [1]). Im Rahmen einer Nachholimpfserie mit Totimpfstoffen (insbesondere tetanustoxoidhaltige Kombinationsimpfstoffe) können an der Impfstelle ausgeprägte Nebenwirkungen (Schmerzen, Schwellung, Rötung, Überwärmung) im Sinne eines Arthus-Phänomens entstehen. Dafür können hohe vorbestehende Serum-Antikörper-Konzentrationen ursächlich verantwortlich sein. In dieser Situation kann deshalb vor weiteren Impfungen eine Antikörperbestimmung gegen Tetanustoxin empfohlen werden. Beträgt der Wert > 0,1 IU/ml, ist von einem ausreichend hohen Schutz auszugehen, und die Nachholimpfserie kann abgebrochen werden. Da die Tetanus-Impfung im Allgemeinen nicht als Einzel-, sondern als Kombinationsimpfung mit Diphtherie und Pertussis (und ggf. weiteren Komponenten) durchgeführt wurde, kann dieser Wert als Surrogatmarker auch für diese Impfungen herangezogen werden. COVID-19 Für Antikörperbestimmungen nach COVID-19-Impfung bei Patienten mit Immundefizienz verweisen wir auf die jeweils aktuellen Empfehlungen der STIKO [8]. Derzeit empfiehlt die STIKO keine serologische Antikörpertestung zur Überprüfung des Impferfolges bei geimpften Personen. Diese Antikörpertestung soll auch dann nicht erfolgen, wenn die Personen älter sind, Vorerkrankungen oder eine Immundefizienz haben. Begründung: Der Wert, der einen sicheren Schutz bedeutet und damit eine oder mehrere Impfstoffdosen unnötig machen würde, ist nicht bekannt. Zudem beruht die Immunantwort gegen SARS-CoV‑2 auch ganz wesentlich auf dem zellulären Arm des Immunsystems, der hier nicht abgebildet wird. Lediglich bei „schwer immundefizienten Personen mit einer erwartbar stark verminderten Impfantwort“ (Verweis auf Tab. 8 der 20. Aktualisierung vom 24. Mai 2022) ≥ 4 Wochen nach der 2. Impfstoffdosis UND ≥ 4 Wochen nach der 3. Impfstoffdosis ist eine quantitative serologische Untersuchung auf spezifische Antikörper gegen das SARS-CoV-2-Spike-Protein empfohlen (Gesamtprotein-S1-Untereinheit oder Rezeptorbindungsdomäne). Mit diesem Vorgehen kann bei initial fehlender oder niedriger Antikörperantwort die Antikörperkinetik (gibt es einen Anstieg?) beurteilt werden. Die Blutentnahme für die erste Antikörpermessung kann am selben Termin durchgeführt werden, an dem die 3. Impfstoffdosis verabreicht wird; das Antikörperergebnis muss aus den o. g. Gründen für die Gabe der 3. Impfstoffdosis nicht abgewartet werden. Sollten auch nach der 3. Impfstoffdosis unverändert sehr niedrige oder keine spezifischen Antikörper messbar sein, verweist die STIKO auf verschiedene weitere Maßnahmen [8]. Vorgehen bei Wunsch von „Titerbestimmungen“ von Patienteneltern Pädiater werden regelmäßig mit dem Wunsch oder der Forderung von Eltern konfrontiert, bei ihrem Kind den individuellen Impfschutz zu messen. Besonders typisch ist der Wunsch, vor oder nach einer anstehenden oder fehlenden 1. oder 2. MMR-Impfung den „Maserntiter“ zu bestimmen. Mit Einführung des Masernschutzgesetzes und dessen Implikationen für den Besuch von Gemeinschaftseinrichtungen hat sich dies intensiviert. Hintergrund ist, dass für die Zulassung in Gemeinschaftseinrichtungen der Nachweis einer Masernimmunität (nicht näher definiert) oder die vollständige Impfung (also 2 Dosen MMR) gefordert wird. Manche Eltern wollen deshalb entweder bereits vor der 1. oder aber nach der 1. MMR-Impfung den „Maserntiter“ bei ihrem Kind bestimmt haben, um im Falle eines positiven Ergebnisses ganz auf die MMR-Impfung bzw. die 2. Dosis verzichten zu können. Wir lehnen dies ab, weil die Spezifität der kommerziell verfügbaren ELISA-Verfahren nicht 100 % beträgt und somit der Schutz gegen Masern beim Kind nicht garantiert werden kann. Hinzu kommt, dass auch der Schutz gegen Mumps (kein zuverlässiges serologisches Korrelat bekannt!) bzw. Röteln nicht gewährleistet ist. Zudem verursacht die Antikörperbestimmung unnötige Kosten und Schmerzen bei der Blutentnahme. Manche besonders besorgte Eltern erbitten die Masern-IgG-Antikörper-Bestimmung nach der 2. MMR-Impfung, um sicher zu sein, dass ihr Kind auch wirklich geschützt ist. Ähnlich wie bei Röteln beträgt die Schutzwahrscheinlichkeit nach 2 Impfdosen > 99 % und ist somit zuverlässiger als eine IgG-Antikörper-Bestimmung. Mit anderen Worten: Nach 2 MMR-Impfungen gibt es mehr falsch-negative als richtig-negative Masern-IgG-Test-Ergebnisse. Aus diesem Grund wird selbst bei schwangeren Frauen (also einer Hochrisikopopulation bei Auftreten von Röteln) seit vielen Jahren keine Röteln-IgG-Antikörper-Bestimmung mehr in der Schwangerschaft empfohlen, wenn 2 dokumentierte Röteln-Impfungen vorliegen. Vielmehr sagt die STIKO dazu [9]:„Liegt der Nachweis über zwei erfolgte Rötelnimpfungen vor, ist von einer Immunität auszugehen, weitere Maßnahmen wie Titerkontrollen sind nicht erforderlich. Der serologische Nachweis von Antikörpern ist nur bei Schwangeren ohne entsprechende Nachweise einer bestehenden Immunität (Ungeimpfte oder einmalig Geimpfte oder Impfanamnese unbekannt) sinnvoll. International wird als schützender Titer ein Wert von 10 bis 15 IU/ml im ELISA-Test angesehen. In Deutschland gilt bislang die Empfehlung der Diagnostik-Kommission der Gesellschaft für Virologie (GfV) und der Deutschen Vereinigung zur Bekämpfung der Viruskrankheiten (DVV), dass bei Werten zwischen 15 IU/ml und 34 IU/ml ein Zweittest herangezogen werden soll. Dafür kann der früher verwendete HHT eingesetzt werden, bei dem Titer ab < 1:8 als ausreichend positiv angesehen werden. Aufgrund der schlechten Standardisierbarkeit der Rötelnteste steht zu vermuten, dass in Zukunft der generelle Nachweis von Anti-Röteln-IgG-Antikörpern, d. h. ein grundsätzlich positives Testergebnis, ausreichend sein wird.“ Deshalb halten wir auch nach der 2. MMR-Impfung den Wunsch zur Bestimmung von Masern‑, Mumps- und Röteln-Antikörpern nicht für sinnvoll und lehnen ihn ab. Stellungnahme der Kommission Die Bestimmung von Antikörperkonzentrationen bzw. Titern nach Impfungen sollte regelhaft auf die von der STIKO definierten Situationen begrenzt und gut begründet sein [10]. Die variable Testgüte mit zu erwartenden unterschiedlichen Ergebnissen aus identischen Seren und die Bedeutung der Prätestwahrscheinlichkeit gilt es dabei zu beachten. Von darüber hinausgehenden Messungen auf individuellen Wunsch der Patienteneltern raten wir ausdrücklich ab, weil sie nicht sinnvoll interpretiert werden können. Mitglieder der Kommission für Infektionskrankheiten und Impffragen des Bündnis Kinder- und Jugendgesundheit e. V. PD Dr. med. U. von Both (München); Dr. med. H. Grundhewer (Berlin); Prof. Dr. med. U. Heininger (Basel; Kommissionssprecher und Federführung); Prof. Dr. med. H.-I. Huppertz (Bremen); Dr. med. A. Iseke (Münster); Prof. Dr. med. M. Knuf (Worms); Prof. Dr. med. G. Ch. Korenke (Oldenburg); Prof. Dr. med. A. Müller (Bonn) Einhaltung ethischer Richtlinien Interessenkonflikt H.-I. Huppertz: Participation on a Data Safety Monitoring Board or Advisory Board: Pfizer on meningococcal vaccines, BioNTech on COVID vaccines, GSK on children’s vaccines. U. Heininger, U. von Both, H. Grundhewer, A. Iseke, M. Knuf, G. Ch. Korenke und A. Müller geben an, dass kein Interessenkonflikt besteht. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. Die Mitglieder der Kommission für Infektionskrankheiten und Impffragen des Bündnis Kinder- und Jugendgesundheit e. V. werden am Beitragsende gelistet. QR-Code scannen & Beitrag online lesen ==== Refs Literatur 1. Ständige Impfkommission Empfehlungen der Ständigen Impfkommission (STIKO) beim Robert Koch-Institut 2022 Epidem. Bull. 2022 4 3 66 2. Niehues T Bogdan C Hecht J Mertens T Wiese-Posselt M Zepp F Impfen bei Immundefizienz: Anwendungshinweise zu den von der Ständigen Impfkommission empfohlenen Impfungen. (I) Grundlagenpapier Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2017 60 674 684 10.1007/s00103-017-2555-4 28466129 3. Ehl S Bogdan C Niehues T Burchard G Baumann U Hecht J Impfen bei Immundefizienz: Anwendungshinweise zu den von der Ständigen Impfkommission empfohlenen Impfungen. (II) Impfen bei 1. Primären Immundefekterkrankungen und 2. HIV-Infektion Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2018 61 8 1034 1051 10.1007/s00103-018-2761-8 30062550 4. Wagner N Assmus F Arendt G Baum E Baumann U Bogdan C Impfen bei Immundefizienz: Anwendungshinweise zu den von der Ständigen Impfkommission empfohlenen Impfungen. (IV) Impfen bei Autoimmunkrankheiten, bei anderen chronisch-entzündlichen Erkrankungen und unter immunmodulatorischer Therapie Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2019 62 4 494 515 10.1007/s00103-019-02905-1 30899964 5. Laws HJ Baumann U Bogdan C Burchard G Christopeit M Hecht J Impfen bei Immundefizienz: Anwendungshinweise zu den von der Ständigen Impfkommission empfohlenen Impfungen. (III) Impfen bei hämatologischen und onkologischen Erkrankungen (antineoplastische Therapie, Stammzelltransplantation), Organtransplantation und Asplenie Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2020 63 588 644 10.1007/s00103-020-03123-w 32350583 6. Kowalzik F Faber J Knuf M Korrelate für Infektionsschutz nach Impfung Monatsschr Kinderheilkd 2017 165 588 595 10.1007/s00112-017-0313-1 7. Ständige Impfkommission (2013) Mitteilung der Ständigen Impfkommission (STIKO) am Robert Koch-Institut (RKI). Epid Bull 36/37. https://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2013/Ausgaben/36_37_13.pdf?__blob=publicationFile 8. https://www.rki.de/DE/Content/Infekt/Impfen/ImpfungenAZ/COVID-19/Impfempfehlung-Zusfassung.html. Zugegriffen: 22.11.2022 9. https://www.rki.de/SharedDocs/FAQ/Impfen/MMR/FAQ-Liste_Roeteln_Impfen.html. Zugegriffen: 22.11.2022 10. Heininger U Plotkin S Titerphilia—The Irresistible Urge to Measure Postimmunization Antibody Values Pediatr Infect Dis J 2022 41 6 490 491 10.1097/INF.0000000000003519 35446805	36471874	PMC9713190	NO-CC CODE	2022-12-02 23:22:56	no	Monatsschr Kinderheilkd. 2022 Nov 30;:1-5	utf-8	Monatsschr Kinderheilkd	2,022	10.1007/s00112-022-01657-8	oa_other
==== Front Cardiovasc Intervent Radiol Cardiovasc Intervent Radiol Cardiovascular and Interventional Radiology 0174-1551 1432-086X Springer US New York 36450993 3319 10.1007/s00270-022-03319-4 Letter to the Editor Transarterial Embolization for Shoulder Injury Related to Vaccine COVID-19 Administration http://orcid.org/0000-0003-4835-7976 Fernández Martínez Ana María [email protected] [email protected] 1 Cuesta Marcos M. Teresa 2 Rodríguez Prieto Joaquín 3 1 grid.411969.2 0000 0000 9516 4411 Vascular and Interventional Radiology, University Hospital of León, Calle Altos de Nava, SN, 24080 León, Spain 2 grid.411969.2 0000 0000 9516 4411 Radiology Department, University Hospital of León, León, Spain 3 grid.411969.2 0000 0000 9516 4411 Physical Medicine and Rehabilitation, University Hospital of León, León, Spain 30 11 2022 13 17 10 2022 13 11 2022 © Springer Science+Business Media, LLC, part of Springer Nature and the Cardiovascular and Interventional Radiological Society of Europe (CIRSE) 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcIntroduction Shoulder injury related to vaccine administration (SIRVA) is a preventable and well-described injury occurring after the administration of a vaccine into anatomic structures adjacent to the deltoid muscle. It causes shoulder pain and limitation of the range of motion due to a mechanical and chemical trauma that triggers an inflammatory response to the vaccine. It is mostly known after influenza vaccination and has also been described after COVID-19 vaccination in this pandemic [1]. The most common diagnoses are adhesive capsulitis (AC) and bursitis, and both manifest with pain and limitation of the shoulder mobility [1, 2]. We present a case of SIRVA due to a COVID-19 vaccination and treated by transarterial embolization (TAE) and physiotherapeutic treatment. Fifty-year-old woman presents painful left shoulder stiffness 24 h after administration of the first dose of COVID-19 vaccine. She refers to a pain of 9 on the visual analogical scale (VAS) with limitation of the mobility especially on internal rotation. Subsequently she develops progressive clinical and functional worsening. After the second dose, one month later, a venous cord and axillary lymphadenopathy appeared, accompanied by increased pain and rapid evolution to loss of mobility. Rehabilitation physicians advise against physiotherapeutic treatment for severe pain, so she is managed with oral analgesia without response. It was decided to perform a magnetic resonance (MR) study where edematous changes and severe thickening of the synovium and the capsule at the axillary recess, moderate obliteration of the fat in the rotator interval and diffuse enhancement after intravenous contrast administration were identified (Fig. 1).Fig. 1 Severe thickening of the synovium and the capsule with a diffuse enhancement after intravenous contrast administration on T1 fat sat gradient echo with MIP reconstruction is visualized (arrows) After these findings, it was decided to perform an angiography of the left shoulder, where areas of pathological blush enhancement were identified in the axillary recess and in the rotator interval—superior capsule (Fig. 2A). An injection of 9 ml (ml) of iodinated contrast media was performed at 3 ml per second through a 6 French guide catheter. Selective microcatheterization of the anterior humeral circumflex, coracoid and acromial arteries, respectively, was performed with a 1.7 F microcatheter (Fig. 2B, C), and they were embolized distally with 2.4 ml of a mixture of imipenem/cilastatin sodium and iodinated contrast media until there was a complete stasis with reflux back along the microcatheter tip. In control angiography, the pathological areas have disappeared except post-vaccination subcutaneous area where blush enhancement persists (Fig. 3). In addition, due to flow redistribution after embolization it was not visualized in the first injection.Fig. 2 A Digital subtraction angiography performed from left subclavian artery where there is a capsular enhancement at the axillary recess (arrow) and the rotator interval—superior capsule (arrowhead). Superselective arteriogram by manual injection from the anterior humeral circumflex artery (B) and from the acromial artery (C) where capsular enhancement (arrow) can be observed Fig. 3 Result after embolization where a normal angiographic pattern can be seen except post-vaccination subcutaneous area where blush enhancement persists (arrow) The patient reported an immediate decrease in pain, being 3 on VAS one week after TAE. She started rehabilitation with great improvement in mobility, and three months after embolization, shoulder mobility has completely recovered. Clinical results have remained stable during one year of follow-up. Many authors have described the importance of hypervascularization of the capsule in the pathophysiology of AC. It is known that angiogenesis is a necessary factor to generate an inflammatory state and Okuno et al. reported the existence of pathological angiographic hypervascularization in all patients in their series [3, 4]. The objective of TAE is to decrease the abnormal vascularization responsible for the inflammatory state that also occurs in SIRVA [1]. Also, TAE has emerged as a therapeutic option in patients with AC refractory to conventional treatment [3–5]. SIRVA is a well-established condition in the medical literature; and there are examples after administration of the COVID-19 vaccine. There are references of arterial embolization in secondary stiff shoulder but not about SIRVA treated by TAE [1, 5]. This case corresponds to a SIRVA of synovitis–capsulitis due to an inflammatory response of the capsule to the vaccine. Transarterial embolization and physiotherapeutic treatment were associated with reduced pain and improved mobility. Funding This study was not supported by any funding. Declarations Conflict of interest The authors declare that they have no conflict of interest. Consent for Publication Consent for publication was obtained for every individual person’s data included in the study. Ethical Approval The study performed was in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Review Board (IRB). Our hospital clinical research ethics committee approved this study. Informed Consent Informed consent was obtained from the patient included in the study. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Bass JR Poland GA Shoulder injury related to vaccine administration (SIRVA) after COVID-19 vaccination Vaccine 2022 40 4964 4971 10.1016/j.vaccine.2022.06.002 35817645 2. Cantarelli Rodrigues T Hidalgo PF Skaf AY Serfaty A Subacromial-subdeltoid bursitis following COVID-19 vaccination: a case of shoulder injury related to vaccine administration (SIRVA) Skelet Radiol 2021 50 2293 2297 10.1007/s00256-021-03803-x 3. Okuno Y Iwamoto W Matsumura N Oguro S Yasumoto T Kaneko T Clinical outcomes of transcatheter arterial embolization for adhesive capsulitis resistant to conservative treatment J Vasc Interv Radiol 2017 28 161 167 10.1016/j.jvir.2016.09.028 28007330 4. Okuno Y Oguro S Iwamoto W Miyamoto T Ikegami H Matsumura N Short-term results of transcatheter arterial embolization for abnormal neovessels in patients with adhesive capsulitis: a pilot study J Shoulder Elb Surg 2014 23 e199 206 10.1016/j.jse.2013.12.014 5. Fernández-Martínez AM Alonso-Burgos A López R Cuesta Marcos MT Baldi S Clinical outcomes of transcatheter arterial embolization for secondary stiff shoulder J Vasc Interv Radiol 2021 32 489 496 10.1016/j.jvir.2020.11.006 33478903	36450993	PMC9713192	NO-CC CODE	2022-12-02 23:22:56	no	Cardiovasc Intervent Radiol. 2022 Nov 30;:1-3	utf-8	Cardiovasc Intervent Radiol	2,022	10.1007/s00270-022-03319-4	oa_other
==== Front Cardiovasc Intervent Radiol Cardiovasc Intervent Radiol Cardiovascular and Interventional Radiology 0174-1551 1432-086X Springer US New York 36450993 3319 10.1007/s00270-022-03319-4 Letter to the Editor Transarterial Embolization for Shoulder Injury Related to Vaccine COVID-19 Administration http://orcid.org/0000-0003-4835-7976 Fernández Martínez Ana María [email protected] [email protected] 1 Cuesta Marcos M. Teresa 2 Rodríguez Prieto Joaquín 3 1 grid.411969.2 0000 0000 9516 4411 Vascular and Interventional Radiology, University Hospital of León, Calle Altos de Nava, SN, 24080 León, Spain 2 grid.411969.2 0000 0000 9516 4411 Radiology Department, University Hospital of León, León, Spain 3 grid.411969.2 0000 0000 9516 4411 Physical Medicine and Rehabilitation, University Hospital of León, León, Spain 30 11 2022 13 17 10 2022 13 11 2022 © Springer Science+Business Media, LLC, part of Springer Nature and the Cardiovascular and Interventional Radiological Society of Europe (CIRSE) 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcIntroduction Shoulder injury related to vaccine administration (SIRVA) is a preventable and well-described injury occurring after the administration of a vaccine into anatomic structures adjacent to the deltoid muscle. It causes shoulder pain and limitation of the range of motion due to a mechanical and chemical trauma that triggers an inflammatory response to the vaccine. It is mostly known after influenza vaccination and has also been described after COVID-19 vaccination in this pandemic [1]. The most common diagnoses are adhesive capsulitis (AC) and bursitis, and both manifest with pain and limitation of the shoulder mobility [1, 2]. We present a case of SIRVA due to a COVID-19 vaccination and treated by transarterial embolization (TAE) and physiotherapeutic treatment. Fifty-year-old woman presents painful left shoulder stiffness 24 h after administration of the first dose of COVID-19 vaccine. She refers to a pain of 9 on the visual analogical scale (VAS) with limitation of the mobility especially on internal rotation. Subsequently she develops progressive clinical and functional worsening. After the second dose, one month later, a venous cord and axillary lymphadenopathy appeared, accompanied by increased pain and rapid evolution to loss of mobility. Rehabilitation physicians advise against physiotherapeutic treatment for severe pain, so she is managed with oral analgesia without response. It was decided to perform a magnetic resonance (MR) study where edematous changes and severe thickening of the synovium and the capsule at the axillary recess, moderate obliteration of the fat in the rotator interval and diffuse enhancement after intravenous contrast administration were identified (Fig. 1).Fig. 1 Severe thickening of the synovium and the capsule with a diffuse enhancement after intravenous contrast administration on T1 fat sat gradient echo with MIP reconstruction is visualized (arrows) After these findings, it was decided to perform an angiography of the left shoulder, where areas of pathological blush enhancement were identified in the axillary recess and in the rotator interval—superior capsule (Fig. 2A). An injection of 9 ml (ml) of iodinated contrast media was performed at 3 ml per second through a 6 French guide catheter. Selective microcatheterization of the anterior humeral circumflex, coracoid and acromial arteries, respectively, was performed with a 1.7 F microcatheter (Fig. 2B, C), and they were embolized distally with 2.4 ml of a mixture of imipenem/cilastatin sodium and iodinated contrast media until there was a complete stasis with reflux back along the microcatheter tip. In control angiography, the pathological areas have disappeared except post-vaccination subcutaneous area where blush enhancement persists (Fig. 3). In addition, due to flow redistribution after embolization it was not visualized in the first injection.Fig. 2 A Digital subtraction angiography performed from left subclavian artery where there is a capsular enhancement at the axillary recess (arrow) and the rotator interval—superior capsule (arrowhead). Superselective arteriogram by manual injection from the anterior humeral circumflex artery (B) and from the acromial artery (C) where capsular enhancement (arrow) can be observed Fig. 3 Result after embolization where a normal angiographic pattern can be seen except post-vaccination subcutaneous area where blush enhancement persists (arrow) The patient reported an immediate decrease in pain, being 3 on VAS one week after TAE. She started rehabilitation with great improvement in mobility, and three months after embolization, shoulder mobility has completely recovered. Clinical results have remained stable during one year of follow-up. Many authors have described the importance of hypervascularization of the capsule in the pathophysiology of AC. It is known that angiogenesis is a necessary factor to generate an inflammatory state and Okuno et al. reported the existence of pathological angiographic hypervascularization in all patients in their series [3, 4]. The objective of TAE is to decrease the abnormal vascularization responsible for the inflammatory state that also occurs in SIRVA [1]. Also, TAE has emerged as a therapeutic option in patients with AC refractory to conventional treatment [3–5]. SIRVA is a well-established condition in the medical literature; and there are examples after administration of the COVID-19 vaccine. There are references of arterial embolization in secondary stiff shoulder but not about SIRVA treated by TAE [1, 5]. This case corresponds to a SIRVA of synovitis–capsulitis due to an inflammatory response of the capsule to the vaccine. Transarterial embolization and physiotherapeutic treatment were associated with reduced pain and improved mobility. Funding This study was not supported by any funding. Declarations Conflict of interest The authors declare that they have no conflict of interest. Consent for Publication Consent for publication was obtained for every individual person’s data included in the study. Ethical Approval The study performed was in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Review Board (IRB). Our hospital clinical research ethics committee approved this study. Informed Consent Informed consent was obtained from the patient included in the study. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Bass JR Poland GA Shoulder injury related to vaccine administration (SIRVA) after COVID-19 vaccination Vaccine 2022 40 4964 4971 10.1016/j.vaccine.2022.06.002 35817645 2. Cantarelli Rodrigues T Hidalgo PF Skaf AY Serfaty A Subacromial-subdeltoid bursitis following COVID-19 vaccination: a case of shoulder injury related to vaccine administration (SIRVA) Skelet Radiol 2021 50 2293 2297 10.1007/s00256-021-03803-x 3. Okuno Y Iwamoto W Matsumura N Oguro S Yasumoto T Kaneko T Clinical outcomes of transcatheter arterial embolization for adhesive capsulitis resistant to conservative treatment J Vasc Interv Radiol 2017 28 161 167 10.1016/j.jvir.2016.09.028 28007330 4. Okuno Y Oguro S Iwamoto W Miyamoto T Ikegami H Matsumura N Short-term results of transcatheter arterial embolization for abnormal neovessels in patients with adhesive capsulitis: a pilot study J Shoulder Elb Surg 2014 23 e199 206 10.1016/j.jse.2013.12.014 5. Fernández-Martínez AM Alonso-Burgos A López R Cuesta Marcos MT Baldi S Clinical outcomes of transcatheter arterial embolization for secondary stiff shoulder J Vasc Interv Radiol 2021 32 489 496 10.1016/j.jvir.2020.11.006 33478903	0	PMC9713196	NO-CC CODE	2022-12-02 23:22:56	no	Datenschutz Datensich. 2022 Nov 30; 46(12):762-766	latin-1	null	null	null	oa_other
==== Front Ann Oper Res Ann Oper Res Annals of Operations Research 0254-5330 1572-9338 Springer US New York 5092 10.1007/s10479-022-05092-6 S.i. : Or for Sustainability in Supply Chain Management Two-echelon fresh product supply chain with different transportation modes http://orcid.org/0000-0002-5764-7732 Yan Bo [email protected] http://orcid.org/0000-0002-9568-4673 Liu Yanping [email protected] Fan Jing [email protected] grid.79703.3a 0000 0004 1764 3838 School of Economics and Finance, South China University of Technology, Guangzhou, China 30 11 2022 124 17 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The fresh product supply chain suffers from the quantity loss and quality loss due to its perishability in long-distance transportation, which affect the health of customers and the sustainable development of supply chain. Using low-cost normal temperature transportation or high-cost cold chain transportation has become a problem for transportation enterprises. This paper aims to investigate the impact of different transportation modes on the supply chain performance. The operation strategies of the supply chain are analyzed under three situations: no coordination contract, wholesale price contract, and revenue-sharing contract. Taking Zhanhua winter jujube as an example, the correctness of theoretical analysis is verified. The main findings are as follows. Supply chain participants, including consumers, can benefit from cold chain transportation. That the cost of cold chain transportation is below the threshold is the basic condition to use cold chain transportation. The retailer has the incentive to encourage the supplier to use cold chain transportation by increasing the wholesale price, but the wholesale price should be set within a certain range. The supplier has the incentive to use cold chain transportation under the revenue-sharing contract, but the revenue-sharing proportion needs to be within a certain range to ensure the retailer's profit. The revenue-sharing contract is superior to the wholesale price contract, and wholesale price contract is superior to no coordination contract. Keywords Value loss Cold chain transportation Fresh product supply chain Wholesale price contract Revenue-sharing contract http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 71871098 Yan Bo ==== Body pmcIntroduction As we all know, fresh products are more prone to corruption than ordinary products. With the rapid development of e-commerce and logistics, human's material life is no longer limited to a certain area, but globalization. People's food, including fresh products such as fruits and vegetables, may come from places of origin thousands of kilometers away. For example, oranges exported from Guangxi, China to Brunei. Cherries from Chile and mangosteen from Thailand are deeply loved by Chinese people. In the process of transportation and storage, these products will not only face the decrease of freshness, which will lead to the decline of customer satisfaction, but also be thrown away because of deterioration, resulting in the loss of quantity. According to the data from "Sustainable Consumption and Production", the twelfth goal of Sustainable Development Goals of United Nations, one third of food products–1.3 billion tons, worth $1 trillion—end up rotting in the dustbins of consumers and retailers each year, or deteriorating due to improper transportation and harvesting every year. One of the specific goal of "Sustainable Consumption and Production" is to halve per capita food waste in the global retail and consumption stages by 2030, and to reduce food losses in production and supply (including post-harvest). This process requires the participation of many parties, including business, consumers, policy makers, researchers, scientists, retailers, media, and development partners. The COVID-19 pandemic reinforces the imperative to improve resource efficiency and promote sustainable lifestyles. Humans should develop strategies to do more and better with less. In recent years, people's attention to life and health makes their requirements for fresh and green foods increasing day by day. The freshness seriously affects the market demand of products. Therefore, the adoption of technical means to improve the freshness of fresh products has become an important event affecting the sustainable development of the supply chain and the national economy and people's livelihood. In fact, the loss of fresh products in circulation in China is much greater than that in other countries (Yang et al., 2017; Yu & Xiao, 2017). The main reason is the gap of modern logistics facilities. With the improvement of consumers' safety awareness, significant progress has been made in the development of cold chain transportation (Mercier et al., 2017). Cold chain transportation provides cold storage conditions for fresh products. It is understood that if the cold chain equipment is used, the freshness-keeping period of fresh products will be longer, which verifies the timeliness of the cold chain and helps to extend the shelf life of fresh products. If fresh products are transported and stored at proper temperature, the loss of quantity and quality can be greatly reduced. However, cold chain transportation has high requirements for logistics enterprises. Enterprises not only need to configure specific refrigeration and control systems, refrigeration vehicles and storage equipment, but also require high power consumption and skills of employees. Therefore, the cost of cold chain transportation is higher than that of normal temperature transportation. Especially for some small and medium-sized enterprises, the cost of cold chain logistics is unbearable. When the supply chain chooses different transportation modes, not only the quality and quantity of fresh products are different, but also the decisions such as the ordering quantity (Bai et al., 2016) and pricing (Banerjee & Agrawal, 2017; Widodo et al., 2018) change accordingly. Use cold chain transportation to ensure the freshness of fresh products or use normal temperature transportation to avoid the cost of cold chain transportation? This is the problem that most enterprises need to face. Based on the above concerns, this paper aims to solve the following problems by using Stackelberg game and backward induction method (Qiu et al., 2020).How different transportation modes affect the quantity and quality of fresh products from the supplier to consumers? What strategies can coordinate supply chain participants to reduce the value loss of fresh products? How to apply these coordination strategies? What are the conditions for the supplier to choose different transportation modes under different coordination strategies? How to encourage the supply chain participants to use cold chain transportation to maximize the profit of the whole supply chain? To address the above problems, we will investigate the impact of different transportation modes on the supply chain. First, a two-echelon fresh product supply chain composed of a supplier and a retailer is established. In the supply chain, the supplier is responsible for the transportation service and has two choices of transportation modes which are normal temperature transportation and cold chain transportation. Second, the value losses of different transportation modes the supplier chooses under three situations are analyzed and compared, respectively. The three situations are no coordination contract, wholesale price contract, and revenue-sharing contract. Finally, Zhanhua winter jujube was taken as an actual case for numerical analysis. Through theoretical analysis and case verification, several conclusions are drawn, and some managerial insights are put forward. To sum up, this paper contributes to the existing research in four aspects.Because using normal temperature transportation or cold chain transportation affects the quantity and quality of fresh products, the impacts of different transportation modes on the supply chain members are analyzed. The supply chain contract is established from two aspects: freshness and quantity loss. The impacts of value loss of the normal temperature transportation and cold chain transportation on supply chain under three situations of no coordination contract, wholesale price contract, and revenue-sharing contract are compared, respectively. The conditions for the supplier to use cold chain transportation under different coordination strategies are obtained. Put forward the profit concession measures under different coordination contracts to encourage the supplier to use cold chain transportation, and provide decision-making guidance for supply chain managers. The rest of this paper is organized as follows. Section 2 gives the literature review and summarizes the literature gap and the contributions of this paper. In Sect. 3, the problem description and assumptions are given. The impacts of value loss on fresh product supply chain under the three situations of no coordination contract, wholesale price contract and revenue-sharing contract are analyzed in Sect. 4, respectively. The model results are verified by a practical case in Sect. 5. Discussion and some managerial insights are put forward in Sect. 6. Finally, Sect. 7 draws conclusions and proposes some future research directions. Literature review There are lots of documents about fresh product supply chain, including pricing, inventory strategy, coordination strategy and so on. This paper considers the choice of different transportation modes in fresh product supply chain under different contracts. Therefore, the studies related to this paper can be divided into two categories: (1) decision-making of fresh product supply chain, (2) coordination contract of fresh product supply chain. Decision-making of fresh product supply chain In view of the perishability of fresh products, many scholars have discussed how to carry out the optimal production (Leung & Ng, 2007; Liu et al., 2015; Zhang et al., 2018) or replenishment strategy (Chakraborty et al., 2018; Huang, 2013; Jaggi et al., 2019; Kiil et al., 2018). Among them, many papers have considered the quantity loss or quality loss in the decision-making process of supply chain (Wu et al., 2018; Yu & Xiao, 2017). Quantity loss was assumed to be constant in the models developed by Hwang and Hahn (2000) and Bai et al. (2016) for deteriorating goods. However, functions were used to express the relationship between quantity loss or quality loss and other factors such as time, which is closer to reality. For example, Herbon et al. (2014) defined quality loss as a quadratic function of time and emphasized the impact of freshness on consumer utility. Controlling the quality loss and quantity loss brought by fresh product wastage has attracted the attention of scholars and researchers in recent years. It is natural to keep products fresh to control the loss of quantity or quality. Some papers analyzed the equilibrium pricing and service level of supply chain for deteriorating products based on freshness-keeping technology investment (Dye & Yang, 2016; Dye et al., 2018; Ma et al., 2019b; Wang & Zhao, 2021). On this basis, some scholars developed optimal management strategies for superior and inferior products (Chen et al., 2021; Huang et al., 2019) or considered the pre-sale strategies (Zhang et al., 2020). However, the aforementioned studies only considered the changes of the quantity or quality of fresh products, and lacked the research on the changes of both. Unlike the above, there are related papers that consider both the quantity loss and quality loss of deteriorating products. Most studies used cold chain transportation as a kind of freshness-keeping service to reduce losses (Aiello et al., 2012; Cai et al., 2013; Mercier et al., 2017; Yu & Xiao, 2017). They focused on the storage or replenishment strategies (Hsu et al., 2010; Lee & Dye, 2012) or pricing and freshness-keeping cost input strategies (Cai et al., 2010; Chang et al., 2016; Yu et al., 2020; Zhang & Wang, 2020) of supply chains that retailers invest in freshness-keeping costs. However, retailers are usually only responsible for keeping products fresh during storage rather than during transportation. It is usual for the third-party logistics service provider (TPLSP) or the supplier to provide freshness-keeping services during transportation (Feng et al., 2021; Liu et al., 2021; Ma et al., 2020; Qiu et al., 2020; Song & He, 2019; Wu et al., 2015; Yan et al., 2020b). Further, Zhang et al. (2015) and Moon et al. (2020) considered the interests of the supply chain of deteriorating goods when both the manufacturer and the retailer invested in freshness-keeping efforts. Wang et al. (2020) discussed the game structure between the retailer and the farmer when the farmer invests more in the production of green fresh products and the retailer invests in the packaging and cold chain transportation. From the above studies, it can be found that whoever provides freshness-keeping service can greatly improve the market value of products. Most of the above studies consider the value loss at a certain stage of the supply chain (such as storage or transportation), but this paper comprehensively considers the value loss of the whole fresh product supply chain. Although the efforts to keep products fresh have not been ignored, there is few studies to compare cold chain transportation with normal temperature transportation, while the choice of transportation mode is important for the supply chain. As far as we know, only Lin et al. (2020) discussed the decision of two transportation modes for vaccines, and concerned about the impact of the two inspection methods of the retailer on the decision of the distributor at the end of transportation. Shui et al. (2021) analyzed the balance between insulation packaging cost and shipping cost when choosing nonrefrigerated vehicles or refrigerated vehicles under the community group purchase model. Coordination contract of fresh product supply chain In the literature, the research on supply chain coordination contracts commonly includes buyback contract, quantity discount contract, cost-sharing contract, revenue-sharing contract, wholesale price contract, and so on. Wu (2013) showed that buyback contract can lead to a higher profit than no buyback in both vertical integration and manufacturer's Stackelberg in competitive supply chains. Chen and Bell (2011) proposed an agreement between the manufacturer and the retailer, which includes two buyback prices, and showed that this agreement can achieve perfect supply chain coordination. Compared with a fixed discount, Venegas and Ventura (2018) found that the non-cooperative model can improve the benefits of the participants when the discount is flexible. Because buyback and quantity discount contracts cannot take the characteristics of short life cycle products into account, they are not suitable for fresh product supply chain. Song and He (2019), Yan et al. (2020b) and Feng et al. (2021) found that cost-sharing contract cannot achieve the coordination of the supply chain. Therefore, the wholesale price contract and the revenue-sharing contract are two common coordination contracts used in fresh product supply chain. The wholesale price contract is a simple and common form of supply chain contracts. When the supply chain is under the decentralized decision-making mode, it can effectively coordinate the supply chain. Cai et al. (2013) and Huang et al. (2019) used the wholesale price contract to coordinate a fresh product supply chain when distributors determine the level of freshness-keeping effort in product transportation. Fang et al. (2020) and Yan et al. (2020a) designed a wholesale price contract to coordinate the fresh product supply chain under the decentralized decision-making mode. Revenue-sharing contract binds the profits of the supplier and the retailer together, which can coordinate the whole supply chain and improve the overall efficiency. There are many papers on coordinating fresh product supply chain that use it (Ma et al., 2019b; Xiao & Xu, 2013; Yang et al., 2014; Zhang et al., 2015; Zheng et al., 2017). For example, Bai et al. (2016) designed a revenue-sharing contract to coordinate the decentralized supply chain for deteriorating items. Yan et al. (2021) proposed a revenue-sharing contract to achieve a win–win situation for the supplier and the retailer in a fresh agricultural product supply chain. As mentioned above, the wholesale price contract and the revenue-sharing contract can achieve good coordination effect in the fresh product supply chain. Therefore, this study analyzes the impact of different transportation modes under three situations: no coordination contract, wholesale price contract and revenue-sharing contract, respectively. Research gap Considering the various studies, Table 1 lists partial recent and relevant papers, and compares them from five aspects to clearly fill the research gap. Table 1 is structured based on columns including (1) quantity loss, (2) quality loss, (3) value loss procedure, (4) comparison of transportation modes, and (5) coordination contracts.Table 1 Comparison between this paper and related papers Authors (year) Quantity loss Quality loss Value loss procedure Comparison of transportation modes Coordination contracts Yang and Tang (2019) – √ Storage – – Zhang et al. (2020) – √ Transportation – – Liu et al. (2021) – √ Transportation – √ Feng et al. (2021) – √ Transportation – √ Lin et al. (2020) – √ Transportation √ – Shui et al. (2021) – √ Transportation √ – Chakraborty et al (2018) √ – Storage – – Huang et al. (2019) √ – Transportation – √ Yan et al. (2020a) √ – Transportation – √ Song and He (2019) √ √ Transportation – √ Zhang and Wang (2020) √ √ Storage – – Yu et al. (2020) √ √ Transportation – – Ma et al. (2020) √ √ Transportation – √ Qiu et al. (2020) √ √ Transportation – √ Yu and Xiao (2017) √ √ Transportation Storage – – Yan et al. (2020b) √ √ Transportation Storage √ √ This paper √ √ Transportation Storage √ √ As shown in Table 1, due to the perishability of fresh products, many scholars have studied the value loss of fresh products over time. In these studies, some documents only considered the quantity or quality loss of fresh products (Chakraborty et al., 2018; Zhang et al., 2020), and some studies considered both quantity loss and quality loss, but only focus on part of the circulation process (Song & He, 2019; Zhang & Wang, 2020). Although Yu and Xiao (2017) focused on the loss of quantity and quality in the whole circulation process, they emphasized the optimal price and service level decisions under different channel leadership and the impacts of channel leadership on supply chain members. Moreover, they set the demand as a linear function. However, in this paper, we focus on the decision-making behavior of supply chain under different transportation modes and coordination contracts. As a whole, there are obvious differences between this paper and the above studies. This paper is closely related to Lin et al. (2020), Shui et al. (2021) and Yan et al. (2020b), which discussed the decision-making of supply chain under different transportation modes. However, Lin et al. (2020) and Shui et al. (2021) only considered the quality loss of products in the transportation process, did not consider the quantity loss in the transportation process, nor consider the value loss in the storage process, let alone the coordination of the supply chain. Moreover, Lin et al. (2020) focused on the impact of the retailer's inspection strategy at the end of transportation on the distributor's transportation modes decision-making. Shui et al. (2021) designed a heuristic algorithm to solve the decision-making of transportation modes. In this paper, Stackelberg game is used to study the decision-making of supply chain with different transportation modes under different contracts. Thus, their research contents and methods are quite different from this paper. Yan et al. (2020b) focused on the formation mechanism of market demand and the equilibrium decision of supply chain resulting from the quantity loss and quality loss of fresh products affecting customer utility. However, our demand function is related to the price and the freshness of products, which is the essential difference. Furthermore, the contracts used to coordinate the supply chain are different. They used revenue-sharing contract and cost-sharing contract, while we use wholesale price contract and revenue-sharing contract. As far as we know, this paper contributes to the literature from four respects. First, the quantity loss and quality loss of fresh products in the whole processes of transportation and storage are considered. Second, the conditions for choosing different transportation modes are discussed. Third, the impacts of different transportation modes on supply chain members are analyzed. Fourth, the decision-making problems under three situations: with no coordinated contract, wholesale price contract and revenue-sharing contract are explored. These are the supplement and extension of the existing research. Problem description and assumptions In this paper, a two-echelon fresh product supply chain is composed of a supplier, a retailer and consumers, in which the supplier is the leader of the Stackelberg game and the retailer is the follower. The structure is illustrated in Fig. 1.Fig. 1 Structure of the fresh product supply chain In this supply chain, there are two transportation modes for the supplier to ship the fresh products to the market, namely cold chain transportation and normal temperature transportation. The cold chain transportation can improve the freshness of fresh products, thereby reducing the loss of quantity and quality, but the supplier must pay the corresponding cost. There is no additional freshness-keeping cost during normal temperature transportation, but the supplier is responsible for the deterioration of the fresh products during long-distance transportation. In the meanwhile, the storage cost borne by the retailer will be affected. As the leader, the supplier first sets the wholesale price according to the transportation mode it chooses and the value loss. Then the retailer determines the retail price according to the freshness of products when they arrive at the market. Because the storage cost varies with the freshness of products, the retailer has the incentive to encourage the supplier to use cold chain transportation. Specifically, Table 2 summarizes the parameters and decision variables used in this paper.Table 2 Symbols and description Symbols Description Subscript "i" i=s,t represents normal temperature or cold chain transportation, respectively cm Supplier's unit production cost ci Transportation cost of per unit product under different transportation modes;ct>cs mi Rate of quantity loss,mt<ms t Sales period of products,t∈[0,T] θit Freshness of products at time t T Duration of the sales period g(θ) Impact of freshness on demand A Potential market demand K Price sensitivity,K>1 Ii Impact of freshness on demand over the whole sales period τi Average time a unit product spends on the shelf before being sold h Storage cost per unit on the shelf wi Wholesale price per unit product pi Retail price per unit product D(p,θ) Demand function Qi Ordering quantity β Revenue-sharing proportion ∏ri,∏si,∏Ti Profit of the retailer, the supplier and the whole supply chain in the decentralized supply chain ∗ Optimal result Superscript "1" Decision under wholesale price contract Superscript "2" Decision under revenue-sharing contract Market demand is influenced by product price and freshness, the following multiplicative demand form is adopted, which is commonly used in the literature (Chernonog & Avinadav, 2019; Ma et al., 2019a),D(p,θ)=Ap-Kg(θ(t)) The decline of the product's freshness means the reduction of perceived value from the perspective of customers. Accordingly, gθt is an increasing function of θ(t). θ(t) decreases with t, and θ(t)∈(0,1). The supplier is responsible for the quality loss, so it should produce and transport adequate quantity Qmi to the market. Suppose I=∫0Tg(θ)dt is the impact of freshness on demand during the whole sales period. T is the duration of the sales period. We assume that T is shorter than the shelf life of the product, so the product will not perish during the sales period, which means there is no quantity loss during the sales period. However, the quality loss always exists in the supply chain. When the quality loss reaches a certain degree, the quantity loss is bound to occur. Let τ=∫0Ttg(θ)dt∫0Tg(θ)dt be the average time a unit spends on the shelf before it is sold. The storage cost borne by the retailer is hτQ. The assumptions in the model are as follows.The residual value of the fresh products is assumed to be 0. Wang et al. (2004) showed that this assumption is reasonable for fresh products. In the sales cycle T, all fresh products can be sold out (Yan et al., 2020b), and the potential consumer market demand A is the market scale, so it is a constant. The supplier and the retailer are risk neutral and pursue their own profit maximization. This is a common assumption (Hua et al., 2011; Qi et al., 2018; Sun, 2013). Impact of value loss Impact of value loss with no coordination contract Value loss with no coordination contract According to Sect. 3, the market demand for the fresh products at time t is1 Di(p,θi(t))=APi-Kg(θi(t)) Therefore, during the sales period T, the total order quantity is2 Qi=∫0TDi(p,θi(t))dt=∫0TAPi-Kg(θi(t))dt The retailer's profit is3 ∏ri=pi-wi-hτQi=APi-K(pi-wi-hτi)Ii When ∂2∏ri∂pi2<0, it means that the retailer has the maximum profit when ∂∏ri∂pi=0. We can obtain the reaction function of pi with respect to wi, that is, pi∗(wi)=K(wi+hτi)K-1. The supplier's profit is4 ∏si=wiQi-cm+ciQimi=Api-Kwi-cm+cimiIi By substituting Pi∗(wi) into Eq. 4, the supplier's optimal profit can be obtained. Because ∂2∏si∂wi2<0, the optimal value of wi exits. Let ∂∏si∂wi=0, the optimal wi∗ can be calculated. pi∗ can also be calculated accordingly. The results are shown in Lemma 1. Lemma 1 For any realized transportation cost ci , the quantity loss mi and the average time spent on the shelf τi , the supplier's optimal wholesale price is wi∗=mihτi+K(cm+ci)(K-1)mi , and the optimal retail price is pi∗=KK-12cm+ci+mihτimi . According to Lemma 1, the wholesale price and the retail price increase with the increase of the transportation cost ci and the average shelf time τi. When the transportation cost borne by the supplier is higher, a higher wholesale price should be set to balance its profit. Correspondingly, consumers must pay more for products. From the expressions of wi∗ and pi∗, we can find that the longer the average time spent on the shelf, the higher the wholesale price and the retail price are. When ∂wi∂mi<0 and ∂pi∂mi<0, the products suffer more quantity loss, the prices set by the supplier and the retailer are higher. By substituting the optimal prices into Eqs. 3 and 4, we obtain the optimal profits of the retailer and the supplier in the decentralized supply chain, as shown in Eqs. 5 and 6.5 ∏ri∗=AIiK1-2K(K-1)2(K-1)micm+ci+mihτiK-1 6 ∏si∗=AIiKK-1-2K1K-1micm+ci+mihτiK-1 We can find ∏ri∗∏si∗=KK-1 in two transportation modes with no coordination contract. The profit ratio of two supply chain participants is related to price sensitivity. Since K>K-1, the retailer's profit is higher than the supplier's profit. The profit of the whole fresh product supply chain is the retailer's profit plus the supplier's profit, that is7 ∏Ti∗=AIi(2K-1)K2K(K-1)2micm+ci+mihτiK-1 Comparative analysis of two transportation modes We can obtain Theorem 1 by comparing the optimal retail price, wholesale price and profits of the supply chain members under cold chain transportation mode and normal temperature transportation mode. Theorem 1 When mt(cm+cs)-ms(cm+ct)<h(τt-τs)K, wt>ws. (2) When mt(cm+cs)-ms(cm+ct)<mtmsh(τt-τs) is satisfied, pt>ps, ∏rt∗>∏rs∗, and ∏st∗>∏ss∗. Theorem 1 shows that the wholesale price, the retail price and profits of the supplier and the retailer under cold chain transportation mode are higher than those under normal temperature transportation mode when mt(cm+cs)-ms(cm+ct)<minh(τt-τs)K,mtmsh(τt-τs). Theorem 2 When cs<ct<cT , the retailer can make more profit when the supplier uses cold chain transportation in the decentralized supply chain. cT is the threshold of cold chain transportation cost. When ∏st∗=∏ss∗, we can get cT=ItIs1K-1mt(cm+cs+mshτs)ms-cm-mthτt. The supplier will use cold chain transportation when cs<ct<cT. In this case, the retailer also benefits from this mode. Therefore, when the cold chain transportation cost is within a certain range, all the supply chain participants can get more profits without any incentive scheme. The retailer has the motivation to encourage the supplier to use cold chain transportation. The threshold of cold chain transportation cost is related to the production cost, quantity loss, normal temperature transportation cost, price sensitivity, and the impact of freshness on demand. Impact of value loss with wholesale price contract Regardless of the transportation mode, the supplier and the retailer sign a wholesale price contract before shipping fresh products. After the supplier decides the wholesale price w, the retailer orders Qi products. Then, the supplier produces and transports Qimi to the retailer. After the contract is signed, the retailer must accept the products even if the freshness of the products is low when they reach the market. Value loss with wholesale price contract The profits of supply chain members with wholesale price contract are the same as those of supply chain members with no coordination contract when w=wt=ws. According to wholesale price contract, the wholesale price w is agreed upon by the supplier and the retailer at the beginning of the contract. Regardless of the transportation mode, the transactions must be carried out in accordance with the wholesale price. The profits of the supply chain are8 ∏ri1=pi-w-htQi=∫0Tpi-w-htAPi-Kg(θi(t))dt=AIiKKK-1w+hτiK-1, 9 ∏si1=wQi-cm+ciQimi=AIiwmi-(cm+ci)miK(w+hτi)K-1-K, 10 ∏Ti1=AIi(wi+mihτi+wimi-cm-ci)KK(wi+mihτi)K-1wi+hτiK-1. Comparative analysis of two transportation modes Theorem 3 When cs≤ct≤CTS , the supplier's profit under cold chain transportation mode is higher than that under normal temperature transportation mode. Proof ∏st1-∏ss1=AItwmt-(cm+ct)mtK(w+hτt)K-1-K-AIswms-(cm+cs)msK(w+hτs)K-1-K. When ∏st1=∏ss1, the cost parameter cTS=(wmt-cm)-mtmsw+hτsw+hτt-K(wms-cm-cs). If cs≤ct<CTS, ∏st1>∏ss1; if CTS<cs≤ct, ∏st1<∏ss1. When the cost of cold chain transportation is within a certain range, the supplier can be guaranteed to obtain more profit by choosing cold chain transportation. For the retailer, cold chain transportation can make more profit, so the retailer encourages the supplier to use cold chain transportation by allowing the supplier to increase the wholesale price. However, the wholesale price must be within a certain range to ensure that the profits of the supplier and the retailer under cold chain transportation mode are greater than those under normal temperature transportation mode before the wholesale price rises. As showed in Theorems 4 and 5. Theorem 4 The supplier uses cold chain transportation when cm+cs<wTS<w . Otherwise, the supplier uses normal temperature transportation. wTS is the lower limit of the wholesale price. Proof ∏st1-∏ss1=AItwmt-(cm+ct)mtK(w+hτt)K-1-K-AIswms-(cm+cs)msK(w+hτs)K-1-K When ∏st1-∏ss1=0, w=wTS. If w<wTS, ∏st1<∏ss1; if w>wTS, ∏st1>∏ss1. Therefore, the supplier should get a reasonable profit share if the retailer wants the supplier to provide cold chain transportation service, which needs to increase the wholesale price. However, the retailer seeks to maximize its own profit, and the wholesale price is constrained by the upper limit. Theorem 5 Under the wholesale price contract, there is an upper limit of wholesale price w¯=ItIs1K-1(w+hτs)-hτt acceptable to the retailer. Proof When the supplier increases the wholesale price to w¯, it will use cold chain transportation. Set the retailer's profit as ∏rt1¯ after the supplier increases the wholesale price to w¯. Let ∏rt1¯=∏rs1, when ∏rt1¯>∏rs1, AItKKK-1w¯+hτtK-1>AIsKKK-1w+hτsK-1, namely, w¯<ItIs1K-1(w+hτs)-hτt. Impact of value loss with revenue-sharing contract Value loss with revenue-sharing contract Revenue-sharing contract is a scheme to redistribute profits among supply chain members. The proportion can be negotiated based on the actual situation, which deepens the trust between the supplier and the retailer. According to the discussion of no coordination contract, the retailer's profit is always greater than the supplier's profit. Therefore, the retailer shares 1-β of its revenue to the supplier to obtain higher quality products. The retailer's profit under the revenue-sharing contract is11 ∏ri2=(βpi-wi-hτi)Api-KIi The supplier's profit is12 ∏si2=wi+(1-β)pi-cm+cimiApi-KIi Then the optimal decisions of the supply chain can be calculated as follows:wi2=mihτi(K2β-K2+K-β)+Kβ(K-1)(cm+ci)mi(K-1)(K-β), pi2=K2β(mihτi+cm+ci)mi(K-β)(K-1), ∏ri2=βmi(K-β)(K-1)1-KK1-2KAIi[mihτi+(Kβ-K+1)(cm+ci)](mihτi+cm+ci)-K, ∏si2=AIiβ-KK-2K(2K2β-K2β2-K2+K-β)mihτi+cm+cimi(K-β)(K-1)1-K, ∏Ti2=∏ri2+∏si2. Comparative analysis of two transportation modes Theorem 6 The retailer's profit increases if the supplier uses cold chain transportation when cs<ct≤CTR , where CTR is the threshold of transportation cost, which meets ∏rt2=∏rs2 . When ∏rs2≤∏rt2 and cs≤ct≤CTR, the retailer's profit increases when the supplier uses cold chain transportation. Therefore, the retailer wants the supplier to choose cold chain transportation mode. The retailer transfers a reasonable profit to the supplier by reducing the revenue-sharing proportion. The incentive scheme encourages the supplier to use cold chain transportation to ensure the freshness of products. However, the retailer should ensure that the profit under cold chain transportation mode is still greater than that under normal temperature transportation mode before transferring profit. Therefore, there is a lower limit of the revenue-sharing proportion. Like the proof of Theorem 4, Theorem 7 can also be obtained. Theorem 7 A proper portion of the retailer's profit β^≤β≤1 should be transferred to the supplier, to encourage the supplier to use cold chain transportation. β^ is the lower limit of revenue-sharing proportion , which satisfies ∏rt2-∏rs2=0 , namely ItIsmsmt1-Kmthτt+cm+ctmshτs+cm+cs-K=mshτs+(Kβ-K+1)(cm+cs)mthτt+(Kβ-K+1)(cm+ct) . If the supplier does not choose cold chain transportation mode under the revenue-sharing contract, the retailer will have to transfer a portion of the profit to the supplier by increasing the revenue-sharing proportion to encourage the supplier to use cold chain transportation. The amount of transferred profit should ensure that the retailer's profit under cold chain transportation mode is higher than that under normal temperature transportation mode before transferring profit. Theorem 8 Under the revenue-sharing contract, the revenue-sharing proportion has an upper limit β¯ , that is, the acceptable upper limit of the retailer, which satisfies ∏rt2¯=∏rs2 . ∏rt2¯ is the retailer's profit after the revenue-sharing proportion is increased to β¯ . Case analysis Zhanhua winter jujube is a unique fruit in Shandong, China. Because of its delicacy and nutrition, it is loved by consumers. Under normal temperature and natural conditions, Zhanhua winter jujube is easy to deteriorate, which affects its freshness and quantity. However, the cost of cold chain transportation is relatively high. The supplier often faces the problem of using normal temperature transportation or cold chain transportation. To help the supplier make the most economical choice, a two-echelon supply chain consisting of a fresh food e-commerce company A and its supplier B is taken as an example in this section. According to the sales data, the monthly market demand for Zhanhua winter jujube is about 700000 kg. Under normal circumstances, when the unit price of Zhanhua winter jujube rises by about 10%, the purchase volume of consumers will be reduced by about 22%. Therefore, the price elasticity of Zhanhua winter jujube is 2.2. The production cost is about 5 yuan/kg, and the storage cost per unit on the shelf is 1 yuan/kg. The transportation price is generally 3 yuan/kg under normal temperature and 5 yuan/kg under cold chain. Under normal temperature, fresh Zhanhua winter jujube can stay on the shelf for 8 days. If the supplier uses cold chain transportation, Zhanhua winter jujube will be sold faster with a shelf life of 4 days. The transportation time from the place of origin to warehouse A is about 2 days, and the quantity loss rate is 0.3 during normal temperature transportation. Under the condition of cold chain transportation at 3 °C-5 °C, the freshness-keeping rate of Zhanhua winter jujube can reach 80%. The wholesale price contract stipulates that the wholesale price is 20 yuan/kg, and the revenue-sharing contract stipulates that the revenue-sharing proportion is 0.9. As a result, the relevant parameters are set in Table 3.Table 3 The relevant parameters A K cm cs ct h τs τt ms mt 700,000 2.2 5 3 5 1 8 4 0.7 0.8 Decisions with no coordination contract By substituting the parameters into the relevant equations in Sect. 4.1, the decision variables and profits under different transportation modes are obtained, and the results are shown in Table 4.Table 4 Comparison of decision variables under two transportation modes with no contract Transportation mode w∗ p∗ ∏s∗ ∏r∗ ∏T∗ Normal temperature 27.62 65.30 2304.39 4224.71 6529.10 Cold chain 26.25 55.46 2523.17 4625.81 7148.98 Difference 1.37 9.84 − 218.78 − 401.10 − 619.88 The threshold of cold chain transportation cost is 5.97. When ct≤5.97, B uses cold chain transportation; otherwise, B uses normal temperature transportation. When ct=5, B uses cold chain transportation even without any incentive mechanism. As shown in Table 4, the profits of both the retailer and the supplier increase. The wholesale price and the retail price under cold chain transportation mode are lower than those under normal temperature transportation mode, which verifies that every supply chain participant, even the consumer, benefits from cold chain transportation. Therefore, A expects B to provide cold chain transportation service. This is consistent with theoretical analysis of Theorems 1 and 2. Decisions under wholesale price contract By substituting the parameters in Table 1 into the equations in Sect. 4.2, when the wholesale price contract stipulates that w=20, the threshold of cold chain transportation cost is 6.12. The cold chain transportation cost of supplier B is 5 which is within this range, so cold chain transportation can be used. This proves that Theorem 3 is effective in practice. In addition, the lower limit of wholesale price can be concluded as 15.59. Figure 2a indicates the relationship between the wholesale price and the supplier's profit under different transportation modes with wholesale price contract. When the wholesale price is greater than 15.59, the supplier's profit will be higher under cold chain transportation mode. As illustrated in Fig. 2b, the retailer's profit is always higher if the supplier uses cold chain transportation. This is consistent with Theorem 4. Under the wholesale price contract, when the contractual wholesale price is greater than the threshold, the supplier can be encouraged to use cold chain transportation by raising the wholesale price.Fig. 2 Relationship between the wholesale price and the profits When the wholesale price contract stipulates that w=20, other parameters remain unchanged. We investigate the upper limit of wholesale price w¯ acceptable to the retailer with the wholesale price contract, where w¯=σw=20σ, which means that the upper limit of wholesale price acceptable to the retailer is σ times of the previous wholesale price. In this case, the retailer allows the supplier to charge σ times higher than the original wholesale price to ensure cold chain transportation. As shown in Fig. 3, when σ<1.98, the retailer's profit under cold chain transportation mode is higher than that under normal temperature transportation mode. If outside this range, the retailer does not encourage the supplier to provide cold chain service. Therefore, the upper limit of the wholesale price is 1.98 times the original price. This is consistent with the analysis of Theorem 5.Fig. 3 Relationship between retailer's profit and σ Decisions with revenue-sharing contract From Fig. 4, as the retailer gradually shares more profit with the supplier, the change trend of its own profit and the supplier's profit is the same, increasing first and then decreasing. Based on the theory, with the increase of revenue-sharing proportion β, the retail price and the wholesale price will increase, and the market demand will correspondingly decrease. When the revenue-sharing proportion is small, the loss of demand reduction is less than the profit increase caused by the rise of the retail price and the wholesale price, resulting in the increase of the profits of the retailer and the supplier. When the revenue-sharing proportion is large, the opposite is true, so the profits of the retailer and the supplier decrease.Fig. 4 Relationship between the revenue-sharing proportion and the profits As shown in Fig. 4a, if the revenue-sharing proportion is greater than the threshold, the supplier's profit under cold chain transportation mode is always higher than that under normal temperature transportation mode. Therefore, the supplier is motivated to use cold chain transportation with the revenue-sharing contract. From Fig. 4b, the retailer's profit under normal temperature transportation mode is greater than that under cold chain transportation mode when β is less than the threshold β^ with the revenue-sharing contract; otherwise, the profit under cold chain transportation mode is greater than that under normal temperature transportation mode. In this case, to ensure that the retailer's profit is higher, the revenue-sharing proportion needs to be greater than the threshold β^. Figure 4b intuitively shows the management implication of Theorem 7. Now let the retailer transfer 10% of the revenue to the supplier, and other parameters remain unchanged. β¯ denotes the transferred profit amount of the retailer, where β¯=λβ=0.9λ. The relationship between the retailer's profit and the amount of transferred profit is shown in Fig. 5.Fig. 5 Relationship between retailer's profit and amount of transferred profits As can be seen from Fig. 5, when β¯≥0.959β, the profit of cold chain transportation after profit transfer is higher than that of normal temperature transportation before profit transfer. Thus, 0.959β≤β¯ is acceptable to the retailer. As can be seen from Table 5, regardless of the existence of coordination contract, the profits of both supply chain members and the total profits of the whole supply chain under the cold chain transportation mode are higher than those under the normal temperature transportation mode. By comparing these three situations, the profits of both supply chain members with coordination contract are higher than those with no coordination contract, and the total profits of the whole supply chain are correspondingly higher. In the case of coordination contract, the profits of supply chain members and the total profits of the whole supply chain with the revenue-sharing contract are higher than those with the wholesale price contract. Therefore, the supply chain with coordination contract is better than the supply chain with no coordination contract, and the revenue-sharing contract can better coordinate the fresh product supply chain than the wholesale price contract.Table 5 Comparison of profits under three situations Transportation mode No coordination contract Wholesale price contract Revenue-sharing contract ∏s∗ ∏r∗ ∏T∗ ∏s1 ∏r1 ∏T1 ∏s2 ∏r2 ∏T2 Normal temperature 2304.39 4224.71 6529.10 2071.58 5639.30 7710.88 3335.96 4594.45 7930.41 Cold chain 2523.17 4625.81 7148.98 2290.00 6106.67 8396.67 3652.68 4815.37 8468.05 Discussion and managerial insights Through the above analysis, this paper draws many conclusions. There are some similarities and differences between these conclusions and existing relevant literature.Cold chain transportation is beneficial to the fresh product supply chain, which is similar to the conclusion of Yan et al. (2020b). Cold chain transportation can not only improve the freshness of products and reduce resource waste, but also improve human food safety. For the supply chain, cold chain transportation can improve the profits of participants under certain situations. In the situation with no coordination contract, Yan et al. (2020b) found that there are two thresholds of cold chain transportation cost for the supplier using cold chain transportation. Different from them, the threshold of cold chain transportation cost has only one upper limit in our model. In the situation with no coordination contract, we find that ∏ri∗∏si∗=KK-1 under two transportation modes. The profit ratio of two supply chain participants is related to price sensitivity, and the retailer's profit is higher than the supplier's profit. This is contrary to the view of Yan et al. (2020b), that is, regardless of other factors, the supplier's profit is always twice the retailer's profit. Whether the supplier uses cold chain transportation depends on the cost of cold chain transportation. This is similar to the conclusion of Lin et al. (2020). The supplier will provide cold chain service only when the cost is lower than the threshold. Wang and Zhao (2021) found that the supplier can be encouraged to invest in cold chain service with the increase of profits, which is consistent with this paper. Increasing the supplier's profit by investing in cold chain service will greatly stimulate the supplier to provide cold chain service. Xiao and Xu (2013) confirmed that the revenue-sharing contract can coordinate the fresh product supply chains. They discussed the coordination of supply chain when the revenue-sharing proportion is in two large ranges. However, the model in this paper obtains the specific value range of revenue-sharing proportion. This paper finds that by formulating the wholesale price contract, the retailer encourages the supplier to use cold chain transportation by increasing the wholesale price, but the wholesale price should be within a certain range. Unlike Yan et al. (2020a), the manufacturer sacrifices the wholesale price to induce the retailer to increase the order quantity. Although both papers use wholesale price contract to coordinate the supply chain, their starting points are different. According to the above analyses, the following management suggestions and improvement strategies are put forward to help the participants of the fresh product supply chain in which the supplier should bear the transportation cost to maximize profit.The fresh product supply chain should use cold chain transportation to reduce food waste and improve customer satisfaction. The supplier should evaluate the cost of cold chain transportation before choosing the transportation mode. It is advantageous only when the cost is below a certain value. Cold chain transportation can increase the profits of the supplier and the retailer, and the whole supply chain can benefit whether there is a coordination contract or not. The wholesale price contract and the revenue-sharing contract can coordinate the supply chain, so that the profits of supply chain members with coordination contract are higher than those with no coordination contract. However, the wholesale price or the revenue-sharing proportion should be set within a certain range when the supplier uses cold chain transportation. Revenue-sharing contract is superior to wholesale price contract, and wholesale price contract is superior to no coordination contract. As a follower of the supply chain with the wholesale price contract, the retailer has the incentive to encourage the supplier to provide cold chain service. With the revenue-sharing contract, the supplier has the incentive to use cold chain transportation. Conclusions When the supplier uses normal temperature transportation, the fresh products will cause quantity loss and quality loss, resulting in lower profits and customer satisfaction for supply chain members. The transportation mode also has a great impact on the retailer, which has always been ignored in the previous literature. However, when the supplier uses cold chain transportation, there will be a game among supply chain members due to the high cost of cold chain. This paper identifies the impact of two transportation modes on the performance of a two-echelon fresh product supply chain, and analyzes the value loss of the two transportation modes under three situations: no coordination contract, wholesale price contract and revenue-sharing contract. The following conclusions can be drawn.The wholesale price and the retail price increase with the increase of the transportation cost and the average shelf time. The higher the transportation cost borne by the supplier, the higher the wholesale price and the retail price. The retailer is motivated to encourage the supplier to use cold chain transportation because it can benefit from cold chain transportation. The supplier uses cold chain transportation only when the cost is lower than the threshold CT, which can maximize its own interests. CT is related to the production cost, quantity losses, normal temperature transportation cost, price sensitivity, and the impact of freshness on demand. With the wholesale price contract, there are two conditions for the supplier to use cold chain transportation. First, the cost of cold chain transportation is required to be lower than the threshold CTS. Second, the wholesale price must be within the specific range of wTS<w<w¯. In this way, both the supplier and the retailer can make more profits than under normal transportation mode; With the revenue-sharing contract, when the cost of cold chain transportation is below the threshold CTR, the supplier will use cold chain transportation. To protect the benefit of both participants, the revenue-sharing proportion should satisfy β^<β<β¯. The profits of both supply chain members and the total profits of the whole supply chain under the cold chain transportation mode are higher than those under the normal temperature transportation mode. The profits of supply chain members with coordination contract are higher than those with no coordination contract. The revenue-sharing contract can better coordinate the fresh product supply chain than the wholesale price contract. Although the conclusions of this paper put forward the conditions and coordination mechanism for supply chain members to promote the supplier to use cold chain transportation, which can provide guidance for supply chain enterprises to better coordinate the supply chain and improve the quality of fresh products, there are still some limitations. This paper considers the supply chain composed of a supplier and a retailer. However, there are many more complex supply chain structures in real life, such as the structure of multiple suppliers and multiple retailers, suppliers or retailers opening online channels, the TPLSP providing cold chain service. In addition, this paper assumes that both the supplier and the retailer are risk neutral. Although this is a general assumption, it does not apply to every member of the supply chain. In fact, many supply chain members are risk averse. Finally, as the leader of Stackelberg game, the supplier takes priority to decision-making in this paper, but the retailer may be the leader in the supply chain, in which many large retailers participate. Based on the above limitations, more topics can be considered in future research. Acknowledgements This work was supported by National Natural Science Foundation of China (71871098). Declarations Conflict of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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==== Front J Gen Intern Med J Gen Intern Med Journal of General Internal Medicine 0884-8734 1525-1497 Springer International Publishing Cham 36451012 7972 10.1007/s11606-022-07972-w Healing Arts A Doctor or a Virus? On Becoming an Asian-American Doctor During the COVID-19 Pandemic http://orcid.org/0000-0002-8483-0647 Au Cherry MD [email protected] grid.240684.c 0000 0001 0705 3621 Department of Internal Medicine, Rush University Medical Center, 1620 W Harrison St, Chicago, IL 60612 USA 30 11 2022 11 12 10 2022 16 11 2022 © The Author(s), under exclusive licence to Society of General Internal Medicine 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcI am learning how to become a doctor in 2018. It’s my third year of medical school, and I am wearing a proud smile as I introduce myself as a “student doctor” and shake the patient’s hand. I start learning the art of medicine. Then, as I approach the end of my third year, whispers of a virus start to circulate. It begins with my family, warning me about the messages they received from WeChat from my family in China. The whispers become headlines and now and the headlines bring panic to the community. During this time, I am at a family medicine practice in rural New Jersey. I still go to the rotation as usual, but now we get barraged with questions about this new virus. Then, I get assigned the task of phone triage when the office limits walk-ins. “I brought my family to a Chinese restaurant; do you think I could get the virus from the workers or was it in the food?”. I answer back in my crisp English; there is no hint of an accent despite Cantonese being my mother tongue. The weight of my skin feels extra heavy on the phone while I am answering her concerns. The next day, I go to the office and immediately get asked to leave the room by a patient. Is it because I am a student or because of my Chinese-Vietnamese appearance? I try not to dwell too much on this thought as I head towards the exit. I have lived long enough to be able to manage my pangs of pain after racial targeting and microaggressions, but this interaction introduced a new anxiety. I worked for years to be given the opportunity to care for others, but now with the associations of Asian-Americans as the vector for disease, would my patients look at me as a doctor or a disease? I learned that same afternoon that students are removed from rotations due to the pandemic, and I spend the next weeks reading headlines of attacks on people who look like me and the spread of COVID-19. I spend my day-to-day trying to hide my Asian features in the street to avoid words of blame and actions of hate. This does not prevent racial slurs hurled at me when I walk down the street or people spitting on me and calling me a virus spreader. Eventually, students get called to return to clinical duties. On my seven-block walk to the hospital from my home, I get statements of appreciation and hate. “Thank you for taking care of our sick” and “Go back to China!” are both said to me during my walk to work. It is an odd dichotomy of being celebrated and vilified within a 15-minute walk. Now, I am in residency. I wear my skin with pride, but it has been considerably thickened from the pandemic. There is now this unspoken bond between Asian-American providers who when we speak about our pandemic experience since I am not alone in this experience. My community is not the first to be blamed for a disease and surely will not be the last. Am I the embodiment of disease or am I a healer? The answer to this question seems to differ depending on who gets asked. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	36451012	PMC9713200	NO-CC CODE	2022-12-02 23:22:56	no	J Gen Intern Med. 2022 Nov 30;:1	utf-8	J Gen Intern Med	2,022	10.1007/s11606-022-07972-w	oa_other
==== Front Datenschutz Datensich Datenschutz und Datensicherheit - DuD 1614-0702 1862-2607 Springer Fachmedien Wiesbaden Wiesbaden 1700 10.1007/s11623-022-1700-x Aufsätze Hat die häufige Videotelefonie in Corona-Zeiten Einfluss auf unser Privatsphäreverständnis genommen? Eine explorative empirische Studie Selzer Annika [email protected] ist Leiterin der Abteilung IT Law & Interdisciplinary Privacy Research am Fraunhofer-Institut für Sichere Informationstechnologie (SIT) und Co-Leiterin des Forschungsbereichs Legal Aspects of Privacy and IT Security am Nationalen Forschungszentrum für angewandte Cybersicherheit ATHENE. Stummer Sarah (LL.M.) [email protected] ist wissenschaftliche Mitarbeiterin der Abteilung IT Law & Interdisciplinary Privacy Research am Fraunhofer-Institut für Sichere Informationstechnologie (SIT) und am Nationalen Forschungszentrum für angewandte Cybersicherheit ATHENE. grid.469848.f 0000 0001 0617 5328 Fraunhofer-Institut für Sichere Informationstechnologie, Darmstadt, Deutschland 30 11 2022 2022 46 12 774777 © Springer Fachmedien Wiesbaden GmbH 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Seit Beginn der Corona-Pandemie konnte man sich mit Geschäftspartnern, Freunden und Familienangehören häufig nur noch virtuell austauschen. Für die meisten Menschen war die Videotelefonie das Mittel der Wahl, um die Kontakte so persönlich wie möglich zu gestalten. Dieser Beitrag untersucht basierend auf zwanzig teilstrukturierten Interviews, inwiefern die Videotelefonie und das damit verbundene ,,Beobachtetwerden‘‘ durch eine Kamera Einfluss auf unser Privatsphäreverständnis genommen hat. issue-copyright-statement© Der/die Autor(en), exklusiv lizenziert an Springer Fachmedien Wiesbaden GmbH, ein Teil von Springer Nature 2022 ==== Body pmc	0	PMC9713201	NO-CC CODE	2022-12-02 23:22:56	no	Datenschutz Datensich. 2022 Nov 30; 46(12):774-777	utf-8	null	null	null	oa_other
==== Front Datenschutz Datensich Datenschutz und Datensicherheit - DuD 1614-0702 1862-2607 Springer Fachmedien Wiesbaden Wiesbaden 1697 10.1007/s11623-022-1697-1 Schwerpunkt Opt-ionen für die elektronische Patientenakte – Einwilligungs- oder Widerspruchsmodell? Dochow Carsten Leiter Personal, Organisation und Datenschutz, Bundesärztekammer, Berlin Autor zahlreicher Fachbeiträge zur Digitalisierung und zum Datenschutz im Gesundheitswesen. https://dochows.de/ Berlin, Deutschland 30 11 2022 2022 46 12 747755 © Springer Fachmedien Wiesbaden GmbH 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Nach mehr als zehnjähriger Planungstätigkeit steht möglichweise ein Systemwechsel für die elektronische Patientenakte an. Gesundheitspolitisch wird von einigen ein Opt-out-Modell favorisiert, unter anderem um die Nutzerzahlen zu erhöhen. Damit geht nicht nur eine technische Umgestaltung einher, sondern die bisher auf einer Einwilligung des Versicherten und damit auf Freiwilligkeit basierende normative Grundkonzeption steht in weiten Teilen zur Disposition. Déjavus zum jahrzehntelangen Streit im Bereich der Organspende scheinen zwangsläufig. Ob nunmehr sehr grundsätzliche Debatten die Einführung der elektronischen Patientenakte noch weiter verzögern werden, ist offen. Der folgende Beitrag setzt sich eher mit den Anforderungen für ein Opt-out-System für die elektronische Patientenakte in Deutschland auseinander und will einige Aspekte kritisch beleuchten. issue-copyright-statement© Der/die Autor(en), exklusiv lizenziert an Springer Fachmedien Wiesbaden GmbH, ein Teil von Springer Nature 2022 ==== Body pmc	0	PMC9713204	NO-CC CODE	2022-12-02 23:22:56	no	Datenschutz Datensich. 2022 Nov 30; 46(12):747-755	utf-8	null	null	null	oa_other
==== Front Voluntas Voluntas Voluntas 0957-8765 1573-7888 Springer US New York 547 10.1007/s11266-022-00547-7 Research Papers Gig Economy Riders on Social Media in Thailand: Contested Identities and Emergent Civil Society Organisations http://orcid.org/0000-0002-0731-8463 Mieruch Yannik [email protected] McFarlane Daniel [email protected] grid.412434.4 0000 0004 1937 1127 School of Global Studies, Thammasat University, Pathumthani, 12120 Thailand 30 11 2022 111 14 11 2022 © International Society for Third-Sector Research 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The emergence of the gig economy has generated a new class of workers who are categorised as independent “partners” instead of employees with rights to labour protection. Triggered by observations of a protest movement by platform-based delivery riders in Thailand, we engaged in seven months of digital ethnographic research of riders’ interactions online to understand the emergence of informal groups facilitating mutual aid and collective action. Civil society research has neglected to analyse such groups within the gig economy. The study finds that social media is a site for the development and contestation of identity narratives. We observed a “Hero” narrative that glorifies delivery riders' independent status and a “Worker” narrative that challenges riders' conditions. We argue that these collective identity narratives crucially facilitate or inhibit the emergence of labour-oriented civil society organisations, thus contributing to third sector research that examines civil society in the Global South. Keywords Gig economy Civil Society Labour Relations Social Media Global South ==== Body pmcIntroduction The way people work, socialise and organise is undergoing rapid transformation in Thailand and globally. The food delivery economy is at the forefront of this change. During the COVID-19 pandemic, the food delivery sector in Thailand grew significantly to US$ 4 billion in 2021 (Momentum Works, 2022). The leading delivery platform Grab captures nearly half of the market in which it competes with foodpanda, LINE MAN, Lalamove and other emerging platforms (Momentum Works, 2022). The impact of these food delivery platforms in Thailand has been striking, particularly in Bangkok, where delivery riders on motorcycles have become a conspicuous part of the urban landscape. These riders are part of the global shift to the gig economy and the digitalisation of work. The rise of the gig economy in Thailand is welcomed by a consumer-focused citizenry who value the expansion of convenience and a labour force eager for independent work in depressed economic times. However, there have been increasing rumblings of discontent in the gig economy. At the end of 2020, we observed a growing labour protest movement in Bangkok by delivery riders (Bangkok Judd, 2020; Post, 2020). Fuelled by frustration with the precarious nature of gig work and a lack of worker rights, the movement reflects the contradictions between workers and capital inherent in the capitalist mode of production (Atzeni, 2010). It is also one of many struggles for better working conditions by gig workers in the region and globally (Alderman, 2015; Buckley, 2020; Gropp, 2019; Tassinari & Maccarrone, 2020; Vasandani, 2021). We began by asking where and how do riders connect and come together. Research on gig workers elsewhere emphasises the importance of physical meeting spaces for the development of collective identities and organising for collective action (Ford & Honan, 2019; Tassinari & Maccarrone, 2020; Wells et al., 2021). Research of gig workers also shows how in the absence of physical meeting spaces online spaces can perform a similar function (Lehdonvirta, 2016; Soriano & Cabañes, 2020; Alex J. Wood et al., 2018). Yet, the role of online spaces has not been analysed among delivery riders. While studies on delivery rider organisation consistently mention social media, they largely treat social media as a tool for communication and mobilisation overlooking potential dynamics of socialisation and identity-building online (for example, see Chesta et al., 2019; Stewart et al., 2020; Tassinari & Maccarrone, 2020). Our early research in Thailand directed us to large and active social media groups and pages managed by riders, some with as many as 80,000 followers. Hence, we started asking questions about what role social media was playing in the formation of worker identities and the emergence of civil society organisations that facilitate collective action. To answer these questions, we engaged in digital ethnographic research observing and analysing online communication among riders. Our research reveals social media as a virtual environment for socialisation and mutual aid, which is consistent with research on online interactions of gig workers in other countries (Lehdonvirta, 2016; Soriano & Cabañes, 2020; Alex J. Wood et al., 2018). Online spaces such as Facebook groups and pages help riders overcome the inherent loneliness and social disconnection of their working conditions while facilitating solidarity and mutual aid. Furthermore, riders also use online spaces as channels to cultivate and express collective identities as well as organising for collective action. On Facebook pages, riders actively express and propagate narratives of themselves as workers with the capacity to unite and campaign for better working conditions. These collective identities provide the foundation for loose social media-based groups of riders to move from leveraging social media for socialising and mutual aid to forming informal labour-focused civil society organisations. The emergence of collective worker identities in online spaces is, however, complicated by the presence of gig economy platform operators. Unlike physical spaces, online spaces are open to platform companies who have a vested interest in obstructing the development of collective worker identities that could form the foundation for organising and collective action. To ensure maximum efficiency in the extension of labour power, capital strives to exert control over the labour process (Braverman, 1998). Increasingly, managerial control is extended to the realm of identities in the form of normative control (Alvesson & Willmott, 2002; Fleming & Sturdy, 2011; Thompson et al., 2004). We observed platforms deliberately attempting to shape the identity of riders as Heroic entrepreneurs operating autonomously. Through adopting labour process theory (LPT), we argue that platform companies’ attempt to shape worker identities is a form of normative control that undermines their capacity for collective action. The narrative of riders as Heroic entrepreneurs is both resisted and consented to by riders on Facebook pages; thus, we argue that social media sites are spaces where riders both project and contest their identities. This process of contestation impacts the formation of new forms of civil society organisations in the Thai gig economy. The remainder of the paper is structured as follows. We start by elaborating on the labour relations in the gig economy, locating our research within third sector literature, discussing our conceptual framework and introducing our methodology. We then present our research findings including analysis of platforms’ exertion of normative control, riders' reaction to normative control and the emergence of new forms of labour-oriented civil society organisations. In conclusion, we discuss the implications of our findings for civil society research and identify areas for further inquiry. Literature, Conceptual Framework and Methodology The Gig Economy The gig economy “involves the exchange of labour for money between individuals or companies via digital platforms that actively facilitate matching between providers and customers, on a short-term and payment by task basis” (Lepanjuuri et al., 2018, p. 4). Leveraging digital technology the platform model employed by gig economy companies differs strongly from traditional business models facilitating flexible operations and reducing overhead costs (Parker et al., 2016). The gig economy is predominantly split into two categories: crowdwork which refers to online-only platforms that leverage a dispersed workforce to complete tasks online; and work-on-demand via app which refers to platforms whose workers perform services offline notably food delivery and transportation services (De Stefano, 2015). This article is concerned with this latter section of the gig economy specifically food delivery platforms in Thailand. A key feature of the gig economy is the categorisation of workers as “partners” rather than employees, which shifts risks to workers creating precarious conditions without social protection (Friedman, 2014). While the dominant neoliberal discourse celebrates workers in the gig economy as entrepreneurs who engage independently in the new digital economy, the perspective of gig economy workers as employed workers with labour protection rights has gained traction globally (De Stefano, 2015; Friedman, 2014). The denial of a proper employment relationship for gig workers, individualised and dispersed working conditions, and the use of algorithmic labour management practices are detrimental to worker organisation and collective action (Johnston & Land-Kazlauskas, 2018). In Thailand, the challenges to gig worker organisation are amplified by weak labour unions which face legal obstacles and state-sanctioned union-busting resulting in consistently low rates of membership (ILOSTAT, 2016; Wannasiri, 2020). Labour movements, Civil Society and Social Media This article is firstly located within scholarship concerned with the emergence of new forms of labour-oriented civil society organisations. Membership-based labour unions have long been central actors in civil society, especially in the Global North (Putman, 1995). However, due to the external pressure of a changing economy and internal failures to adapt, they have faced a decline in importance (Anjum, 2012; Kumar, 1993). Yet, in both the Global North and South, labour issues persist, and demand for labour organisations remains as worker rights are squeezed under neoliberal policies and new forms of digitally mediated labour relations (De Stefano, 2015; Mosoetsa & Williams, 2012). In response, alternative forms of labour movements have emerged in the context of the gig economy (Chesta et al., 2019; Panimbang, 2021; Tassinari & Maccarrone, 2020; Alex J. Wood et al., 2018). What are the dynamics of power and control that shape the emergence of these organisations is a driving question behind the research for this article. Secondly, this article is located within a discourse that considers alternative conceptions of civil society. Scholarship on civil society has tended to focus on institutionalised forms of civil society and neglect the diversity of informal civil society activities, which dominate urban politics in much of the Global South (Bayat, 1997). This focus reflects the evolution of the concept of civil society in liberal democracies of the Global North where freedom of association exists and formal forms of civil society are not restricted or co-opted by the state (Anjum, 2012; Kumar, 1993). However, research on civil society in the Global South, post-soviet countries and countries in economic crisis illustrates a great variety of alternative and informal forms of civil society organisations and civic engagement (Hannah, 2007; Krasynska & Martin, 2017; Schak & Hudson, 2003; Sotiropoulos, 2004; Urinboyev & Eraliev, 2022; Wells-Dang, 2012). In the Thai context, Chua (2018) argues that there is no clear distinction between institutionalised civil society organisations and the state. Institutionalised NGOs often depend on the state for funds and are arguably ideologically similar. Hence, some scholars argue that large sections of civil society in Thailand tend not to adhere to traditional notions of civil society organisations and it is informal and locally rooted sections of civil society that consistently challenge state power and elite interests (Vichit-Vadakan, 2003; Walker, 2012). Hence, it is important we adopt a definition of civil society that accounts for the conditions of the digitally connected world in which gig workers live and the political and social circumstances in the Global South, particularly in Thailand. Chambers and Kopstein (2006, p. 364) describe civil society as “a sphere in which individuals come together and form groups, pursue common enterprises, share interests, communicate over important and sometimes not so important matters”. Reflecting traditional understandings of civil society as voluntary associations they categorise this form of civil society as “apart from the state” in contrast to civil society against the state, in support of the state, in dialogue with the state, in partnership with the state or beyond the state. The informal groups of delivery riders we observed initially coming together voluntarily on social media sites for mutual aid constitute a form of civil society apart from the state. However, we observed a shift within these groups going beyond mutual aid and developing a common identity that facilitates collective action towards a common goal. According to Chambers and Kopstein (2006, p. 365), “in civil society individuals come together to pursue particularistic ends”. When these particularistic goals challenge the state, corporations and other elite interests, civil society actions take on a political dimension. Therefore, following Wells-Dang (2012, p. 24) we conceive civil society as “a political process of collective action and alliance building”. Through this process, civil society actors “articulate their interests and make demands; defend their rights vis-a-vis the state and others; and meet their needs directly, without depending on state agencies” (Uphoff & Krishna, 2004, p. 359). It is through these lenses we explore how delivery drivers transition from individuals that come together for mutual aid to becoming civil society actors. Thirdly, this article speaks to a discourse that examines the role of social media within civil society. New forms of networked communication such as social media decentralise power, enable the seamless formation and growth of groups with common interests, and empower civil society actors to circumvent traditional hierarchal state-centric power structures (Castells, 2015; Castells & Cardoso, 1996). Hence, authors such as McNutt et al., (2018, p. 27) argue that social media could trigger a change in the way the third sector is organised, which "might revolutionise non-profit research and theory”. Given the prevalence of digital technology in all sectors of society, it is crucial that non-profit and third sector research considers new forms of civil society that are emerging online. Virtual civil society organisations are particularly salient in societies where conventional civil society is weak and there is little trust in the state (Beissinger, 2017; Papachristopoulos & Zafiropoulou, 2016). Analysis of the pro-democracy movement in Thailand during the COVID-19 pandemic reveals how activists leveraged social media to galvanise support, engage in decentralised coordination and tactics as well as build collective narratives (Auethavornpipat & Tanyag, 2021; Sinpeng, 2021). Yet, according to McNutt et al. (2018) research on civil society tends to focus on the use of information communication technologies, including social media, as tools for administration, marketing and fundraising (for example, see Lai & Fu, 2021; Tian et al., 2021; Zhou & Pan, 2016). As a result, scholarship on the emergence of virtual civil society organisations and associations that do not conform to traditional conceptions of civil society is often neglected. By examining the dynamics of power and control that shape how groups on social media develop collective identities and form particularistic goals, we intend to address critical questions on how new forms of civil society organisations, particularly labour-oriented organisations, are emerging in the digital age. Conceptual Framework: Labour Process Theory (LPT) Labour process theory (LPT) focuses on issues of control over the labour process and worker agency in the face of such control (Lucio & Stewart, 1997). Rooted in critical Marxist thinking within the sociology of work and critical management studies, LPT frames managerial control over the labour process as a key mechanism for ensuring the most efficient transformation of labour power into a commodity for the maximisation of profit (Braverman, 1998). Scholars examining the gig economy have frequently adopted LPT as a framework to understand the control exerted by platforms over gig workers through mechanisms such as algorithmic control, the “gamification” of work, incentive structures and information inequalities (Gandini, 2019; Gerber & Krzywdzinski, 2019; Veen et al., 2020; Alex J Wood et al., 2019). Studies in Thailand have also adopted LPT to demonstrate that platform companies exert control over delivery riders’ labour process (Teerakowitkajorn & Tularak, 2020). These scholars reject the notion that delivery riders are independent “partners” instead framing them as “workers” and platform companies as “employers” with the capacity to exercise power over the labour process. Recently traditional forms of control have been supplemented by normative control, which shifts the focus of control from behaviours and outputs towards crafting worker identities to align with company objectives (Alvesson & Willmott, 2002; Thompson et al., 2004). Previous studies have found evidence of different forms of normative control in the gig economy (Ens, 2019; Fleming & Sturdy, 2011). In our research, we focus specifically on identity regulation as a form of normative control (Rennstam, 2017). According to Rennstam (2017), identity regulation puts the naturally occurring socialisation process of identity formation under managerial control through measures that construct specific personal characteristics or skills as ideal characteristics of a "good" worker in the given domain. According to Stewart et al. (2020), platform companies project a public narrative of the gig economy as an opportunity for flexible and self-determined work unattainable in traditional employment and gig workers as entrepreneurs that flourish in this working environment. They argue that this narrative supports the justification for platforms to avoid compliance with legal employment requirements. Power and agency are rarely one-sided. LPT scholars consistently highlight how workers are not only objects of control but retain the agency to actively resist or consent to regimes of control (Burawoy, 1982; Thompson & Smith, 2017). Several studies show how gig workers consent to normative control and adopt platform companies’ identity narratives (Josserand & Kaine, 2019; Soriano & Cabañes, 2020; Stewart et al., 2020). However, the studies by Stewart et al. (2020) and Josserand and Kaine (2019) also reveal workers contesting the identity narratives propagated by platform companies. Stewart et al. (2020) further argue that identity narratives that contest dominant platform narratives play a key role in the emergence of collectivism among delivery riders. In this study we are interested in how collective identities facilitate the emergence of alternative forms of labour-oriented civil society organisations. Methodology: Digital Ethnography The research for this paper builds on a rich tradition of applying ethnographic methods to studying the sociology of work in offline and online field sites (Braverman, 1998; Burawoy, 1982; Lehdonvirta, 2016; Soriano & Cabañes, 2020). Since our focus is on interactions and communication within online spaces, such as social media, our primary method of research is digital ethnography, which has been adopted by scholars from various disciplines including third sector research (Boellstorff et al.,2012; Kozinets, 2010; Oreg & Babis, 2021; Pink et al., 2015). While digital ethnography may incorporate both online and offline field sites, we adopt netnographic methods, which is a sub-category of digital ethnography that focuses solely on online field sites (Kozinets, 2010). Within the literature on the gig economy, scholars such as Soriano and Cabañes (2020) and Lehdonvirta (2016) have studied the online interactions of crowdworkers including identity-building processes and the role of online forums for the organisation of collective action. Due to the exploratory nature of the study and the fluidity of online spaces, we started the research without a clearly defined field site. As a starting point, we used identifiable Facebook pages that were used to mobilise riders to participate in protests and then expanded to other social media pages and sites. Although other sites of research were included, most observations and data collection were concentrated on the three Facebook rider managed pages with the most followers and the three public rider Facebook groups with the most members. The pages include two pages dedicated to riders from all platforms and one page dedicated to Grab riders. Followers on these pages range from 13,000 to 86,000. The three groups observed are dedicated to three of the largest platforms (Grab, Foodpanda, Lalamove) and membership ranges from 19,000 to 78,000. We also observed and analysed the presence of platform companies online. Netnographic research in the form of non-participant observation was conducted for approximately seven months starting in early 2021. The research was carried out using our personal Facebook profiles which allowed for a deep immersion in the field site as the posts would appear on our Facebook feed throughout the day as they would for riders. The collected data were coded and grouped around emerging themes building on narrative analysis (Bernard, 2017). The nature of Facebook as a virtual field site raises new ethical concerns for the researcher (Sveningsson, 2004; Willis, 2019). Most importantly, acquiring informed consent from participants is difficult if not impractical (Hudson & Bruckman, 2004). However, like public spaces offline informed consent is not necessary in public online settings (Willis, 2019). Willis (2019) argues that for an online space to be considered public it must be public on a technological level and members of the community must perceive the space as public. Applying this criterion, we identify the Facebook pages and groups in our study as public places and as such informed consent from participants was not required. Nevertheless, we consistently applied measures to ensure the anonymity of research subjects by masking quotes and omitting the names of informants and groups. Findings Platform Projections of a Hero Identity Narrative A feature of identity regulation, as a form of normative control, is the provision of a set of vocabulary to workers which supports the propagation of an identity narrative that encapsulates the characteristics and values that align workers with their employers’ objectives (Rennstam, 2017). Grab recruits its delivery riders online with slogans such as: “Partner with us to empower your livelihood and more”; “Take pride in satisfying millions of our hungry customers every day”; and “Be your own boss”. The use of the terms “empower” and “take pride” as well as the notion of being one’s own boss imply that working as a rider both requires and allows for a specific set of values such as pride in individual capabilities and independence. Describing riders as “Heroes” is a particularly pervasive feature of platform companies' efforts to construct rider identities. The LINE MAN platform offers riders a Hero version of the brand’s rider jacket if they can regularly achieve a designated volume of deliveries. In one advertising campaign, riders are further directed to identify as heroes by saying the slogan “LINE MAN Food Hero fights for every meal to make sure every meal is delicious” to customers when they make deliveries. Grab also produces rider jackets featuring Marvel superheroes and incorporates the Hero identity in their points system that ranks riders based on delivery performance metrics and customer ratings by making “Hero” the highest attainable rank (Fig. 1).Fig. 1 Grab superhero jacket The Hero narrative portrays riders as hard-working and service-minded individuals who give their best to deliver goods and services while overcoming considerable difficulties. For instance, one of Grab’s online video campaigns shows riders delivering food to a boat, making merit for an old couple that cannot go out and dealing with complaining customers. Another video campaign advertises the Grab Mart service, which involves riders delivering groceries to customers. The campaign features singing riders choosing the freshest products for their customers. While these campaigns are directed at consumers, they also capture the attention of riders, evident in riders mentioning the videos in several posts shared across rider Facebook groups and pages. These campaigns support the formation of a rider identity with the values of grit, a hard-working ethic and a service mindset. The projection of the Hero narrative and the associated vocabulary presents riders with the values and characteristics associated with a “good” rider within the context of precarious and individualised working conditions, thus functioning as a form of identity regulation. Rider Adoption of the Hero Narrative Posts made on rider Facebook groups and pages reveal that the Hero narrative and the associated characteristics are widely embraced and consented to by riders. Riders actively portray themselves as hardworking individuals that fight to overcome difficulties within the context of precarious working conditions while earning a living and providing for their families. One widely shared post is an artist's depiction of riders as superheroes. Riders representing each of the platforms are illustrated as distinct superheroes in a form that nearly mirrors the platforms' use of superhero imagery (Fig. 2).Fig. 2 Riders as superheroes By embracing the Hero narrative riders’ consent to the normative control of platforms, effectively internalising the values and norms of behaviour that platforms see as desirable. In some cases, riders enforce these values and norms of behaviour among themselves. One example observed is a reaction to a post in which a rider complained about a negative experience with a customer. Instead of expressing support, fellow riders assigned blame to the rider who made the post by citing the rider’s lack of service-mindedness. In another case, a page administrator created a live stream of them accompanying a rider to apologise to a restaurant for rude behaviour. Many posts showcase hardships throughout the day, but these hardships are often framed as obstacles to overcome with a fighter's mindset. One rider posted the following:“People ask me about working in the sun. Isn't it hot? Aren't you afraid of getting black skin? My Answer: “I’m more afraid of starving to death." […] I decided […] to drive for Grab because I thought it provided me more freedom. […] When I tried it, it was a little bad at first. […] I felt very upset. But I met many fellow riders who gave me a lot of advice. […] During the Covid-19 pandemic it's not easy. […] It is very tiring, but I'm proud! #ThankYouGrabForGivingMeThisOpportunity. I am writing this to encourage all Grab brothers and sisters. Fight on for our (Grab) family ”. The rider recognises the difficult working conditions but refrains from complaining and instead expresses gratitude to Grab for the opportunity to earn a living. The tough working conditions are portrayed as an individual challenge and the ability to overcome them as a source of personal pride. The post is also a call to all riders in the Grab community to fight on to overcome their individual challenges. One widely shared video portrayed a rider who was missing one hand, but through hard work and determination, was able to make a living as a rider. The video received widespread acclaim in comments that affirmed the necessity to fight hard to succeed as a rider reinforcing the notion that success or failure ultimately depends on individual effort. By adopting the Hero narrative riders adjust to the trying working conditions. If they are the Hero of the story, then the effort necessary to be a successful rider is a choice rather than forced on them by platforms. The Hero narrative thus effectively masks the real social relations of production based on exploitation and unequal power between riders and platforms. Riders as Workers: Contesting the Hero Narrative The Hero identity narrative, while significant, is not uncontested. Some riders resist the framing of their hardships as individual challenges. In social media posts, riders highlight their common conditions by complaining about the arbitrariness and unfairness of the platform's algorithm and negative customer reviews. They challenge unfair and unexplainable bans or deactivations from using a platform or the general inefficiency of the platform’s app. Most of these posts receive both understanding and encouragement from other riders in the comments section suggesting recognition of common grievances. In contrast to the Hero narrative, these posts and comments display a recognition that riders are dependent on platforms, which take advantage of them as workers and have little concern for the problems they face. A post by a foodpanda rider conveys this sentiment:"We work for the foodpanda system, so the platform doesn't care if the rider has one problem or another. They only care about the number of orders in each region. But we must accept the system. In the end, there is no use to complain about it, we must use the app to make money anyway". The rider’s post asserts their status as workers and dependency on the platform. This sense of dependence on the platform and helplessness is shared by other riders who note that any attempt to protest would be futile given the dominant narrative. One rider posted: "In fact, if we create too much fuss, we become a bad person in the eyes of the public. In the end, we have to accept the system that paints us as being in the wrong". Some riders also draw the connection to the Thai labour market and labour protection laws, which is evident in the following post:"I feel pity for Thai workers because the law doesn't help with anything. Bowing to foreign companies to exploit us working like slaves. However, it's a Thai habit to let things slide without knowing how to fix or change it. That's how it works in our own country, being left to be taken advantage of". These posts portray a narrative of riders as exploited and powerless workers. The use of terms such as "exploitation", "being taken advantage of" and "working as slaves" signifies an acknowledgement of the power imbalance between riders and platforms and implies the collective nature of these power structures. In some rare cases, riders address the classification of the riders as “partners” as a source of their exploitation. Shared across different rider Facebook groups, one post presented an analysis of riders' classification as partners:"[…] The `partner' status […] is nothing other than the platform company's excuse to extort workers and evade worker protection legislation. In addition, […] flexible employment gives workers no bargaining power with the company. […] Because riders on the platform are workers they deserve compensation, welfare rights, protection, and the company honouring their responsibilities. They are not independent partners who don’t have the power to negotiate like we have been deceived to believe […]" As part of the contestation of rider identities, the post is a clear call to riders to recognise themselves as workers with rights. From Mutual Aid Groups to Labour-Oriented Civil Society Organisation The Hero and Worker narratives generate contrasting perceptions of rider working conditions and identities. On the one hand, the Hero narrative highlights gig work as empowering and self-determined. On the other hand, the Worker narrative conveys sentiments of powerlessness and dependence on the platforms. While the Hero narrative constructs riders willing to accept their precarious working conditions as individual challenges to overcome, the counter Worker narrative recognises the precarious conditions as intentionally created by platforms. The latter's acknowledgement of these structural conditions often comes with expressions of powerlessness that contrast with the empowerment promised by the Hero narrative, but it also raises questions about the potential of resistance through collective action. Despite the contesting identity narratives, we observed riders throughout the groups and pages frequently coming together to extend solidarity and mutual aid. Cases of mutual aid range from answering questions and sharing tips on how to deal with work-related challenges to more direct actions such as assisting in the case of accidents and the collection of donations for the families of riders that had fatal traffic accidents. Most rider pages were founded with a focus on providing mutual aid and often take the lead in providing the most extensive and organised help to their fellow riders. Scholars in a Marxist tradition argue that mutual aid and solidarity are fundamental forms of collectivism and the basis for the development of more direct forms of collective action (Atzeni, 2010; Fantasia, 1989). Some riders who adopt the Worker identity narrative recognise collective action as a potential avenue to addressing their many grievances. One rider jokingly hypothesised about the potential of a countrywide strike: "Just a funny thought what if riders all over the country stop working in protest?" Another rider took a more assertive position: "If protests are ineffective, let's try to agree to stop working for a day or two. That will probably shake up the people at the top". These posts indicate that some riders can overcome the sense of disempowerment woven through the Worker narrative to establish an optimistic view that collective action can challenge the power of platforms. We also observed a shift towards collective action at an organisational level. One case is a group of riders who managed a rider Facebook page. It transitioned from an informal group focused on mutual aid for Lalamove riders to an organisation resembling a union that champions all riders’ rights. It changed its name from Lalamove Freedom Riders to Freedom Riders Union and began displaying characteristics of a more formal union organisation including the collection of member fees and organising social activities. Over the following months, the group evolved into a nationwide network with different organisations in various provinces that organise protests and strikes. Another group behind a page dedicated to Grab riders engaged in a dialogue with representatives from Grab and the Ministry of Labour. While these attempts resulted in little to no tangible improvements in riders’ working conditions, the adoption of these strategies marks a shift away from localised mutual aid groups towards forms of civil society organisations with specific goals. Throughout the social media pages and groups, however, there was evidence of opposition to direct collective action soliciting conflict between riders. Some riders expressed bewilderment and opposition to them, exemplified in the following comment: “Why do you protest? If you feel that the company gives you too little, then just don’t work. They don’t force you to work, do they? I’m really confused when I see this”. Emphasising that riders are not forced to work for the platform exemplifies the Hero narrative’s characterisation of riders as autonomous and independent entrepreneurs. Another rider goes further by stressing that riders are independent entrepreneurs as per their classification as “partners” while highlighting the futility of protests and strikes due to the large pool of potential future riders:“You should understand first that Grab is not your boss, and we are not employees. Grab is a connection service. Whoever wants to use the service is free to use it and whoever doesn't is free not to. It's not compulsory. Let's be honest. Everyone looks out for themselves, so why should the company care about the riders? And why would we want to waste time protesting because people who are in a worse position than us are ready to join Grab every day?” In response to comments such as these, another rider commented: “It is called working for the collective. Do you understand that sometimes one voice is not enough? We need many voices to be successful. But an ass licker like you wouldn’t understand”. These kinds of rider conflicts speak to the contestation of rider identities. Under the worker identity, riders see collective action as empowering, while riders who adopt the Hero narrative see collective action as an affront to their individual liberty and autonomous entrepreneurial lives. This shows that while the Worker narrative facilitates collective action the Hero narrative undermines these efforts. Therefore, the potential of these emergent civil society organisations to organise and garner the support of workers to collectively campaign for better working conditions and protection is contingent on the broad adoption of the Worker identity narrative among riders. Conclusion This study demonstrates that riders in Thailand use social media to overcome the challenges associated with their individualised working conditions. Organisations that leverage social media to facilitate mutual aid among riders have emerged and increasingly developed features of unions. Our research extends the argument of Stewart et al. (2020) who argue that riders contesting platform narratives forms the basis the development of collectivism among riders. We argue that contested identity narratives not only constrict or facilitate the development of collectivism but also the development of labour-oriented civil society organisations. Our findings further demonstrate that social media is an important space for the development and contestation of identity narratives. Therefore, we argue that social media can play a role in the emergence of new forms of civil society beyond functioning as mere tools for organisational communication and logistics. In the Thai context, these findings are mirrored in scholarship on Thai social movements that highlights the growing importance of social media to galvanise support as well as build collective narratives (Auethavornpipat & Tanyag, 2021; Sinpeng, 2021). Thus, civil society scholars in Thailand should take social media and other online spaces seriously when considering the changing Thai civil society landscape. This study further contributes to a discourse that examines alternative forms of labour organisations in the gig economy which has so far neglected to analyse the role of social media as an arena of identity formation among work-on-demand via app workers such as delivery drivers. Additionally, we argue that it is important for third sector research to consider these newly emergent forms of labour movements as part of civil society. Our findings suggest that the underlying processes of identity formation on social media can provide insights into emerging civil society organisations within the gig economy and beyond. This study thus contributes to a discourse within third sector research that argues for broadening the conception of civil society to include informal, online and other alternative forms of civil society organisations. This study was limited to research in online settings, and we suggest that future research considers the interaction between offline and online settings. Attention should be directed towards how the contestation of identities among riders online translates into an offline environment and how offline settings influence the processes we observed online. The research was also limited to public social media groups and pages, but preliminary ethnographic research we have conducted indicates the extensive use of the LINE messenger app among riders who form closed group chats to facilitate solidarity and mutual aid in a local terrain. We suggest that further research could shed light on the relationship between these small and localised groups with larger organisations on Facebook and how identities may also be contested within these closed groups. Furthermore, we discovered cautionary evidence that an external formal civil society organisation has played a role in the process of organisation, specifically facilitating formalisation and capacity building of rider organisations. This suggests that while rider organisations defy traditional notions of civil society organisations, they do not emerge in a vacuum but rather engage in relationships with wider civil society. Hence, we suggest a closer analysis of the relationships between these new organisations and other civil society actors, particularly examining the influence of these external civil society actors and movements on the adoption of the Worker identity narrative among riders. Despite these unanswered questions, our research provides important insights into the dynamics of identity development among gig workers and the emergence of new forms of labour-oriented civil society organisations that champion workers’ rights in rapidly changing economies. Declarations Conflict of interest The authors declare that they have no conflict of interest. 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==== Front J Fr Ophtalmol J Fr Ophtalmol Journal Francais D'Ophtalmologie 0181-5512 1773-0597 Elsevier Masson SAS. S0181-5512(22)00407-7 10.1016/j.jfo.2022.11.001 Erratum Erratum to “Optic neuropathy in a COVID-19 patient” [J. Fr. Ophtalmol. 45 (2022) 1271–1273] Erratum à « Neuropathie optique chez un patient atteint de COVID-19 » [J. Fr. Ophtalmol. 45 (2022) 1271–1273]Borrego-Sanz L. Guemes-Villahoz N. * Fernández-Tresguerres F. Santos-Bueso E. Ophthalmology Department, Hospital Clínico San Carlos; Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), IdISSC, Calle del Prof Martín Lagos, s/n, 28040 Madrid, Spain ⁎ Corresponding author. 1 12 2022 1 12 2022 © 2022 Elsevier Masson SAS. All rights reserved. 2022 Elsevier Masson SAS Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcThe publisher regrets that there is an error in the spelling of the last name of an author. The correct last name is Guemes-Villahoz, and it appears as Guermes-Villahoz (extra “r”).	36464528	PMC9713487	NO-CC CODE	2022-12-02 23:23:04	no	J Fr Ophtalmol. 2022 Dec 1; doi: 10.1016/j.jfo.2022.11.001	utf-8	J Fr Ophtalmol	2,022	10.1016/j.jfo.2022.11.001	oa_other
==== Front Endocrinol Diabetes Nutr (Engl Ed) Endocrinol Diabetes Nutr (Engl Ed) Endocrinologia, Diabetes Y Nutricion 2530-0180 SEEN and SED. Published by Elsevier España, S.L.U. S2530-0180(22)00229-3 10.1016/j.endien.2021.12.014 Original Article Severity of new-onset type 1 diabetes in children and adolescents during the coronavirus-19 disease pandemic Gravedad al debut de la diabetes tipo 1 en niños y adolescentes durante la pandemia por la enfermedad por coronavirus-19Rivero-Martín María José a⁎ Rivas-Mercado Carmen María b Ceñal-González-Fierro María Jesús b López-Barrena Nuria c Lara-Orejas Emma d Alonso-Martín Daniel e Alfaro-Iznaola Cristina f Alcázar-Villar María José a Sánchez-Escudero Verónica g González-Vergaz Amparo g a Servicio de Pediatría, Hospital Universitario de Fuenlabrada, Fuenlabrada, Madrid, Spain b Servicio de Pediatría, Hospital Universitario de Móstoles, Móstoles, Madrid, Spain c Servicio de Pediatría, Hospital Universitario de Getafe, Getafe, Madrid, Spain d Servicio de Pediatría, Hospital Universitario Infanta Cristina, Parla, Madrid, Spain e Servicio de Pediatría, Hospital Universitario del Tajo, Aranjuez, Madrid, Spain f Servicio de Pediatría, Hospital Universitario Rey Juan Carlos. Móstoles, Madrid, Spain g Servicio de Pediatría, Hospital Universitario Severo Ochoa, Leganés, Madrid, Spain ⁎ Corresponding author. 1 12 2022 12 2022 1 12 2022 69 10 810815 2 9 2021 15 12 2021 © 2022 SEEN and SED. Published by Elsevier España, S.L.U. All rights reserved. 2022 SEEN and SED Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction ß-pancreatic cells are susceptible to SARS-CoV-2 infection and replication; this could lead to infection-related diabetes or precipitate the onset of type 1 diabetes. This study aimed to determine the severity at diagnosis, analyzing clinical and epidemiological features at debut in children under 16 years of age in the context of the SARS-CoV-2 pandemic. Material and methods A retrospective observational multicenter study was carried out in 7 hospitals of the public health network located in the south of our community. The severity at debut is compared with that of the two previous years (2018 and 2019). The level of statistical significance is set at p < 0.05. Results In 2020, 61 patients debuted at the 7 hospital centres. The mean age was 10.1 years (SD: 2.6), 50.8% older than 10 years. The clinical profile at diagnosis was ketoacidosis in 52.5% compared to 39.5% and 26.5% in the previous two years (p < 0.01). The mean pH (7.24 vs 7.30/7.30) and excess of bases (−11.9 vs −7.43/−7.9) was lower than in the previous two years, and the glycated haemoglobin higher (11.9 vs 11/10.6), p < 0.05. At least 10% of the patients had a positive history of SARS-CoV-2 infection. Conclusions There has been an increase in the frequency of diabetic ketoacidosis in type 1 diabetes onset during the first year of the COVID-19 pandemic. Introducción Las células ß-pancreáticas son susceptibles a la infección y replicación de SARS-CoV-2, lo que podría conducir a una diabetes relacionada con infección o precipitar el debut de una diabetes tipo 1. El objetivo de este estudio ha sido determinar la gravedad al diagnóstico, analizando características clínicas y epidemiológicas en el contexto de la pandemia por SARS-CoV-2 en menores de 16 años. Material y métodos Se lleva a cabo un estudio multicéntrico observacional retrospectivo en7 hospitales de la red pública de sanidad ubicados en el sur de nuestra comunidad. Se compara la gravedad al debut con la de los dos años previos (2018 y 2019). Se fija el nivel de significación estadística en una p < 0,05. Resultados En 2020 61 pacientes debutaron en los 7 centros hospitalarios. La edad media fue 10.1 años (DE: 2.6), 50.8% mayores de 10 años. La forma clínica del debut fue cetoacidosis en el 52.5% frente al 39.5% y 26.5% en los dos años previos (p < 0.01). El pH medio (7.24 vs 7.30/7.30) y exceso de bases (−11.9 vs −7.43/−7.9) fue menor que en los dos años anteriores y la hemoglobina glicada mayor (11.9 vs 11/10.6), p < 0.05. Al menos el 10% de los pacientes tenían antecedentes positivos de infección por SARS-CoV-2. Conclusiones Durante el primer año de pandemia COVID-19 ha habido un aumento en la frecuencia de cetoacidosis diabética como forma de debut. Keywords Type 1 diabetes Diabetic ketoacidosis Children Adolescent COVID-19 Palabras clave Diabetes tipo 1 Cetoacidosis Niños Adolescente COVID-19 ==== Body pmcIntroduction Type 1 diabetes (T1DM) is an autoimmune disease involving different triggering factors: genetic, socioeconomic and environmental.1 Against the backdrop of the COVID-19 pandemic, several studies showing the susceptibility of pancreatic beta cells to SARS-CoV-2 infection and replication have been published,2 which could lead to infection-related diabetes according to the WHO classification or precipitate the onset of T1DM.3 Ketoacidosis is one of the most severe complications of T1DM that is especially prevalent in the paediatric population, particularly children under five years of age.4 Global incidence rates vary widely from country to country and range between 15% and 80% for newly-diagnosed T1DM.5 In Spain, data published to date show a frequency of 40% of newly-diagnosed patients under 15 years of age.6 In Germany and Italy7 an increased incidence of ketoacidosis in children and adolescents at the onset of T1DM has been reported.8 Some publications in Spain suggest the same findings.9 Some of the hypotheses proposed to explain this phenomenon are the relationship with the strict lockdown imposed in the first few months of the pandemic, the fear of the population going to medical centres, or the role the infection itself could have in acting as an accelerator or trigger.10, 11 This study aimed to determine the clinical severity of patients under 16 years of age at the onset of diabetes against the backdrop of the SARS-CoV-2 pandemic and to compare this with the two previous years as any possible relationship with COVID-19 infection. Material and methods A retrospective, observational, multicentre study was conducted to determine the clinical and epidemiological characteristics of patients <16 years of age with new-onset T1DM in 2020, coinciding with the COVID-19 pandemic. Severity at onset was compared with the two previous years (2018 and 2019). Seven public hospitals located in the south of the Community of Madrid took part: Hospital Universitario de Fuenlabrada, Hospital Universitario Severo Ochoa (Leganés), Hospital Universitario de Móstoles, Hospital Universitario Rey Juan Carlos (Móstoles), Hospital Universitario de Getafe, Hospital Universitario del Tajo (Aranjuez) and Hospital Universitario Infanta Cristina (Parla). The first case of COVID-19 in Spain was reported on 31 January 2020 and in the Community of Madrid on 25 February 2020. Inclusion criteria- Being under 16 years of age at the onset of T1DM. - Living in the catchment area of the participating sites in the 3 months before onset. - Clinical follow-up at any of the study's participating sites. Exclusion criteria: not meeting any of the inclusion criteria. Age, severity and clinical form of presentation, time since onset of cardinal symptoms, lab test parameters (blood insulin, C-peptide, haemoglobin A1c and venous pH), association with other autoimmune diseases and SARS-CoV-2 infection (PCR and/or IgG and IgM serology) were analysed upon admission. The criteria for the diagnosis of diabetic ketoacidosis were taken from the ISPAD Consensus Guidelines12 (blood glucose >200 mg/dl, venous pH < 7.13 or bicarbonate <15 mmol/l, ketonaemia [blood ß-hydroxybutyrate ≥3 mmol/l] or moderate or severe ketonuria). Three age groups were established: 0−4 years, 5−9 years and 10−15 years. The software EPIDAT 4.0. was used. Quantitative variables are expressed as mean and standard deviation. If the variable does not fit a normal distribution, it is expressed as the median and interquartile range (IQR). Qualitative variables are expressed as frequencies. Quantitative variables were compared using the Student's t-test and analysis of variance (ANOVA). In contrast, qualitative variables were compared using the Chi-squared test and applying Fisher's exact test when required. The level of statistical significance was defined as p < 0.05. The study was approved by the Hospital Universitario de Fuenlabrada Independent Ethics Committee and seconded by the Principal Investigator. Results In 2020, 61 patients were diagnosed with T1DM at the seven hospitals. Some 24.6% of the patients came from a single centre. The mean age was 10.1 years (SD: 2.6; IQR: 7.2–13.5). Regarding age group, 36.1% of patients were aged 0–4 years, 13.1% 5–9 years and 50.8% >10 years. In total, 54.1% of the study population were female. Overall, 75.4% were of Spanish origin, 11.5% from North Africa, 6.5% from Eastern Europe and one patient was from Latin America. Some 31.1% of cases occurred in the third quarter of the year and 24.6% in the first (Fig. 1 ).Figure 1 Monthly distribution of new-onset T1DM cases and new COVID-19 cases in the Community of Madrid (new cases × 104). Figure 1 In 2018 and 2019, data were collected from 43 and 34 patients, respectively, with a mean age of 8.7 years (SD: 3.9) and 8.8 years (SD: 3.8), which was lower than the mean age recorded in 2020 (p < 0.05). In total, 69.8% and 79.4%, respectively, were of Spanish origin. Signs and symptoms The most common symptom was polyuria (83.6%), followed by polydipsia (80.3%). In both cases, approximately half of patients reported a time since onset of 2 weeks or less (51% and 55%, respectively). Some 59.2% reported weight loss; the least common symptom (34.4%) was polyphagia. The initial clinical form was ketoacidosis in 52.5% of patients and ketosis without acidosis in 32.8%. In total, 16.4% required admission to the paediatric intensive care unit (PICU), with no significant differences in presentation between the different age groups (Table 1 ). However, in contrast to the findings for previous years ketoacidosis was reported in just 39.5% and 26.5% of patients respectively (p < 0.01). Table 2 details the initial clinical forms in 2020 and the previous two years.Table 1 Severity of presentation by age group. Table 12020 0−4 years (n = 8) 5−9 years (n = 22) 10−15 years (n = 31) Ketoacidosis 75% 54.5% 45.2% Mean pH (SD) 7.16 (0.21) 7.24 (0.12) 7.23 (0.14) ICU 37.5% 13.6% 12.9% SD, standard deviation; ICU, intensive care unit. Table 2 Form of presentation (comparison by year). Table 2 2018 (n = 43) 2019 (n = 34) 2020 (n = 61) Stat. sig. (p) Ketoacidosis 17 (39.5%) 9 (26.5%) 32 (52.5%) p = 0.008 Ketosis 14 (32.6%) 14 (41.2%) 21 (34.4%) Hyperglycaemia 12 (27.9%) 9 (26.5%) 3 (4.9%) Other 0 (0%) 2 (5.9%) 5 (8.2%) Stat. sig., Statistical significance. In total, 34.4% received subcutaneous insulin from the onset, and in those who required IV infusion, the mean time was 13 h (SD: 9.3 h). It was maintained for >24 h in 9.8% of patients. In 2018 and 2019, the most common symptom was also polyuria, reported by 95.3% and 91.2% of patients, respectively. Time since onset was 2 weeks or less in 45.2% and 55% of cases. Table 3 details the symptoms and time since onset.Table 3 Cardinal symptoms and time since onset. Table 3 Polyuria Polydipsia Polyphagia 2020 (n = 61) 2019 (n = 34) 2018 (n = 43) 2020 (n = 61) 2019 (n = 34) 2018 (n = 43) 2020 (n = 61) 2019 (n = 34) 2018 (n = 43) No 6 (9.8%) 3 (8.8%) 2 (4.7%) 9 (14.7%) 9 (26.5%) 9 (21%) 36 (59.1%) 15 (44.1%) 19 (44.2%) Yes 0−2 51 (83.6%) 31 (91.2%) 41 (95.3%) 49 (80.3%) 25 (73.5%) 34 (79%) 21 (34.4%) 16 (47.1%) 24 (55.8%) weeks 26 (51%) 14 (45.2%) 22 (55%) 27 (55.1%) 14 (56%) 19 (55.9%) 15 (71.4%) 8 (50%) 11 (45.8%) 2−4 weeks 16 (31.4%) 8 (25.8%) 9 (21.9%) 14 (28.6%) 5 (20%) 7 (20.6%) 3 (14.3%) 5 (31.2%) 4 (16.7%) 1−2 months 7 (13.7%) 5 (16.1%) 8 (19.5%) 5 (10.2%) 4 (16%) 7 (20.6%) 2 (9.5%) 1 (6.2%) 8 (33.3%) >2 months 0 2 (5.9%) 2 (6.4%) 0 1 (4%) 1 (2.9%) 0 1 (6.2%) 1 (4.2%) Clinical chemistry, autoimmunity and related conditions Acid-base balance at diagnosis: in 2020, the mean pH was: 7.24 (SD: 0.01), the mean bicarbonate was 14.7 mEq/l (SD: 7.4) and the mean base excess was –11.9 (SD: 9.33). The mean glycated haemoglobin (DCCT) was 11.9% (SD: 2.2) and the mean C-peptide was 0.62 ng/mL (SD: 0.5; median 0.5 ng/mL and IQR: 0.3−0.7) (see Table 4 ).Table 4 Acid-base balance and pancreatic reserve. Table 4 2018 (n = 43) 2019 (n = 34) 2020 (n = 61) Stat. sig. (p) Mean pH (SD) 7.30 (0.11) 7.30 (0.15) 7.24 (0.01) p = 0.002 Mean bicarbonate mEq/l (SD) 17.5 (6.74) (n = 42) 16.99 (7.74) (n = 31) 14.7 (7.4) (n = 54) p = 0.14 BE mEq/l (SD) (n = 41) –7.9 (7.9) (n = 29) –7.43 (9) (n = 53) –11.9 (9.33) p = 0.035 Mean HbA1c % (SD) (n = 32) 10.6 (2.8) (n = 39) 11 (2.5) (n = 57) 11.9 (2.2) p = 0.04 Mean C-peptide ng/mL (SD) (n = 42) 0.78 (0.55) (n = 30) 0.8 (0.47) (n = 53) 0.62 (0.5) p = 0.192 BE, base excess; HbA1c, glycated haemoglobin; SD, standard deviation; Stat. sig., statistical significance. In 2020, the mean pH at diagnosis was lower than in the previous two years (p < 0.01), as was the mean base excess (p < 0.05), while mean glycated haemoglobin was higher (p < 0.05). T1DM-related antibodies were positive in all cases except one, in which they were not measured. Around 10% of patients (6/61) also had thyroiditis at diagnosis, while three patients had coeliac disease-related antibodies. Coronavirus associated with severe acute respiratory syndrome Fig. 1 shows new-onset diabetes cases, and the incidence of COVID-19 in the Community of Madrid superimposed. In total, the onset of 85.2% (n = 52) of patients diagnosed in 2020 was after the first reported case of COVID-19 in the Community of Madrid. An RT-PCR for SARS-CoV-2 was performed in 40 patients upon admission, which was positive in three cases. Of those patients with negative RT-PCR, one had positive IgG serology, and in another case, both parents were infected at the time. Therefore, five patients (9.6%) had a positive history. Discussion 2020 saw an increase in the severity of the onset of T1DM in children under 16 years of age (higher frequency of ketoacidosis, lower pH, lower base excess [BE] and higher glycated haemoglobin), as has been reported in other countries.13, 14 Although the association with lockdown and the fear of going to medical centres has been one of the hypotheses proposed to explain this8 the time since onset of symptoms was <2 weeks in slightly more than 50% of cases, with no differences compared to the previous years, suggesting that there was no delay in receiving health care, which leads us to believe that there must be other factors involved in this greater severity at onset. Moreover, no differences have been found in symptom duration between patients presenting with or without ketoacidosis.4 One feature of healthcare in the Autonomous Community of Madrid was the partial closure of primary care during the first months of the pandemic to support the care of patients with COVID-19 at the Hospital de Emergencias Enfermera Zendal, as well as the subsequent restriction of face-to-face consultations in favour of telephone appointments. This lack of primary care15 could have affected aspects such as severity of presentation.16 Although no significant differences were found, there was a tendency in our study towards lower C-peptide values in 2020 compared to the two previous years (0.62 vs 0.78/0.8) (ng/mL), a finding that goes hand in hand with the higher incidence of ketoacidosis17 and would translate into a lower residual capacity for endogenous insulin production. During the months of March, April and May (21 March to 6 May), emergency paediatric care was redistributed to two main hospitals in the Community of Madrid. However, some patients continued to go to their nearest referral hospital. More cases in our population were recorded in 2020 than in 2018 or 2019. Patient displacement to other hospitals could be one of the reasons why the mean age of our patients was greater in 2020 than in the two previous years (10.1 years versus 8.7 and 8.8 years; p < 0.05) 9 even though other series have recorded a younger age at diagnosis during the COVID-19 pandemic.18 Children under five years of age are the most susceptible to ketoacidosis and the most likely to require intensive care, which was confirmed by our data (75% ketoacidosis). However, no significant differences were found due to the small sample size. Data from the literature regarding SARS-CoV-2-precipitated T1DM in children and adolescents are contradictory.7, 19, 20 In our series, of the 40 patients with onset during the COVID-19 pandemic and who underwent some kind of diagnostic test for SARS-CoV-2 infection, up to 12.5% of cases were in some way associated with SARS-CoV-2 infection. In December 2020, the overall prevalence in Spain among 16-year-old adolescents was around 5%, according to the ENECOVID seroprevalence study21 almost half the figure obtained from our sample. Although it is impossible to conclude the small sample size and specific geographical area, we believe these data should be analysed as part of the national figures to confirm this result. Contrary to this finding, no parallelism was observed between the COVID-19 incidence graphs in the Community of Madrid and newly-diagnosed T1DM (Fig. 1). One aspect to analyse in future studies is the severity of T1DM cases in patients with a history of infection. Owing to the small number of cases, this could not be evaluated in our study. The limitations of this paper are those characteristic of a retrospective study, namely lost and/or missing data. The time since the onset of initial symptoms is not very precise due to the subjective and retrospective nature of the data collection. It is also impossible to confirm whether these symptoms may have gone unnoticed, been attributed to lockdown, or even been detected earlier due to stricter control. Even so, given that most of the variables analysed are biochemical parameters, interpretation and selection bias can be ruled out, rendering these results valid. We cannot determine the incidence of new-onset T1DM as some diagnosed cases will have been seen at other centres, meaning that these data are not real and may underestimate the actual incidence. Conclusions In 2020, 52.5% of patients had ketoacidosis at onset, representing an increase in this form of presentation and therefore an increase in severity. Between 10% and 12.5% of patients had a positive history of SARS-CoV-2 infection. A national registry of newly-diagnosed T1DM cases, their severity and their possible association with SARS-CoV-2 infection are just one of the measures that need to be implemented as part of the ketoacidosis prevention policies. Conflicts of interest The authors declare that they have no conflicts of interest. ==== Refs References 1 Rewers M. Ludvigsson J. Environmental risk factors for type 1 diabetes Lancet. 387 10035 2016 2340 2348 27302273 2 Lima-Martínez M.M. Carrera Boada C. Madera-Silva M.D. Marín W. Contreras M. COVID-19 and diabetes: a bidirectional relationship Clin Investig Arterioscler. 33 3 2021 151 157 3 Müller J.A. Groß R. Conzelmann C. Krüger J. Merle U. Steinhart J. SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas Nat Metab. 2021 10.1038/s42255-021-00347-1 Epub ahead of print. PMID: 33536639 4 Usher-Smith J.A. Thompson M.J. Sharp S.J. Walter F.M. Factors associated with the presence of diabetic ketoacidosis at diagnosis of diabetes in children and young adults: a systematic review BMJ. 7 2011 d4092 5 Große J. Hornstein H. Manuwald U. Kugler J. Glauche I. Rothe U. Correction: incidence of diabetic ketoacidosis of new-onset type 1 diabetes in children and adolescents in different countries correlates with Human Development Index (HDI): an updated systematic review, meta-analysis, and meta-regression Horm Metab Res. 50 3 2018 e2 29523008 6 Oyarzabal Irigoyen M. García Cuartero B. Barrio Castellanos R. Torres Lacruz M. Gómez Gila A.L. González Casado I. Ketoacidosis at onset of type 1 diabetes mellitus in pediatric age in Spain and review of the literature Pediatr Endocrinol Rev. 9 3 2012 669 671 22523835 7 Rabbone I. Schiaffini R. Cherubini V. Maffeis C. Scaramuzza A and the diabetes study group of the italian society for pediatric endocrinology and diabetes. Has COVID-19 delayed the diagnosis and worsened the presentation of type 1 diabetes in children? Diabetes Care 43 11 2020 2870 2872 32778554 8 Kamrath C. Mönkemöller K. Biester T. Ketoacidosis in children and adolescents with newly diagnosed type 1 diabetes during the COVID-19 pandemic in Germany JAMA. 324 8 2020 801 804 10.1001/jama.2020.13445 32702751 9 Güemes M. Storch-de-Gracia P. Enriquez S.V. Martín-Rivada Á Brabin A.G. Argente J. Severity in pediatric type 1 diabetes mellitus debut during the COVID-19 pandemic J Pediatr Endocrinol Metab. 33 2020 1601 1603 33151178 10 Boddu S.K. Aurangabadkar G. Kuchay M.S. New onset diabetes, type 1 diabetes and COVID-19 Diabetes Metab Syndr. 14 6 2020 2211 2217 33395782 11 Gentile S. Strollo F. Mambro A. Ceriello A. COVID-19, ketoacidosis, and new-onset diabetes: might we envisage any cause-effect relationships among them? Diabetes Obes Metab 22 2020 2507 2508 32790021 12 Wolfsdorf J.I. Glaser N. Agus M. Fritsch M. Hanas R. Rewers A. ISPAD Clinical Practice Consensus Guidelines 2018: diabetic ketoacidosis and the hyperglycemic hyperosmolar state Pediatric Diabetes 19 Suppl 27 2018 155 177 29900641 13 Loh C. Weihe P. Kuplin N. Placzek K. Weihrauch-Blüher S. Diabetic ketoacidosis in pediatric patients with type 1- and type 2 diabetes during the COVID-19 pandemic Metabolism. 122 2021 154842 14 Gregg E.W. Sophiea M.K. Weldegiorgis M. Diabetes and COVID-19: population impact 18 months into the pandemic Diabetes Care. 44 9 2021 1916 1923 34244333 15 Albañil Ballesteros M.R. Pediatría y COVID-19 Rev Pediatr Aten Primaria 22 2020 125 128 16 McGlacken-Byrne S.M. Drew S.E.V. Turner K. Peters C. Amin R. The SARS-CoV-2 pandemic is associated with increased severity of presentation of childhood onset type 1 diabetes mellitus: a multi-centre study of the first COVID-19 wave Diabet Med. 38 9 2021 e14640 17 Szypowska A. Skórka A. The risk factors of ketoacidosis in children with newly diagnosed type 1 diabetes mellitus Pediatric Diabetes 12 2011 302 306 21129138 18 Dilek S.Ö Gürbüz F. Turan İ Celiloğlu C. Yüksel B. Changes in the presentation of newly diagnosed type 1 diabetes in children during the COVID-19 pandemic in a tertiary center in Southern Turkey J Pediatr Endocrinol Metab. 34 10 2021 1303 1309 34291625 19 Domínguez J.A. Tello M.V. Tasayco J. Coronado A. Cetoacidosis diabética severa precipitada por COVID-19 en pacientes pediátricos: reporte de dos casos Medwave 21 3 2021 e8176 34081683 20 Messaaoui A. Hajselova L. Tenoutasse S. Anti-SARS-CoV-2 antibodies in new-onset type 1 diabetes in children during pandemic in Belgium J Pediatr Endocrinol Metab. 34 10 2021 1319 1322 34280962 21 Pollán M. Pérez-Gómez B. Pastor-Barriuso R. Oteo J. Hernán M.A. Pérez-Olmeda M. (ENE-COVID Study Group) Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study Lancet. 396 10250 2020 535 544 32645347	36464601	PMC9713488	NO-CC CODE	2022-12-16 23:17:55	no	Endocrinol Diabetes Nutr (Engl Ed). 2022 Dec 1; 69(10):810-815	utf-8	Endocrinol Diabetes Nutr (Engl Ed)	2,022	10.1016/j.endien.2021.12.014	oa_other
==== Front Am Behav Sci Am Behav Sci ABS spabs The American Behavioral Scientist 0002-7642 1552-3381 SAGE Publications Sage CA: Los Angeles, CA 10.1177/00027642221138272 10.1177_00027642221138272 Article Big Shots: A Social Media Campaign to Honor Local Heroes who Promote COVID-19 Vaccine Literacy and Increase Vaccine Acceptance Schub Tanja 1 Swan-Potras Lauren 1 Rabin Kenneth 1 1 CUNY Graduate School of Public Health & Health Policy, New York, NY, USA Lauren Swan-Potras, CUNY Graduate School of Public Health & Health Policy, 55 W 125th St., New York, NY, 10027, USA. Email: [email protected] 30 11 2022 30 11 2022 00027642221138272© 2022 SAGE Publications 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Big Shots is a graduate student-developed social media campaign that aims to promote COVID-19 vaccine literacy and build vaccine confidence through the power of storytelling. Here we describe the development of the partnerships underlying the campaign and detail the campaign’s achievements thus far, including its recognition and celebration (to date) of 12 individuals and groups who have broken down barriers to COVID-19 vaccination in their communities. The ongoing campaign may serve as a model to guide future “grassroots” social media campaigns aimed at addressing public health issues. health communication vaccine hesitancy social media campaign narrative communication theory vaccine literacy CDC Foundation edited-statecorrected-proof typesetterts1 ==== Body pmcIntroduction As the COVID-19 vaccine rollout began in the United States in late 2020 and early 2021, it was unclear how many people would be willing to take the shot. In December 2020, 34% of U.S. adults reported that they would get the vaccine as soon as possible, 39% planned to “wait and see,” 9% said they would only get the vaccine if they were required to do so, and 15% said they would “definitely not” get the vaccine. Although the proportion of U.S. adults reporting that they have been vaccinated increased to 73% in February 2022, 4% continue to “wait and see,” while 3% report willingness to get the vaccine only if required and 16% persist in their unwillingness to get the vaccine under any circumstances (KFF, 2022). Factors that contributed to early COVID-19 vaccine hesitancy and continue to hamper efforts to increase vaccination rates include concerns related to the speed of the vaccine development process; fear of adverse effects of the vaccines, including possible adverse effects on fertility; mistrust of government and the medical establishment; and the belief that vaccination against COVID-19 is not necessary or effective (Diaz et al., 2021; King et al., 2021; McFadden et al., 2022). Some of the beliefs underlying vaccine hesitancy stem from anti-vaccine rhetoric spread via social media and elsewhere; however, for some, vaccine hesitancy traces back to legitimate experience- and history-based reasons for mistrust of the government and health-care system (Dada et al., 2022). Politicization of the vaccine and lack of consistent messaging from a trusted source have also exacerbated the problem, leading to increased or sustained vaccine hesitancy (Bolsen & Palm, 2022; Ratzan et al., 2020). Countering these barriers is crucial to increasing vaccine uptake and may require efforts to combat misinformation and address mistrust, while also increasing access (Dada et al., 2022). Storytelling as a public health messaging strategy is gaining in popularity (McCall et al., 2019) and first-person narration may be an effective way to increase vaccine confidence and uptake (Dada et al., 2022; Finnegan et al., 2018). In a study of Chinese college students, narrative messages were more effective than non-narrative messages in persuading study participants to get vaccinated (Ye et al., 2021). Additionally, storytelling may be particularly powerful when the stories are being told by trusted messengers. For example, Black physicians and other health-care providers—a group likely to be viewed as trusted messengers in the Black community—have successfully used social media to disseminate COVID-19 vaccine information and convince previously vaccine-hesitant individuals to get the shot (Dada et al., 2022). Storytelling has also been shown to engage with harder-to-reach audiences and, by sharing relatable experiences, can help inspire and model positive behaviors (Kreuter et al., 2010). This background underlies the development of Big Shots, a unique campaign to promote vaccine literacy and build vaccine acceptance using the power of storytelling. Developed and implemented by students and faculty at the City University of New York Graduate School of Public Health & Health Policy (CUNY SPH), the Big Shots campaign has celebrated “local heroes,” or “Big Shots,” (including community members, local health-care providers, and high school students) who have broken down barriers to COVID-19 vaccination in their communities. The individuals recognized by the campaign have aided their communities in several ways, including setting up vaccination sites, helping older adults find vaccination appointments, transporting people to vaccination sites, and correcting misinformation about vaccine safety and effectiveness, while also inspiring others to find their own way to contribute to the effort to increase COVID-19 vaccination rates. Lessons from the development of Big Shots could guide future social media campaigns aimed at addressing public health issues. Program Development Beginning in the early stages of the COVID-19 pandemic, CUNY SPH was invested in building vaccine literacy and promoting immunization programs through its work with the global initiative COVID-19 New Vaccine Information, Communication and Engagement (CONVINCE); (Larson et al., 2020). Interest in further countering vaccine hesitancy through communication efforts led to discussions among CUNY SPH faculty members regarding additional proactive steps that could be taken to make positive change happen. Recognition that local media outlets around the country were publishing inspiring, but one-off stories of everyday people taking action to encourage and help others to get vaccinated resulted in the development of the Big Shots campaign as a way of cumulatively honoring such people to amplify the message and help to increase vaccination rates. These faculty members relied on their professional and academic experience in health communication, health literacy, social marketing, behavior change, and public relations to conceptualize a campaign where the recognition of the important work of these “heroes” could be collected, amplified, and celebrated. Where contemporaneous campaigns were vaccine message driven, Big Shots was created to be story driven. The campaign founders obtained funding from the CDC Foundation and leveraged a relationship with a former student to obtain pro bono creative assistance from a health communications and marketing firm (McCann Global Health) in the form of graphics, social media templates, and logos. Big Shots was created to be run and directed by graduate students at CUNY SPH. As such, graduate students, including students in a new Master of Science (MS) in Health Communication for Social Change program at CUNY SPH, were recruited to work on the campaign, as part of their fieldwork requirements or as volunteers. Early in its development, Big Shots partnered with various national and international organizations that are recognized as leaders in vaccine advocacy, including Made to Save and the Vaccine Confidence Project. Made to Save, an initiative of Civic Nation, was a national grassroots campaign that increased COVID-19 vaccine awareness by empowering community organizations (About Made to Save, n.d.). Made to Save provided the Big Shots campaign with access to their community of grassroot organizations committed to increase vaccine awareness and uptake and amplified the Big Shots campaign through their social media pages and internal emails, newsletters, and other forms of communication. The Vaccine Confidence Project, an interdisciplinary research group at the London School of Hygiene and Tropical Medicine dedicated to addressing vaccine hesitancy and misinformation, provided web and social media support for Big Shots by hosting the campaign videos on their platform, thereby increasing campaign visibility. These partnerships were based on a shared goal of confronting vaccine hesitancy and misinformation. It was also important to the design, tone, and goals of the campaign to rely on cross-promotion and amplification, as well as utilizing relationships with other organizations. Campaign in Action The Big Shots campaign was launched on July 1, 2021, with a national online awards show, which was hosted by celebrity chef and TV personality Carla Hall and honored eight Big Shots from around the country. These Big Shots—whose efforts included participating in COVID-19 vaccine clinical trial recruitment, finding vaccine appointments for local older adults, setting up a vaccination clinic, and providing neighbors with accurate vaccine-related information—were identified by a CUNY SPH student who organized the Big Shots award show as part of the field work component of her Master of Public Health (MPH) degree program. This student found potential Big Shots by scouring local and national news sources, looking for inspiring stories and especially those highlighting the stories and work of those in communities with higher vaccine hesitancy and/or lower vaccination rates. She then reached out, vetted them, and organized their acceptance of the awards. The awards show was promoted using social media accounts and a website set up for this purpose. The Big Shots campaign did not end with the launch of the award show. Indeed, Big Shots was designed to function in two phases: an awards show to introduce the campaign and an ongoing campaign with peer-nominated awardees. Big Shots can still be nominated by colleagues, neighbors, friends, or others via the Big Shots website. Individuals who are nominated are assessed for their acceptability by a CUNY SPH graduate who is currently running the program and contacted via email if they are chosen for inclusion. If an awardee accepts, they are asked to provide materials for use on the website and in social media posts, including several photos and an optional video. These materials are then combined and edited by CUNY SPH graduate students and posted to the social media accounts and Big Shots website. Thus far, the campaign has recognized individuals and organizations in California, Nebraska, Kentucky, Illinois, Oklahoma, New York, and Maryland. To date, Big Shots participants have not received any compensation beyond recognition of their good works. Although there has been discussion among program directors regarding the possibility of future monetary compensation, such as in the form of a gift card, no decisions have been made regarding this potential plan. Since its launch, the campaign has exceeded its original goal of 1,000 unique visitors, with over 2,500 views from 1,200 unique visitors from around the globe, reaching as far as Thailand, Pakistan, and South Korea. Big Shots has had many nominations from communities across the country. Although the campaign has received fewer than the 100 nominations originally anticipated, more nominations are typically received after the announcement of each awardee, suggesting that with an increased presence, the initial goals may be reached or exceeded. Additionally, each time the Big Shots campaign shared content, online engagement with the campaign increased through likes and shares from the past awardees’ social media accounts. This is significant, in that it suggests that Big Shots continues to be a viable tool to recognize and support voluntarism in vaccine advocacy and the improvement of vaccine literacy, not just through identifying and honoring new Big Shot awardees, but by adding to the ripple effect of multiple, credible, independent programs in the same space. Big Shots content has been amplified and shared with partner organizations, including Made to Save, Teens for Vaccines, and various organizations at CUNY. The number of people following the campaign on Instagram has increased by 20% over the last few months, coinciding with an increase of posts and engagement. In addition, the campaign’s Twitter following has grown and content has been retweeted several times. The Big Shots campaign has been covered in several publications, most notably The Nation’s Health, a publication of the American Public Health Association. Lessons Learned The launch of the Big Shots campaign has provided valuable lessons about the potential utility of social media campaigns aimed at addressing public health issues. It demonstrated that truly grassroots public health campaigns are possible, especially when they are delivered via social media. Social media campaigns require minimal equipment (a smartphone or laptop computer and an account with a graphic design website) and minimal technological expertise, and allow content to be viewed, amplified, and engaged with, both nationally and internationally. The campaign also illustrated that, even during the time of political division and rampant misinformation, controversial topics can be addressed in a celebratory and nonjudgmental tone to build the trust and authenticity needed to reach vaccine-hesitant or hard-to-reach communities, while avoiding messaging that may be regarded as patronizing. The broad definition of what makes a Big Shot allows the campaign to be used, embraced, and applied to various communities. A Big Shot nomination can be seen as a lens through which to see and understand communities; a way to align with that community’s values and priorities. Big Shots has also proved to be a useful teaching tool for graduate students at CUNY SPH. The inception of the Big Shots campaign coincided with the introduction of a new Health Communication for Social Change MS degree program at CUNY SPH. This program provides students with the tools needed to advance public health through health communication, social marketing, and other behavior-change strategies using various multimedia formats (CUNY Graduate School of Public Health & Health Policy, 2022). Both CUNY SPH and the MS in Health Communication for Social Change provided an ideal environment to develop and implement the Big Shots campaign. Program directors P. Christopher Palmedo, PhD, MBA and Scott C. Ratzan, MD, MPA coordinated with former students and faculty members in the development and implementation of Big Shots. Students in both this program, as well as in MPH programs, gained valuable, real-world experience in building the campaign, from the ground up, with the supervision and mentorship of established health communicators, practitioners, and public relations experts. The success of Big Shots has been hampered by a limited budget and small team size. A larger team with greater time commitments would allow for increased content creation, nominee and media outreach, and other coordinating efforts that could maintain momentum and inspire nominations. A larger team might also allow Big Shots to grow its brand, such as by adding subcategories that focus on specific communities and share their stories in engaging and interesting ways. It is easy for a small, grassroots campaign like Big Shots to be overshadowed by larger campaigns run by larger organizations that compete for user engagement and audience. Studies have shown that narrative or storytelling-driven campaigns can help promote single-event behavior change, such as vaccine uptake (Kim et al., 2020; Lee et al., 2018). Narrative Communication Theory suggests that the inherent logic of storytelling will influence, shape, and change attitudes. Through transportation and identification, narrative storytelling can help influence and facilitate behavior change, especially when the story being told is culturally relevant to the priority audience (Bokhour et al., 2016). Big Shots is similarly harnessing the inherent power of storytelling, identification, and transportation by sharing and uplifting the stories and experiences of Big Shots. Furthermore, as social media and digital communication techniques become more accessible and normalized, storytelling can similarly be embraced by public health practitioners and communications. Next Steps The Big Shots campaign continues to accept nominations. Two partnerships, with Made to Save and the American College Health Association (a well-established organization of college health professionals committed to advancing the health and wellness needs of 20 million students at over 800 higher education institutions), have helped increase the campaign’s reach and impact. Similarly, Big Shots continues to search for and facilitate other partnerships to increase impact and reach. The Big Shots campaign—as well as the grassroots, nonjudgmental Big Shots approach—could be used in various communities, by various community-based organizations, and perhaps for other vaccination efforts. Although Big Shots was created within the context of COVID-19, its peer nomination and recognition template could be used as a model for other vaccination awareness efforts such as those focused on childhood vaccinations and the human papillomavirus (HPV) vaccine. Furthermore, Big Shots can be used in other countries and communities outside of the United States. Further study and utilization of this model is warranted. Author Biographies Tanja Schub is a graduate student in the Health Communication for Social Change MS program at the CUNY Graduate School of Public Health & Health Policy with an expetected graduation date of December 2022. She has worked in various capacities in biomedical and medical publishing, including as a writer and editor. Lauren Swan-Potras is a graduate of the CUNY, Graduate School of Public Health and Health Policy Health Communication for Social Change program. Her background is in storytelling and public health, investigating the use of narrative in effective health messaging. She is currently the Managing Editor of the Journal of Health Communication, and has experience in social media campaigns, live performance, and storytelling. She holds an MS from CUNY SPH and a BFA in Drama from New York University, with a Minor in American and British Literature. Dr. Kenneth Rabin is a Senior Scholar at the City University of New York, Graduate School of Public Health and Health Policy. He retired after a 30-year career in health care communications consulting, preceded by an academic appointment as associate professor and director of the graduate public relations program at American University in Washington, DC, and as a Foreign Service Information Officer in the former US Information Agency. He holds a BA Degree cum laude in English from Cornell; an MA in teaching from Yale University; an MA in Literature from the University of North Carolina, Chapel Hill, and a PhD in higher educational administration from the George Peabody College of Vanderbilt University, Nashville, TN. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ==== Refs References About Made to Save. (n.d.). Made to Save. https://madetosave.org/about Bolsen T. Palm R. (2022). Politicization and COVID-19 vaccine resistance in the U.S. Progress in Molecular Biology and Translational Science, 188 (1 ), 81–100. 10.1016/bs.pmbts.2021.10.002 35168748 Bokhour B. G. Fix G. M. Gordon H. S. Long J. A. DeLaughter K. Orner M. B. Pope C. Houston T. K. (2016). Can stories influence African-American patients’ intentions to change hypertension management behaviors? A randomized control trial. Patient Education and Counseling, 99 (9 ), 1482–1488. 10.1016/j.pec.2016.06.024 27387121 CUNY Graduate School of Public Health & Health Policy. (2022). MS in health communication for social change. https://sph.cuny.edu/academics/degrees-and-programs/masters-programs/ms-in-health-communication-for-social-change/ Dada D. Djiometio J. N. McFadden S. M. Demeke J. Vlahov D. Wilton L. Wang M. Nelson L. E. (2022). Strategies that promote equity in COVID-19 vaccine uptake for Black communities: A review. Journal of Urban Health: Bulletin of the New York Academy of Medicine, 99 (1 ), 15–27. 10.1007/s11524-021-00594-3 35018612 Diaz P. Zizzo J. Balaji N. C. Reddy R. Khodamoradi K. Ory J. Ramasamy R. (2021). Fear about adverse effects on fertility is a major cause of COVID-19 vaccine hesitancy in the United States. Andrologia. Advance online publication. 10.1111/and.14361 Finnegan G. Holt D. English P. M. Glismann S. Thomson A. Salisbury D. M. Bogaerts H. Bonanni P. (2018). Lessons from an online vaccine communication project. Vaccine, 36 (44 ), 6509–6511. 10.1016/j.vaccine.2018.05.007 29921491 KFF. (2022). KFF COVID-19 vaccine monitor. https://www.kff.org/coronavirus-covid-19/dashboard/kff-covid-19-vaccine-monitor-dashboard/ Kim M. Lee H. Kiang P. Aronowitz T. Sheldon L. K. Shi L. Allison J. J. (2020). A storytelling intervention in a mobile, web-based platform: A pilot randomized controlled trial to evaluate the preliminary effectiveness to promote human papillomavirus vaccination in Korean American college women. Health Education & Behavior, 47 (2 ), 258–263. 10.1177/1090198119894589 31958991 King W. C. Rubinstein M. Reinhart A. Mejia R. (2021). Time trends, factors associated with, and reasons for COVID-19 vaccine hesitancy: A massive online survey of US adults from January-May 2021. PLoS One, 16 (12 ), e0260731. 10.1371/journal.pone.0260731 Kreuter M. W. Holmes K. Alcaraz K. Kalesan B. Rath S. Richert M. McQueen A. Caito N. Robinson L. Clark E. M. (2010). Comparing narrative and informational videos to increase mammography in low-income African American women. Patient Education and Counseling, 81 (Suppl. ), S6–S14. 10.1016/j.pec.2010.09.008 Larson H. J. Lee N. Rabin K. H. Rauh L. Ratzan S. C. (2020). Building confidence to CONVINCE. Journal of Health Communication, 25 (10 ), 838–842. 10.1080/10810730.2021.1884149 33719882 Lee H. Kim M. Cooley M. E. Kiang P. N. Kim D. Tang S. Shi L. Thiem L. Kan P. Peou S. Touch C. Chea P. Allison J. (2018). Using narrative intervention for HPV vaccine behavior change among Khmer mothers and daughters: A pilot RCT to examine feasibility, acceptability, and preliminary effectiveness. Applied Nursing Research, 40, 51–60. 10.1016/j.apnr.2017.12.008 McCall B. Shallcross L. Wilson M. Fuller C. Hayward A. (2019). Storytelling as a research tool and intervention around public health perceptions and behaviour: A protocol for a systematic narrative review. BMJ Open, 9 (12 ), e030597. 10.1136/bmjopen-2019-030597 McFadden S. M. Demeke J. Dada D. Wilton L. Wang M. Vlahov D. Nelson L. E. (2022). Confidence and hesitancy during the early roll-out of COVID-19 vaccines among Black, Hispanic, and undocumented immigrant communities: A review. Journal of Urban Health: Bulletin of the New York Academy of Medicine, 99 (1 ), 3–14. 10.1007/s11524-021-00588-1 34940933 Ratzan S. C. Gostin L. O. Meshkati N. Rabin K. Parker R. M. (2020). COVID-19: An urgent call for coordinated, trusted sources to tell everyone what they need to know and do. NAM Perspectives, 2020 , 10.31478/202003a. 10.31478/202003a Ye W. Li Q. Yu S. (2021). Persuasive effects of message framing and narrative format on promoting COVID-19 vaccination: A study on Chinese college students. International Journal of Environmental Research and Public Health, 18 (18 ), 9485. 10.3390/ijerph18189485 34574406	0	PMC9713517	NO-CC CODE	2022-12-02 23:23:05	no	Am Behav Sci. 2022 Nov 30;:00027642221138272	utf-8	Am Behav Sci	2,022	10.1177/00027642221138272	oa_other
==== Front Eur J Ophthalmol Eur J Ophthalmol EJO spejo European Journal of Ophthalmology 1120-6721 1724-6016 SAGE Publications Sage UK: London, England 36448253 10.1177/11206721221143008 10.1177_11206721221143008 Editorial The impact of the pandemic highlights the urgent need for myopia guidelines: The clinicians’ role https://orcid.org/0000-0001-8575-4888 Németh János 1 Aclimandos Wagih A 23 Tapasztó Beáta 1 Jonas Jost B 45 Grzybowski Andrzej 67 Nagy Zoltán Zsolt 1 1 Department of Ophthalmology, 37637 Semmelweis University , Budapest, Hungary 2 King's College Hospital, London, UK 3 European Society of Ophthalmology, Rotterdam, The Netherlands 4 Department of Ophthalmology, 9144 Heidelberg University , Mannheim, Germany 5 Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland 6 Department of Ophthalmology, 49674 University of Warmia and Mazury , Olsztyn, Poland 7 Institute for Research in Ophthalmology, Foundation for Ophthalmology Development, Poznan, Poland János Németh, Dept. of Ophthalmology, Semmelweis University, Mária utca 39., H-1085 Budapest, Hungary. Email: [email protected] 29 11 2022 29 11 2022 1120672122114300812 5 2022 30 10 2022 © The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Myopia is already one of the leading causes of permanent vision impairment, including blindness, and the COVID-19 pandemic has exacerbated the global myopia-related burden among children owing to home confinement, increased screen time (e-learning), and decreased outside activities. To reverse the rising trend of myopia and myopia-related blindness, collaborative efforts are required. There is a wealth of evidence-based medicine (EBM) data on the epidemiology of myopia and effective interventions, but very little has been published on the clinicians’ roles and responsibilities. However, this aspect is critical because preventing the onset and progression of myopia necessitates extensive health promotion and advocacy efforts among decision-makers. Only broad medical expert collaboration can bring about the necessary changes in children's lifestyle and education. This article discusses clinicians’ critical roles in preventing the onset and progression of myopia. Myopia pathologic myopia COVID-19 pandemic blindness preventive medicine screening public eye health advocacy health promotion edited-statecorrected-proof typesetterts19 ==== Body pmcEven before the COVID-19 pandemic, the number of people with myopia in 2020 was estimated to be 2.6 billion globally, and it was expected to rise up to 4.9 billion by 2050, unless preventive actions and interventions were taken.1 Myopia is an important health-care problem due to its well-known complications and the natural course of high myopia; myopia is already one of the most common causes for irreversible vision impairment including blindness.1,2 Without setting guidelines and the prompt establishment of national reforms, the existing large number of myopes is bound to rise considerably. The children population is the most crucial one, as myopia is increasing the greatest among the young generation. Recent studies found that a) in Hungary, the prevalence of myopia was three times higher (57%-61%) in younger age groups (18–35y) than in older ones3; b) in France, the highest prevalence of myopia was also found in the age groups between 10–39y, with a similarly high peak (52.4%) in those aged 20–29y4; and c) the European Eye Epidemiology Consortium found the highest rates of myopia (around 40%) in younger age groups.5 Preventing myopia and its progression became all the more important, since home confinement during the COVID-19 pandemic has exacerbated the worldwide burden of myopia.6 Studies exhibited an 1.4 to 3.6 times increase in myopia progression during the COVID-19 lockdown,7–10 especially in younger children,6,10,11 even in children under low-dose (0.05% or 0.025%) atropine therapy.11 Studies showed that the statistically most important risk factors for an increased myopia progression were the increased digital device screen time of children (in association with a significant increase in e-learning using smartphones, tablets or computers) and the decreased time spent on outdoor activities (and reduced sunlight exposure) due to home confinement.6,7,9–14 The digital screen time of children increased 1.3 to 7.8 times during the COVID-19 lockdown as compared to the pre-COVID time7,11–14 while the outdoor activities decreased by 48.6% to 62.8%.7,8,11,14 The COVID-19 epidemic presented an unwelcome chance to test the impact of behavioral changes and myopia in the real world.13 Based on these new findings, it is critical to address the environmental risk factors for myopia, even in children undergoing therapy to slow down myopic progression.11 As children's physical activity during the first COVID-19 UK lockdown was significantly reduced,15 promoting safe physical outdoors exercises for all children is critical not only for myopia control but also for the children's overall health and development.12,15,16 There is a wealth of evidence-based knowledge on myopia, including its onset, progression, risk factors, screening and detection, follow-up, and interventions for myopia control.2,17 The significant increase in the number of articles published about myopia in general is not matched with information and discussion about the roles and responsibilities of specialists in the prevention of myopia and reduction of its progression. This article provides an overview of the required action. 1. Primary prevention refers to procedures used to prevent the onset of a disease, in this instance myopia. The highest level of evidence supports the need for children to spend more time outdoors and do less near vison activities (level I. evidences, Elsevier) to prevent or at least delay the onset of myopia.6–14,18,19 Strong evidence (level III. evidences, Elsevier) has shown the major preventive nature of increased reading distance (more than 20–30 cm),20 limitation of continuous reading (less than 30–45 min),21 and use of incandescent or fluorescent light bulbs (instead of LED lamps).22 Using natural light or extremely bright light bulbs (over 3000 Lux) indoors is beneficial for myopia prevention.20 Children who used television and projectors for online or e-learning had significantly less myopic shift than those who used tablets and mobile phones, suggesting that television and projectors may be superior options for online learning.14 Preventing the onset of myopia requires health promotion. Medical experts have a dual role. There is a need to present current information and necessary interventions in a manner that would encourage decision makers and politicians to undertake actions and decisions that accomplish environmental changes. To modify the environmental factors surrounding education necessitates action from the Ministries of Education, including the provision of evidence-based facts about myopia to children and parents as well as to those in charge of initiating and achieving lifestyle changes among children. Eye care specialists should play a key role in information dissemination and advocacy since they are more familiar with the epidemiological and scientific facts about the myopia epidemics and feasible preventative methods. Further concrete actions of clinicians might be:Regular teaching of nurses and social services Education of nursery school staff about the importance of light and safe outdoor activities (socially distant during the pandemic) Education of school teachers and healthcare officers about proper reading distance, light intensity, and the importance of recess and outdoor activities Regular teaching of optometrists who deal with pediatric groups Regular teaching of students, showing them spectacles and how they will see if they develop myopia Provision of leaflets using simple layman terms can be made available at schools and GP practices Discussions and workshops about the national and regional possibilities of myopia prevention in the national and regional ophthalmological societies Development of a national plan for myopia prevention Organization of national, regional and local media campaigns on myopia prevention. “Save our children's sight” might be a campaign slogan. Undertaking actions to increase the awareness of the problem among local, regional and national health authorities 2. Secondary preventive strategies involve detecting and addressing an existing disease before symptoms arise. Secondary prevention in the case of myopia aims to keep pre-myopes from advancing to myopia. Pre-myopia describes a state of refractive error, ranging from ≤ + 0.75 D to >−0.50 D, in children when a combination of baseline refractive error, age, and other quantifiable risk factors provides a sufficient likelihood of future development of myopia (International Myopia Institute definition,23 adopted by the European Ophthalmological Society2). Screening and monitoring pre-myopes to avoid progression to myopia are secondary preventative methods. Scientific data suggests that pre-myopes can be identified at preschool age by active population screening, which includes cycloplegic refractometry, evaluation of binocular vision (level I. evidence, Elsevier) and ocular axial length measurements.2,17,24 Following the identification of pre-myopes, monitoring is required, which involves both monitoring of axial eye length growth and monitoring of changes in refractive error measured under cycloplegic conditions. Intervention is recommended in cases of abnormal binocularity or progression of ocular axial elongation. Secondary prevention needs active screening and follow-up programs. The task of eye care personnel is to submit evidence on unmet needs and appropriate ways of such screening campaigns to decision makers/politicians so that the Ministries of Health and Education can develop mass screening programs for preschool children. There is also a need to raise public awareness by disseminating information through various media (news, radio, television, online, etc). Eye care and medical professionals need to prepare appropriate messages for the public and to be prepared to undertake screening, follow-up, and interventions for pre-myopes. Further concrete actions of a clinician might be:Regular teaching of nurses and social services who perform the vision screening Pediatric ophthalmologist or optometrist checking the binocular vision of preschool children and young schoolchildren regularly Clinicians should educate those who examine children about the importance of axial length and its measurement Creation of screening protocols Advocacy in local, regional, and national health care and education authorities to develop and perform screening programs for screening and monitoring pre-myops 3. Tertiary prevention is the management of a condition after it has been diagnosed in order to delay or stop its progression. Tertiary prevention strategies are myopia controlling methods that slow the progression of myopia. Also based on the landmark “Atropine for the Treatment of Myopia 2” study by Chia and colleagues,25 the primary evidence-based therapies are as follows (Level of evidence by Cochrane 2020 in brackets, M: Moderate, L: Low)26: low-concentration atropine eye drops (M), multifocal spectacle design (M), contact lenses with power profiles that generate a peripheral myopic defocus (L), and orthokeratology, which employs corneal gas-permeable contact lenses to flatten the central cornea, resulting in midperipheral steepening and peripheral myopic defocus, during overnight wear to eliminate daytime myopia (M). Concerning the low-dose atropine, studies showed that the 0.05% atropine concentration to be the most effective one,27 especially in young children.28 Some of these measures can be combined and may be useful.2,17,26 Tertiary prevention to avoid progression to high myopia necessitates active work by eye care professionals. There is an urgent need to advocate the appropriate assessment procedures as well as potential myopia control interventions. Campaigning for proper funding of necessary myopia examinations, follow-up examinations, interventions, optical equipment, and eye drops in the healthcare budget is required by all stakeholders. Concrete actions of clinicians might be:Organizing training courses for eye care specialist on the application of myopia control methods suggested by the latest literature Providing flowcharts to assist in selecting the best myopia control method for a child Definition of the timing of control examinations in a protocol Selection of the most appropriate intervention individualized for the children Advocacy for proper funding of anti-myopia examinations, therapies and equipments There are examples of such successful projects and programs of implementation of myopia guidelines (Annex, supplemental material).17,29,30 Because the myopia epidemic began in East Asia, there are lessons to be learnt from the colleagues in that world region. There are currently active governmental programs to combat the increase in myopia which can be implemented in other regions of the world, including Europe, where myopia is already on the rise. Supplemental Material sj-docx-1-ejo-10.1177_11206721221143008 - Supplemental material for The impact of the pandemic highlights the urgent need for myopia guidelines: The clinicians’ role Click here for additional data file. Supplemental material, sj-docx-1-ejo-10.1177_11206721221143008 for The impact of the pandemic highlights the urgent need for myopia guidelines: The clinicians’ role by János Németh, Wagih A Aclimandos, Beáta Tapasztó, Jost B Jonas, Andrzej Grzybowski and Zoltán Zsolt Nagy in European Journal of Ophthalmology AG: member of Nevakar, GoCheckKids and Thea Advisory Boards. Recipient of grants from CooperVision, Hoya, and lecture honoraria from Thea. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ORCID iD: János Németh https://orcid.org/0000-0001-8575-4888 Supplemental material: Supplemental material for this article is available online. ==== Refs References 1 Holden BA Fricke TR Wilson DA , et al. 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Near work related parameters and myopia in Chinese children: the Anyang Childhood Eye Study. PLoS One 2015; 10 : e0134514.26244865 22 Pan CW Wu RK Liu H , et al. Types of lamp for homework and myopia among Chinese school-aged children. Ophthalmic Epidemiol 2018; 25 : 250–256.29281362 23 Flitcroft DI He M Jonas JB , et al. IMI - Defining and classifying myopia: a proposed set of standards for clinical and epidemiologic studies. Invest Ophthalmol Vis Sci 2019; 60 : M20–M30.30817826 24 McCullough S Adamson G Breslin KMM , et al. Axial growth and refractive change in white European children and young adults: predictive factors for myopia. Sci Rep 2020; 10 : 15189.32938970 25 Chia A Chua WH Cheung YB , et al. Atropine for the treatment of childhood myopia: safety and efficacy of 0.5%, 0.1%, and 0.01% doses (atropine for the treatment of myopia 2). Ophthalmology 2012; 119 : 347–354.21963266 26 Walline JJ Lindsley KB Vedula SS , et al. Interventions to slow progression of myopia in children. Cochrane Database of Syst Rev 2020; 1 : CD004916.31930781 27 Yam JC Li FF Zhang X , et al. Two-year clinical trial of the low-concentration atropine for myopia progression (LAMP) study: phase 2 report. Ophthalmology 2020; 127 : 910–919.32019700 28 Li FF Zhang Y Zhang X , et al. Age effect on treatment responses to 0.05%, 0.025%, and 0.01% atropine: low-concentration atropine for myopia progression study. Ophthalmology 2021; 128 : 1180–1187.33422558 29 Petty AD Wilson G . Reducing the impact of the impending myopia epidemic in New Zealand. N Z Med J 2018; 131 : 80–85. PMID: 30543614. 30 Jan C Li L Keay L , et al. Prevention of myopia, China. Bull World Health Organ 2020; 98 : 435–437.32514219	36448253	PMC9713518	NO-CC CODE	2022-12-02 23:23:05	no	Eur J Ophthalmol. 2022 Nov 29;:11206721221143008	utf-8	Eur J Ophthalmol	2,022	10.1177/11206721221143008	oa_other
==== Front Am J Lifestyle Med Am J Lifestyle Med spajl AJL American Journal of Lifestyle Medicine 1559-8276 1559-8284 SAGE Publications Sage CA: Los Angeles, CA 10.1177_15598276221140864 10.1177/15598276221140864 Pharmacy Review Potential Benefit of Vitamin D Supplementation in COVID-19 Stafford Andrea PharmD candidate White Nicole D. PharmD, CDCES, NBC-HWC, DipACLM 6216 Creighton University School of Pharmacy and Health Professions , Omaha, NE, USA Nicole White, PharmD, CDCES, NBC-HWC, DipACLM, School of Pharmacy and Health Professions, Creighton University, 2500 California Ave, Omaha, NE 68178, USA; e-mail: [email protected] 29 11 2022 29 11 2022 15598276221140864Copyright © 2022 The Author(s) 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Vitamin D is an important nutrient in the body that plays a vital role in immune system function. Several epidemiologic studies have shown that low vitamin D levels are found in a large percentage of COVID-19 patients with acute respiratory failure and that vitamin D levels may predict mortality in COVID-19 infection. Based on these findings, vitamin D supplementation may be an effective approach to preventing and/or treating COVID-19. Potential underlying mechanisms and clinical trial data evaluating the impact of supplementation in humans are described below. COVID-19 vitamin D supplements mechanism of action immune function edited-statecorrected-proof typesetterts10 ==== Body pmc “Cathelcidin is a peptide produced by neutrophils and mucosal cells in the respiratory tract with direct antimicrobial effects on bacteria, viruses, and fungi.” Introduction In recent years, COVID-19 has been the cause of serious illness and death. COVID-19 is caused by a virus known as severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2.1 Signs and symptoms of COVID-19 are far reaching and can impact a multitude of body systems. Most commonly, patients experience cough, fatigue, headache, fever, chest pain, and sore throat but complications such as shortness of breath, pneumonia, heart problems, acute kidney injury, organ failure, and progressive respiratory failure can also occur.1-3 Additionally, some individuals experience long-term effects following COVID-19 infection. This condition, termed long COVID, can include a wide range of ongoing health problems, which last weeks, months, or longer.4 Due to the broad range of symptoms, complications, and lack of treatment in the early phases of the pandemic, many individuals looked to supplements with hopes to prevent and/or treat COVID-19. Over the counter supplement use by Americans increased 29% during the pandemic.5 Of those who started supplements during this time, 65% reported a desire to enhance immunity or protect themselves from COVID-19.5 A number of supplements have been proposed for the prevention or treatment of COVID-19. Among the most popular are zinc, vitamin C, vitamin D, elderberry, echinacea, and melatonin.2,3 Vitamin D will be described here. Vitamin D is an important nutrient in the body that helps maintain bone health and plays a vital role in immune system function, muscle function, and brain cell activity.6 Vitamin D is found naturally in some foods and added to others. It can also be produced endogenously in the skin upon exposure to ultraviolet (UV) radiation. The amount of Vitamin D produced or consumed can vary based on location and lifestyle; therefore, supplementation may be needed to ensure optimal health and prevent deficiency. Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D (25(OH)D) concentration <30 ng/mL, occurs in approximately 1 billion people worldwide, and has been associated with a number of conditions, including an increased risk of respiratory infections.2,7 Populations at greatest risk of Vitamin D deficiency include older adults, patients with obesity, diabetes, and those with heart, liver, or kidney disease.7 Many individuals at greatest risk of vitamin D deficiency are also at increased risk of severe illness and death from COVID-19.8 Several epidemiologic studies have found that low vitamin D levels are found in a large percentage of COVID-19 patients with acute respiratory failure and that vitamin D levels may predict mortality in COVID-19 infection.9,10 Based on these findings, vitamin D supplementation presents a plausible approach to the prevention and treatment of COVID-19. Potential underlying mechanisms and clinical trial data evaluating the impact of supplementation in humans are described below. Potential Underlying Mechanisms Vitamin D is a fat-soluble vitamin synthesized in the skin via UV radiation or acquired through diet and/or supplementation.11 UV radiation converts 7-dehydrocholesterol in the epidermis to cholecalciferol.11 In the liver, cholecalciferol hydroxylates to form 25-hydroxyvitamin D3, which is then converted into calcitriol, the active metabolite of vitamin D in the kidneys.11 Most of the physiological effects of vitamin D in the body are related to calcitriol, mediated through the vitamin D receptor (VDR). Vitamin D receptors are expressed by cells in the brain, heart, skin, gonads, prostate, and breast, as well as numerous types of immune cells including macrophages, monocytes, and dendritic cells.11-13 Studies show that vitamin D plays a role in the regulation of both the innate and adaptive immune system.11-15 In the innate immune system, vitamin D may function as an anti-inflammatory on macrophages, decreasing inflammatory stimuli. Vitamin D may also have an anti-oxidative effect on monocytes, reducing oxygen radical formation, and minimizing damage by pathogens. In the adaptive immune system, vitamin D plays a role in decreasing antigen presentation, modulating T-lymphocyte response, or inducing cell death of activated B-lymphocytes. As mentioned previously, adequate levels of vitamin D have been linked to a decrease in respiratory tract infections as well as a decrease in severe COVID-19 cases.2,9,10,14 This association is thought to be mediated, in part, by a vitamin D-induced increase in cathelcidin.15 Cathelcidin is a peptide produced by neutrophils and mucosal cells in the respiratory tract with direct antimicrobial effects on bacteria, viruses, and fungi. It also plays a protective role in epithelial/alveolar permeability. In cases of severe COVID-19, high levels of circulating cytokines and chemokines released from the infected lungs (cytokine storm), may be responsible for acute respiratory distress, multiple organ failure, and death. Vitamin D is thought to suppress this immune system overactivity, thereby reducing the risk of damage to vital organs. Clinical Trial Data The number of interventional studies evaluating the impact of vitamin D on COVID-19 is growing rapidly. As of October 28, 2022, 41 clinical trials have been completed and 23 are underway, according to Clinicaltrials.gov.16 The most recent systematic review and meta-analysis of interventional trials was published in May 2022.17 The study included nine randomized controlled trials (RCTs) and fourteen non-randomized studies of intervention (NRISs) with 5,870,189 total subjects. Studies were divided into three categories based on the outcomes assessed: primary, secondary, and tertiary prevention. The primary prevention analysis evaluated the association between vitamin D supplementation and risk of incident COVID-19. Vitamin D supplementation did not significantly reduce the risk of testing positive for COVID-19, regardless of regimen (bolus vs non-bolus). Only two of the studies included provided baseline 25(OH)D levels; thus, no subgroup analysis evaluating in impact of baseline vitamin D status was performed. The secondary prevention analysis, which assessed the association between vitamin D supplementation and COVID-19 related outcomes in mild (ambulatory) COVID-19 cases, was unable to aggregate data. Only one study evaluated the risk of hospitalization and while two studies reported on severity of symptoms, the methods used to measure symptoms differed, and therefore, the data could not be pooled. The tertiary prevention analysis, which assessed the impact of vitamin D supplementation in patients hospitalized with COVID-19, found a significant reduction in ICU admission, need for mechanical ventilation, and COVID-19 mortality in those patients supplemented with vitamin D. Protective effects of vitamin D appear stronger when non-bolus regimens were used and in patients with baseline vitamin D status ≥25 nmol/L. Since the completion of the aforementioned meta-analysis, a number of interventional studies evaluating the impact of vitamin D supplementation on COVID-19 outcomes have been published.18-24 All but two of the studies23,24 found benefits to vitamin D supplementation in patients hospitalized with COVID-19. Of note, bolus dosing was used in both studies that found no benefit, and in the Mariani et al study, baseline 25(OH)VitD levels >30 ng/mL. Varying doses and dosing regimens were explored in those studies with favorable effects of vitamin D supplementation. Two studies compared high- vs low-dose vitamin D supplementation in hospitalized patients.18,21 The first compared alfacalcidiol 1mcg/day orally, to cholecalciferol 200 000 IU intramuscularly once.18 The second study compared oral cholecalciferol 2000 iu/day to cholecalciferol 10000 iu/day.21 Both studies found significant improvements in outcomes with the higher dose. Conclusion Clinical trial data suggests that vitamin D supplementation, when used in addition to best care available, is associated with reduced ICU admission, need for mechanical ventilation, and death in patients hospitalized with COVID-19. Further investigation is needed to determine the best dose and regimen, as well as underlying mechanisms of action in the acute setting. Insufficient evidence exists to determine whether vitamin D supplementation reduces the risk of acquiring COVID-19, particularly in patients with vitamin D deficiency. Likewise, additional studies are needed to determine whether vitamin D supplementation improves outcomes in ambulatory patients with mild-moderate COVID-19. Based on the prevalence of vitamin D deficiency, the low risk of adverse effects associated with supplementation, and the stronger protective benefits observed from supplementation in hospitalized patients with baseline vitamin D levels ≥25 nmol/L, it may be worthwhile for patients desiring protection from severe COVID-19 to consider a daily vitamin D supplement. Appropriate dosing depends on the baseline vitamin D status. For patients with vitamin D deficiency, eight weekly doses of oral ergocalciferol 50 000 IU, or 5000 IU of oral cholecalciferol daily have been recommended.7,25 Once a target vitamin D level is reached, 800–3000 IU of cholecalciferol per day have been recommended to maintain adequate levels.7,25 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ==== Refs References 1 Centers for Disease Control and Prevention. Basics of covid-19. Centers for Disease Control and Prevention; 2021. https://www.cdc.gov/coronavirus/2019-ncov/your-health/about-covid-19/basics-covid-19.html. Accessed October 25, 2022. 2 Moscatelli F Sessa F Valenzano A , et al. COVID-19: Role of nutrition and supplementation. Nutrients. 2021;13 (3 ):976. doi:10.3390/nu13030976. PMID: 33803015; PMCID: PMC8002713.33803015 3 Office of dietary supplements - dietary supplements in the time of COVID-19. Ods.od.nih.gov. https://ods.od.nih.gov/factsheets/COVID19-HealthProfessional/. Updated May 13, 2022. Accessed October 4, 2022. 4 Centers for Disease Control and Prevention. Long COVID or post-COVID conditions; 2022. Available at: https://www.cdc.gov/coronavirus/2019-ncov/long-term-effects/index.html. Accessed October 25, 2022. 5 Three in ten Americans increased supplement use since onset of pandemic. EurekAlert!; 2021. https://www.eurekalert.org/news-releases/807544 6 Bouillon R Manousaki D Rosen C , et al. The health effects of vitamin d suplemetation: Evidence from human studies. Nat Rev Endocrinol. 2022;18 (2 ):96-110.34815552 7 Holick MF . The vitamin d deficiency pandemic: Approaches for diagnosis, treatment and prevention. Rev Endocr Metab Disord. 2017;18 :153-165.28516265 8 Gao YD Ding M Dong X , et al. Risk factors for severe and critically ill COVID-19 patients: A review. Allergy. 2021;76 (2 ):428-455.33185910 9 Carpagnano GE Lecce VD Quaranta VN , et al. Vitamin D deficiency as a predictor of poor prognosis in patients with acute respiratory failure due to COVID-19. J Endocrinol Invest. 2020;1-7. doi:10.1007/s40518-020-0137-x. 10 Karahan S Katkat F . Impact of serum 25(OH) Vitamin D level on mortality in patiaens with COVID-19 in Turkey. J Nutr Health Aging. 2020. doi:10.1007/s12603-020-1479-0. 11 Mora JR Iwata M von Andrian UH . Vitamin effects on the immune system: Vitamins A and D take centre stage. Nat Rev Immunol. 2008;8 (9 ):685-698. doi:10.1038/nri2378. PMID: 19172691; PMCID: PMC2906676.19172691 12 Bikle DD . Vitamin D: Production, metabolism and mechanisms of action. In: Feingold KR Anawalt B Boyce A , et al. , eds. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000. Available from: https://www.ncbi.nlm.nih.gov/books/NBK278935/. Updated December 31 2021. 13 Martens PJ Gysemans C Verstuyf A Mathieu AC . Vitamin D’s effect on immune function. Nutrients. 2020;12 (5 ):1248. doi:10.3390/nu12051248 . PMID: 32353972; PMCID: PMC7281985.32353972 14 Hansdottir S Monick MM . Vitamin D effects on lung immunity and respiratory diseases. Vitam Horm. 2011;86 :217-237. doi:10.1016/B978-0-12-386960-9.00009-5. PMID: 21419273; PMCID: PMC3559187.21419273 15 Banerjee A Ganguly U Saha S , et al. Vitamin D and immune-pathology of COVID-19: many interactions but uncertain therapeutic effects. Expert Rev Anti-Infect Ther. 2021. doi:10.1080/1478721.2021.1905519. 16 Clinicaltrials.gov. Vitamin D and COVID-19. Available at: https://clinicaltrials.gov/ct2/results?term=vitamin+D&cond=COVID-19&Search=Apply&recrs=e&age_v=&gndr=&type=Intr&rslt=. Accessed October 28, 2022. 17 Hosseini B El Abd A Ducharme FM . Effects of vitamin D supplementation on COVID-19 related outcomes: A systematic review and meta-analysis. Nutrients. 2022;14 :2134. doi:10.3390/nu14102134.35631275 18 Sarhan N Abou Arda AE Sarhan RM , et al. Evidence for the efficacy of high dose vitamin D on the hyperinflammation state in moderate-to-severe COVID-19 Patients: A randomized clinical trial. Medicina. 2022;58 (10 ):1358.36295519 19 Annweiler C Beaudenon M Gautier J , et al. High -dose versus standard-dose vitamin D supplementation in older adults with COVID-19 (COVID-TRIAL): A multicenter, open-label, randomized, controlled, superiority trial. PLoS Med. 2022;19 (5 ):e1003999.35639792 20 De Niet S Tremege M Coffiner M , et al. Positive effects of vitamin D supplementation in patients hospitalized for COVID-19: A randomized, double-blind, placebo-controlled trial. Nutrients. 2022;14 (15 ):3048.35893907 21 Torres M Casado G Vigon L , et al. Changes in the immune response against SARS-CoV-2 in individuals with severe COVID-19 treat with high dose of vitamin D. Biomed Pharmacother. 2022;150 :112065. 22 Karonova TL Golovatyuk KA Kudryavtsev IV , et al. Effect of cholecalciferol supplementation on the clinical features and inflammatory markers in hostilzed COVID-19 patients: A randomized, open-label, single-center study. Nutrients. 2022;45 (13 ):2602. 23 Mariani J Antonietti L Tajer C , et al. High-dose vitamin D versus placebo to prevent complications in COVID-19 patients: Multicenter randomized controlled clinical trial. PLoS One. 2022;17 (5 ):e0267918.35622854 24 Cannata-Andia JB Diaz-Sottolano A Fernandez P , et al. A single-oral bolus of 10,000 IU of cholecalciferol at hospital admission did not improve outcomes in the COVID-19 disease: The COVID-VIT-D- a randomized multicenter international clinical trial. BMC Med. 2022;20 (1 ):83.35177066 25 Bordelon P Ghetu MV Langan R . Recognition and management of vitamin D deficiency. Am Fam Physician. 2009;80 (8 ):841-846.19835345	0	PMC9713520	NO-CC CODE	2022-12-02 23:23:05	no	Am J Lifestyle Med. 2022 Nov 29;:15598276221140864	utf-8	Am J Lifestyle Med	2,022	10.1177/15598276221140864	oa_other
==== Front Am J Lifestyle Med Am J Lifestyle Med spajl AJL American Journal of Lifestyle Medicine 1559-8276 1559-8284 SAGE Publications Sage CA: Los Angeles, CA 10.1177_15598276221140864 10.1177/15598276221140864 Pharmacy Review Potential Benefit of Vitamin D Supplementation in COVID-19 Stafford Andrea PharmD candidate White Nicole D. PharmD, CDCES, NBC-HWC, DipACLM 6216 Creighton University School of Pharmacy and Health Professions , Omaha, NE, USA Nicole White, PharmD, CDCES, NBC-HWC, DipACLM, School of Pharmacy and Health Professions, Creighton University, 2500 California Ave, Omaha, NE 68178, USA; e-mail: [email protected] 29 11 2022 29 11 2022 15598276221140864Copyright © 2022 The Author(s) 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Vitamin D is an important nutrient in the body that plays a vital role in immune system function. Several epidemiologic studies have shown that low vitamin D levels are found in a large percentage of COVID-19 patients with acute respiratory failure and that vitamin D levels may predict mortality in COVID-19 infection. Based on these findings, vitamin D supplementation may be an effective approach to preventing and/or treating COVID-19. Potential underlying mechanisms and clinical trial data evaluating the impact of supplementation in humans are described below. COVID-19 vitamin D supplements mechanism of action immune function edited-statecorrected-proof typesetterts10 ==== Body pmc “Cathelcidin is a peptide produced by neutrophils and mucosal cells in the respiratory tract with direct antimicrobial effects on bacteria, viruses, and fungi.” Introduction In recent years, COVID-19 has been the cause of serious illness and death. COVID-19 is caused by a virus known as severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2.1 Signs and symptoms of COVID-19 are far reaching and can impact a multitude of body systems. Most commonly, patients experience cough, fatigue, headache, fever, chest pain, and sore throat but complications such as shortness of breath, pneumonia, heart problems, acute kidney injury, organ failure, and progressive respiratory failure can also occur.1-3 Additionally, some individuals experience long-term effects following COVID-19 infection. This condition, termed long COVID, can include a wide range of ongoing health problems, which last weeks, months, or longer.4 Due to the broad range of symptoms, complications, and lack of treatment in the early phases of the pandemic, many individuals looked to supplements with hopes to prevent and/or treat COVID-19. Over the counter supplement use by Americans increased 29% during the pandemic.5 Of those who started supplements during this time, 65% reported a desire to enhance immunity or protect themselves from COVID-19.5 A number of supplements have been proposed for the prevention or treatment of COVID-19. Among the most popular are zinc, vitamin C, vitamin D, elderberry, echinacea, and melatonin.2,3 Vitamin D will be described here. Vitamin D is an important nutrient in the body that helps maintain bone health and plays a vital role in immune system function, muscle function, and brain cell activity.6 Vitamin D is found naturally in some foods and added to others. It can also be produced endogenously in the skin upon exposure to ultraviolet (UV) radiation. The amount of Vitamin D produced or consumed can vary based on location and lifestyle; therefore, supplementation may be needed to ensure optimal health and prevent deficiency. Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D (25(OH)D) concentration <30 ng/mL, occurs in approximately 1 billion people worldwide, and has been associated with a number of conditions, including an increased risk of respiratory infections.2,7 Populations at greatest risk of Vitamin D deficiency include older adults, patients with obesity, diabetes, and those with heart, liver, or kidney disease.7 Many individuals at greatest risk of vitamin D deficiency are also at increased risk of severe illness and death from COVID-19.8 Several epidemiologic studies have found that low vitamin D levels are found in a large percentage of COVID-19 patients with acute respiratory failure and that vitamin D levels may predict mortality in COVID-19 infection.9,10 Based on these findings, vitamin D supplementation presents a plausible approach to the prevention and treatment of COVID-19. Potential underlying mechanisms and clinical trial data evaluating the impact of supplementation in humans are described below. Potential Underlying Mechanisms Vitamin D is a fat-soluble vitamin synthesized in the skin via UV radiation or acquired through diet and/or supplementation.11 UV radiation converts 7-dehydrocholesterol in the epidermis to cholecalciferol.11 In the liver, cholecalciferol hydroxylates to form 25-hydroxyvitamin D3, which is then converted into calcitriol, the active metabolite of vitamin D in the kidneys.11 Most of the physiological effects of vitamin D in the body are related to calcitriol, mediated through the vitamin D receptor (VDR). Vitamin D receptors are expressed by cells in the brain, heart, skin, gonads, prostate, and breast, as well as numerous types of immune cells including macrophages, monocytes, and dendritic cells.11-13 Studies show that vitamin D plays a role in the regulation of both the innate and adaptive immune system.11-15 In the innate immune system, vitamin D may function as an anti-inflammatory on macrophages, decreasing inflammatory stimuli. Vitamin D may also have an anti-oxidative effect on monocytes, reducing oxygen radical formation, and minimizing damage by pathogens. In the adaptive immune system, vitamin D plays a role in decreasing antigen presentation, modulating T-lymphocyte response, or inducing cell death of activated B-lymphocytes. As mentioned previously, adequate levels of vitamin D have been linked to a decrease in respiratory tract infections as well as a decrease in severe COVID-19 cases.2,9,10,14 This association is thought to be mediated, in part, by a vitamin D-induced increase in cathelcidin.15 Cathelcidin is a peptide produced by neutrophils and mucosal cells in the respiratory tract with direct antimicrobial effects on bacteria, viruses, and fungi. It also plays a protective role in epithelial/alveolar permeability. In cases of severe COVID-19, high levels of circulating cytokines and chemokines released from the infected lungs (cytokine storm), may be responsible for acute respiratory distress, multiple organ failure, and death. Vitamin D is thought to suppress this immune system overactivity, thereby reducing the risk of damage to vital organs. Clinical Trial Data The number of interventional studies evaluating the impact of vitamin D on COVID-19 is growing rapidly. As of October 28, 2022, 41 clinical trials have been completed and 23 are underway, according to Clinicaltrials.gov.16 The most recent systematic review and meta-analysis of interventional trials was published in May 2022.17 The study included nine randomized controlled trials (RCTs) and fourteen non-randomized studies of intervention (NRISs) with 5,870,189 total subjects. Studies were divided into three categories based on the outcomes assessed: primary, secondary, and tertiary prevention. The primary prevention analysis evaluated the association between vitamin D supplementation and risk of incident COVID-19. Vitamin D supplementation did not significantly reduce the risk of testing positive for COVID-19, regardless of regimen (bolus vs non-bolus). Only two of the studies included provided baseline 25(OH)D levels; thus, no subgroup analysis evaluating in impact of baseline vitamin D status was performed. The secondary prevention analysis, which assessed the association between vitamin D supplementation and COVID-19 related outcomes in mild (ambulatory) COVID-19 cases, was unable to aggregate data. Only one study evaluated the risk of hospitalization and while two studies reported on severity of symptoms, the methods used to measure symptoms differed, and therefore, the data could not be pooled. The tertiary prevention analysis, which assessed the impact of vitamin D supplementation in patients hospitalized with COVID-19, found a significant reduction in ICU admission, need for mechanical ventilation, and COVID-19 mortality in those patients supplemented with vitamin D. Protective effects of vitamin D appear stronger when non-bolus regimens were used and in patients with baseline vitamin D status ≥25 nmol/L. Since the completion of the aforementioned meta-analysis, a number of interventional studies evaluating the impact of vitamin D supplementation on COVID-19 outcomes have been published.18-24 All but two of the studies23,24 found benefits to vitamin D supplementation in patients hospitalized with COVID-19. Of note, bolus dosing was used in both studies that found no benefit, and in the Mariani et al study, baseline 25(OH)VitD levels >30 ng/mL. Varying doses and dosing regimens were explored in those studies with favorable effects of vitamin D supplementation. Two studies compared high- vs low-dose vitamin D supplementation in hospitalized patients.18,21 The first compared alfacalcidiol 1mcg/day orally, to cholecalciferol 200 000 IU intramuscularly once.18 The second study compared oral cholecalciferol 2000 iu/day to cholecalciferol 10000 iu/day.21 Both studies found significant improvements in outcomes with the higher dose. Conclusion Clinical trial data suggests that vitamin D supplementation, when used in addition to best care available, is associated with reduced ICU admission, need for mechanical ventilation, and death in patients hospitalized with COVID-19. Further investigation is needed to determine the best dose and regimen, as well as underlying mechanisms of action in the acute setting. Insufficient evidence exists to determine whether vitamin D supplementation reduces the risk of acquiring COVID-19, particularly in patients with vitamin D deficiency. Likewise, additional studies are needed to determine whether vitamin D supplementation improves outcomes in ambulatory patients with mild-moderate COVID-19. Based on the prevalence of vitamin D deficiency, the low risk of adverse effects associated with supplementation, and the stronger protective benefits observed from supplementation in hospitalized patients with baseline vitamin D levels ≥25 nmol/L, it may be worthwhile for patients desiring protection from severe COVID-19 to consider a daily vitamin D supplement. Appropriate dosing depends on the baseline vitamin D status. For patients with vitamin D deficiency, eight weekly doses of oral ergocalciferol 50 000 IU, or 5000 IU of oral cholecalciferol daily have been recommended.7,25 Once a target vitamin D level is reached, 800–3000 IU of cholecalciferol per day have been recommended to maintain adequate levels.7,25 The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ==== Refs References 1 Centers for Disease Control and Prevention. Basics of covid-19. Centers for Disease Control and Prevention; 2021. https://www.cdc.gov/coronavirus/2019-ncov/your-health/about-covid-19/basics-covid-19.html. Accessed October 25, 2022. 2 Moscatelli F Sessa F Valenzano A , et al. COVID-19: Role of nutrition and supplementation. Nutrients. 2021;13 (3 ):976. doi:10.3390/nu13030976. PMID: 33803015; PMCID: PMC8002713.33803015 3 Office of dietary supplements - dietary supplements in the time of COVID-19. Ods.od.nih.gov. https://ods.od.nih.gov/factsheets/COVID19-HealthProfessional/. Updated May 13, 2022. Accessed October 4, 2022. 4 Centers for Disease Control and Prevention. Long COVID or post-COVID conditions; 2022. Available at: https://www.cdc.gov/coronavirus/2019-ncov/long-term-effects/index.html. Accessed October 25, 2022. 5 Three in ten Americans increased supplement use since onset of pandemic. EurekAlert!; 2021. https://www.eurekalert.org/news-releases/807544 6 Bouillon R Manousaki D Rosen C , et al. The health effects of vitamin d suplemetation: Evidence from human studies. Nat Rev Endocrinol. 2022;18 (2 ):96-110.34815552 7 Holick MF . The vitamin d deficiency pandemic: Approaches for diagnosis, treatment and prevention. Rev Endocr Metab Disord. 2017;18 :153-165.28516265 8 Gao YD Ding M Dong X , et al. Risk factors for severe and critically ill COVID-19 patients: A review. Allergy. 2021;76 (2 ):428-455.33185910 9 Carpagnano GE Lecce VD Quaranta VN , et al. Vitamin D deficiency as a predictor of poor prognosis in patients with acute respiratory failure due to COVID-19. J Endocrinol Invest. 2020;1-7. doi:10.1007/s40518-020-0137-x. 10 Karahan S Katkat F . Impact of serum 25(OH) Vitamin D level on mortality in patiaens with COVID-19 in Turkey. J Nutr Health Aging. 2020. doi:10.1007/s12603-020-1479-0. 11 Mora JR Iwata M von Andrian UH . Vitamin effects on the immune system: Vitamins A and D take centre stage. Nat Rev Immunol. 2008;8 (9 ):685-698. doi:10.1038/nri2378. PMID: 19172691; PMCID: PMC2906676.19172691 12 Bikle DD . Vitamin D: Production, metabolism and mechanisms of action. In: Feingold KR Anawalt B Boyce A , et al. , eds. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000. Available from: https://www.ncbi.nlm.nih.gov/books/NBK278935/. Updated December 31 2021. 13 Martens PJ Gysemans C Verstuyf A Mathieu AC . Vitamin D’s effect on immune function. Nutrients. 2020;12 (5 ):1248. doi:10.3390/nu12051248 . PMID: 32353972; PMCID: PMC7281985.32353972 14 Hansdottir S Monick MM . Vitamin D effects on lung immunity and respiratory diseases. Vitam Horm. 2011;86 :217-237. doi:10.1016/B978-0-12-386960-9.00009-5. PMID: 21419273; PMCID: PMC3559187.21419273 15 Banerjee A Ganguly U Saha S , et al. 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High -dose versus standard-dose vitamin D supplementation in older adults with COVID-19 (COVID-TRIAL): A multicenter, open-label, randomized, controlled, superiority trial. PLoS Med. 2022;19 (5 ):e1003999.35639792 20 De Niet S Tremege M Coffiner M , et al. Positive effects of vitamin D supplementation in patients hospitalized for COVID-19: A randomized, double-blind, placebo-controlled trial. Nutrients. 2022;14 (15 ):3048.35893907 21 Torres M Casado G Vigon L , et al. Changes in the immune response against SARS-CoV-2 in individuals with severe COVID-19 treat with high dose of vitamin D. Biomed Pharmacother. 2022;150 :112065. 22 Karonova TL Golovatyuk KA Kudryavtsev IV , et al. Effect of cholecalciferol supplementation on the clinical features and inflammatory markers in hostilzed COVID-19 patients: A randomized, open-label, single-center study. Nutrients. 2022;45 (13 ):2602. 23 Mariani J Antonietti L Tajer C , et al. High-dose vitamin D versus placebo to prevent complications in COVID-19 patients: Multicenter randomized controlled clinical trial. PLoS One. 2022;17 (5 ):e0267918.35622854 24 Cannata-Andia JB Diaz-Sottolano A Fernandez P , et al. A single-oral bolus of 10,000 IU of cholecalciferol at hospital admission did not improve outcomes in the COVID-19 disease: The COVID-VIT-D- a randomized multicenter international clinical trial. BMC Med. 2022;20 (1 ):83.35177066 25 Bordelon P Ghetu MV Langan R . Recognition and management of vitamin D deficiency. Am Fam Physician. 2009;80 (8 ):841-846.19835345	0	PMC9713529	NO-CC CODE	2022-12-02 23:23:05	no	Ann Hepatol. 2022 Dec 1; 27:100834	latin-1	Ann Hepatol	2,022	10.1016/j.aohep.2022.100834	oa_other
==== Front Ann Hepatol Ann Hepatol Annals of Hepatology 1665-2681 1665-2681 Published by Elsevier España, S.L. S1665-2681(22)00179-X 10.1016/j.aohep.2022.100837 100837 Article Manifestations of SARS-COV-2 in patients with chronic liver disease Jiménez-Partida AE Jiménez-Luévano MA Jiménez-Partida MA Bravo-Cuellar A Cortes-Aguilar Y Regional Hospital “Dr. Valentín Gómez Farías” Gastroenterology Service. ISSSTE. Mexico 1 12 2022 12 2022 1 12 2022 27 100837100837 Copyright © 2022 Published by Elsevier España, S.L. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction and Objectives This study aimed to analyze the degree of severity of SARS-CoV-2 infection in patients with the previous chronic liver disease through clinical, laboratory and histological variables. Materials and Methods From November 2021 to July 2021, at the Valentín Gómez Farías Hospital, a Gastroenterology service, 70 patients were treated with prior informed consent and endorsed by the ethics committee. For this study, 51 individuals with chronic liver disease and diagnosis of SARS-CoV-2 were included: 25 with steatohepatitis and 26 with liver cirrhosis. The following findings were observed: Results Histological findings: • Micro vesicular steatosis. • Mild mortal and lobular inflammatory activity. • High viral load in the vascular endothelium (48 to 53%) and cytopathic effect of the SARS-CoV-2 virus. • Ischemia due to hypoperfusion mainly due to myocardial injury. • Immune hyperactivation. • Drug-reactive liver injury. • Apoptosis Discussion The COVID-19 pandemic is more severe in vulnerable patients, mainly older adults, male gender and comorbidities such as hypertension, diabetes, nephropathy, heart disease, lung disease, immunosuppression and patients with liver disease. Of these, 60% have severe symptoms and a mortality of 34%. Conclusions COVID-19 is the leading cause of death in Mexico. High-risk entities in this viremia are of great global prevalence. Steatohepatitis (NASH) and liver cirrhosis predispose high mortality and complications, possibly evidenced by these clinical evaluations and hepatic laboratory tests. Funding The resources used in this study were from the hospital without any additional financing Declaration of interest The authors declare no potential conflicts of interest. ==== Body pmcTable 1. Demographic, biochemical and symptomatology characteristics of the two groupsPrevious pathologies Steatohepatitis Liver cirrhosis Age 55.64 60,84 Gender 72% women - 28% men 42% women – 58% men BMI 30.76 27.84 Comorbidities Overweight/obesity 100% 100% DM 2 31% 20% Alcoholism 0% 27% Autoimmune disease 0% 4% Laboratory AST 42.24 56.76 ALT 50.25 69.90 DHL 308.2 315.6 Platelets 170 100.38 Ferritin 496.24 592.5 D-dimer 530.54 1,064 Lymphocytes 35.2 29.96 ESR 32.32 30.06 PCR 49 47.03 Oxygen saturation 85.24 85.69 Clinic: Cough 16 (64%) 14 (54%) Dyspnea 13 (52%) 12 (46%) Pneumonia 5 (20%) 8 (32%) Asthenia 6 (24%) 6 (24%) Fever 2 (8%) 13 (52%) Headache 3 (12%) 8 (31%) Anosmia 1 (4%) 1 (4%) Shivers 1 (4%) 1 (4%) Arthralgia 3 (12%) 2 (8%) Diarrhea 1 (4%) 2 (8%)	0	PMC9713530	NO-CC CODE	2022-12-02 23:23:05	no	Ann Hepatol. 2022 Dec 1; 27:100837	utf-8	Ann Hepatol	2,022	10.1016/j.aohep.2022.100837	oa_other
==== Front Ann Hepatol Ann Hepatol Annals of Hepatology 1665-2681 1665-2681 Published by Elsevier España, S.L. S1665-2681(22)00180-6 10.1016/j.aohep.2022.100838 100838 Article Pilot study: management with pentoxifylline in patients with chronic liver disease and COVID-19 Jiménez-Luévano MA Jiménez-Partida AE Jiménez-Partida MA Bravo-Cuellar A Cortes-Aguilar Y Regional Hospital “Dr. Valentín Gómez Farías”. Gastroenterology Service. ISSSTE. Mexico 1 12 2022 12 2022 1 12 2022 27 100838100838 Copyright © 2022 Published by Elsevier España, S.L. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction and Objectives This study aimed to improve the response to adjuvant treatment with pentoxifylline in patients with SARS-CoV-2 and previous chronic liver disease. Materials and Methods In the ISSSTE hospital of Zapopan for eight months, 51 patients with moderate to severe SARS-CoV-2 and chronic liver disease, 26 with cirrhosis and 25 with steatohepatitis were evaluated, with prior informed consent and endorsed by the ethics committee. They were administered pentoxifylline 400 mg for 28 days, in addition to supportive measures such as paracetamol 750 mg, celecoxib 100 mg, or anticoagulants (enoxaparin) in patients with D-dimer > 600 mg/dL and supplemental oxygen in patients with saturation < 90. Clinical, laboratory and mortality variables were analyzed. The trial was approved by the research ethics committee, and informed consent was obtained. Results They were patients with Covid-19 plus cirrhosis and steatohepatitis. They survived 100%, after 28 days of driving, in addition to avoiding admission to intensive care. Discussion Pentoxifylline is a methylxanthine with antioxidant, hemorheological, anti-inflammatory and immunomodulatory properties since it inhibits NF-KB (via JAK/STAT and 1KB), pro-inflammatory cytokines, phosphodiesterase, in addition to stimulating anti-inflammatory cytokines, interferon-gamma, growth factors, TGF beta and granulocyte growth factor. Also, antiviral, as in Japanese encephalitis virus, vaccine virus, Rotavirus, HPV, respiratory syncytial virus, HIV, HCV, etc. Conclusions This viremia is severe in vulnerable groups, particularly liver diseases. It is inferred that Pentoxifylline may be alternative management, as manifested in this group of patients who managed to survive. So, we suggest multicenter and randomized studies to know their real benefit. Funding The resources used in this study were from the hospital without any additional financing Declaration of interest The authors declare no potential conflicts of interest. ==== Body pmcTable 1. Demographic, biochemical and symptomatology characteristics of the two groupsPrevious pathologies Steatohepatitis Liver cirrhosis Age 55.64 60,84 Gender 72% women - 28% men 42% women – 58% men BMI 30.76 27.84 Comorbidities Overweight / obesity 100% 100% DM 2 31% 20% Alcoholism 0% 27% Autoimmune disease 0% 4% Laboratory AST 42.24 56.76 ALT 50.25 69.90 DHL 308.2 315.6 Platelets 170 100.38 Ferritin 496.24 592.5 D-dimer 530.54 1,064 Lymphocytes 35.2 29.96 ESR 32.32 30.06 PCR 49 47.03 Oxygen saturation 85.24 85.69 Clinic: Cough 16 (64%) 14 (54%) Dyspnea 13 (52%) 12 (46%) Pneumonia 5 (20%) 8 (32%) Asthenia 6 (24%) 6 (24%) Fever 2 (8%) 13 (52%) Headache 3 (12%) 8 (31%) Anosmia 1 (4%) 1 (4%) Shivers 1 (4%) 1 (4%) Arthralgia 3 (12%) 2 (8%) Diarrhea 1 (4%) 2 (8%)	0	PMC9713531	NO-CC CODE	2022-12-02 23:23:06	no	Ann Hepatol. 2022 Dec 1; 27:100838	utf-8	Ann Hepatol	2,022	10.1016/j.aohep.2022.100838	oa_other
==== Front Health Promot Pract Health Promot Pract HPP sphpp Health Promotion Practice 1524-8399 1552-6372 SAGE Publications Sage CA: Los Angeles, CA 36448342 10.1177/15248399221135589 10.1177_15248399221135589 Resources, Frameworks, and Perspectives Addressing HIV and Homelessness During COVID-19: A Community-Based Demonstration Project https://orcid.org/0000-0002-8425-9196 Kay Emma Sophia PhD, MSW 12 Bruce Josh MPH 2 Foster-Hill Sherri BS 2 Rygiel Anne MSW 3 Batey D. Scott PhD, MSW 1 1 The University of Alabama at Birmingham, Birmingham, AL, USA 2 Birmingham AIDS Outreach, Birmingham, AL, USA 3 Firehouse Ministries, Birmingham, AL, USA Emma Sophia Kay, Birmingham AIDS Outreach, Birmingham, AL 35233, USA; e-mail: [email protected] 30 11 2022 30 11 2022 15248399221135589© 2022 Society for Public Health Education 2022 Society for Public Health Education This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. People experiencing homelessness are at increased risk for HIV, and people with HIV (PWH) experiencing homelessness are more likely to experience suboptimal HIV health outcomes than PWH with stable housing. Within Alabama, a state prioritized in the Ending the HIV Epidemic initiative, Jefferson County consistently has the highest number of new HIV diagnoses as well as a high percentage of the state’s homeless population. To address the twin epidemics of both HIV and homelessness within the high-priority setting of Jefferson County, Alabama, this 1-year community-based project, Ending the HIV Epidemic: Addressing HIV Health and Homelessness (AH3), sought to increase HIV testing and linkage to care among this population by placing a full-time case manager trained in HIV testing and case management at a homeless shelter. Results demonstrated that HIV testing was highly acceptable: 733 individuals were offered a test, and only 2.7% (n = 20) declined. Nine previously diagnosed, out of care PWH and one newly diagnosed PWH were identified through AH3 testing efforts. Of these, five (50%) were linked to care at a local HIV clinic. The remaining five PWH left the shelter before they could be linked to care. Just 10 shelter guests expressed interest in taking PrEP (just 1.4% of guests who tested negative for HIV), and only one of these linked to PrEP care. Future health promotion programs are needed to address mental health and other ancillary needs among this population, as well as programs that provide access to PrEP and other HIV prevention services. HIV/AIDS health promotion access to health care community organization edited-statecorrected-proof typesetterts1 ==== Body pmcLinkage to HIV care (LTC) and receipt of antiretroviral therapy (ART), in addition to ongoing retention in care, are necessary for people with HIV (PWH) to achieve viral suppression, the principal indicator of HIV health and ultimate goal of HIV medical treatment. However, PWH who lack stable housing are significantly less likely than stably housed PWH to achieve optimal health HIV outcomes, including lower likelihood of ART adherence, retention in care, and viral suppression (Wainwright et al., 2019). The Southern United States, a region prioritized in the EHE (Ending the HIV Epidemic) initiative, has the highest rates of HIV incidence and HIV-related mortality in the nation. Within the State of Alabama (AL), one of the seven Southern states prioritized in the EHE initiative, Jefferson County consistently has the highest number of new HIV diagnoses in the state as well as a high percentage of the state’s homeless population. On any given day, 3,351 individuals experienced homelessness in AL in 2020, as reported by the Department of Housing and Urban Development (HUD)’s annual count of both sheltered and unsheltered homeless persons (U.S. Interagency Council on Homelessness, 2019), while one-fourth (n = 848; 25%) of these individuals were in the Birmingham area (U.S. Department of Housing and Urban Development, 2021b), the area which also accounted for 24% of all new state HIV infections in 2021 (Alabama Department of Public Health, 2021). Given that people experiencing homelessness are at increased risk for HIV and PWH experiencing homelessness are more likely to experience suboptimal HIV health outcomes than PWH not experiencing homelessness, it is imperative to increase access to testing and HIV care for this vulnerable population. To address the twin epidemics of both HIV and homelessness within the high-priority setting of Jefferson County, AL, this 1-year community-based project, funded by the University of Alabama at Birmingham (UAB)’s Center for AIDS Research (CFAR) and the UAB 1917 Clinic’s 340B funds earned through the Ryan White Part C Grant, Ending the HIV Epidemic: Addressing HIV Health and Homelessness (AH3), had three primary objectives that aligned with the broader goals of the EHE initiative while explicitly focusing on persons experiencing homelessness: (a) increase access to HIV testing for adults experiencing homelessness; (b) facilitate LTC for PWH experiencing homelessness; and (c) facilitate linkage to pre-exposure prophylaxis (PrEP) for those at increased risk for HIV. This article describes the formation of the community-based partnerships and shelter-based HIV services, HIV testing and LTC rates, lessons learned, and implications for health promotion practice. Methods Formation of a Community Partnership AH3 was the result of a partnership between two well-established nonprofit organizations in Jefferson County that serve low-income community members. The first community partner, which provides temporary shelter, housing, case management, and crisis intervention to those who identify as homeless, upholds a Housing First model based on the principles of harm reduction and serves approximately 5,000 people per year. People who are eligible to receive services from the shelter are defined as “literally homeless,” which, according to HUD’s definition, is an “individual or family who lacks a fixed, regular, and adequate nighttime residence” (U.S. Department of Housing and Urban Development, 2021a). Men can stay in the shelter overnight, while anyone (including men, women, and children) can access day program services, including meals, showers, and clothing services. The other community partner, an AIDS service organization (ASO), serves more than 1,000 PWH per year and provides HIV testing, LTC, and multiple ancillary services for PWH (e.g., case management, transportation, food boxes, and counseling). The shelter partner is a vital touchpoint for people experiencing homelessness locally and, by leveraging the ASO partner’s experience in serving PWH, can identify and serve homeless persons not previously diagnosed with HIV or those with previous diagnoses who are not in care. An additional organization involved in this project, a university-affiliated HIV clinic with more than 4,000 active PWH patients, provides in-house LTC support and HIV primary-care services. The project was reviewed by UAB’s Institutional Review Board and was determined to be program evaluation and, therefore, not human subjects research. Procedures Building Rapport Since the shelter had not previously provided HIV-related services to persons experiencing homelessness, the project leveraged the ASO partner’s expertise in community-based HIV testing and outreach by placing a full-time case manager at the shelter with this requisite extensive experience. It was important to build rapport slowly and intentionally with shelter clients (“guests”) so that they would feel more comfortable once the project began. Therefore, the ASO case manager (hereafter referred to as the “AH3 Health Coordinator”) began working at the shelter about 2 months before the program officially started, allowing the guests to get accustomed to her presence and slowly warm-up to the idea of a shelter-based HIV testing initiative. This strategy proved fruitful, as the AH3 Health Coordinator reported anecdotally that, throughout the project period, many of the guests would come sit in her office to simply talk. All adults accessing shelter services, whether this included overnight shelter or daytime services, were eligible to participate in AH3. HIV Testing Procedure All new guests met with the AH3 Health Coordinator in a private office upon entry into the shelter, who administered a COVID-19 test and provided a brief discussion on HIV 101, PrEP, and condom use. If the guest agreed to be tested for HIV, they were given a rapid test and testing form (with signed consent) to complete. HIV test results were provided after 20 min. Linkage to HIV Care If the HIV test result was positive, or if the AH3 Health Coordinator identified a guest who had previously been diagnosed with HIV but had been out of care, the AH3 Health Coordinator immediately initiated the LTC process using Anti-Retroviral Treatment and Access to Services (ARTAS), a Centers for Disease Control and Prevention (CDC)-recognized, evidence-based intervention that draws on strength-based case management to link PWH to care (Gardner et al., 2005). The AH3 Health Coordinator worked with the guest to make an appointment with an HIV primary-care provider at the university-affiliated HIV clinic within 30 days of diagnosis, the CDC-recognized metric for successful LTC (within 30 days of initial contact, for previously diagnosed PWH). The AH3 Health Coordinator continues to provide intensive case management up to the day of the appointment; is available to provide transportation to the appointment, if needed; and checks in periodically with the guest thereafter. Results During the project period, AH3 facilitated HIV testing, LTC, and linkage to ancillary services for persons experiencing homelessness in Jefferson County. Between 26 January 2021 and 22 December 2021, 100% of shelter guests were offered an HIV test, such that n = 713 unique individuals received an HIV test. Only 2.7% (n = 20) declined an HIV test, the most common reason for which was that the guest already knew their status (n = 14; 70% of people who refused HIV tests). A total of nine previously diagnosed, out-of-care PWH and one newly diagnosed PWH were identified through project HIV testing efforts, for a positivity rate of 1.4% among newly and previously diagnosed, out-of-care PWH. An additional four PWH were identified through testing, all of whom were already aware of their diagnosis and care. Based on these data, which illustrate a 2% HIV positivity rate among all persons who received an HIV test through program efforts, AH3 has the potential to identify up to 16 newly or previously diagnosed PWH out of the 848 individuals experiencing homelessness on any given night in the Birmingham area. Of the nine previously diagnosed but out of care and one newly diagnosed PWH identified in AH3, four attended their primary-care appointment at the university-affiliated HIV clinic partner within 30 days, and one PWH attended their primary-care appointment after 42 days of meeting with the AH3 Health Coordinator. The remaining five PWH left the shelter before they could be linked to HIV primary care. Figure 1 illustrates guests’ passage through the HIV care continuum during the project period. Figure 1 AH3 HIV Care Continuum, 26 January 2021 Through 22 December 2021 Note. AH3 = Addressing HIV Health and Homelessness. Finally, 100% of shelter guests who tested negative for HIV and who met criteria for PrEP were counseled on PrEP services and offered a referral to the local health department, but only 10 expressed interest in taking PrEP (just 1.4% of guests who tested negative for HIV), and only one actually linked to PrEP care. Implications for practice Shelter-based HIV testing and LTC has the potential to positively impact regional EHE efforts to diagnose and relink PWH to care, particularly for PWH with limited resources, such as those experiencing homelessness. Almost all guests approached for testing in AH3 agreed to take an HIV test, with under a 3% opt-out rate. While LTC was more challenging and only 50% of PWH identified in the project were linked to HIV primary care, the lessons learned in our first year of this project will inform future efforts. For example, mental health and substance use disorders are common among PWH experiencing homelessness (Marcus et al., 2018), and we found that this was the case for PWH in AH3, as well. A recent systematic review found that the odds of maintaining retention in HIV care are significantly less likely for PWH with mental health challenges, underscoring the need for mental health care and services that take all health needs into account (Rooks-Peck et al., 2018). We also found that knowledge of and interest in PrEP was extremely low among persons experiencing homelessness. Just over 1% of the HIV-negative individuals approached for HIV testing were interested in PrEP, and, among those referred, only one attended their PrEP appointment at a local health department. This finding is supported by the literature, which shows that PrEP knowledge among persons experiencing homelessness is extremely low, especially in the U.S. South. To our knowledge, there are just two published studies on providing PrEP services to persons experiencing homelessness (Biello et al., 2021; Gregg et al., 2020). Thus, future iterations of AH3 will also focus on expanding PrEP care to persons experiencing homelessness. Finally, we learned that the provision of COVID-19 testing alongside HIV testing helped minimize potential stigma around HIV testing. Since AH3 launched during the COVID-19 pandemic, all incoming shelter guests had to obtain a COVID-19 test as standard protocol. This operating procedure proved fruitful in several different ways. First, since all incoming guests had to meet with the AH3 Health Coordinator at least once to get their COVID-19 test, all guests also had the opportunity to be asked about whether they would like to receive an HIV test. Second, the fact that all incoming guests met with the AH3 Health Coordinator whether or not they received HIV testing served to protect privacy and reduce potential stigma around HIV testing. Implications for practice and research Shelter-based HIV testing and LTC programs, such as AH3, have the potential to increase successful passage through the HIV care continuum for people experiencing homelessness. In particular, the high rate of HIV testing acceptability (over 97%) demonstrates the success of this shelter-based HIV testing and LTC model. Providing testing services other than HIV (e.g., COVID-19) is an acceptable and feasible way to destigmatize HIV testing for persons in high-proximity, low-privacy settings such as homeless shelters. Future health promotion programs are needed that help address mental health and other ancillary needs among this population, as well as those that provide access to PrEP and other HIV prevention services, to ensure that PWH are successfully linked to care and that people experiencing homelessness who would benefit from PrEP are able to access these services. In addition, the paucity of extant literature on HIV and homelessness underscores the need for additional research that explores the intersection between homelessness and HIV-related health outcomes. ORCID iD: Emma Sophia Kay https://orcid.org/0000-0002-8425-9196 ==== Refs References Alabama Department of Public Health. (2021). HIV cases among persons residing in Alabama at diagnosis by public health district and county. https://www.alabamapublichealth.gov/hiv/assets/4th_qtr_2021.pdf Biello K. B. Bazzi A. R. Vahey S. Harris M. Shaw L. Brody J. (2021). 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(2019). Socioeconomic, behavioral, and clinical characteristics of persons living with HIV who experience homelessness in the United States, 2015-2016. AIDS and Behavior, 24 , 1701–1708. 10.1007/s10461-019-02704-4	36448342	PMC9713533	NO-CC CODE	2022-12-02 23:23:06	no	Health Promot Pract. 2022 Nov 30;:15248399221135589	utf-8	Health Promot Pract	2,022	10.1177/15248399221135589	oa_other
==== Front Phlebology Phlebology spphl PHL Phlebology 0268-3555 1758-1125 SAGE Publications Sage UK: London, England 36441941 10.1177_02683555221141824 10.1177/02683555221141824 Original Article The risk of harm whilst waiting for varicose veins procedure https://orcid.org/0000-0002-2378-6232 Bootun Roshan 12 Burrows Mandy 2 Chowdhury Mohammed M 23 https://orcid.org/0000-0002-3585-6728 Stather Philip W 24 Al-Jundi Wissam 25 1 Vascular Trainee, East of England Deanery, United Kingdom, and Honorary Research Fellow, Section of Vascular Surgery, Imperial College London , UK 2 Department of Vascular Surgery, 156671 Norfolk and Norwich University Hospital , UK 3 NIHR Clinical Lecturer in Vascular Surgery, Department of Vascular Surgery, 2153 Cambridge University Hospitals , University of Cambridge, UK 4 Clinical Associate Professor, Norwich Medical School, University of East Anglia , UK 5 Honorary Senior Lecturer, Norwich Medical School, University of East Anglia , UK Roshan Bootun, Department of Vascular Surgery, Norfolk and Norwich University Hospital, Norwich, NR4 7UY, UK. Email: [email protected] 28 11 2022 28 11 2022 02683555221141824© The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Introduction Varicose veins (VV) negatively impact quality of life (QoL) and have risks of major complications including bleeding, ulceration and phlebitis. During the COVID-19 pandemic, the VSGBI (Vascular Society of Great Britain and Ireland) and GIRFT (Get It Right First Time) classified VVs as lowest priority for intervention. Objective This study aims to determine harm caused and the impact on the QoL on patients waiting for their VVs procedures for more than 1 year. Methods This was a prospective study conducted at the Norfolk and Norwich University Hospital (NNUH). Patients with VVs awaiting intervention for >1 year were included in the study. Patients with CEAP C6 disease were considered to be too high risk to be invited for treatment during the Covid-19 pandemic. Patients were sent QoL questionnaires and underwent a telephone consultation to assess harm. Both generic (EQ-VAS and EQ-5D) and disease-specific (AVVQ and CIVIQ-14) instruments were utilised. There were no control groups available for comparison. Results 275 patients were identified (37.1% male) with median time on waiting list of 60 weeks (IQR 56–65). 19 patients (6.9%) came to major harm, including phlebitis (3.6%), bleeding (1.8%) and ulceration (1.8%). Fifty-two patients (18.9%) had minor harm, including worsening pain (12.7%) and swelling (6.2%). 6.9% reported psychological harm. Rising CEAP stage was also associated with worsening level of harm in patients with C5-6 disease (p < 0.0001). Only 8.7% stated they would decline surgery during the pandemic. 104 QoL questionnaires were returned. Median EQ-VAS and EQ-5D was 75 (IQR: 60–85) and 0.685 (0.566–0.761), respectively. Median AVVQ score was 23.2 (14.9–31.0) and CIVIQ-14 score was 33 (21–44). ConclusionsThis study highlights the impact of delaying VVs surgery during a pandemic. A significant rate of both major and minor as well as psychological harm was reported. In addition, VVs had a significant detriment to quality of life. varicose vein waiting list COVID-19 pandemic quality of life edited-statecorrected-proof typesetterts10 ==== Body pmc What this paper adds: This study looked at the effect of waiting for more than 1 year on our varicose veins patients and found significant occurrence of clinical as well as psychological harm. Introduction Varicose veins are a common condition affecting approximately a third of the population.1 Involving mostly the lower limb, they cause a number of symptoms ranging from achiness and swelling to more serious complications such as ulceration, bleeding and phlebitis.2,3 They have a negative impact on the quality of life (QoL) of patients, which is, in turn, improved by treatment of the condition.4,5 In the UK, diagnosis and management of this condition are guided by the National Institute for Health and Care Excellence (NICE) guidelines.6 Along with a set of referral criteria, these guidelines also include recommendations regarding treatment, with endothermal ablation methods being the ‘gold standard’ management option. Treatment of this condition and its sequelae consume approximately 2% of the national health expenditure.7 However, because of its low mortality, it is considered by many to be benign and of low priority for treatment and, therefore, often does not attract the same level of urgency compared to other vascular conditions. This chronic situation has unfortunately been severely exacerbated by the emergence of the global coronavirus disease 19 (COVID-19) pandemic. Indeed, the pandemic led to the unprecedented cancellation of elective procedures in the United Kingdom, putting the whole National Health Service (NHS) under a tremendous amount of strain, all in a bid to protect patients from viral transmission and offer support to the wider pandemic response.8 The CovidSurg Collaborative report also projected the cancellation or postponement of more than 28 million elective procedures in 2020.8 At the height of this pandemic, new ideas and pathways were being devised to address these issues, with tier classification of surgical procedures stratified according to their urgency increasingly being advocated.9 The intended objective of these novel systems were to maintain optimal patient outcomes, preserve essential equipment and resources needed to handle the volume of critically ill patients, and uphold crucial public health guidelines for physical distancing.9,10 To that end, in 2020, the Vascular Society of Great Britain and Ireland (VSGBI) and the Get It Right First Time (GIRFT) initiative released guidance for the resumption of vascular services with vein surgery being classified as the lowest priority (Priority 4: Surgery that can usually be delayed for more than 3 months).11,12 This inevitably led to increased waiting times for varicose vein patients with a large number having their procedures delayed for longer than 3 months. As such, this current study aims to investigate the effect of waiting for intervention on patients with varicose veins. Methods This study was conducted according to STROBE guidelines.13 This was a prospective study conducted at the Norfolk and Norwich University Hospital (NNUH). All participants were managed in an outpatient setting between March and June 2021. All patients waiting for longer than 1 year for varicose veins procedures were identified from the waiting list database and were contacted as part of clinical governance to determine if they had come to harm. These patients were included on the waiting list after being deemed to meet the criteria for treatment as per the NICE Guidelines.1 After their clinic review, patients are prescribed Class II compression stockings, if there are no contra-indications, until they have their varicose vein procedures. No venotonic drugs were prescribed. Patients were also invited to participate in this study (via telephone), and complete telephone and postal questionnaires. Verbal consent and completion of questionnaires was deemed adequate informed consent. The study was registered with the local NNUH institutional audit department – to ensure all data collection was in line with local committee ethical standards and after review by local R&D department the study was deemed exempt from ethical approval owing to the minimal risk and nonidentifiable nature of the study. Patients were de-identified and analysed anonymously. Once verbal informed consent has been obtained, they were sent QoL questionnaires and had a telephone consultation. Both generic (EQ-VAS and EQ-5D) and disease-specific (AVVQ and CIVIQ-14) instruments were utilised (See Supplementary Appendix 1). The telephone interview was used to assess for any clinical progression of their symptoms and the level of harm (low/major/psychological) experienced while on the waiting list. Data collection on each patient included age, gender, symptomatology, CEAP classification, delay of intervention (days), proposed planned procedure, changes in symptoms, psychological impact, and level harm. Furthermore, complications specific to bleeding, new ulceration, and phlebitis were also collected. All patients were contacted by the same research nurse (MB) to minimise risk of bias. Study size was dependent on the number of patients on the waiting list. Statistical analysis All data were analysed to determine distribution of data with parametric and non-parametric statistical tests carried out accordingly. The tests included calculation of means (with standard deviation), median (with interquartile range) and Pearson’s Chi-squared for examining proportions. Analysis and diagrammatic representations were undertaken using Microsoft® Excel for Mac (Version 16.53) (Redmond, Washington, USA) and Stata for Mac (College Station, Texas, USA). Results In total, 275 patients were identified and provided informed consent. One patient passed away while on the waiting list. One patient was waiting for foam sclerotherapy, while all the others were on the list for endovenous thermal ablation with radiofrequency (RFA) (first line treatment according to the NICE Guidelines). The mean age was 57.2 (standard deviation, SD: ±16) years and 62.9% of the sample were females. The median time on the waiting list was 60 weeks (interquartile range [IQR]: 56–65 weeks) and the majority of patients (91.7%) were classified as CEAP Clinical class C2-C4 (see Table 1). The C6 patients were not prioritised as they were deemed too high risk to attend during the COVID-19 pandemic.Table 1. Baseline patient characteristics. Males (%) 37.1 Age (years)* (SD) 57.2 (16) Clinical CEAP class (%) C2S 25.0 C3 43.8 C4 22.9 C5 6.3 C6 2.1 Time on waiting list (weeks)§ (IQR) 60 (56–65) IQR: interquartile range; SD: standard deviation; (* mean; § median). All patients had a telephone interview while 104 patients returned their QoL questionnaires (37.8% response rate). A little over a third of patients (36.5%) felt that their symptoms had worsened since they have been added to the waiting list (Figure 1). Fifty-two patients (18.9%) suffered minor harm with worsening symptoms of pain and leg swelling, while 20 patients (7.2%) suffered what was considered to be major harm with phlebitis (3.6%), bleeding (1.8%) and ulceration (1.8%). 19 patients (6.9%) also experienced psychological harm as reported by the patient (Figure 2). Further analysis was undertaken to determine whether initial CEAP stage was associated with an increased risk of adverse events. Table 2 shows that with rising CEAP stage, increasing proportions of patients developed harm (3.8% of those with CEAP stage C2-4 disease compared to 60% for those with C5-6 disease; Pearson’s Chi-square, X2 = 49.0; p < 0.0001).Figure 1. Change in patients’ symptoms since added to the waiting list. Figure 2. Harm experienced by patients since added to the waiting list. Table 2. Development of harm with baseline CEAP classification. Higher proportion of patients develop harm with rising CEAP classification (Pearson’s Chi-square, X2 = 49.0; p < 0.0001). CEAP classification Harm No harm Total C2S-4 5 126 131 C5-6 9 6 15 Total 14 132 146 Generic QoL was also affected with a median EQ-VAS of 75 (IQR: 58–81) and EQ-5D of 0.674 (IQR: 0.566–0.761) (Figure 3). In addition, the median disease-specific QoL scores were AVVQ 24 (IQR: 15–31) and CIVIQ-14 33 (IQR: 23–44) (Figure 4).Figure 3. Generic quality of life of patients on the waiting list. (a) EQ-VAS (b) EQ-5D. On direct questioning, 91.3% of patients stated that they would have been happy to attend hospital to undergo their varicose veins treatment during the Covid-19 pandemic.Figure 4. Disease-specific quality of life for patients on the waiting list. (a) Aberdeen Varicose Veins Questionnaire (AVVQ) (b) Chronic Lower Limb Venous Insufficiency Questionnaire (CIVIQ-14). Discussion This study demonstrates the impact of delaying VVs surgery, particularly in the context of a pandemic. Along with showing worsening of patients’ symptoms, a significant number also developed major clinical and psychological harm. Predictably, worsening levels of harm were also observed with rising baseline CEAP classification, typically with patients with C5-6 disease having worse outcomes with recurrent ulceration. The implication, therefore, is that varicose veins are not a completely benign condition after all with waiting for intervention having a detrimental impact on the morbidity and QoL of patients. This state of affairs has been documented previously with Oudhoff et al. (2007) demonstrating that patients on waiting lists suffered from decreased health as well as an impaired psychological and social life.14 Progression of vein disease is a recognised phenomenon with progression of venous reflux found in nearly a third of patients following a median follow-up of 19 months15 which would fit with the observations in this study. In the long-term follow-up for the Edinburgh Vein Study, progression of venous disease was noted in 57.4% of patients after a mean follow-up period of 13 years (rate of progression: 4.3% per year) and approximately 1 in 3 of those with only varicose veins developed chronic venous insufficiency (CVI).16 In our study, we found that the progression for more advanced disease was more significant with 60% of those in stage C5-6 disease developing harm after a median waiting time of greater than 1 year. When GIRFT and VSGBI released the recommendations regarding resumption of vascular services following the COVID-19 pandemic, the intention was not to pit different vascular conditions against each other, but rather rightly to treat the more urgent and potentially life- and limb-threatening conditions first to reduce morbidity and mortality. As the number of active COVID-19 cases were decreasing and some of the COVID-19 restrictions were being eased, more vascular procedures were being carried out. However, it became increasingly evident that some patients with venous diseases (e.g. those with bleeding/ulcerations) were being overlooked. Hence, in the VSGBI’s fourth Edition of the Provision of Vascular Surgery (2021), patients with venous bleeding or ulcerations have been elevated to the ‘Expedited Pathway’ (time-critical outpatient pathway) for an intervention within days so as to prevent any further deterioration.17 Only time will tell what effect these changes will have on the management of these venous complications, especially as the waiting list of patients on the National Health Service reached 6 million in November 2021.18 The reasons for the long waiting lists are often multifactorial with organisational and staffing levels often to blame. Hence, tackling these would require a combination of increases in resources available (human/infrastructure) as well as more efficient surgical pathways. This study highlights the importance of finding effective solutions for managing patients with VVs whenever an embargo is implemented on elective procedures with low priority. Such solutions can include conducting telephone consultation follow up with patients to emphasise the importance of conservative measures including compression stockings. Such reassessments can also help identify patients who are coming to harm and prioritise them for interventions. This could include patients with more advanced clinical presentation (i.e. C5-6 disease) who have already been identified as being more likely to develop harm. During such circumstances, office-based procedures under local anaesthesia and outside operating rooms may be appropriate, such as treating incompetent truncal veins only and utilisation of foam sclerotherapy for residual veins, thus avoiding the need for stripping or multiple phlebectomies which often require general anaesthesia and the associated resources required to achieve that. Additionally undertaking multiple staged procedures such as a single leg at a time, can maintain a high proportion of procedures under local anaesthesia. Future research is required to examine these recommendations. Despite the situation with the COVID-19 pandemic, it was rather surprising to find that more than 90% of the patients interviewed were willing to attend for their surgery should they have been on offer during that time. It could be deemed a missed opportunity to address the waiting list for this group of patients, but with an ever-evolving picture during the pandemic with unpredictable infection rates and staff/resource availability, it could be argued that this would have achieved very little, with cancellations/rescheduled procedures becoming the norm rather than the exception. Limitations One of the main weaknesses of this study is the absence of baseline QoL scores from the time the patients were reviewed in clinic and added onto the waiting list. This is especially important as it would help determine the degree and rate of deterioration while waiting for the varicose vein procedures. Unfortunately, this is not common practice and, therefore, patients were not provided with these questionnaires. Moreover, the nature of the assessment tools used with self-completed QoL questionnaires and telephone interviews on their own poses a different type of problem. Indeed, the study would have benefitted from the addition of a clinical score (e.g. the Venous Clinical Severity Score, VCSS), which would have offered a more objective element to the evaluation done and would likely have strengthened the findings of the study. A degree of recall bias may also be present with patients having an altered recollection of the intensity and severity of their symptoms. Another limitation of the study is the modest response rate of around 38%. This could have introduced a response bias whereby patients suffering harm would be more agreeable to take part of the study. In addition, the absence of a control group prevents the comparison of our cohort to other patients waiting a long time for their procedures. It is also likely that some of the psychological harm effects noted are secondary to the Covid-19 pandemic and its societal implications rather than purely the effect of being on the waiting list for varicose veins treatment.19 Conclusion This study demonstrates the negative effect of waiting on patients with varicose veins and the need to identify patients most at risk of coming to harm so that any potential detriment is addressed urgently. This study has led the department to update the hospital advice leaflet with key signs of deterioration (bleeding, ulceration and phlebitis) and contact details should these occur in order to expedite treatment where required. Supplemental Material Supplemental Material - The risk of harm whilst waiting for varicose veins proceduresj-pdf-1-phl-10.1177_02683555221141824 Click here for additional data file. Supplemental Material for The risk of harm whilst waiting for varicose veins procedure by Roshan Bootun, Mandy Burrows, Mohammed M Chowdhury, Philip W Stather, and Wissam Al-Jundi in Phlebology Acknowledgements The authors would like to thank the clinical and non-clinical staff in the Department of Vascular Surgery for their support and advice with this study. ORCID iDs Roshan Bootun https://orcid.org/0000-0002-2378-6232 Philip W Stather https://orcid.org/0000-0002-3585-6728 Author Contributions: - Conception and design of study: RB, MB, MMC, PWS, WAJ - Data collection: MB - Data analysis and interpretation: RB, MB, PWS - Drafting and revision of manuscript: RB, MMC, PWS, WAJ - Reading and final approval of manuscript: RB, MB, MMC, PWS, WAJ. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. Guarantor: Mr Wissam Al-Jundi. Supplemental Material: Supplemental material for this article is available online. ==== Refs References 1 Evans CJ Fowkes FG Ruckley CV , et al. Prevalence of varicose veins and chronic venous insufficiency in men and women in the general population: Edinburgh Vein Study. J Epidemiology Community Health 1999; 53 : 149–153. 2 Bradbury A Evans C Allan P , et al. What are the symptoms of varicose veins? Edinburgh vein study cross sectional population survey. Br Medical Journal 1999; 318 : 353–356. 3 Raetz J Wilson M Collins K . Varicose Veins: Diagnosis and Treatment. Am Fam Physician 2019; 99 : 682–688.31150188 4 Darvall KAL Bate GR Adam DJ , et al. Generic Health-related Quality of Life is Significantly Worse in Varicose Vein Patients with Lower Limb Symptoms Independent of Ceap Clinical Grade. Eur J Vasc Endovascular Surg 2012; 44 : 341–344. DOI: 10.1016/J.Ejvs.2012.06.022. 5 MacKenzie RK Paisley A Allan PL , et al. The effect of long saphenous vein stripping on quality of life. J Vascular Surgery 2002; 35 : 1197–1203. 6 NICE Clinical Guideline Centre. Varicose veins in the legs - the diagnosis and management of varicose veins (Clinical guideline 168). 7 Davies AH . The Seriousness of Chronic Venous Disease: A Review of Real-World Evidence. Adv Ther 2019; 36 : 5–12. DOI: 10.1007/s12325-019-0881-7 30758738 8 COVIDSurg Collaborative . Elective surgery cancellations due to the COVID-19 pandemic: global predictive modelling to inform surgical recovery plans. Br Journal Surgery 2020; 107 : 1440–1449. DOI: 10.1002/bjs.11746. 9 Stahel PF . How to risk-stratify elective surgery during the COVID-19 pandemic? Patient Saf Surg 2020; 14 . DOI: 10.1186/s13037-020-00235-9 10 Ross SW Lauer CW Miles WS , et al. Maximizing the Calm before the Storm: Tiered Surgical Response Plan for Novel Coronavirus (COVID-19). J Am Coll Surg 2020; 230 : 1080–1091. DOI: 10.1016/j.jamcollsurg.2020.03.019.e1083 32240770 11 Vascular Society of Great Britain & Ireland. Resumption of less urgent and elective (endo-) vascular surgery, 2020. 12 COVID-19 response: Guidance on the resumption of less urgent/elective vascular and endovascular surgery. 13 Cuschieri S . The STROBE guidelines. Saudi J Anaesth 2019; 13 : S31–S34. DOI: 10.4103/sja.SJA_543_18 30930717 14 Oudhoff JP Timmermans DR Knol DL , et al. Waiting for elective general surgery: impact on health related quality of life and psychosocial consequences. BMC Public Health 2007; 7 . DOI: 10.1186/1471-2458-7-164. 15 Labropoulos N Leon L Kwon S , et al. Study of the venous reflux progression. J Vascular Surgery 2005; 41 : 291–295. DOI: 10.1016/j.jvs.2004.11.014. 16 Lee AJ Robertson LA Boghossian SM , et al. Progression of varicose veins and chronic venous insufficiency in the general population in the Edinburgh Vein Study. J Vasc Surg Venous Lymphat Disord 2015; 3 : 18–26. DOI: 10.1016/j.jvsv.2014.09.008 26993676 17 Vascular Society of Great Britain & Ireland. Provision of Services for People with Vascular Disease 2021, 2021. 18 NHS England . Statistical Press Notice: NHS referral to treatment (RTT) waiting times data - November 2021, 2022. 19 Vindegaard N Benros ME . COVID-19 pandemic and mental health consequences: Systematic review of the current evidence. Brain Behav Immun 2020; 89 : 531–542. DOI: 10.1016/j.bbi.2020.05.048 32485289	36441941	PMC9713534	NO-CC CODE	2022-12-02 23:23:06	no	Phlebology. 2022 Nov 28;:02683555221141824	utf-8	Phlebology	2,022	10.1177/02683555221141824	oa_other
==== Front J Intensive Care Med J Intensive Care Med JIC spjic Journal of Intensive Care Medicine 0885-0666 1525-1489 SAGE Publications Sage CA: Los Angeles, CA 36448250 10.1177/08850666221142265 10.1177_08850666221142265 Original Research Thromboelastography Parameters do not Discriminate for Thrombotic Events in Hospitalized Patients With COVID-19 https://orcid.org/0000-0002-4688-1523 Kartiko Susan MD, PhD 1 Koizumi Naoru PhD 23 Yamane David MD 4 Sarani Babak MD 1 Siddique Abu B. PhD 3 https://orcid.org/0000-0003-4103-1025 Levine Andrea R. MD 5 Jackson Amanda M. MD 6 https://orcid.org/0000-0002-5871-5186 Wieruszewski Patrick M. PharmD 7 https://orcid.org/0000-0003-1051-098X Smischney Nathan J. MD, MSc 8 Khanna Ashish K. MD, FCCP, FCCM, FASA 910 https://orcid.org/0000-0003-1750-3416 Chow Jonathan H. MD 11 for the CRUSH COVID Investigatorsa 1 Department of Surgery, 43989 George Washington University School of Medicine and Health Sciences , Washington, DC, USA 2 43989 George Washington University School of Medicine and Health Sciences , Washington, DC, USA 3 George Mason University, Schar School of Policy and Government, Fairfax, VA, USA 4 Department of Emergency Medicine, Anesthesiology and Critical Care Medicine, 43989 George Washington University School of Medicine and Health Sciences , Washington, DC, USA 5 Division of Pulmonary and Critical Care, Department of Medicine, 12264 University of Maryland School of Medicine , Baltimore, MD, USA 6 Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Madigan Army Medical Center, Joint Base Lewis–McChord, WA, USA 7 Departments of Anesthesiology and Pharmacy, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, USA 8 Department of Anesthesiology and Perioperative Medicine, Division of Critical Care Medicine, Mayo Clinic, Rochester, MN, USA 9 Department of Anesthesiology, Section on Critical Care Medicine, Wake Forest School of Medicine, Atrium Health Wake Forest Baptist Medical Center, Winston-Salem, NC, USA 10 Outcomes Research Consortium, Cleveland, OH, USA 11 Department of Anesthesiology and Critical Care Medicine, 43989 George Washington University School of Medicine and Health Sciences , Washington, DC, USA aA full list of the CRUSH COVID Investigators is provided in the Supplemental material. Susan Kartiko, Department of Surgery, George Washington University School of Medicine and Health Sciences, 2150 Pennsylvania Ave, NW, Washington, DC 20038, USA. Email: [email protected] 29 11 2022 29 11 2022 08850666221142265© The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Background Coronavirus disease 2019 (COVID-19) is associated with a prothrombotic state; leading to multiple sequelae. We sought to detect whether thromboelastography (TEG) parameters would be able to detect thromboembolic events in patients hospitalized with COVID-19. Methods We performed a retrospective multicenter case–control study of the Collaborative Research to Understand the Sequelae of Harm in COVID (CRUSH COVID) registry of 8 tertiary care level hospitals in the United States (US). This registry contains adult patients with COVID-19 hospitalized between March 2020 and September 2020. Results A total of 277 hospitalized COVID-19 patients were analyzed to determine whether conventional coagulation TEG parameters were associated with venous thromboembolic (VTE) and thrombotic events during hospitalization. A clotting index (CI) >3 was present in 45.8% of the population, consistent with a hypercoagulable state. Eighty-three percent of the patients had clot lysis at 30 min (LY30) = 0, consistent with fibrinolysis shutdown, with a median of 0.1%. We did not find TEG parameters (LY30 area under the receiver operating characteristic [ROC] curve [AUC] = 0.55, 95% CI: 0.44-0.65, P value = .32; alpha angle [α] AUC = 0.58, 95% CI: 0.47-0.69, P value = .17; K time AUC = 0.58, 95% CI: 0.46-0.69, P value = .67; maximum amplitude (MA) AUC = 0.54, 95% CI: 0.44-0.64, P value = .47; reaction time [R time] AUC = 0.53, 95% CI: 0.42-0.65, P value = .70) to be a good discriminator for VTE. We also did not find TEG parameters (LY30 AUC = 0.51, 95% CI: 0.42-0.60, P value = .84; R time AUC = 0.57, 95%CI: 0.48-0.67, P value .07; α AUC = 0.59, 95%CI: 0.51-0.68, P value = .02; K time AUC = 0.62, 95% CI: 0.53-0.70, P value = .07; MA AUC = 0.65, 95% CI: 0.57-0.74, P value < .01) to be a good discriminator for thrombotic events. Conclusions In this retrospective multicenter cohort study, TEG in COVID-19 hospitalized patients may indicate a hypercoagulable state, however, its use in detecting VTE or thrombotic events is limited in this population. COVID-19 TEG hypercoagulable state VTE fibrinolysis shutdown edited-statecorrected-proof typesetterts19 ==== Body pmcIntroduction Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). As of October 2022, COVID-19 has infected more than 626 million people and led to more than 6.5 million deaths.1 While the majority of COVID-19 cases are mild, approximately 5% to 32% of infected patients were admitted to the intensive care unit (ICU) in the United States (US).2–4 The hallmark symptom of COVID-19 is acute respiratory distress, ranging from pneumonia to acute respiratory distress syndrome (ARDS). Studies have shown that COVID-19 is also associated with a prothrombotic state; leading to multiple sequelae. In the European and Chinese studies, 20% to 30% of COVID-19 patients were found to have thrombotic complications, consisting mostly of venous thromboembolic (VTE) disease,5,6 while a US study showed that the overall thrombosis rate was found to be 9.5%.7 Elevated D-dimer, a marker of increased fibrin formation and degradation, is associated with thrombosis and higher mortality in COVID-19 patients.8–10 Thromboelastography (TEG, Haemonetics) is a whole-blood coagulation assay that measures the dynamics of clot formation, clot strength, and clot dissolution. It is commonly used to guide transfusion of hemostatic products in hemorrhaging patients. It is also used to identify hypercoagulable patients at risk for thrombosis.11–13 A study of 44 COVID-19-positive patients showed that a TEG lysis at 30 min (LY30) of 0% and D-dimer value greater than 2600 ng/mL were associated with a markedly elevated risk of renal failure, VTE and thrombotic events.14 However, another study of 64 COVID-19-positive patients was not able to show a correlation between TEG parameters and VTE.15 The objective of our study was to evaluate the association between TEG parameters and other common coagulation parameters and thromboembolic events in hospitalized COVID-19 patients. We sought to detect whether TEG parameter would be able to detect thromboembolic events in patients hospitalized with COVID-19 Patients and Methods Study Design This was a retrospective case–control study of 277 patients hospitalized with COVID-19. These patients were abstracted from the multicenter Collaborative Research to Understand the Sequelae of Harm in COVID (CRUSH COVID) registry of patients admitted between March 2020 and September 2020. Eight tertiary centers, The George Washington University Hospital, University of Arkansas, Mayo Clinic, University of Southern California, University of Maryland Medical Center, Wake Forest Baptist Medical Center, University of Mississippi Medical Center, and Northeast Georgia Health System, contributed data to the registry. This was managed with the Research Electronic Data Capture (REDCap) application.16 The study was reviewed and approved by the Institutional Review Board at the University of Maryland Medical Center, Baltimore (IRB HP-00084946), which served as the central IRB for all sites. The requirement for written informed consent was waived by the IRB. Study Population All adult patients admitted to the 8 participating hospitals from March 2020 to September 2020 diagnosed with COVID-19 were eligible for the study. Patients were included if they had laboratory confirmed COVID-19 as measured from a polymerase chain reaction test. Patients were excluded if they did not have a TEG measurement performed during their hospitalization. Data Collection Demographic variables collected included age, race, sex, past medical history, body mass index (BMI), and a history of smoking or vaping (both active and former). Comorbidity variables abstracted included hypertension, diabetes mellitus (DM), end-stage renal and liver diseases (ESRD and ESLD), cancer, HIV, cardiovascular disease (CVD), cerebrovascular accident (CVA), and coronary artery disease (CAD). VTE surveillance, detection, and diagnosis were done at the discretion of the clinicians and practice may slightly differ across the different institutions. Kaolin TEG values and other laboratory values were obtained at clinicians’ discretion and TEG measurements were done based on each institutions’ policy. Heparinase was used to measure TEG values in patients on heparin based on each institution's policy. In the event of multiple TEG or laboratory samples, analysis was done based on the initial values. Since TEG measurements were obtained at the discretion of the clinician, the time to TEG measurement from initial hospital admission is not uniform. Case Definitions The study's primary outcome was the area under the receiver operating characteristic (ROC) curve for VTE. The secondary outcome was the ROC for the composite endpoint of VTE, ischemic stroke, and myocardial infarction, herein defined as “thrombotic events”. TEG indices utilized in the analysis included reaction time (R time), alpha angle (α), K time, maximum amplitude (MA), and LY30. In addition, the clotting index (CI), which is computed as CI = –0.2454R + 0.0184 K + 0.1655MA–0.0241α–5.0220, was assessed in order to provide an overall assessment of coagulation.15 Additional coagulation factors evaluated were D-dimer and fibrinogen levels measured at the time of hospital admission. Statistical Analysis Data analysis was performed using STATA (Version 14, StataCorp). This was a nested case–control study of COVID-19-positive patients whereby the patient was stratified based on whether they experienced the primary or secondary outcome. Demographic and laboratory values were compared using Student's t-test or the Wilcoxon–Mann–Whitney test for continuous variables, depending on the normality of the distribution. A chi-square test or Fisher's exact test was utilized for nominal variables depending on the sample size. The ROC curves were generated for each outcome of interest for all TEG parameters, the initial D-dimer and fibrinogen level. Delong's test was performed to examine whether the area under the ROC curve (AUC) was statistically different from 0.5. Using Youden's J statistic, the optimal threshold for each value in detecting each outcome was identified, and the sensitivity and specificity were calculated. Statistical significance was defined by p < .05 for all analyses. Results Study Population In total, 277 hospitalized COVID-19 patients met study inclusion criteria. Sixty percent (n = 165) of patients were male and 80% (n = 223) of patients had a BMI ≥ 25 kg/m2. Outside of being overweight, the most common preexisting conditions were hypertension (n = 165; 59.6%) and DM (n = 125, 45.1%). One hundred and seventy-nine (65%) patients were admitted to the ICU, and their hospital length of stay was considerably longer with the median days of 24 days (Q1 = 11, Q3 = 41). The median length of stay among those who were not admitted to ICU was 5 days (3, 8). In-hospital mortality occurred in 25.6% (n = 71) of patients. The median qSOFA (quick Sepsis Related Organ Failure Assessment) score for these patients was 1 (0, 2). Twenty-eight patients (10.1%) developed VTE and 43 patients (15.7%) developed renal failure requiring hemodialysis. The median day from admission to VTE diagnosis was 15 days (8, 29). The composite secondary outcome of thrombotic events was detected in 22.4% (n = 62) of patients. Fibrinolysis shutdown (Ly30 = 0) was observed in 83.8% (n = 232) of the population. A hypercoagulable state (CI > 3.0) was observed in 54.2% of the sample (n = 150) (Table 1). The median time to TEG measurement was 4 days (inter quartile range [IQR]: 0-20). Thirty-nine percent (n = 108) of patients received therapeutic level heparin dosing and 8.3% (n = 23) received therapeutic level enoxaparin dosing. Nine percent (n = 25) received no prophylactic or therapeutic anticoagulation and the rest received prophylactic dosing. Table 1. Characteristics of Patients. Variable Data (n = 277) Age, years, median (IQR) 58 (45–69) Sex, n (%) Male 165 (59.6%) Female 112 (40.4%) Ethnicity, n (%) White 121 (44.2%) Black/African American 64 (23.4%) Hispanic 85 (31%) Other 4 (1.5%) BMI, n (%) Underweight (BMI < 18.5) 7 (2.5%) Normal (18.5 ≤ BMI < 25) 47 (17%) Overweight (25 ≤ BMI < 30) 60 (21.7%) Obese (BMI ≥ 30) 163 (58.8%) Comorbidity, n (%) Coronary artery disease (CAD) 47 (17%) Hypertension 165 (59.6%) Diabetes mellitus (DM) 125 (45.1%) Congestive heart failure (CHF) 56 (20.2%) HIV 3 (1.1%) Active cancer 22 (7.9%) Cardiovascular disease (CVD) 14 (5.1%) Renal disease 60 (21.7%) Liver disease 24 (8.7%) History of smoking/vaping 25 (9.0%) Acute respiratory distress syndrome (ARDS) 73 (26.4%) Pneumonia 215 (77.6%) Clotting index (CI) >3 150 (54.2%) CI ≤3 (normal) 127 (45.8%) Outcome, n (%) VTE 28 (10.1%) HD 43 (15.7%) Fibrinolysis shutdown 232 (83.7%) Thrombotic event 62 (22.4%) Death 71 (25.6%) Abbreviations: BMI, body mass index; HD, hemodialysis; HIV, human immunodeficiency virus; IQR, interquartile range; VTE, venous thromboembolic. Conventional Coagulation Tests and Ability to Differentiate VTE and Thrombotic Events The median values of the conventional coagulation parameters were outside the reference range for most conventional coagulation tests except for platelet count (Tables 2 and 3). The median D-dimer concentration in our population was 1140 ng/mL, (605-3670) which is much higher than the reference range (≤250 ng/mL). The median fibrinogen was 590 mg/dL (443-730). When comparing patients with VTE to those without, the median D-dimer concentration was 3790 ng/mL (1032.5-4660) compared to 1090 ng/mL (576-2470) (P = .9). When comparing fibrinogen concentration between patients with VTE to those without, the median fibrinogen concentration was 686 mg/dL (446-800) compared to 580 mg/dL (441.5-728) (P = .09) (Table 2). Table 2. Coagulation Markers and Venous Thromboembolic (VTE) Events. Coagulation parameters, median (IQR) All patients No VTE VTE P value Platelet count, 109/L 210 (162–277) 210 (163–275) 197 (143–295) .08 Prothrombin time, s 14.7 (13.3–15.65) 15.1 (13.5–15.8) 13.8 (12.1–14.7) .58 Activated partial thromboplastin time (aPTT), s 29.95 (27–33.5) 29.9 (27–33.5) 30.9 (28–33.5) .69 Fibrinogen, mg/dL 590 (443–730) 580 (441.45–728) 686 (446–800) .09 D-dimer, ng/mL 1140 (605–3670) 1090 (576–2470) 3790 (1032.5–4660) .90 International normalized ratio (INR) 1.18 (1.1–1.3) 1.18 (1.1–1.3) 1.12 (1.1–1.3) .97 C-reactive protein (CRP) level 11 (4.66–22.75) 10.25 (4.19–21) 20.35 (10.1–34.2) .34 Viscoelastic index Reaction time (R time) (min) 5.4 (4.4–6.9) 5.4 (4.4–6.9) 5.3 (4.25–6.75) .72 K time (min) 1.4 (1.1–1.8) 1.4 (1.1–1.8) 1.2 (1.05–1.75) .60 α, ° 69.8 (65–74.2) 69.6 (63.9–73.9) 72.05 (66.1–75.25) .35 Maximum amplitude, mm 68.5 (61.3–74) 68.8 (61.4–74.3) 67.8 (60.15–72.05) .45 Clot lysis at 30 min (LY30), % 0.1 (0–1.1) 0.1 (0–1) 0.25 (0–1.15) .29 Clotting index (CI) 3.26 (2.03–4.21) 3.26 (2.02–4.23) 3.10 (2.10–4.13) .04 Abbreviations: IQR, interquartile range; α, alpha angle. Table 3. Coagulation Markers and Thrombotic Events. Coagulation parameters, median (IQR) Total (n = 277) No thrombotic (n = 215) Thrombotic (n = 62) P value Platelet count, 109/L 210 (162–277) 226 (181–283) 148 (100–207) .29 Prothrombin time, s 14.7 (13.3–15.65) 14.4 (13.2–15.2) 15.3 (13.9–16.17) .34 Activated partial thromboplastin time (aPTT), s 29.95 (27–33.5) 29.2 (27–32.1) 33 (28.1–38.35) .27 Fibrinogen, mg/dL 590 (443–730) 603 (470–744) 480 (372–699) .26 D-dimer, ng/mL 1140 (605–3670) 1043 (554–2330) 1770 (820–4000) .66 International normalized ratio (INR) 1.18 (1.1–1.3) 1.18 (1.08–1.28) 1.2 (1.1–1.4) .61 C-reactive protein (CRP) level 11 (4.66–22.75) 11.25 (4.51–21.55) 9.61 (5.21–24.6) .30 Viscoelastic index Reaction time (R time) (min) 5.4 (4.4–6.9) 5.3 (4.3–6.6) 5.8 (4.6–8.1) .70 K time (min) 1.4 (1.1–1.8) 1.3 (1.1–1.8) 1.65 (1.2–2.5) .01 α, ° 69.8 (65–74.2) 70.5 (65.5–74.5) 68 (60.7–73.1) .61 Maximum amplitude, mm 68.5 (61.3–74) 69.7 (63.5–75.1) 62.7 (58–71.2) .36 Clot lysis at 30 min (LY30), % 0.1 (0–1.1) 0.1 (0–1.1) 0.1 (0–1.1) .52 Clotting index (CI) 3.26 (2.03–4.21) 3.50 (2.26–4.31) 2.32 (1.28–3.66) .50 Abbreviations: IQR, interquartile range; α, alpha angle. When comparing patients with thrombotic events to those without, the median D-dimer concentration was 1770 ng/mL (820-4000) compared to 1043 ng/mL (554-2330) (P = .66). When comparing fibrinogen concentration between patients with thrombotic events to those without, the median fibrinogen concentration was 480 mg/dL (372-699) compared to 603 mg/dL (470-744) (P = .26) (Table 3). When examining the ability of D-dimer or fibrinogen to discriminate VTE, our AUC analysis found that D-dimer had only a fair ability to discriminate (D-dimer AUC = 0.66, 95% CI: 0.54-0.78, P value = .004). Fibrinogen was not able to discriminate VTE well (fibrinogen AUC = 0.57, 95% CI: 0.45-0.69, P value = .21) (Figure 1a and b). Calculation of the optimal point for the detection of VTE for D-dimer was 1642 ng/mL, which was associated with a sensitivity of 53% and specificity of 69% (Figure 1c). Figure 1. Receiver operating characteristic (ROC) curve and analysis for venous thromboembolic (VTE) events. (a) ROC curve for VTE. (b) VTE—ROC analysis summary. (c) VTE—threshold values are obtained using Youden's J statistic. D-dimer and fibrinogen were able to detect thrombotic events with fair discrimination (D-dimer, AUC = 0.57, 95%CI: 0.48-0.67, P value = .05; fibrinogen, AUC = 0.61, 95% CI: 0.52-0.70, P value = .01) (Figure 2a and b). Calculation of the optimal point for the detection of thrombotic event for D-dimer was 3090 ng/mL and for fibrinogen level was 487 mg/dL resulting in sensitivities and specificities of 61% and 77% for D-dimer and 53% and 68% for fibrinogen, respectively (Figure 2c). Figure 2. Receiver operating characteristic (ROC) curve and analysis for thrombotic event. (a) ROC curve for thrombotic event. (b) Thrombotic event—ROC analysis summary. (c) Thrombotic event—threshold values are obtained using Youden's J statistic. TEG Measurements and VTE All median TEG coagulation values were within the reference range except for LY30. The median LY30 value in our population was 0.1% (0-1.1), which is below the lower range of normal of 0.8%. The median CI in our population was 3.26 (2.03-4.21), indicating a hypercoagulable state. The median α was 69.8° (65.0-74.2); the median K time was 1.4 min (1.1-1.8); the median MA was 68.5 mm (61.3-74.2); and the median R time was 5.4 min (4.4-6.9). Among patients with a VTE, the median LY30 was 0.25% (0-1.15) compared to patients without a VTE whose median LY30 was 0.1% (0-1) (Table 2). This difference was not statistically significant (P = .29). Similarly, when comparing the α (P = .35), MA (P = .45), and R time (P = .72) among patients with and without a VTE, these differences were not statistically significant. When examining the ability of any TEG parameter to detect VTE, the AUC analysis found that none of the TEG parameters were able to accurately discriminate for VTE (LY30 AUC = 0.55, 95% CI: 0.44-0.65, P value = .32; α AUC = 0.58, 95% CI: 0.47-0.69, P value = .17; K time AUC = 0.58, 95% CI: 0.46-0.69, P value = .67; MA AUC = 0.54, 95% CI: 0.44-0.64, P value = .47; R time AUC = 0.53, 95% CI: 0.42-0.65, P value = .70) (Figure 1a and b). TEG Measurements and Thrombotic Events When comparing TEG values in patients with and without thrombotic events, there were no differences in TEG values. The median LY30 of patients without thrombotic events was 0.1% and the median LY30 for patients with thrombotic events was 0.1% (0-1.1) (P = .52) (Table 3). Similarly, when comparing the α (P = .61), MA (P = .36), and R time (P = .70) among patients with and without thrombotic events, these differences were not statistically significant. However, when comparing patients with thrombotic events to those without, the median K time was 1.65 min (1.2-2.5) compared to 1.3 min (1.2-2.5) (P = .01). When examining the ability of any TEG parameter to discriminate thrombotic events, the AUC analysis found that LY30 and R time were not able to accurately discriminate for thrombotic events (LY30 AUC = 0.51, 95% CI: 0.42-0.60, P value = .84); R time AUC = 0.57, 95%CI: 0.48–0.67, P value .07). The α, K time, and MA had fair discrimination for thrombotic events (α AUC = 0.59, 95%CI: 0.51-0.68, P value = .02; K time AUC = 0.62, 95% CI: 0.53-0.70, P value = .07; MA AUC = 0.65, 95% CI: 0.57-0.74, P value < .01) (Figure 2a and b). Calculation of the optimal point for the detection of thrombotic event for MA was 66.1 mm resulting in sensitivities and specificities of 46% and 61%. The optimal point for the detection of thrombotic event for α level was 69.50°, with sensitivities and specificities of 68% and 49%. The optimal point for the detection of thrombotic event for K time was 1.4 min, with sensitivities and specificities of 65% and 53% (Figure 2c). Discussion Patients with COVID-19 have been reported to have a 20% to 30% incidence of thrombotic and thromboembolic events.5,6 Autopsies performed on COVID-19 decedents have revealed higher VTE (58%) and microscopic thrombi in observed lungs (53%) than those detected in live COVID-19 patients.17,18 COVID-19 associated hypercoagulability is associated with multiple changes in traditional coagulation tests and viscoelastic coagulation parameters. The most significant change in traditional coagulation parameters is reported to be D-dimer, which is commonly elevated in cases of severe disease. Given the number of patients with COVID-19 with a D dimer over 2600 ng/mL without any identifiable DVT/PE, it is not surprising that D dimer is neither a sensitive nor specific marker for VTE. However, we may not be able to see microthrombi, which are part of the ARDS pathophysiology seen after autopsy but cannot be detected in live patients.17,18 In this multicenter study, we report coagulation and TEG profiles of hospitalized COVID-19 patients and analyze the utility of TEG in discriminating VTE and thrombotic events. We did not find that either traditional coagulation tests or TEG parameters were able to discriminate VTE events or other thrombotic events with a high degree of accuracy. To our knowledge, this is the largest study to date examining TEG in hospitalized COVID-19 patients. Previous studies suggested that TEG may be used to identify patients at risk for VTE or other thrombotic events.14–16,19–21 A study by Wright et al suggested that D-dimer and LY30 could be used to predict of VTE. Their study reported that patients with LY30 = 0 and D-dimer > 2600 ng/mL had a VTE rate of 50% compared with 0% for patients with neither risk factor.14 Our results are not consistent with their finding. We found that TEG, as a whole, was not able to discriminate patients who had VTE to those who did not. We also found that only α and MA were able to discriminate thrombotic event with fair accuracy, which resulted in fairly low sensitivities and specificities. D-dimer also had a fair ability to discriminate for VTE with low sensitivity and specificity. As for thrombotic events, D-dimer and fibrinogen both had a fair ability to discriminate thrombotic events with low sensitivities and specificities. Overall, our findings do not support the use of TEG parameters, D-dimer or fibrinogen to discriminate for patients who are at increased risk for VTE or thrombotic events. Both TEG and traditional coagulation tests have some limitations that may account for these findings. For example, TEG and traditional coagulation assays do not measure primary hemostasis and cannot readily demonstrate enhanced platelet adhesion that occurs in COVID-19 from elevated von Willebrand factor.22 TEG parameters also may not reflect changes in native endothelium that creates a procoagulant state in COVID-19. Our population had similar coagulation parameters when compared to other cohorts.14,15,19–22 We found that, despite prophylactic anticoagulation, our VTE rate was 10.1%, which is similar to what is previously reported in the literature.8,14–18 Based on TEG measurements, we found that 83.8% of our patients was in fibrinolysis shutdown with a median LY30 of 0.1%, which is consistent with multiple previous studies that investigated the TEG parameters in hospitalized COVID-19 patients.14,15,20,21 Fibrinolysis shutdown may occur in COVID-19 patients because patients have increased release of plasminogen activator inhibitor-1.23 In our study, median R time, the speed of fibrin polymerization (α), and the MA were all within the normal range, which is generally consistent with prior studies.14,15,19–21 Our study also demonstrated that 22.4% of hospitalized COVID-19 patients met the composite endpoint of having a thrombotic event. Fifty-four percent of the population had a hypercoagulable profile as defined by CI greater than 3. Despite these findings suggestive of a hypercoagulable state; TEG parameters, D-dimer and fibrinogen level were not found to effectively discriminate VTE or thrombotic events in COVID-19 patients. Although this study benefits from its multicenter design and sample size, it has several limitations. First, this is a retrospective observational study with limitations that are inherent in observational studies including residual confounding. Some of our patients may have active cancer, may have been pregnant or have other anticoagulation abnormalities that may affect the TEG values, D-dimer and fibrinogen level used in this analysis. Next, as a retrospective study, there was no uniformity in the timing of TEG measurement within the patients’ disease course. VTE surveillance and detection were done at the discretion of the clinicians and the diagnosis of VTE and thrombotic event may not follow exactly the same criteria across the different institution. Additionally, there is no uniformity among the patients in receiving therapeutic anticoagulation, which would alter the results of coagulation measurements. Conclusion In this multicenter retrospective case–control study, TEG parameters in hospitalized patients with COVID-19 were suggestive of a hypercoagulable state, however, were not accurate discriminators of VTE or thrombotic events in this population. The role of TEG in screening for VTE and other thrombotic events in COVID-19 patients remains unclear and randomized controlled trials will ultimately be needed to confirm the utility of viscoelastic testing. However, we do not recommend the use of TEG to predict VTE and thrombotic events in COVID-19 patients. Supplemental Material sj-docx-1-jic-10.1177_08850666221142265 - Supplemental material for Thromboelastography Parameters do not Discriminate for Thrombotic Events in Hospitalized Patients With COVID-19 Click here for additional data file. Supplemental material, sj-docx-1-jic-10.1177_08850666221142265 for Thromboelastography Parameters do not Discriminate for Thrombotic Events in Hospitalized Patients With COVID-19 by Susan Kartiko, Naoru Koizumi, David Yamane, Babak Sarani, Abu B. Siddique and Andrea R. Levine, Amanda M. Jackson, Patrick M. Wieruszewski, Nathan J. Smischney, Ashish K. Khanna, Jonathan H. Chow, in Journal of Intensive Care Medicine The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Ashish K. Khanna, MD FCCP FCCM FASA is a consultant for Potrero Medical, Edwards Lifesciences, Philips North America, GE Healthcare, Hillr-Rom, Trevena Pharmaceuticals, and Caretaker Medical. AKK is on the executive advisory board for Medtronic and Retia Medical. AKK receives support from the Wake Forest CTSI via NIH/NCATS KL2 for a trial of continuous portable hemodynamic and saturation monitoring on hospital wards and the Wake Forest Hypertension and Cardiovascular Sciences Research Center. The Department of Anesthesiology at the Wake Forest School of Medicine has received funding support from Edwards Lifesciences, Trevena Pharmaceuticals, Retia Medical, Caretaker Medical, and Potrero Medical. Michael A. Mazzeffi, MD MPH is a consultant for Hemosonics Corporation and Octapharma. Jonathan Chow, MD is a consultant of La Jolla Pharmaceutical Company, outside the scope of the submitted work. Funding: The authors received no financial support for the research, authorship, and/or publication of this article. ORCID iDs: Susan Kartiko https://orcid.org/0000-0002-4688-1523 Andrea R. Levine https://orcid.org/0000-0003-4103-1025 Patrick M. Wieruszewski https://orcid.org/0000-0002-5871-5186 Nathan J. Smischney https://orcid.org/0000-0003-1051-098X Jonathan H. Chow https://orcid.org/0000-0003-1750-3416 Supplemental Material: Supplemental material for this article is available online. ==== Refs References 1 World Health Organization. Coronavirus disease (COVID-19) outbreak. https://www.who.int/emergencies/diseases/novel-coronavirus-2019. Published 2020. Accessed on December 17, 2021. 2 Richardson S Hirsch JS Narasimhan M , et al. Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York city area. JAMA. 2020;323 (20 ):2052.32320003 3 Kim L Garg S O’Halloran A , et al. Risk factors for intensive care unit admission and in-hospital mortality among hospitalized adults identified through the US coronavirus disease 2019 (COVID-19)-associated hospitalization surveillance network (COVID-NET). 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Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients. medRxiv [Preprint]. 2020 December 5:2020.08.29.20184358. doi: 10.1101/2020.08.29.20184358 . Update in: Sci Rep. 2021 January 15; 11(1):1580. PMID: 32909005.	36448250	PMC9713537	NO-CC CODE	2022-12-02 23:23:06	no	J Intensive Care Med. 2022 Nov 29;:08850666221142265	utf-8	J Intensive Care Med	2,022	10.1177/08850666221142265	oa_other
==== Front Int J Low Extrem Wounds Int J Low Extrem Wounds IJL spijl The International Journal of Lower Extremity Wounds 1534-7346 1552-6941 SAGE Publications Sage CA: Los Angeles, CA 36426538 10.1177/15347346221141173 10.1177_15347346221141173 Case Report Pyoderma Gangrenosum Triggered by COVID-19 Vaccination in a Patient with Ulcerative Colitis: A Case Report https://orcid.org/0000-0002-7812-3428 Kim Yoon-Chung MD, PhD 1 Shim Hyung Sup MD, PhD 2 Jeong Howon MD 1 Park Yune-Jung MD, PhD 3 1 Department of Orthopaedic Surgery, St. Vincent's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea 2 Department of Plastic and Reconstructive Surgery, St. Vincent's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea 3 Division of Rheumatology, Department of Internal Medicine, St. Vincent's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Yune-Jung Park, Division of Rheumatology, Department of Internal Medicine, St. Vincent's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea 93, Jungbu-daero, Paldal-gu, Suwon-si, Gyeonggi-do, 16247, Republic of Korea. Email: [email protected] 25 11 2022 25 11 2022 15347346221141173© The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Pyoderma gangrenosum (PG) is a rare inflammatory skin disease that is difficult to diagnose. PG may be an extra-intestinal manifestation of ulcerative colitis (UC). In recent times, coronavirus disease (COVID-19) vaccines have caused various adverse cutaneous reactions. However, to the best our knowledge, combinations thereof have not been reported. We encountered a case of PG triggered by COVID-19 vaccination in a patient with UC. A 40-year-old woman developed severe pain and an abscess in the dorsum of the left foot after receiving the first dose of the messenger RNA (mRNA)-based Pfizer/BioNTech BNT162b2 COVID-19 vaccine. Severe painful ulcers with purulent necrosis and gaseous gangrene progressed rapidly along the extensor tendons and muscles to the toes and ankle. Although surgical debridement can worsen PG by triggering pathergy, we nonetheless performed wide debridement including partial extensor tenotomy with abscess drainage to prevent progression to pyogenic ankle arthritis and to rescue the toes. Antibiotics, corticosteroids, and anticoagulants were prescribed during surgical wound management via negative pressure therapy. After the lesion improved, the skin and soft tissue defect were covered using a superficial circumflex iliac artery perforator free flap and a split-thickness skin graft. The patient was satisfied with the foot salvage, and could walk unaided (without a brace or cane) from 8 weeks after the final surgery. PG may be rare even in UC patients, but mRNA-based COVID-19 vaccines may find an immunosuppressive niche. A high level of caution and suspicion of skin manifestations after vaccination is essential. foot pyoderma gangrenosum ulcerative colitis Pfizer/BioNTech BNT162b2 COVID-19 vaccine edited-statecorrected-proof typesetterts19 ==== Body pmcIntroduction Pyoderma gangrenosum (PG), a neutrophilic auto-inflammatory dermatosis, is a rare skin disease. Although the etiology or pathogenesis is unknown, PG is thought to be an immune reaction induced by inappropriate neutrophil activity that triggers neutrophilic dermatosis followed by rapid development of painful, cutaneous necrotic ulcerations.1 PG is characterized by painful pustules or nodules that become ulcerated. PG may be an extra-intestinal manifestation of ulcerative colitis (UC).2 The incidence varies from 0.4 to 2.6% in patients with inflammatory bowel disease.3 PG may be combined with rheumatological or autoimmune diseases, or hematological malignancies. PG commonly affects the lower extremities and trunk, but can involve any part of the body including the perineal region, peristomal sites, and previous surgical incisions. Since December 2019, when a novel coronavirus disease (COVID-19) was first reported in Wuhan, Hubei Province, China,4,5 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has spread across the globe. The rapid development of various COVID-19 vaccines has helped to drastically decrease the devastating burden of the pandemic. Like other vaccines, COVID-19 vaccines can cause a variety of adverse events, including injection site pain, erythema, induration or edema, fatigue, headache, fever, chills, myalgia, arthralgia, lymphadenopathy, and hypersensitivity reactions.6–8 Several cutaneous manifestations after messenger RNA (mRNA)-based COVID-19 vaccination have been reported, including urticaria, eczematous dermatitis, and granulomatous inflammation.9,10 Herein, we report a 40-year-old woman with UC who presented with a rapidly progressing PG lesion of one foot dorsum after the first dose of the Pfizer/BioNTech BNT162b2 COVID-19 vaccine. Informed consent was obtained from the patient for publication of this report and the accompanying images. Case Report A 40-year-old woman visited our emergency department with left foot pain and purulent discharge from a dorsal foot wound. She had no fever and her vital signs were stable. Laboratory findings revealed a hemoglobin level of 8.6 g/dL; slight thrombocytosis (557 × 109/L; normal 150-450 109/L); a normal white blood cell count; an elevated C-reactive protein level (11.70 mg/dL; normal 0–0.3 mg/dL); a positive anti-PR3 antibody reaction (73.8 IU/mL; normal <20 IU/mL); a slightly elevated fibrinogen level (413.9 mg/dL; normal 160–410 mg/dL); and an elevated D-dimer level (1.64 mg/L FEU; normal 0–0.55 mg/L FEU). Antinuclear and antineutrophil cytoplasmic antibody status, and the prothrombin and activated partial thromboplastin times, were normal. On physical examination, the wound was 7 × 7-cm in dimension with a central skin defect and internal pus. The margin was violaceous with palpating gas crepitus, and the soft tissue was very friable. The lesion was extremely painful and progressed rapidly (Figure 1). She had been diagnosed with UC at our hospital 2 years prior. However, she ceased follow-up on her own initiative 19 months prior when her gastrointestinal symptoms became tolerable on medications. Even after arbitrary cessation of medication including mesalazine and methylprednisolone, she had only intermittent bloody stools for the recent 2 months. She had received the first dose of the messenger RNA-based Pfizer/BioNTech BNT162b2 COVID-19 vaccine 2 weeks prior. The left foot pain and swelling commenced 1 week later. She visited a local clinic and received a steroid injection into the painful left foot dorsum. However, the swelling worsened. Ulcerative vesicles and pustules appeared 2 days prior to the emergency department visit. Radiography revealed air (gas) in the dorsal soft tissue and magnetic resonance imaging showed a mixture of infectious soft tissue and abscesses. Figure 1. The left foot. A 7 × 7-cm ulcerative lesion with a purulent discharge, a violaceous margin, and gaseous crepitus. We performed an emergency operation for exploration, debridement, and abscess drainage. The operative findings included skin and subcutaneous necrosis, and a peritendinous infection along the extensor tendons. All five metatarsophalangeal joints were involved; a diffuse progressive infection was evident. All infected extensor tendon sheathes were excised and debrided; we performed second and third extensor digitorum tenotomy and dorsalis pedis arterial ligation. An infectious, medial dorsal cutaneous branch of superficial peroneal nerve underwent neurectomy; and an infectious, intermediate dorsal cutaneous branch of superficial peroneal nerve was subjected to neurolysis. All obvious abscesses were maximally drained with contiguous debridement and irrigation, followed by negative pressure wound therapy (NPWT) using a vacuum-assisted closure system (V.A.C.TM, KCI®, San Antonio, Texas, USA) for 14 days. After confirming that the infection was grossly controlled (Figure 2) and that the laboratory inflammatory findings had normalized, we referred the patient to our plastic surgery team. A superficial circumflex iliac artery perforator free flap combined with a split-thickness skin graft was performed (Figure 3). Biopsies were performed at the ulcer edge consistent with diagnostic criteria according to the referenced Delphi consensus.11 The biopsy of the ulcer revealed erosion, hemorrhagic necrosis, and abscess formation of/in the epidermis, as well as neutrophilic aggregations with necrosis in the upper dermis and subcutis, consistent with acute suppurative inflammation including PG (Figure 4). Four tissue cultures were performed from different sites of initial purulent wound prior to debridement surgery, and the blood culture was also performed. All microbiological studies, including aerobic and anaerobic microbes in blood and tissues, were negative. Interferon-gamma release assay was negative and skin biopsies showed no tuberculoid granulomas or other characteristic histopathologic features of tuberculosis. Sigmoidoscopy to follow-up the untreated UC revealed complete loss of the vascular pattern, diffuse nodular mucosal swelling, and erosion, but fortunately nothing worse than had been observed at the last follow-up 2 years prior. Figure 2. Negative pressure wound therapy was performed after consecutive necrotic tissue debridement with abscess drainage and irrigation. Figure 3. A superficial circumflex iliac artery perforator free flap and a split-thickness skin graft were placed. Figure 4. Histopathology revealed erosion and hemorrhagic necrosis of the epidermis, and neutrophil infiltration into the dermis and subcutis [hematoxylin-eosin, original magnifications x40 for (a) and x100 for (b)]. During the above treatment, the patient received methylprednisolone 40 mg/day intravenously for 5 days and was then changed to oral prednisolone that was progressively tapered every 3 days. The initial intravenous antibiotics were ceftriaxone 4 g/day and clindamycin 1800 mg/day, with a change to cefobactam 6 g/day after 2 weeks. Since she received high-dose glucocorticoid therapy, prophylactic antibiotic treatment was maintained to prevent secondary infection. Rivaroxaban 20 mg was administered throughout the entire hospital stay until the laboratory hypercoagulation profile normalized. After confirming that the wound had healed and that the flap was stable (about 2 weeks after surgery), the patient commenced active ankle range-of-motion exercises and was allowed to walk with crutches (without weight-bearing) for 4–6 weeks in a boot. After 8 weeks, she could walk with full weight-bearing, without crutches or a cane. Twelve weeks after flap surgery, she could walk on her own in wide-toe boxed shoes. She was satisfied with the foot salvage despite sequelae of toe stiffness and sensory loss of the foot dorsum. Discussion PG is an uncommon, ulcerative auto-inflammatory dermatosis. The initial clinical signs include painful skin nodules, which usually occur new or after minor trauma, then rapidly progress to large ulcers with eroded margins and sloughy necrotic bases.12,13 Local treatments include limb elevation, dressings, wet compresses, and topical steroids. Systemic treatments include corticosteroids, thiopurines, cyclosporine, infliximab, and intravenous antibiotics.2,14–16 Although PG is not associated with UC disease activity, PG can be an extra-intestinal manifestation in patients with acute severe UC.2 The previously diagnosed UC of our patient had not recently been treated, but the sigmoidoscopic findings had not worsened. We suggest that the PG ulcer was not associated with UC activity, being rather immunologically triggered by the COVID-19 vaccine. Although rapid vaccine development has saved many lives, the vaccines cause several side-effects, including skin manifestations. Several studies over the past 2 years have reported skin side-effects after COVID-19 vaccination, including PG.17–21 To the best of our knowledge, a PG ulcer triggered by COVID-19 vaccination in a patient with UC has not been reported. During the treatment of our patients, the following diagnoses should be differentiated: Infectious diseases such as tuberculosis, rheumatic diseases (eg, sarcoidosis, vasculitis etc), and other immune dysregulation diseases. We excluded bacterial or tuberculosis infection by repeated bacterial cultures, interferon-gamma release assays, and tissue biopsies. Autoinflammatory phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) should also be differentiated when the patients have untypical skin lesions with gaseous gangrene. APLAID is an autosomal dominant chromosomal disease characterized by recurrent blistering skin lesions and inflammatory cell infiltration into the joints, eyes, and gastrointestinal tract.22–24 If the patient has recurrent respiratory tract infections, skin disorders, inflammatory symptoms such as arthritis, abdominal pain, low serum immunoglobulin (Ig)G, IgA, and IgM levels, ALPAID should be considered as a differential diagnosis. However, clinically and serologically, the possibility of these disease was low, so they were excluded. The literature states that PG wound debridement is usually contraindicated because this can exacerbate the PG ulcers. However, in real clinical situations, it is not uncommon for patients with PG to undergo surgical debridement after visiting the emergency department rather than visiting a dermatologist due to a rapidly progressing disease.1,25,26 Our patient had received a steroid injection into the foot before visiting our emergency department. We hypothesize that the injection served as the initial minor trauma, which induced the PG ulcer that could be exacerbated via surgical debridement. Jin et al27 reported that a combination of intravenous immunoglobulin and intermittent NPWT completely healed a refractory PG ulcer. Our patient was referred to an internal medicine expert and a plastic surgeon of our hospital, and received systemic treatments (corticosteroids with antibiotics) combined with local wound management. The PG lesion healed completely. We report this case for two reasons. First, PG may be a rare adverse event even in patients with UC, but COVID-19 vaccination may find an immunosuppressive niche that clinicians should consider, especially in the current unprecedented pandemic situation. Second, caution should be exercised when determining injection therapy for musculoskeletal pain in patients with underlying autoimmune diseases or immunosuppressive conditions. No author has any potential conflict of interest in terms of the research, authorship, and/or publication of this article. Ethical approval: This article is not a human or animal study. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ORCID iD: Yoon-Chung Kim https://orcid.org/0000-0002-7812-3428 ==== Refs References 1 Maverakis E Marzano AV Le ST , et al. Pyoderma gangrenosum. Nat Rev Dis Primers. 2020;6 (1):81. doi: 10.1038/s41572-020-0213-x 33033263 2 Jena A Sachan A Singh AK Sharma V . Multifocal pyoderma Gangrenosum at presentation in a patient with acute severe ulcerative colitis. Inflamm Bowel Dis. 2022;28 (6):e84-e85. doi: 10.1093/ibd/izab309 34893838 3 States V O’Brien S Rai JP , et al. Pyoderma Gangrenosum in inflammatory bowel disease: A systematic review and meta-analysis. Dig Dis Sci. 2020;65 (9):2675-2685. doi: 10.1007/s10620-019-05999-4 31925675 4 Hui DS, Azhar EI, Madani TA, et al. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health - the latest 2019 novel coronavirus outbreak in Wuhan, China. Int J Infect Dis. 2020;91 :264-266. doi: 10.1016/j.ijid.2020.01.009 31953166 5 Huang C Wang Y Li X , et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan. China. The lancet. 2020;395 (10223):497-506. doi: 10.1016/S0140-6736(20)30183-5 6 Castells MC Phillips EJ . Maintaining safety with SARS-CoV-2 vaccines. N Engl J Med. 2021;384 (7):643-649. doi: 10.1056/NEJMra2035343 33378605 7 Polack FP Thomas SJ Kitchin N , et al. Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N Engl J Med. 2020;383 (27):2603-2615. doi: 10.1056/NEJMoa2034577 33301246 8 Baden LR El Sahly HM Essink B , et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med. 2021;384 (5):403-416. doi: 10.1056/NEJMoa2035389 33378609 9 McMahon DE Amerson E Rosenbach M , et al. Cutaneous reactions reported after Moderna and Pfizer COVID-19 vaccination: A registry-based study of 414 cases. J Am Acad Dermatol. 2021;85 (1):46-55. doi: 10.1016/j.jaad.2021.03.092 33838206 10 Magro C Crowson AN Franks L , et al. The histologic and molecular correlates of COVID-19 vaccine-induced changes in the skin. Clin Dermatol. 2021;39 (6):966-984. doi: 10.1016/j.clindermatol.2021.07.011 34920834 11 Maverakis E Ma C Shinkai K , et al. Diagnostic criteria of ulcerative pyoderma Gangrenosum: A Delphi consensus of international experts. JAMA Dermatol. 2018;154 (4):461-466. doi: 10.1001/jamadermatol.2017.5980 29450466 12 Ruocco E Sangiuliano S Gravina AG Miranda A Nicoletti G . Pyoderma gangrenosum: An updated review. J Eur Acad Dermatol Venereol. 2009;23 (9):1008-1017.19470075 13 Weizman AV Huang B Targan S , et al. Pyoderma gangrenosum among patients with inflammatory bowel disease: A descriptive cohort study. J Cutan Med Surg. 2014;18 (5):361. doi:10.2310/7750.2013.13103 14 Argüelles-Arias F Castro-Laria L Lobatón T , et al. Characteristics and treatment of pyoderma gangrenosum in inflammatory bowel disease. Dig Dis Sci. 2013;58 :2949-2954.23828140 15 Chatzinasiou F Polymeros D Panagiotou M Theodoropoulos K Rigopoulos D . Generalized pyoderma Gangrenosum associated with ulcerative colitis: Successful treatment with infliximab and azathioprine. Acta Dermatovenerol Croat. 2016;24 (1):83-85.27149138 16 Mikail M Wilson A . Infliximab treatment for large, multifocal, abdominal pyoderma gangrenosum associated with ulcerative colitis: A case report. SAGE Open Med Case Rep. 2020;8 :2050313-20964113. doi: 10.1177/2050313X20964113 17 Mohd AB Mohd OB Ghannam RA Al-Thnaibat MH . COVID-19 Vaccine: A possible trigger for pyoderma Gangrenosum. Cureus. 2022;14 (5):e25295. doi: 10.7759/cureus.25295 35755518 18 Barry M AlRajhi A Aljerian K . Pyoderma Gangrenosum induced by BNT162b2 COVID-19 vaccine in a healthy adult. Vaccines (Basel). 2022;10 (1):87.35062748 19 Toyama Y Kamiya K Maekawa T Komine M Ohtsuki M . Pyoderma gangrenosum following vaccination against coronavirus disease-2019: A case report. Int J Dermatol. 2022;61 (7):905-906. doi: 10.1111/ijd.16255 35567368 20 Hung YT Chung WH Tsai TF Chen CB . Haemorrhagic bullous pyoderma gangrenosum following COVID-19 vaccination. J Eur Acad Dermatol Venereol. 2022;36 (8):e611-e613. doi: 10.1111/jdv.18132 35398933 21 Franceschi J Darrigade AS Sanchez-Pena P Legrain-Lifermann V Milpied B . Pyoderma gangrenosum after mRNA-based SARS-CoV-2 vaccine. J Eur Acad Dermatol Venereol. 2022;36(12):e969-e970. doi: 10.1111/jdv.18389 Online ahead of print. 22 Ombrello MJ Remmers EF Sun G , et al. Cold urticarial, immunodeficiency and autoimmunity related to PLCG2 deletions. N Engl J Med. 2012;366 :330-338.22236196 23 Zhou Q Lee GS Brady J , et al. A hypermorphic missense mutation in PLCG2, encoding phospholipase C gamma2, causes a dominantly inherited autoinflammatory disease with immunodeficiency. Am J Hum Genet. 2012;91 (4):713-720.23000145 24 Neves JF Doffinger R Barcena-Morales G , et al. Novel PLCG2 mutation in patient with APLAID and cutis laxa. Front Immunol. 2018;9 :2863. doi: 10.3389/fimmu.2018.02863 30619256 25 Long CC Jessop J Young M Holt PJ . Minimizing the risk of post-operative pyoderma gangrenosum. Br J Dermatol. 1992;127 (1):45-48. doi:10.1111/j.1365-2133.1992.tb14826.x 1637694 26 Zuo KJ Fung E Tredget EE Lin AN . A systematic review of post-surgical pyoderma gangrenosum: Identification of risk factors and proposed management strategy. J Plast Reconstr Aesthet Surg. 2015;68 (3):295-303. doi:10.1016/j.bjps.2014.12.036 25589459 27 Jin SY Chen M Wang FY Wang F . Applying intravenous immunoglobulin and negative-pressure wound therapy to treat refractory pyoderma Gangrenosum: A case report. Int J Low Extrem Wounds. 2021;20 (2):158-161. doi:10.1177/1534734620940459 32734793	36426538	PMC9713538	NO-CC CODE	2022-12-02 23:23:06	no	Int J Low Extrem Wounds. 2022 Nov 25;:15347346221141173	utf-8	Int J Low Extrem Wounds	2,022	10.1177/15347346221141173	oa_other
==== Front Ethnicities Ethnicities spetn ETN Ethnicities 1468-7968 1741-2706 SAGE Publications Sage UK: London, England 10.1177_14687968221139497 10.1177/14687968221139497 Article Health inspector ratings of Asian restaurants during the early COVID-19 pandemic https://orcid.org/0000-0003-2444-4354 Cherng Hua-Yu Sebastian Moreno Martha 5894 New York University , New York, NY, USA Liu Jia-Lin 14669 California State University , Los Angeles, CA, USA Hua-Yu Sebastian Cherng, New York University, 246 Greene Street, 302W, New York, NY 10012-1126, USA. Email: [email protected] 29 11 2022 29 11 2022 14687968221139497© The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. The COVID-19 pandemic has devastated the restaurant industry, with Asian restaurants having perhaps suffered the most, as many reported business losses well before shelter-in-place orders were announced. Media outlets argue that this decline in business reflects biases that are linked to the China- and food-related origin of COVID-19. However, discrimination against Asian Americans and their cuisine is not new, as it is rooted in a long and history of assimilation and racism. Overlooked in this body of literature, as well as in conversations on the impacts of COVID-19 on Asian restaurants, is the role of how government institutions shape these biases against a cuisine that has hundreds of years of history in the US yet remains distinctly ‘foreign’. In this study, we use 3-years of New York City restaurant health inspection data to examine trends in citation scores before and after the onset of the news of the COVID-19 pandemic. Using a synthetic control approach, we find that Asian restaurants uniquely received more citations after news of the pandemic became pervasive in the US. We end by discussing the implications of this finding for the history of Asian cuisine in the US, theoretical frameworks to understand assimilation, and the restaurant industry. Asian American racism inequality immigration restaurant ratings edited-statecorrected-proof typesetterts10 ==== Body pmcIntroduction As one of the worst global health crises in human history, the COVID-19 pandemic has also devastated many industries and perhaps none more so than restaurants. While virtually all restaurants in the US have suffered substantial economic losses, early reports suggest that Asian restaurants, which can encompass a number of global cuisines, were impacted the most. The Pew Research Center found that the highest increase in unemployment observed between February and May of 2020 was among Asian Americans, from 2.5 to 20.3% (Pew Research Center, 2020). Moreover, numerous media outlets and empirical studies reported that Asian restaurants had less business starting as early as January 2020 (Alcorn, 2020; Shen-Berro, 2020; Yi et al., 2022). Estimates of food businesses that closed due to COVID showed that Chinese enclaves in both Brooklyn and Manhattan were the ones with the highest figures, with 16% in Sunset Park and 27% in Chinatown (Yi et al., 2022). These articles stated that the reason for the significant decline in business, even before any state and local governments issued shelter-in-place orders, was the public perception that the coronavirus1 causing COVID-19 originated in China due to food (sold in wet markets). This argument was also used to explain why Asian restaurants experienced vandalism during this time. However, the notion that Asian cuisine is unclean and disease-bearing is rooted in a racist US history that spans hundreds of years. The expansion of the US economy in the 1800s brought many Chinese men to the US to seek work opportunities, which posed a threat to other non-Chinese workers, mostly white men, who were competing for such jobs. With growing tension and hostility, the Chinese Exclusion Act of 1882 legitimized the illegality and otherness of Chinese immigrants. During this time, many Chinese immigrants retreated into Chinatowns and opened Chinese restaurants. Because of their low prices and convenience, Chinese restaurants attracted a diverse clientele, and chefs began to make Chinese American food that catered to the white palette. At the same time, long-standing racism towards Chinese immigrants transformed into stereotypes of Chinese cuisine as unclean and unhealthy. While scholarship has largely focused on Chinese American cuisine, similar stereotypes have also affected the food of other Asian groups (Ku, 2014; Ku et al., 2013). The study of Asian cuisine not only provides insight into its unique history, but also into our theoretical understanding of the assimilation of Asian Americans into the US mainstream. Many cuisines brought over by Asian Americans, such as Chinese and Filipinx immigrants, predate theories of immigration, such as straight line (Park and Burgess, 1921) and segmented assimilation (Portes and Zhou, 1993) theories. Straight line assimilation theory argues that as immigrants become more integrated into the US mainstream through education and occupation, they abandon their markers of ethnicity, such as language and culture (Park and Burgess, 1921; Zhou, 1999). Yet, other scholars have pinpointed the complexity of this process through the framework of segmented assimilation, stating that there are three possible adaptation patterns that occur among contemporary immigrants and their next generation: upward mobility into the white middle class, downward mobility into the underclass, or the preservation of immigrants’ values and solidarity through the establishments of ethnic capital (Portes and Zhou, 1993; Zhou, 1999). Studying how food and cuisine shape these trajectories has not been a focus of research using these frameworks and applying them can help us understand both how different types of cuisines are received by the white mainstream and the broader social standing of ethnic groups within US racial hierarchies. Within the historical context and sociological analyses frame our investigation of the impacts of the COVID-19 pandemic on the economic livelihoods of Asian restaurants, there is one key actor that remains overlooked: the governmental health inspector. As individuals that enforce institutional policies regarding food safety, health inspectors play an important role in conveying to the public the level of compliance restaurants have with food safety regulations, and are particularly important in jurisdictions where restaurant grades must be prominently displayed for customers. Health inspectors are also official agents of the State, and any biases they manifest could result in systematic disadvantage against restaurants in ways that exacerbate existing inequalities. Moreover, these inequalities are structural ones given that representatives of the State are responsible for carrying out and enforcing laws. This study seeks to understand how these potential biases shaped the livelihoods of Asian restaurants. Focusing on New York City, home to some of the largest enclaves of Asian restaurants in the US, we examine trends in health inspection scores before and after news of the pandemic became widespread in the US. The COVID-19 pandemic and the restaurant industry The COVID-19 pandemic devastated the economy and particularly the restaurant industry. From the authors’ own calculations using US Bureau of Labor Statistics data, the number of employees in the restaurant industry in April 2020 declined by 46.1% compared to April 2019 (see Appendix Table 1). This decline equates to a loss of approximately 5.1 million jobs in the restaurant industry (see Appendix Table 2). Additionally, the decline occurred primarily in April 2020, as the number of jobs in the restaurant industry during this time was approximately half of the number in the preceding month. Employment data also suggests that Asian Americans were affected more than other groups. Data from the New York Bureau of Labor showed that in the 6-week period ending on 9 May 2020, unemployment claims filed by Asian Americans were 56 times higher than for the same period in 2019, which was also much higher than the growth in claims filed by white, Black, and Latinx individuals, which were 15, eleven, and 16 times higher, respectively (Klein, 2020). Moreover, a nation-wide report from the Pew Research Center (2020) found that the highest increase in unemployment was among Asian Americans, from 2.5% to 20.3% between February and May of 2020. News reports suggest that the effects of the pandemic were felt by Asian restaurants as early as January (Alcorn, 2020; Shen-Berro, 2020). For example, the owner of San Francisco’s famous Golden Gate Fortune Cookie Company when describing his business said “‘It’s dead. We have no people coming.’ He estimated that he had seen an 80% drop in foot traffic to his shop since the start of the outbreak” (Ho, 2020). One Washington Post article argued that “Asian-owned businesses are facing a crippling pileup of pressures during the coronavirus pandemic, advocates and researchers say — pressures that won’t all ease with the country’s piecemeal reopening. They’ve been hurting longer, seeing their group’s unemployment rate skyrocket faster and struggling like many minority-owned shops to access government aid, often in industries with especially uncertain roads to recovery” (Knowles and Bellware, 2020). Decreased business was not the only form of hardship that Asian Americans in the restaurant industry experienced. A number of Asian restaurants also reported being defaced with racist graffiti and other forms of vandalism during the pandemic, such as Douglas Kim’s Jeju Noodle Bar in New York City (Adams, 2020). One restaurant owner described this dynamic from the perspective of Asian American customers: “It was people’s fear of coming to Manhattan’s Chinatown...but also Asian families’ fears of going out...of being punched, spat on or glared at because of her race or mask on the subway (Knowles and Bellware, 2020).” This restaurant-specific discrimination was undoubtedly linked to reports of spikes in complaints of anti-Asian harassment and discrimination across the US and the globe (Gover et al., 2020; Phillips, 2020). Li and Nicholson (2021) argued that the surge in anti-Asian discrimination reflected historic notions of “Yellow Peril”, or the notion of East Asians as an ever-present threat against whiteness. One initiative, Stop AAPI Hate, collected 1843 self-reported incidents of anti-Asian discrimination from March 19 to 13 May 2020 (Turton, 2020). History of anti-Asian racism and food In order to understand contemporary anti-Asian sentiments, it is important to place these events in the history of Anti-Asian discrimination. While not common in popular discourse of Asian restaurants and the COVID-19 pandemic, the roots of anti-Asian racism and food date back to the late 1800s. Before 1882, the California Gold Rush brought the first wave of Chinese immigrants to the US and by the latter half of the 19th Century as many as 300,000 individuals were working in various sectors, such as agriculture, mining, transportation, and manufacturing (Barbas, 2003; Godoy, 2016; Takaki, 1998). Many non-Chinese, often white workers felt threatened by how new incoming Chinese laborers worked for lower wages and anti-Chinese sentiment soon escalated into violence (Carter, 2018; Godoy, 2016). For example, in 1871, at least 18 Chinese immigrants were beat, shot, and hung by a mob of mostly white Americans in Los Angeles – known as “the most lethal example of racial violence ever recorded in the city” - yet, the white attackers were tried and eventually released with very little penalty (Chung, 2014; Johnson, 2011; Lee, 2013). The US government not only did not provide relief and support, but in 1882, President Chester A. Arthur signed the Chinese Exclusion Act, the very first law that prohibited the entry of one specific ethnic group to the US and denied existing US residents any pathway to citizenship (Godoy, 2016; Gyory, 1998; Kil, 2012; Lee, 2002). The law also legitimized ways for others to persecute and force out Chinese immigrants from the general labor market, which contributed to the formation of segregated ethnic enclaves and labor niches (Barbas, 2003; Mendelson, 2016). By 1924, the Immigration Act of 1924 denied all Chinese and other Asian immigrants the right to become naturalized citizens and the ownership of land (Lee, 2002; Ngai, 1999). With such severe limitations, Chinese immigrants had to establish new work opportunities for themselves. Restaurant work within Chinatowns became one of the main job opportunities for Chinese immigrants (Chin, 2005; Shah, 2001; Siu and Tchen, 1988). These ethnic enclaves were some of the poorest and most segregated neighborhoods in large cities (Anderson, 1987; Lin, 1998; Yan and Santos, 2009). Chinese immigrants opened up new restaurants in Chinatowns where they adapted and learned to create dishes for non-Chinese patrons (Mendelson, 2016). Chinese restaurants became a symbol of “abundance” and “democracy” as eating fusion dishes such as “chop suey” and “chow mein” became not only a privilege to the wealthy, but one for the masses to be enjoyed on a regular basis (Barbas, 2003; Chen, 2014: p.3; Liu, 2015). Still, the phenomenon of inexpensive Chinese American food shaped anti-Chinese sentiments, which now stereotyped Chinese immigrants as subservient restaurant workers (Barbas, 2003). The perception of Chinese workers as cheap restaurant laborers and Chinatowns as poor and unclean soon formed a new racist rhetoric about Chinese food. As Chen (2014) stated in his book Chop Suey, USA: the Story of Chinese Food in America, “the lowly position of Chinese food in mainstream America’s restaurant-market hierarchy has mirrored the inferior status of China both as a culture and as a supplier of cheap labor in the economy” (p.3). Such anti-Chinese discrimination affected how white consumers saw other types of Asian cuisines as well. Many of these stereotypes formed the basis of racist propaganda, which claimed that Chinese restaurants used rats, cats, and dogs as their preferred meat (Liu, 2015). The media was ruthless in their writings and descriptions. In 1853, the Daily Alta California announced:If there is one class of nasty foreigners. . . more ill-favored, unfortunate, and forlorn among us, than another, it certainly must be the Chinese…Rats, lizards, mud-terrapins, rank and indigestible shellfish, and such small deer, have been and continue to be, the food of the “no ways partickler” Celestial, where flour, beef, and bacon, and other fare suitable to the stomachs of “white folk” abound. It is not to be wondered at, therefore, that the habits of the Chinese in California should excite ineffable disgust, and turn the stomachs of the stoutest Anglo-Saxon (Liu 2015: p.29). Alexander Young of the Daily Alta California stated the following in 1872:“Superficial artists have insisted that the reason why the Chinese are attached to California, notwithstanding the ill-treatment to which they have been subjected there, is the abundance and superior quality of the rats of the golden gate.” (Liu, p.31) Similar descriptions of Chinese immigrants and their cuisine also infiltrated the advertising world. “Rough on Rats,'' a pest control product, had an advertisement that showcased “a Chinese man with his mouth open ready to eat a rodent…personified…the Chinese man being an effective rodent exterminator” (Liu 2015: p.40). The American Restaurant magazine stated in 1927 that “eating chop suey in a Chinese-run, rather than white-owned restaurants…is a definite way to contract a disease – if not commit ‘chop-suey-cide’” (Barbas, 2003: p.632). In its early formation, this rhetoric was largely propagated among only white Americans. Wong Chin Foo, a Chinese civil rights activist in the late 1800s once answered during a public lecture that “I never knew that rats and puppies were good to eat until I was told by American people” (p.36). These stereotypes of Chinese food continue today: for example, in 2007, despite having no evidence, New York television station CW 11 broadcasted in their news that a Chinese restaurant in Brooklyn served mouse meat. While the restaurant passed their next health inspection, the owners received numerous threats and the television station never apologized (Liu, 2015). More recently, rhetoric over the cleanliness and safety of Chinese and Asian American food has centered on the use of monosodium glutamate, or MSG. Unscientifically thought to cause “The Chinese Restaurant Syndrome” (Mosby, 2009), MSG, and more broadly Chinese and Asian American cuisine, has been blamed for a host of symptoms ranging from headache and fatigue to dry mouth. Despite decades of scientific studies that have found no statistical links between the consumption of MSG and these symptoms (Freeman, 2006), its use (or supposed use) still is associated with uncleanliness in the white consciousness (Huang, 2021; Yeung et al., 2020). Theoretical frameworks The history of Asian cuisine in the US also allows us to understand better the theoretical tools that focus on immigration. Common theories of immigration, particularly in sociology, stem from analysis of either southern, central, and eastern European immigrants at the turn of the 20th century (e.g. straight-line assimilation theory, Park and Burgess, 1921) or racially and ethnically diverse populations that arrived as a result of the Immigration Act of 1965 (e.g. Portes and Zhou, 1993). However, Asian cuisines in the US transcend these timelines, and they remain simultaneously one of the most popular foods while also bearing the marker of ‘ethnic’ difference. For example, one recent study of reviews of restaurants on Yelp, an online platform for business and product reviews, found that customers of Chinese restaurants evoked mentions of ethnicity more than any Mexican, Italian, and American restaurants (Boch et al., 2020). This dynamic shapes more than just the reception of food to the mainstream, but also the lives of Asian Americans themselves. For example, one ethnographic study of Chinese American restaurant owners found that opening Chinese restaurants helped solidify their ethnic identities (Liu and Lin, 2009). These identities were influenced by an acknowledgement of both past and current histories of Chinese American cuisines and were a form of resistance to a “wholesale assimilation” that was devoid of ethnic identities. Finally, understanding how policies beyond immigration laws shape assimilation trajectories is also vital, as scholars have long argued that policies that seemingly are “race-neutral” are enacted in ways that reinforce existing inequalities (Crenshaw, 1989). Research question Given their recent economic devastation, history of discrimination, and potential to further our theoretical understanding of immigrant assimilation, Asian restaurants, and how they have fared during the COVID-19 pandemic, are important subjects of investigation. Moreover, it is vital to understand the role that institutions, such as health inspection, play in shaping the outcomes of these restaurants. We ask the following research question: did patterns of health inspection citations of Asian restaurants, compared to other restaurants, in New York City change when news of the COVID-19 pandemic became widespread in the US? Data To answer our research question, we use data from NYC Open data, which is provided by the New York City Department of Health and Mental Hygiene (DOHMH).2 The dataset contained violation citations from every inspection conducted up to 3 years prior to the most recent inspection for restaurants in active status on the date the data is accessed. When an inspection results in more than one violation, values for associated fields are repeated for each additional violation record. Each establishment is identified by a unique record ID. It is important to note that we only included restaurants that had active status in February of 2020 in order to analyze the period between December 2019 and February 2020. We chose the cutoff of December 2019 to mark the month before public attention to the COVID-19 pandemic started in the US. Using Google Trends to examine google searches in the US on terms such as “Coronavirus”, we saw that searches for these words grew rapidly during the week of January 19–25, 2020.3 We chose the latter cutoff of February 2020 to take into consideration that a number of restaurants closed during and after March 2020. Therefore, the period of December 2019 to February 2020 represents the months after the news of COVID-19 began and restaurants were still open. The data we used in this study is at the restaurant type/cuisine level. The first step of our analysis was to create a dataset aggregating the citation information by type of cuisine. For each cuisine, we calculate the average citations and number of records (the number of times a restaurant was graded). We then reshaped the data so that each cuisine was a row and each column represented monthly information covering the period between January 2017 and February 2020. We also calculated cumulative citations, which represent the average of the monthly citations up to the corresponding month. For example, the cumulative rating for October 2019 was the average of ratings from January 2017 to October 2019.4 We decided to use the cumulative ratings as the outcome for our synthetic control analysis (described below) as it was a better measure of sustained increase or decrease in the ratings of certain cuisines than the raw monthly averages, which we explain further in the results section. Focusing on when news of the COVID-19 outbreak started to rise in the US, we analyzed potential changes in the cumulative ratings of restaurants. To create the synthetic control group, we included as covariates the (1) cumulative citations, as they speak to a sustained increase or decrease in citations, (2) monthly ratings, as they indicate whether restaurants had a month with a particularly high number of citations, which helps us factor in for the seasonality that can arise in citations and (3) number of records, or how many times in a month a restaurant was cited. We modeled the synthetic controls using data from January 2017 until (and including) December 2019 and then predicted values for January and February 2020. We did this using a causal framework: the pre-treatment condition (before January 2020) is not contaminated by our “treatment” or shock, which in this case is when news of COVID-19 began in the US. Our donor pool for the synthetic control group included American, Caribbean, Latin, Italian, and Mexican restaurants. Please see Appendix Table 3 for descriptive statistics of variables used for the synthetic controls. We only included these cuisines as they were the ones with sufficient citation information during the period analyzed.5 Analytic strategy To determine whether there was an effect of the news of the COVID-19 pandemic on food inspector citations of Asian restaurants, we used a comparative case study approach: we compared citations for restaurants of Asian cuisine, which we argue were particularly affected by COVID-19 news, to the citations of comparison or control groups.6 One of the main concerns when using a comparative case study approach is how to select correctly a control group. One approach given our data and research question is to use the difference in differences (DID) method, which relies on the parallel trends assumption. For this study, this assumption requires us to find a comparison group that has a similar trend in citations to Asian restaurants before January 2020. If this comparison cuisine existed, we could implement a difference in differences (DID) approach to evaluate and compare the average change over time for Asian restaurants and the control cuisine. To find a potential comparison group of this type, we visually analyzed the citations’ trends and did not find any cuisine for which we could make the parallel trend assumption. For this reason, we used the synthetic control method developed by Abadie and Gardeazabal (2003). The synthetic control method is a statistical tool that combines matching (in a similar way as propensity scores techniques do) and DID approaches by creating a weighted combination from a pool of controls, called the synthetic control, which is then compared to the treatment group. This method has several benefits: it can account for the effects of other confounders that change over time as long as the covariates are not related to the intervention; it uses the data available to create a better control group (i.e. one that most resembles the treatment group before intervention); and it is data driven, meaning that the researcher does not select the control group, as the synthetic control is obtained from the data structure and covariates used in the model (Abadie and Gardeazabal, 2003). In mathematical terms, let us assume we have J+1 different cuisines. Without loss of generality, we assume that the first one is Asian cuisine with a total of J possible controls. Let YitN be the outcomes that would be observed for cuisine i at time t in absence of COVID-19 news and let YitI be the outcome for cuisine i at time t with the news of COVID-19. Let T0 be the first month when the COVID-19 news started to surface, so for 1 ≤t ≤T0 we have that YitN=YitI. We are interested in estimating the following equation for t>T0(1) αit=YitN−YitI Since one of our assumptions is that only Asian cuisine restaurants were affected by the COVID-19 news (we further discuss this assumption in our discussion section) then equation (1) becomes(2) α1t=Y1tN−Y1tI And because the second term of equation (2) is observed, then we are only interested in estimating YitN. Suppose that we can express YitN with a factor model so that Equation 3 becomes(3) YitN=δt+θtZi+λtμi+εit Where δt is an unknown common parameter, Zi is a (r x 1) vector of observed covariates not related to the spread of COVID-19 news, θt is a (1 x r) vector of unknown parameters, λt is a (1 x F) vector of unobserved common factors, μi is an (F x 1) vector of unknown factor loadings and εit are the error terms for each cuisine. The particular vectors of weights W=(w2, …wJ+1)′ such that each wj≥0 and ∑wj=1 represent a different solution for a potential synthetic control, which is a weighted average of cuisines. If there exists a vector W=(w2, …, wJ+1)′ such that Equation 4 becomes(4) ∑j=2J+1wjYj1=Y11, …, ∑j=2J+1wjYjT0=Y1T0and ∑j=2J+1wjZj=Z1 Then we can use such vector of weights W* to estimate our synthetic control group as the weighted average of the other cuisines and we can proceed to estimate equation (2). Results To address our research question we first analyzed, descriptively, restaurant ratings by cuisine. From Figure 1, we see that some cuisines had decreases in the number of citations from December 2019 to January 2020, while other cuisines, such as Asian, Caribbean, and Italian, showed consistent increases in citations until February of 2020. It is also clear from Figure 1 that monthly citations vary greatly within cuisines, which makes it difficult to examine whether the number of citations after December 2019 changed due to systematic biases held by potential inspector bias or random noise. For example, in December 2019, Asian restaurants had an average citation count of 11.5, while in January 2020 the citation count was 11.8. Moreover, Asian restaurants had lower citation numbers in that 6-month period; therefore, it is unclear whether the increase seen in later months is simply due to the comparison with December. Given our interest in whether citation numbers in the months after December 2019 were substantially different from prior months, we calculate the citations’ cumulative average, which we present in Figure 2. The cumulative average helps address spikes in certain months that may affect the distribution in a way that precludes meaningful analysis by providing information on sustained increases or decreases in the number of citations. When investigating this alternative measure descriptively, we see from Figure 2 that there is still an increase starting in December of 2019 that is more pronounced for Asian restaurants vis àvis other restaurants. To examine in a more robust manner whether January 2020 and February 2020 citations were significantly different from earlier ratings, we used the technique of synthetic control. As mentioned in the methods section, the synthetic control approach relies on predicting a counterfactual or control group for the treatment group as a function of the other cuisines’ covariates (cumulative citations, monthly citations, and records) before intervention (December 2019). The first step of our analysis was to create a synthetic control for Asian cuisine. The second step was a robustness check of our estimates to investigate whether other non-Asian cuisines also showed systematic increases in the number of citations after December 2019. Figure 3 shows the results of our synthetic control for Asian restaurants. We can see that the synthetic control for Asian restaurants closely mirrors the actual trend for Asian restaurants until December 2019 (shown by a vertical red dotted line), which shows that the model estimated a robust synthetic control for the number of citations received by Asian restaurants. However, after December 2019, a gap emerges between the synthetic control and actual citation count and widens throughout January and February 2020. This pattern shows that the number of citations for Asian restaurants were higher than predicted in January and February 2020, which is when news of coronavirus started in the US. As mentioned earlier, we smoothed the actual monthly scores as the raw data reflects too much variation (e.g. has too much noise). As a robustness check, we calculated a 3-month moving average of the scores and analyzed the synthetic control for this trend. The results hold in this case are shown in Appendix Figure 1. To confirm that a gap was only observed for Asian restaurants, we compared the synthetic controls and actual numbers of citations for other cuisines, which is commonly referred to as a placebo test. Figure 4 shows the synthetic controls and actual citation numbers for Italian, Mexican, Latin, and Asian cuisines. From Figure 4, we can see that patterns for other cuisines do not resemble the one for Asian cuisines. The synthetic control for Italian cuisine is parallel with the observed line from across December 2019 to February 2020, while the citations for Mexican and Latin restaurants are lower than their synthetic predictions. To illustrate the gaps between synthetic controls and actual citation numbers for the other cuisines, we turn to Figure 5. Here, we see that only Asian restaurants had more citations in January and February 2020 than predicted. Lines that are close to zero prior to December of 2019 indicate synthetic controls that mirrored closely the actual citation numbers. We were able to find robust synthetic controls for many cuisines, but were unable to calculate robust controls for Caribbean and American cuisines. Yet for Caribbean and American cuisines, the gap between actual and synthetic does not change before and after December 2019.7 In terms of the extent of the gaps for each cuisine and their corresponding synthetic control, Asian cuisine had an increase of 0.66% in citations in February of 2020. For Latin, Italian and Mexican restaurants, the percentages were of −0.29%, −0.73% and −0.69%, respectively. These negative values show that the synthetic control predicted fewer citations for all cuisines, except Asian cuisines. Figure 1. Monthly citations, by cuisine. Figure 2. Cumulative citations, by cuisine. Figure 3. Actual versus synthetic control citations for Asian cuisine. Figure 4. Actual versus synthetic control of number of citations for Asian, Italian, Latin, and Mexican cuisines. Figure 5. Gaps in number of citations for Asian, American, Caribbean, Italian, Latin, and Mexican cuisines (Actual - Synthetic control). Note: Our donor pool for the synthetic controls included American, Caribbean, Latin, Italian and Mexican restaurants since they were the only ones with sufficient citation information during the period of time analyzed. Discussion The premise of this study was to examine whether Asian restaurants received more citations after news of the coronavirus causing COVID-19 spread in the US, which would suggest biases on the part of health inspectors. Our findings show that in January and February 2020, Asian restaurants - and no other types of restaurants - received more citations than would have been otherwise predicted. Many discussions of anti-Asian racism and food in the US, both contemporary and those that reach back hundreds of years, have constructed Asian cuisine as foreign, unclean, and disease-bearing. A symphony of voices, from politicians and business owners to everyday individuals, contribute to these stereotypes. However, there is less evidence that discrimination against Asian food establishments is codified in structures and institutions. In other words, there is no mention of the origins of food in health code laws. This is similar to how many laws in the US do not include language of race/ethnicity or identity. However, scholars have argued that a law or policy is racist if the enactment of it produces racial/ethnic inequalities. For example, Crenshaw’s (1989) work on intersectionality focused on the racial, gender, and class consequences of various laws. Here, the results of this study suggest how health inspectors who subscribe to these stereotypes may perpetuate and exacerbate racism against Asian food establishments. By giving biased citations, health inspectors likely deter customers; however, for Asian restaurants, the effects may be multiplicative, as clientele are likely primed to think that such establishments are already unclean. It is also worthwhile to imagine how biases may have manifested during health inspections after news of the coronavirus causing COVID-19 began in the US. Descriptions in documents describing the health inspection process detail significant interactions between restaurant employees (referred to as “operators”) and health inspectors (New York City Department of Health and Mental Hygiene, 2020). For example, at the end of the inspection, health inspectors are required to “review the results of the sanitary inspection with the operator, explain violations and suggest ways to correct them and improve food safety (New York City Department of Health and Mental Hygiene, 2016).” They also provide “answers to reasonable questions about the inspection and instructions for viewing results...in non-English languages” with the aid of language assistance programs (New York City Department of Health and Mental Hygiene, 2014). Often these conversations involve food service operators explaining how certain violations may be, in fact, in compliance with regulations. These exchanges are particularly important for restaurants that serve food that may be unfamiliar to the health inspector despite their rigorous training. It may be the case that news of the outbreak shaped these interactions; health inspectors may have asked for less explanation of violations from operators of Asian restaurants and spent less time investigating potential violations before giving citations. Our findings shed light on how the current discrimination against Asian restaurants in the US fits into broader discourses of the history of Asian American cuisine and frameworks of immigrant assimilation. Unpacking the theoretical roots of stereotypes of Asian food, and its intersections with other stereotypes of Asian Americans, can reveal how Asian cuisine is both stigmatized as a longstanding ‘other’ and appropriated into the mainstream. The notion that Asian Americans are “forever foreign” aligns well with notions of Asian cuisine as being distinct from ‘American’ cuisine. Yet the trope of Asian Americans as Model Minorities, who excel in science and math, and are obedient and servile, is seemingly antithetical to stereotypes of Asian food. It may be the case that the longstanding stereotype of Asian cuisine as disease-baring simply supersedes notions of the Model Minority, or that both can coexist in the imagination of the racist mainstream. Our study also revealed the potential consequences of biases against Asian cuisine as unclean and unsafe; however, this is not the only type of racism that affects this type of cuisine. Much attention has been paid to how Asian cuisines in the US have been appropriated by mainly white chefs. This appropriation not only asserts power over and ownership of Asian cuisines, but also creates the idea that white appropriators can ‘elevate’ Asian food. This sentiment was found in an advertisement in 1927 from a white-owned Chinese restaurant that boasted that “the dishes are not made by a Chinaman, which only means that the food is cleaner’’ (Barbas, 2003: p.678). A similar sentiment was echoed in April 2019, when a journalist for the New York Times wrote about a white-owned Chinese restaurant called Lucky Lee’s in New York City (Otterman, 2019). The purpose for opening the restaurant was that “...she [the owner] wanted to open up a Chinese restaurant where her and her food-sensitive clients could eat – where the lo-mein wouldn’t make people feel ‘bloated and icky’ the next day, or one where the food wasn’t ‘too oily’ or salty.” Notably, the cuisine was also touted as being MSG-free. Such discrimination and appropriation extend to other Asian cuisines as well (Consul, 2016). For example, in 2016, the renowned American food magazine Bon Appétit not only posted a video that showed a young white chef teaching the proper way of eating Vietnamese pho, which the magazine deemed as “the new ramen,” but also published another white chef’s modified recipe of the Filipinx dessert Halo-Halo. The appropriation of Asian cuisines by the white mainstream also complicates notions of assimilation. Many assimilation theories measure the level of group integration with markers of social distance. However, it is not sufficient to describe Asian cuisines appropriated by the white mainstream as a sign of integration: Asian cuisine is both ‘exotic’ and enticing while simultaneously in need of refining by the white mainstream. We framed our study using theories of discrimination and assimilation, but our findings are not fully described by any one framework. The biased health inspection grades reflect in some ways the centuries of discrimination against Asian cuisines. This construction stands seemingly opposed to the notion of “cultural advantage” that is evoked to explain high Asian American achievement, which is also used to uphold historic notions of anti-Blackness. Even the appropriation of Asian cuisine reflects aspects of both of these theories: whiteness desires Asian cuisine but also advertises its version as “cleaner” and better. It is notable that the direct messaging of an “improvement” on Asian cuisine is often absent in other forms of cultural appropriation in which cultural products are wholly stolen and resold for profit. Instead, appropriated Asian cuisine is explicitly advertised using stereotypes and is still marketed as Asian cuisine. This dynamic complicates assimilation frameworks that describe different pathways of mobility and the preservation (or lack there of) of ethnic capital. The marginalization of Asian cuisine cements its position in the periphery, yet the desire for whiteness to appropriate it while evoking the same racist stereotypes, reflects a process that is more complicated than direct assimilation. This study has limitations. Comparative case studies are widely used in the social sciences but one of their main limitations is the researcher’s own bias when selecting a proper comparison group. In this study, we try to address this issue by using a synthetic control method that relies on the data structure for the selection of a proper comparison group. But synthetic control methods have limitations linked to the availability of data. First, the selection of the synthetic Asian cuisine group is dependent on the available pretreatment covariates, which in our case is limited. Second, the pool of donors is limited by cuisines that have complete information during the period of the analysis. We try to isolate the potential effect of the news of COVID-19 on health inspectors racial bias, but we recognize that our estimates of consistent increases in citations may also be due to other factors associated with the pandemic for which we had no information. However, given our placebo tests’ results, we argue that these factors, whatever they may be, were uniquely detrimental to Asian restaurants. Despite these limitations, the results of this study suggest that anti-Asian sentiments can further racial inequalities that arise from biased enforcement of institutional rules. Future directions of research should further investigate the extent to which Asian restaurants have been devastated by the COVID-19 pandemic. In terms of addressing these inequalities, in order to ensure that Asian restaurant owners have access to “knowledgeable, fair and impartial inspectors who enforce agency rules uniformly” (New York City Department of Health and Mental Hygiene, 2014), it is imperative that health inspectors engage in conversations of racial stereotypes and food. An inspection of public-facing documents found no mention of any type of training that is required of health inspectors (New York City Department of Health and Mental Hygiene, 2014, 2016, 2020). Moving forward, health inspectors should be trained to address their own potential biases, particularly in a context as diverse as New York City. Researchers should also partner with public agencies to continue to utilize data to inform practice. ORCID iD Hua-Yu Sebastian Cherng https://orcid.org/0000-0003-2444-4354 Notes Appendix Table 1. Year over year percent change in restaurant employment. Source: US Bureau of Labor Statistics 2020. Appendix Table 2. Number of jobs in restaurant industry. Source: US Bureau of Labor Statistics 2020. Appendix Table 3. Descriptive statistics of variables used for the synthetic controls. Appendix Figure 1. Actual vs. synthetic control citations for Asian cuisine using a 3-month moving average. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. 1 We distinguish in this manuscript between the coronavirus that causes COVID-19 and the COVID-19 disease as our analyses focused on a time when the term COVID-19 was not yet used. 2 Data pulled from https://www.google.com/url?q=https://data.cityofnewyork.us/Health/Restaurant-Grades/gra9-xbjk&sa=D&ust=1595883211144000&usg=AFQjCNGvu-KtuWNuxdhdQFas64upZNgx4w 3 https://trends.google.com/trends/explore?geo=US&q=coronavirus 4 To illustrate the configuration of the final dataset, we give the example of Mexican cuisine: one row represents Mexican restaurants where the first three columns are the ratings, the number of records, and the cumulative ratings for January 2017, the next three values are the ratings, the number of records and the cumulative ratings of February 2017 and so on until February of 2020. 5 As a robustness check we ran the same analysis for a shortened period of time (starting in January 2019 rather than 2017) to include more cuisines, with a total pool of 16 different cuisines. The results were in the same direction as the ones presented here. 6 As stereotypes of Asian cuisine encompass a number of cuisines with origins in Asia, we consider as Asian any restaurant that is classified as Chinese, Japanese, Asian, Thai, Vietnamese or Korean. We do not include Indian restaurants as we argue, and literature has shown, that stereotypes of Chinese cuisines are often more similar with those of other East and Southeast Asian cuisines than South Asian cuisines. Depending on the number of restaurants on each cuisine, we create a weighted average for scores of our constructed Asian restaurants group. Results for this constructed category of cuisine mirrored those calculated for each individual cuisine. 7 For both American and Caribbean cuisines no conclusions can be drawn regarding their synthetic controls since it assigned a weight of 1 to Italian and Latin cuisines respectively, meaning that their comparison groups are not a weighted average of other cuisines but rather only one cuisine that resembles them the most. ==== Refs References Abadie A Gardeazabal J (2003) The economic costs of conflict: A case study of the Basque Country. American Economic Review 93 (1 ): 113–132. Adams E (2020) Racist Graffiti Scrawled on Michelin-Starred West Village Korean Restaurant. Eater. https://ny.eater.com/2020/4/13/21218921/jeju-noodle-bar-racist-graffiti-harrasment-coronavirus-nyc (April 13, 2020). Alcorn (2020) Coronavirus Anxiety Is Devastating Chinese Businesses in New York City. 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==== Front Nutr Health Nutr Health NAH spnah Nutrition and Health 0260-1060 2047-945X SAGE Publications Sage UK: London, England 36445072 10.1177/02601060221139628 10.1177_02601060221139628 Review Article Unwinding the potentials of vitamin C in COVID-19 and other diseases: An updated review Mehta Nikhil Pokharna Purvi https://orcid.org/0000-0003-2440-5398 Shetty Saritha R Department of Pharmaceutics, 539694 Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management , SVKMs NMIMS. Mumbai, India Saritha R Shetty, Department of Pharmaceutics, Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKMs NMIMS. Mumbai, 400056, India. Email: [email protected] 29 11 2022 29 11 2022 02601060221139628© The Author(s) 2022 2022 SAGE Publications This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Background: The discovery of vitamin C (ascorbic acid) is related to the ancient history of persistent research on the origins of the haemorrhagic disease scurvy. Vitamin C is an important nutrient that aids in a variety of biological and physiological processes. Scientists have been researching the function of vitamin C in the prevention and ailment of sepsis and pneumonia for decades. This has created a potential platform for applying these results to individuals suffering from severe coronavirus infection (COVID-19). Vitamin C's ability to activate and enhance the immune system makes it a promising treatment in the present COVID-19 pandemic. Vitamin C also aids in the activation of vitamin B, the production of certain neurotransmitters, and the transformation of cholesterol into bile acids. Hence, vitamin C is used for the treatment of many diseases. Aim: This review highlights the Vitamin C investigations that are performed by various researchers on patients with COVID 19 infection, the clinical studies and their observations. The authors have additionally updated information on the significance of vitamin C insufficiency, as well as its relevance and involvement in diseases such as cancer, wound healing, iron deficiency anaemia, atherosclerosis and neurodegenerative disorders. Here, we discuss them with the references. Methods: The method used in order to perform literature search was done using SciFinder, PubMed and ScienceDirect. Results: There is a potential role of vitamin C in various diseases including neurodegenerative disorders, COVID-19 and other diseases and the results are highlighted in the review with the help of clinical and preclinical data. Conclusion: More research on vitamin C and the undergoing clinical trials might prove a potential role of vitamin C in protecting the population from current COVID-19 pandemic. Vitamin C scurvy antioxidant neurodegenerative COVID-19 edited-statecorrected-proof typesetterts19 ==== Body pmcIntroduction Micronutrients are essential for the immune system's proper functioning and play an important part in supporting health and nutritional well-being (Cámara et al., 2021). Vitamins are necessary for the human body for several biochemical and physiological activities. Vitamins are categorised according to their solubility: (1) water soluble such as B and C complexes and (2) fat-soluble vitamins A, D, E and K (Chambial et al., 2013). Vitamin C is a water-soluble, thermolabile vitamin that is necessary for human health. Vitamin C is found in two forms in living organisms as ascorbic acid (AA)-reduced form and dehydroascorbic acid-oxidised form (Kocot et al., 2017b). The biosynthesis and catabolism of vitamin C is shown in Figures 1 and 2. Despite rising public concern over the malnutrition of micronutrients in low-income and middle-income countries which leads to bad health, greater morbidity and death rates, the worldwide status and prevalence of vitamin C deficiency has not been fully documented (Rowe and Carr, 2020). Humans like the majority of animals do not possess the enzyme L-gluconolactone oxidase which is accountable for vitamin C production, hence, enough vitamin C intake is critical (Langlois and Lamontagne, 2019). This enzyme's deficiency is the product of a mutation that happened about 40 million years ago (Nishikimi et al., 1994). Many regulatory authorities of the countries have decided to increase the recommended dose of vitamin C intake in their countries as a result of growing evidence that increasing the vitamin C intake improves long-term health outcomes (Dickinson et al., 2009; Khan and Iqbal, 2006). Figure 1. Catabolism of ascorbic acid. Figure 2. Biosynthesis of L-ascorbic acid in animals. Vitamin C is necessary for a variety of physiological activities. It contributes to the production and metabolism of tyrosine, tryptophan and folic acid as well as the hydroxylation of proline, glycine, lysine, carnitine and catecholamines (Chambial et al., 2013). Vitamin C is a required nutrient for human life as well as a cofactor in the production of collagen (Caritá et al., 2020). It makes cholesterol conversion to bile acids easier, lowering blood cholesterol levels. Vitamin C has various applications in chronic diseases. Vitamin C is a potent antioxidant that contributes to the maintenance of good health and illness prevention. Vitamin C also helps to prevent sepsis-related coagulation problems by lowering pro-inflammatory responses, improving the function of the epithelial barrier, improving alveolar fluid clearance and reducing pro-inflammatory responses (Bharara et al., 2016). Patients who are critically ill and who are suffering from sepsis or multiple organ failure do have lower amounts of vitamin C. They require around 20 to 30 times the amount of vitamin C required by the average population (Carr et al., 2017). The review highlights the promising role of vitamin C in various diseases such as COVID-19, central nervous system (CNS) disorder to name a few. The method adopted in order to gather information were from different search engines including SciFinder, Sciencedirect and PubMed. This different search engines were used to find out the appropriate literature regarding vitamin C, COVID-19, CNS disorders and other diseases using keywords like vitamin C deficiency, CNS disorders, cardiovascular diseases, ascorbic acid, immunity, toxicity, mechanism of action and clinical trials. The Introduction section covers the various aspects of vitamin C with citation of 15 articles. The mechanism of action and role of vitamin C in immunity covers the citation of 28 articles. The section of vitamin C and toxicity part covers the citation of 19 articles. The treatment of critically ill patients consists of citation of seven articles. The disease part unravels the role of vitamin C in various diseases contains citation of 91 articles. The nutritional strategies of vitamin C in various drug delivery systems consist of citation of five articles. This review attempts to summarise diverse shreds of evidence in the context of therapeutic roles of vitamin C in various clinical aspects like scurvy, and CNS disorders to name a few. For example, scurvy is a syndrome, caused due to insufficiency of vitamin C for a long time defined by the weakening of the collagenous structure which leads to poor wound healing and impaired immunity (Sunil Kumar et al., 2017). Vitamin C deficiency is also reported in smokers, alcoholics as well as mentally ill patients (Lewis et al., 1998). According to research, vitamin C deficiency is linked to the development of neurological diseases such as Huntington's, Parkinson's, Alzheimer's, multiple sclerosis, and amyotrophic lateral sclerosis, as well as mental disorders like anxiety, depression and schizophrenia (Kocot et al., 2017a). Vitamin C treatment is likely to enhance outcomes in patients with COVID-19 infection, based on findings from clinical trials of people with pneumonia and sepsis, as well as preliminary observational and interventional investigations of patients with COVID-19 infection (Carr and Rowe, 2020). Globally, we observe the presence of low vitamin C in most of the common health conditions. Various studies from basic science to the clinical application of vitamin C have reported to have strong association with vitamin C in acute and chronic conditions. In this article, focus is to discuss the potentials of vitamin C in COVID-19 and many other diseases. Mechanism of action of vitamin C Vitamin C is a co-substrate for a variety of enzymes that are essential for the body's optimal functioning. The mechanism of action of vitamin C is depicted in Figure 3. Vitamin C is predominantly converted to its ionised form ascorbate (ASC) at physiological pH. Because of their poor hydrophobicity, ASC and dehydroascorbate (DHA) have to rely on interactions with the transporter molecules which are anchored in the cell membrane to cross biological membranes. Sodium ascorbate co-transporter 1 (SVCT1) and SVCT2, both are the glycoproteins that are responsible for the transportation of ASC into the cell, which are recognised as SVCTs (Daruwala et al., 1999). SVCT1 is only present within epithelial cells where the amount of ASC transported is more than that of the amount which is required by the cells (Liang et al., 2001). Enterocytes can take up ASC and DHA via the SVCT and glucose transporters (GLUTs), respectively (Malo and Wilson, 2000). Although they have different cellular sites, enterocytes express GLUT1, GLUT2 and GLUT3 (Harris et al., 1992; Helliwell et al., 2000; Mesonero et al., 1994). The apical brush-border membrane contains GLUT3, while the basolateral membrane has GLUT1 and GLUT2 which are located on both sides of the membrane. Vitamin C is thought to enter the bloodstream via endothelial discontinuities and circulate with the blood throughout the body, where it is usually identified as the ASC anion (Wilson, 2005). Both ASC, as well as DHA, are absorbed through the length of the small intestine (duodenum, jejunum and ileum) (Malo and Wilson, 2000; Mellors et al., 1977; Stevenson, 1974). ASC is absorbed differently than glucose, with ASC being faster absorbed in almost the distal segments of the small intestine and absorbed in lesser amounts in proximal portions. The jejunum absorbs DHA better, while the ileum's distal parts absorb only a small quantity (Malo and Wilson, 2000). Several glucose transporters have showed that DHA competes with glucose for transfer (Corpe et al., 2013; Vera et al., 1993). DHA is transported by facilitated diffusion, which allows it to travel along the concentration gradient. DHA is converted to ASC as soon as it crosses through the membrane, therefore, this gradient is maintained (Asard et al., 2005; Hughes and Maton, 1968; Rumsey et al., 2000; Washko et al., 1993). It was established that vitamin C transport is satiable, concentration, energy, temperature, as well as dependent on sodium and is mediated by two distinct components. The blood–brain barrier prevents ASC from entering the brain because it is not permeable to ASC and lacking the SVCT2 expression (Agus et al., 1997; García et al., 2005; Qiao and May, 2008). On the other hand due to the expression of GLUT1, DHA easily passes through the blood–brain barrier (Agus et al., 1997; Farrell et al., 1992). Renal glomeruli filters and remove ASC from the blood and excrete it into the urine (Friedman et al., 1940). Consequently, a considerable part of vitamin C is being reabsorbed throughout proximal tubules, depending on the individual's vitamin C status (Bowers-Komro and McCormick, 1991; Rose, 1986). Figure 3. (a) Humans cannot synthesise vitamin C due to lack of L-gluconolactone oxidase enzyme. Plants can synthesise ascorbate due to presence of L-gluconolactone oxidase enzyme. (b) Mechanism of action of vitamin C and (c) ascorbate molecule is impermeable to blood–brain barrier and lacks SVCT2 expression. ASC: ascorbate; DHA: dehydroascorbate; GLUT: glucose transporter; SVCT: sodium ascorbate co-transporter. Role of vitamin C in immunity The immune system is made up of a complex network of specialised cells, tissues, organs, proteins and chemicals that protect the host from foreign pathogens (Kellie and Al-Mansour, 2017). It is separated into epithelial barriers, as well as cellular and humoral components of innate (non-specific) and acquired (specific immunity) (Kellie and Al-Mansour, 2017). For more than a half century of study and research, vitamin C has been proven to be crucial in various areas of the immune system, most notably immune cell activity. Vitamin C is a water-soluble antioxidant found in extracellular fluid and cell cytoplasm, where it performs a variety of functions, including immune homoeostasis (De la Fuente et al., 2020). As it is quickly utilised during the defensive activities, there is high concentration of vitamin C in leukocytes which gets decreased during infections and stress. Vitamin C supplementation in the form of diet or gram doses has been demonstrated to improve neutrophil chemotactic capacity in healthy volunteers (Bozonet et al., 2015). In another study, the intake of diet rich with vitamin C, vitamin E and others showed improvement in the peritoneal leukocyte activities, restoring their redox equilibrium, and extended the life span of the animals in prematurely aged mice (Alvarado et al., 2006). Vitamin C's anti-histamine impact, according to Johnston et al. (1992), is linked to increased chemotaxis. After phagocytosis and microbial annihilation, neutrophils go into a process called cell apoptosis, which involves planned cell death. This procedure aids macrophages in phagocytosis and evacuation of dead neutrophils from inflammatory sites, allowing inflammation to be resolved faster and tissue damage to be reduced. Vitamin C is thought to prevent the oxidant-sensitive caspase-dependent apoptotic pathway after neutrophil activation. In vitro, macrophages did not phagocyte these vitamin C-deficient neutrophils, and they remained in inflammatory sites in vivo. Furthermore, giving vitamin C to sick animals leads to decrease in the amount of neutrophils in the lungs (Vissers and Wilkie, 2007). Vitamin C deficiency Because of random genetic alterations that happened throughout evolution, humans had lost their capacity to generate ASC in their livers (Nishikimi and Yagi, 1991). The polymorphism of single nucleotide in the SLC23A1 and SLC23A2 genes, which have been associated to human illnesses, are used to study the impact of genetic variants on vitamin C pharmacokinetics (Granger and Eck, 2018; Padayatty and Levine, 2016). Reduced renal reabsorption was determined to be the strongest proof for numerous common single nucleotide polymorphisms (SNPs) of SLC23A1 lowering circulating vitamin C levels (Corpe et al., 2010; Michels et al., 2013; Monteiro-grillo, 1990). Crohn's disease (Amir Shaghaghi et al., 2014), non-Hodgkin lymphoma (Skibola et al., 2008), pre-term birth (Erichsen et al., 2006) and severe periodontitis (De Jong et al., 2014) all have been linked to SLC23A1 polymorphisms. Sodium-dependent transporter 1 (SVCT-1) is the primary regulator of vitamin C absorption, and it is quickly saturated, resulting in low vitamin C absorption per meal (Savini et al., 2008). More vitamin C is lost when AA gets converted to dehydroascorbic acid, which can then be later converted to AA or undergoes further oxidative breakdown (Washko et al., 1993). Several studies have revealed the relationship between the lifestyle-related diseases as well as vitamin C deficiency. In animal research, vitamin C deprivation is linked to reduced brain growth and hippocampal function. The recommended dietary allowance of vitamin C is depicted in Table 1. Table 1. RDAs of vitamin C. Category RDA per day Infants (underage of 6 months) 40 mg Infants (underage of 12 months) 50 mg Children (underage of 8 years) 25 mg Children (underage of 13 years) 45 mg Male(13–17 years) 75 mg Female(13–17 years) 65 mg Adult male 90 mg Adult female 75 mg Pregnant women 85 mg Lactating women 120 mg Smokers Requires 35 mg/day more vitamin C as compared to non-smokers RDA: recommended dietary allowance. Toxicity There are several complaints for vitamin C when it is consumed in high doses, such as diarrhoea and intestinal distension or gas (Ohno et al., 2009). Vitamin C at large levels has also been proven to have the following outcomes; for increase in calcium, iron and manganese urinary excretion (Ohno et al., 2009); to enhance iron absorption; to enhance urinary oxalate or uric acid levels, for a small segment of population; and to change the several standard laboratory values. In individuals with the no prior symptoms or indications of renal impairment, evidence shows that the intravenous vitamin C is unlikely to cause any type of damage (Casciari et al., 2005). The people who are suffering from glucose-6-phosphate insufficiency, the administration of vitamin C intravenously or high oral dose of vitamin C can be a cause of haemolysis (Levine et al., 1999). According to Campell and Jack, one patient died from significant tumour necrosis and haemorrhaging followed by the initial dose of the ASC (intravenous). As a result, it is recommended that treatment should begin with the modest dose and be administered via a gradual drip infusion (Campbell and Jack, 1979). It's important to remember that, when taken in large dosages, vitamin C is a drug with adverse effects, just like any other. In the human body, vitamin C is partially converted to oxalate (Hellman and Burns, 1958). As per Figure 4, vitamin C elevates oxalate levels in urine in a dose-dependent manner, raising concerns about its safety and urinary stone development is a possibility. In the setting of hyperoxaluria, there could be the supersaturation of calcium oxalate which will be resulting in the formation of crystal nuclei in the urine. The crystals can pass swiftly in the healthy individuals but intratubular retention is thought to occur in areas of injured and tubular epithelium that regenerates, it is where the molecules with potential binding capacity of crystal are expressed (Cossey et al., 2013; Verkoelen and Verhulst, 2007). Patients who are suffering from sepsis, higher dose of vitamin C may be beneficial, so there should be a careful risk–benefit analysis of its extended administration in patients who are suffering from COVID-19 as well as kidney dysfunction and it should be carried on case-to-case basis (Fontana et al., 2020). Figure 4. Toxicity of vitamin C. Treatment of critically ill patients The patients who are critically ill, vitamin C insufficiency is prevalent in them, and patients with septic shock having the lowest levels. This is most likely due to the higher metabolism caused along by septic shock's heightened inflammatory response (Carr et al., 2017). The plasma levels of vitamin C regularly play with the scurvy levels in conditions like sepsis, trauma, after major surgery and burn patients, and in any situation caused or characterised by severe systemic oxidative and inflammatory stress (Carr et al., 2017). The increased oxidative stress which is produced by the during critical illness increases antioxidant consumption and decreases antioxidant recycling, especially vitamin C. While other species boost their vitamin C production in response to stress, humans are not able to enhance the serum levels like other stress hormones. So due to this, the insufficiency exists (Langlois and Lamontagne, 2019). Administration of high-dose intravenous (IV) AA reduces demand for fluid, there is occurrence of wound oedema and weight gain in the initial phase of burn injury while simultaneously improving renal and lung function (Tanaka et al., 2000). Patients in the intensive care unit (ICU) are frequently given enteral nutrition or parenteral nutrition if they cannot get enough enteral nutrition (Yamazaki et al., 2011). The presence or lack of a working intestine, as well as the patient's haemodynamic condition, dictate the route of feeding for the patient – enteral or parenteral (Chan et al., 1999) In the case of patient having fluid restriction and organ failure, nutritional requirements are usually altered (Chan et al., 1999). Enteral and parenteral nutrition, which provide 100 to 200 mg of vitamin C per day on average, are insufficient for critically ill patients. A regular intake of vitamin C of at least 2 to 3 g is likely to be required for these patients (Carr et al., 2017). The effects of AA supplementation were first discovered in a number of animal models of sepsis and ischaemia reperfusion. The therapy of AA in a sepsis murine model was responsible for enhanced microvascular perfusion, improved blood flow and blood pressure with dosages ranging from 10 to 200 mg/kg provided either as a preventative measure or after an injury (Secor et al., 2010; Tyml et al., 2008). In septic mice, the AA infusion was responsible for enhancing the blood flow of capillary, the barrier function of microvascular and arteriolar response to vasoconstrictors (Armour et al., 2001; Wu et al., 2004). So, the supplementation of vitamin C is useful for the critically ill patients. One study revealed that despite receiving conventional ICU nutritional care, approximately 70% of the critically ill patients were suffering from hypovitaminosis C, with a substantial percentage of patients having the vitamin C insufficiency (32%). Patients suffering with septic shock cause further vitamin C depletion in the blood, with approximately 90% of patients having hypovitaminosis C and 40% having hypovitaminosis B. Vitamin C deficiency is likely due to increased inflammatory pathway activation in response to infection, as demonstrated by higher C-reactive protein levels in these patients (Carr et al., 2017). Diseases The role of vitamin C in various diseases is depicted in Figure 5. Figure 5. Various pathways and mechanism through which vitamin C acts in various diseases. 5HT: 5-hydroxytryptamine; cGMP: cyclic guanosine monophosphate; DOPA: dihydroxyphenylalanine; GABA: gamma amino butyric acid; LDL: low-density lipoprotein. COVID-19 COVID-19 is been reported as one of the most serious public health threats (Zhang et al., 2021). This outbreak began with an animal to human transmission, and severe atypical pneumonia was the immediate cause of mortality (Muscogiuri et al., 2020). The COVID-19 pandemic has caused widespread alarm and action, posing a significant risk to public health. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19 results in common cold and has main impact on the respiratory tract and human immune system (Callender et al., 2020). Patients who are aged as well as those with pre-existing respiratory, cardiac or diabetic disease seems to be at a greater risk of rapidly progressing to the severe and critical stages (Weiss and Murdoch, 2020). The search for a coronavirus-specific drug has led to a dead end, with most of the studied medications, including corticosteroids, appearing to be beneficial to patients in addition to normal therapy. Despite the encouraging published results, the vaccination process is taking its time. As a result, there is a pressing need to explore new avenues for potential treatments. When considering the possible function of vitamin C against the novel coronavirus, there are various animal studies that are relevant (Hemilä, 2017). The clinical trials data of vitamin C in COVID-19 is given in Table 2. Vitamin C aids in the downregulation of cytokines, protects the endothelium from oxidative stress, and has significant role in tissue healing, which is especially crucial during COVID-19's critical phase (Holford et al., 2020). Vitamin C also prevents the production of inflammatory cytokines. An immunological hyper-reaction, involving Interleukin-6 (IL-6) and endothelin-1, appears to be driving the progression of COVID-19 pneumonia to respiratory failure. Patients infected with COVID-19 should benefit from vitamin C, as vitamin C has been given the importance of IL-6 in severe COVID-19 and vitamin C's capacity to prevent IL-6 from increasing in a variety of (pro)inflammatory disorders (Mrityunjaya et al., 2020). Vitamin C has long been thought to help sepsis patients retain normal lung function by improving broncho-alveolar function, alveolar fluid clearance, and reduction of neutrophil sequestration. A higher amount of IV vitamin C (10–20 g/day) was successfully administered in 50 patients with severe COVID-19 infection in a recent Chinese study, and the oxygenation index was stabilised, and all of the patients were cured and discharged after a defined length of time (Farjana et al., 2020). Vitamin C studies delayed and eased upper respiratory tract infections that occurred over the course of vitamin C treatment, according to a meta-analysis of 29 randomised trials including 11,306 patients (Hemilä and Chalker, 2020). In addition, studies have shown that combining antiviral, antioxidant and immunomodulatory effects of vitamin C and quercetin are synergistic. As a result, incorporating vitamin C into a dietary supplement can aid to relax and boost the immune system while also acting as an anti-inflammatory and antioxidant against COVID-19 infection (Colunga Biancatelli et al., 2020a). High-dose vitamin C treatment can help patients with viral pneumonia and acute respiratory distress syndrome who have a severe COVID-19 infection by lowering inflammation and pathogen infectivity and virulence, increasing immune defence, minimising tissue and organ damage, and improving overall prognosis (Wessels et al., 2020). Supplementing with vitamin C can drastically minimise the need for corticosteroids, antibiotics and antiviral medications. Vitamin C enhances the innate immune response, which aids in the prevention of viral infections. Table 2. List of clinical trials for intervention of vitamin C in COVID-19 infections. Serial number Title Country Participants Interventions NCT number Status 1 Coronavirus 2019 (COVID-19) – using ascorbic acid and zinc supplementation (COVID A to Z) The United States 214 Ascorbic acid and zinc gluconate NCT04342728 Completed 2 Administration of intravenous vitamin C in novel coronavirus infection (COVID-19) and decreased oxygenation (AVoCaDO) The United States 20 50 mg/kg L-ascorbic acid of infusion given every 6 h for 4 days (16 total doses) NCT04357782 Completed 3 High-dose vitamin C treatment in critically ill patients with COVID-19 Turkey 78 The daily administration of 6 g of vitamin C intravenously in 4 equal doses every 6 h NCT04710329 Completed 4 Lessening organ dysfunction with vitamin – COVID-19 (lovit-covid) Canada 800 Vitamin C Control: saline or 5% dextrose solution NCT04401150 Recruiting 5 The effect of melatonin and vitamin C on COVID-19 The United States 150 (1) 10 mg melatonin, at bedtime (2) 1000 mg vitamin C, at bedtime Placebo at bedtime NCT04530539 Recruiting 6 The study of quadruple therapy zinc, quercetin, bromelain and vitamin C on the clinical outcomes of patients infected with COVID-19 Saudi Arabia 60 1) Zinc:50 mg 2) Quercetin:500 mg 3) Bromelin:500 mg Vitamin C:1000 mg NCT04468139 Recruiting 7 Use of ascorbic acid in patients with COVID-19 Italy 500 10 g* of vitamin C intravenously in addition to conventional therapy NCT04323514 Recruiting 8 Safety study of early infusion of vitamin C for the treatment of novel coronavirus acute lung injury (SAVE EVICT CORONA-ALI) The United States 60 50 mg/kg intravenous vitamin C infusion every 6 h for up to 96 h NCT04344184 Recruiting 9 A study of hydroxychloroquine, vitamin C, vitamin D and zinc for the prevention of COVID-19 (HELPCOVID19) The United States 600 Experimental: hydroxychloroquine, vitamin C, vitamin D, zinc Placebo: vitamin C, vitamin D, zinc NCT04335084 Recruiting 10 Role of mega dose of vitamin C in critical patients with COVID-19 infection Pakistan 15 Experimental: the dose was 30 g a day (10 g TDS) for 2 days with standard treatment Placebo: distilled water in the same dose with same standard treatment NCT04682574 Recruiting 11 International alliance study of therapies to prevent progression of COVID-19 Australia 200 Vitamin C, Hydroxychloroquine Azithromycin Zinc citrate Vitamin D3, vitamin B12 NCT04395768 Recruiting 12 Vitamin C infusion for the treatment of severe 2019-ncov-infected pneumonia China 56 Experimental: 12g vitamin C was infused in the experimental group twice a day for 7 days Placebo: 50 ml sterile water for injection will be infused in the placebo comparator group twice a day for 7 days NCT04264533 Terminated NCT: National Clinical Trial number. Mechanism of action of vitamin C in COVID-19 With reference to COVID-19, vitamin C has various roles including downregulation of cytokines, endothelium protection from oxidative stress as well as a critical role in tissue repair (May and Harrison, 2013; May and Qu, 2011). The oxidative stress and genes critical to the inflammatory response including IL-1, IL-8, tumour necrosis factor alpha, as well as intercellular adhesion molecule 1 is mediated through the nuclear factor kappa B (NF-κB) pathway activation (Sen and Packer, 1996). Vitamin C causes reduction in the oxidative stress as well as inflammation through activation of NF-κB pathway (Chen et al., 2014). In COVID-19, there is downregulation of interferon type 1 expression (Blanco-Melo et al., 2020). Vitamin C is responsible to upregulate the expression of these important enzymes which acts as a defence against the infection (Colunga Biancatelli et al., 2020b). The concentration of vitamin C is much higher in adrenal glands than in other body organs. Vitamin C is released from the adrenal glands in response to stress and increasing the plasma level of vitamin C (Padayatty et al., 2007). Vitamin C is also responsible for the cortisol production which eventually increases the anti-inflammatory response and cytoprotective effects of glucocorticoids (Barabutis et al., 2017; Kodama et al., 1994). Clinical trials and nutritional status of vitamin C in COVID-19 The main reason responsible for lung injury in people suffering from COVID-19 is the oxidative stress and the free radicals which are generated by the immune system of the body. The antioxidant status of the patients can be improved by administration of vitamin C (“The Financial Express,” 2020). There are number of studies conducted preclinically on animals which has provided the benefits and proved the efficiency of vitamin C in order to treat infections (Hemilä, 2017). A pilot study was conducted on critically ill patients suffering from sepsis. The patients were administered with IV vitamin C (50 and 200 mg/kg). The results showed that the patients administered with IV vitamin C had lower levels of pro-inflammatory markers and lower scores of Sequential Organ Failure Assessment as compared to the patients administered with placebo (Fowler et al., 2014). Another study titled ‘COVID A to Z’ showed a statistically significant difference in the recovery rate between the vitamin C and usual care arms (P value = 0.025). The time to reduce the symptoms by 50% was 1.2 days less in vitamin C arm in comparison with the usual arm (Hemilä et al., 2021). There are various other studies which do highlight the combination of vitamin C, hydrocortisone and thiamine which had efficient and fruitful results in sepsis (Fujii et al., 2020). There are number of clinical trials that have been announced since the start of the COVID-19 pandemic, so that the vitamin C's therapeutic benefits can be evaluated. The clinical trials are registered under National Institute of Health Clinical Trials in order to evaluate the efficiency of vitamin C in COVID-19 as alone treatment or in combination with other supplements. The randomised controlled trial was conducted in patients suffering from septic shock and the results were compared with the combination of vitamin C (6 g/day), hydrocortisone (200 mg/day) and thiamine (400 mg/day) to hydrocortisone alone. The results revealed that the combination therapy did not affect the duration of the attack but it decreases the Sequential Organ Failure Assessment scores (Fujii et al., 2020). This signifies that vitamin C has a major role in prevention and management of COVID-19. The dose of vitamin C for healthy individuals is 200 mg/day. There is increase in the requirement of dose during infections, that is, 1 to 2 g/day (Abobaker et al., 2020). The clinical trials ongoing may provide clear picture as well as evidence for the role of vitamin C in COVID-19. Central nervous system Vitamin C is useful for treatment of wide range of neurological and mental diseases as an adjuvant. High quantities of unsaturated fatty acids and a rapid rate of cell metabolism make the brain a particularly sensitive organ to oxidative stress and free radical activity. As an antioxidant, AA functions by scavenging reactive oxygen and nitrogen species produced during normal cell metabolism. As per Figure 6, vitamin C participates in CNS signal transduction via neurotransmitters, which is a non-antioxidant action. Vitamin C is hypothesised to play a part in this process by affecting neurotransmitter binding to receptors and controlling their release. Furthermore, several neurotransmitters, including catecholamines, require vitamin C as a cofactor in their production. Figure 6. Non-oxidant action of vitamin C in central nervous system (CNS). Anxiety Anxiety is a natural response that detects and prepares a person for an actual or possible threat (McNaughton and Corr, 2004). Glutamate is an excitatory neurotransmitter that plays a role in anxiety-related behaviour modulation. An elevation in glutamate signalling in the brain is linked to anxious behaviour, whereas a decrease in glutamate signalling reduces anxiety-related behaviour (Swanson et al., 2005). Patients who suffer from anxious types of depression seem to be more prone to experience depression symptoms that are severe, such as exhaustion, guilt and feelings of worthlessness (Goldberg et al., 2014). Furthermore, at a physiological level, AA shields neuronal cells against glutamate-induced excitotoxicity. The importance of internal dietary AA levels for mental wellness is critical. Dianae B. Fraga reported that in diverse animal models of anxiety, the single dose's effects of diazepam (positive control), AA or ketamine, were studied. Mice were given ketamine, AA or diazepam before mice being tested in the open field test, elevated plus maze test, marble burying test and light–dark preference test (Fraga et al., 2018). The anxiogenic and anxiolytic effects of various medications are commonly assessed using the light–dark test (Bourin and Hascoët, 2003; Gallo et al., 2014). The hypothesis behind this study is that mice avoid brightly lit surroundings and engage in spontaneously exploratory action in relation to mild stressors such as novelty and light. In the light–dark preference test, in the light region vitamin C increased latency time and duration, but had no influence on mice's performance in the marble burying test. The findings imply that AA combined with ketamine may have an anxiolytic effect, opening up a new therapy option for anxiety disorders (Fraga et al., 2018). Through cyclic guanosine monophosphate (cGMP)-mediated signalling, nitric oxide (NO) has also been shown to behave as a post-synaptic or pre-synaptic retro-grade messenger (Wang et al., 2005). The phosphorylation of synaptophysin, the protein that induces glutamate release by fusing glutamate-containing granules to the membrane of pre-synaptic nerve terminals, activates the cGMP-dependent protein kinase G pathway (Wang et al., 2005). According to Vaibhav et al., AA, an NO signalling pathway and N-methyl-D-aspartic acid receptor modulator, resulted in an anxiolytic effect in mice. When mice were administered with AA, the amount of nitrite in their brains was reduced. Anxiogenic reactions were generated by pre-treatment with NO donor and sildenafil which were followed by AA treatments (Walia et al., 2019). As a result, modulation of NO signalling was implicated in the action of AA as an anxiolytic in mice. An inherent vitamin C deficiency in SMP30/GNL knockout mice resulted in anxiety and anorexia, according to Koizumi et al. (2016). Depression According to World Health Organization, there are millions of people worldwide who are affected by depression. Dopamine, noradrenaline and serotonin neurotransmission disturbances may all play an important role in the illness. Depression is defined by loss of interest and persistent sorrow in the activities you enjoy the most, in addition, for at least 2 weeks, you will be unable to carry out daily activities. In addition, those who suffer from depression are likely to have multiple symptoms such as: loss of energy; changes in the appetite; change of sleeping pattern; worry; poor focus; lack of direction; restlessness; thoughts of worthlessness, regret or hopelessness; a feeling of unworthy, remorse and thoughts of suicide and self-harm (Estimates, 2014). Long-term treatment with a multivitamin that included enough vitamin C enhanced mood in persons with low plasma vitamin C levels (Helmeste and Tang, 1998). Exhaustion and psychosomatic idiosyncrasy can result from a vitamin C deficit (Vahdat Shariatpanaahi et al., 2007). The stimulation of 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2A/2C receptors at post-synaptic sites has also been connected to AA's antidepressant-like activity (Binfaré et al., 2009). Furthermore, a 3-month AA treatment promoted remission of depression symptoms in 50 depressive patients who were experiencing acute depression. Amr et al. (2013) and co-workers hypothesised that AA's antidepressant activity benefits are unrelated to its antioxidant characteristics. Furthermore, investigations have shown that depressed patients have reduced levels of inhibitory neurotransmitter amino butyric acid in their brains, as well as changes in the subunit makeup of its major receptor (gamma amino butyric acid (GABA)-A) (Luscher et al., 2011). Preclinical evidence suggests that current depression medication may help to compensate for GABAnergic deficits (Luscher et al., 2011). AA's antidepressant-like effect may be due to GABA-A receptor activation and maybe the GABA-B receptor inhibition (Rosa et al., 2016). When taken with AA at a sub-effective dose, muscimol (GABA-A agonist) produced an antidepressant-like synergistic effect, but baclofen (GABA-B agonist) reduced AA's antidepressant-like effect (Rosa et al., 2016). Another study conducted in New Zealand in young adult males also suggested the possible relation with the vitamin C status as well as the mood state. The study is cross-sectional, and evidence of causality will require further well-conducted intervention studies. There are several physiological reasons for vitamin C's good influence on mood, including its role in brain function and homoeostasis (Pullar et al., 2018). Antioxidant vitamin intake, according to one study, could be a good dietary choice for preventing depression and anxiety disorders (Farhadnejad et al., 2020). Parkinsonism Parkinsonism is a neurodegenerative disease which is marked by tremor, stiffness and bradykinesia in the muscles. Oxidative stress is assumed being the cause of neurodegenerative diseases like Parkinsonism (Niedzielska et al., 2016). Furthermore, this ailment is hyposmia, sleep problems and depression are all linked to non-motor symptoms (Massano and Bhatia, 2012; Schapira et al., 2017). Parkinsonism is marked by the degeneration of dopaminergic neurons in substantia nigra and formation of Lewy bodies within the brain (DeMaagd and Philip, 2015). In investigations on humans, vitamin C deficiency has already been linked with Parkinson's disease (Medeiros et al., 2016; Quiroga et al., 2014; Senarath Yapa, 1992). Vitamin C has been studied extensively in a variety of Parkinson's disease in vivo and in vitro models, and it has been shown to have beneficial effects (Kocot et al., 2017b). The Drosophila model was utilised by Huynh Man Anh et al. to study the duration and dose-dependent effects of vitamin C therapy on Parkinsonism symptoms caused by the knockdown of Drosophila ubiquitin C-terminal hydrolase. In this model, they discovered that vitamin C was found to improve Parkinsonism-like characteristics, which are characterised by neurodegeneration as well as locomotive abnormalities (Man Anh et al., 2019). On the contrary, high dose of AA had deleterious impacts on eating behaviour and motor capacity. Long-term vitamin C medication, for example, may protect against dopaminergic neuron degeneration. Vitamin C can boost dihydroxyphenylalanine synthesis (DOPA). Seitz et al. discovered that incubating the cell line of human neuroblastoma with AA resulted in more production of DOPA in dose-dependent manner. Furthermore, after treatments with AA, tyrosine hydroxylase gene expression increased thrice (Seitz et al., 1998). Alzheimer's disease Vitamin C insufficiency has been linked to the neuropathology of Alzheimer's disease as well as normal aging. The neuropathology of Alzheimer's disease is characterised by oxidative stress (Praticò et al., 2002; Praticò and Sung, 2004). In neurodegeneration, disturbance of AA homoeostasis has been well-documented (Covarrubias-Pinto et al., 2015). In the CNS, AA is an important antioxidant that is produced by glial cells within the synaptic cleft and then as an antioxidant defence, neurons absorb it to keep synaptic function as well as neuronal metabolism and in check. The astrocytes and neurons interaction has been discovered to be a key mechanism for AA recycling, as well as a participant in the brain's antioxidative defence (Dringen et al., 1999). Parenteral administration of AA has nootropic characteristics in amyloid protein precursor/presenilin-1 transgenic mice, according to a prior study, without affecting the Alzheimer's like traits of acetylcholinesterase activity, oxidative stress or plaque deposition (Harrison et al., 2009). Dixit et al. (2015) reported that the results of their study conducted imply that as age increases, vitamin C deficiency may accelerate the start of oxidative stress in the brain, as well as play a key role in amyloid production, oligomerization and deposition. Javier et al. reported that during the early asymptomatic phases of Alzheimer's disease, the cerebrovascular response to hypoxia can be altered by vitamin C preventing beta secretase 1 production and amyloid β deposition while lowering the thickness of the cerebrovascular basement membrane (Frontiñán-Rubio et al., 2018). Schizophrenia Schizophrenia is a life-altering, chronic illness that causes functional impairment in the social, cognitive and emotional arenas. Positive symptoms (hallucinations and delusions) co-exist with negative symptoms (emotional blunting and apathy) and cognitive impairment (Brown and Roffman, 2014). Negative symptoms and cognitive deficiencies, on the other hand, do not usually react to antipsychotic therapy (Brown and Roffman, 2014). The case reported by Reuven Sandyk was about a patient suffering from schizophrenia since 17. The patient has been profoundly psychotic and has only partially responded to numerous psychotropic medicines. He has had practically total remission of psychotic symptoms since starting vitamin C therapy in 1987, and he is now able to work part-time. Even his relapse in early 1989 was much milder than previous relapses. Finally, his capacity to work part-time after being released from the hospital attests to the significant improvement in his mental state. This demonstrates the usefulness of vitamin C when used as a supplement in the treatment of a subset of persistent schizophrenia patients who do not have responded to the conventional treatments (Sandyk and Kanofsky, 1993). Dakhale et al. also reported that vitamin C supplementation when combined with atypical antipsychotics is responsible for the decrease in oxidative stress and improves schizophrenia outcomes. Forty-five patients took part in the study having schizophrenia. It was an 8-week, non-crossover, prospective, double-blinded trial. The primary finding of this study is that vitamin C supplementation with atypical antipsychotics resulted in a substantial drop in serum malondialdehyde and rise in plasma levels of AA in comparison to placebo with atypical antipsychotics (Dakhale et al., 2005). Wound healing AA is responsible for every aspect of wound healing including cellular apoptosis, synthesis of collagen, collagen synthesis and bone formation as well as antioxidant activities (Anderson, 2005). When wounding takes place, there is a drop in tissue and plasma levels. By using high-dose vitamin C supplements early in process of healing appears to be advantageous, especially if people who are already scorbutic (Long et al., 2003). Major difficulties caused by AA deficiency have traditionally been noted in scurvy patients; however, these defining traits are not always present in today's patients. AA deficit is more likely in patients having vascular disease, the elderly, pregnant women, smokers, drug abusers and persons who are underweight. According to the case study, oral vitamin C supplementation increased wound healing in those who had previously suffered from the deficit. The amount of AA in the blood was below than the standard value, that is, 25 µmol/L in 65 of 180 patients throughout a 21-month period (36%). Patients and their clinicians noticed a significant and rapid recovery of vast and intricate wounds after starting supplementation (1000 mg/day) (Bikker et al., 2016). Because exact AA levels can only be determined through laboratory testing, doctors must take a pragmatic approach and be on the watch for any visible indicators of deficit in patients with a case of acute chronic wounds (Moores, 2013). Iron deficiency anaemia Iron deficiency anaemia is characterised by a decrease in red blood cells formation as a result of a total body iron deficiency (Brugnara, 2002). It is critical for the structure as well as the function of cells. Dietary iron is made up of both haeme and non-haeme iron. The divalent metal transporter-1 transports non-haeme ferrous iron into enterocytes (Frazer and Anderson, 2005). The basolateral membrane protein ferroportin is responsible for iron exiting enterocytes (Himmelfarb, 2007). Fe3+ is transported by transferrin from the bloodstream to haematopoietic organs and other tissues, including the liver (Graham et al., 2007). Macrophages in the reticuloendothelial system destroy aged red blood cells (Himmelfarb, 2007). Ferroportin is a protein that is responsible for release of iron from macrophages into the bloodstream. Iron is absorbed mostly in the region of duodenum and upper jejunum of intestine, where Fe2+ can be transferred into mucosal epithelial cells of small intestine. AA has shown to boost iron availability and availability from non-haeme iron sources (Hallberg, 1981). Vitamin C reduces synthesis of hepcidin and gives protection against anaemia caused by iron deficiency through altering the erythropoietin receptor in HepG2 cells as well as altering the bioavailability of iron (Chiu et al., 2012). Furthermore, ASC taken orally had a significant positive impact on haematological markers in the anaemic haemodialysis patients (Sirover et al., 2008). Darius et al. reported the ASC's effect on cellular iron absorption from Fe2-Tf and they demonstrate that the physiological ASC concentrations considerably stimulate Tf-dependent iron uptake by variety of human cells (Lane et al., 2013). On contrary, randomised clinical trial reported that the findings call into question the recommendation that vitamin C supplements be taken in conjunction with iron orally in order to boost efficacy and accelerate anaemia recovery. In a moderately acidic environment, iron is absorbed as ferrous salt (Fe2+). Vitamin C is thought to help in calcium absorption. However, based on the findings of this clinical investigation, vitamin C may not be as beneficial as previously assumed (Li et al., 2020). Atherosclerosis In the pathophysiology of vascular diseases, the endothelium plays a critical role (Iaccarino et al., 2004; Ross, 1986). Atherosclerosis is a degenerative condition of disease in which the major, as well as medium-sized arteries, are affected which is marked by loss of flexibility of the arterial wall and narrowing of the lumen (Rafieian-Kopaei et al., 2014). Localised thickening of the tunica intima causes the lumen to narrow. Myocardial infarction and ischaemic stroke are the most common symptoms of atherosclerosis. The antioxidant properties of vitamin C give an idea that it may help prevent cardiovascular disease (Moser and Chun, 2016). According to epidemiological data, antioxidant content in fruits and vegetables lowers the incidence of cardiovascular disease (CVD), which could be explained by the antioxidant's role in preventing oxidative alterations to low-density lipoprotein (LDL) (Salvayre et al., 2016). Scavenger receptors recognise oxidised LDL and integrate it into plaque (Li and Mehta, 2005). As a result, vitamin C's ability to prevent LDL oxidation may help to prevent atherosclerosis, potentially lowering CVD risk. Endothelial NO generation has been demonstrated to increase with AA, leading to increased vasodilation and lower blood pressure (d’Uscio et al., 2003). Furthermore, vitamin C may help make plaques more stable if atherosclerosis has been established by preventing vascular smooth muscle cell death (Siow et al., 1999). Cancer The debate over vitamin C's anti-cancer capabilities and its usefulness in palliative care has raged on very much (Zasowska-Nowak et al., 2021). Because of the development of resistance by cancer cells as well as toxicity, most chemotherapeutic drugs have restrictions on their usage (Ng et al., 2015). At high therapeutic levels, vitamin C has been demonstrated to have a particular influence on the cytotoxicity of many types of cancer cells (Pawlowska et al., 2019). There are number of processes through which vitamin C prevent and cure cancer. The processes are: boosting the immune system, increasing collagen production, blocking enzymes to prevent metastasis (spreading), prevention of cancer-causing viruses, the deficit of vitamin C in the patients suffering from cancer is treated, post-surgery wound healing in people with cancer improves chemotherapy efficiency, lowering chemotherapy toxicity, avoiding free radical damage and neutralising certain carcinogens (Chambial et al., 2013). Vitamin C's cytotoxic effects have been attributed to its capacity for the generation of reactive oxygen species (ROS) that has been linked to activating p53 which is tumour suppressor protein and the master regulator of DNA repair and death (Liu et al., 2015). Complementary and alternative medicine practitioners routinely administer vitamin C intravenously to treat infections, cancer and tiredness. Vitamin C's bioavailability and anti-cancer properties are known to be influenced by how it is administered in the body. Because high-dose IV has been already demonstrated in various multiple clinical studies to enhance the quality of life and health, it may be considered a palliative treatment (Zasowska-Nowak et al., 2021). SVCT1 does not appear to be important in cancer. According to various studies, SVCT2 plays a significant role in malignancies. The first discovered that when compared to normal cells, breast tumours exhibit greater levels of SVCT2 expression (Roa et al., 2020). Overexpression of this transporter enhanced chemosensitivity to the high dose of AA, resulting in to increase in ROS production as well as cell death (Hong et al., 2013). Cancer cells, on the other hand, become resistant to the therapy when siRNA targeting SVCT2 is used. As a result, SVCT2 may have a role in ASC-mediated cancer cell killing (Hong et al., 2013). Table 3 represents the list of clinical trials for interventions of AA in other disease conditions. Table 3. List of clinical trials for interventions of vitamin C (ascorbic acid) in other health conditions. Serial number Title Study type Population Interventions NCT number Status Key findings 1 Micronutrient to prevent noise-induced hearing loss Interventional study 70 participants Beta-carotene, vitamins C and E, magnesium NCT00808470 Completed Supplement had no effect on noise-induced changes in hearing 2 Quantitative in vivo biomarkers of oxidative stress in diabetes Interventional study 42 participants Vitamin C NCT00845130 Completed 3 Prophylaxis to reduce post-operative atrial fibrillation in cardiac surgery Interventional study 304 participants Beta blockers, amiodarone, ascorbic acid NCT00953212 Completed 4 ACTS trial Interventional study 205 participants Vitamin C, vitamin B1, hydrocortisone NCT03389555 Completed The combination did not show significant reduction in the SOFA score 5 Intravenous vitamin C in the treatment of viral infection especially in the treatment of shingles Interventional study 68 participants IV vitamin C NCT00921934 Completed The results showed that there was decline in the pain score which provide evidence that IV vitamin C is beneficial in treatment of viral infection 6 A study of vitamin C in the treatment of liver cancer to determine if it is safe and effective Interventional study 5 participants Ascorbic acid & sorafenib NCT01754987 Completed The safety data indicates no toxicity 7 A phase 2 pharmacokinetic study of 6r-bh4 alone or 6r-bh4 with vitamin C in subjects with endothelial dysfunction Interventional study 52 participants Sapropterin dihydrochloride and vitamin C NCT00532844 Completed 8 The effect of high-dose vitamin C on the liver function in chronic hepatitis patients Interventional study 10 participants High-dose vitamin C NCT01413360 Completed 9 Liposomal encapsulated vitamin C in complex regional pain syndrome Interventional study 66 participants Liposomal vitamin C versus vitamin C versus placebo NCT04204200 Completed There was no benefit to patients and no significant difference in time to heal the fracture 10 Ascorbic acid (vitamin C) infusion in human sepsis Interventional study 24 participants Ascorbic acid NCT01434121 Completed Ascorbic acid significantly reduced the proinflammatory biomarkers C-reactive protein and procalcitonin 11 Vit C-AF Interventional study 20 participants Vitamin C NCT03148236 Completed High dose of vitamin C is safe and well tolerated at the time of AF ablation and is associated with a blunted rise in C-reactive protein 12 CITRIS-ALI Interventional study 170 participants Infusion: vitamin C NCT02106975 Completed The infusion of ascorbic acid did not significantly improve the organ dysfunction scores 13 Study of high-dose vitamin C on outcome in cardiac surgery patients Interventional study 57 participants Drug: ascorbic acid Placebo: 5% Dextrose water or normal saline NCT01167569 Completed 14 VCSIP of infant lung infection Interventional study 252 participants Vitamin C NCT01723696 Completed 15 Does vitamin C reduce finger stiffness after distal radius fractures? Interventional study 126 participants 500 mg vitamin C, 1 pill per day for 6 weeks versus 1 placebo pill for 6 weeks NCT02216812 Completed There was no difference in the finger stiffness 16 VICTAS Interventional study 501 participants Vitamin C, thiamine hydrocortisone NCT03509350 Completed 17 Vitamin C supplementation intervention Interventional study 36 participants Vitamin C 500 mg versus vitamin C 1000 mg versus placebo NCT04036110 Completed Patients with CHF acute intravenous administration of vitamin C enhances platelet responsiveness to the anti-aggregatory effects of NO donors and improves endothelial function ACTS: Ascorbic acid, corticosteroids and thiamine in sepsis; AF: atrial fibrillation, CHF: congestive heart failure, CITRIS-ALI: vitamin C infusion for treatment is sepsis-induced acute lung injury; IV: intravenous; NCT: National Clinical Trial number; NO: nitric oxide; SOFA: Sequential Organ Failure Assessment; VCSIP: vitamin C to decrease effects of smoking in pregnancy; VICTAS: vitamin C, thiamine and steroids in sepsis; Vit C-AF: vitamin C in atrial fibrillation ablation. Nutritional strategies for delivery of vitamin C The hydrophilic character as well as the instability of vitamin C has led to its less utilisation. But, during recent years there are various types of studies conducted in order to improve the therapeutic outcome using vitamin C which includes nanoparticles and microparticles (Jang and Lee, 2008), liposomes (Hope et al., 1986), micelles (Sutradhar and Amin, 2014), microemulsion (Sawant et al., 2011) and orally disintegrating films (Garcia et al., 2018). This not only improved the therapeutic efficiency of vitamin C but also the drug targeting (Caritá et al., 2020). Future perspective AA is a vital and necessary component of human health. In humans, it is required for a number of physiological activities. Vitamin C is also been found to aid in the transportation of therapeutic agents in the body. Vitamin C has indeed been linked to a number of health advantages which includes antioxidant, anti-atherogenic, anti-carcinogenic and treatment of various neurodegenerative disorders. In recent decades, AA has been thoroughly studied and discovered to be an excellent antioxidant and cofactor in the synthesis of collagen. In preclinical and preliminary trials, vitamin C has been shown to increase organ function amid oxidative stress and inflammation. There are a certain number of ways in which AA reduces inflammation as well as oxidative damage has sparked a surge in interest in its medicinal use. The biggest limitation of the clinical trials of AA is that the study is not conducted on a large number of patients. Smaller groups of studies are conducted which cannot produce the results which can prove something concrete. The patients who are at high risk of COVID-19 suffering from vitamin C deficiency should be encouraged to start supplementation of vitamin C. There are several preclinical and clinical studies reported which highlights the role of vitamin C in COVID-19. There is more data required from the clinical trials which would prove the efficiency of vitamin C in COVID-19. The pending COVID-19 clinical trials may prove the potential benefits of vitamin C in COVID-19. Conclusion There is high prevalence of deficiency of vitamin C worldwide. Vitamin C may not be the first-line treatment, but may be considered as a safe, adjuvant as well as inexpensive nutritional supplement therapy for many diseases as mentioned in this review article. The public health sector should take an account of vitamin C deficiency and should do necessary things for the public in order to reduce the patients suffering from deficiency of vitamin C. Supplemental Material sj-png-1-nah-10.1177_02601060221139628 - Supplemental material for Unwinding the potentials of vitamin C in COVID-19 and other diseases: An updated review Click here for additional data file. Supplemental material, sj-png-1-nah-10.1177_02601060221139628 for Unwinding the potentials of vitamin C in COVID-19 and other diseases: An updated review by Nikhil Mehta, Purvi Pokharna and Saritha R Shetty in Nutrition and Health Acknowledgements All the figures are partially created with ‘Biorender.com’. Abbreviations 5-HT 5-hydroxytryptamine AA ascorbic acid APP/PSEN 1 amyloid protein precursor/presenilin-1; ARDS acute respiratory distress syndrome ASC ascorbate cGMP cyclic guanosine monophosphate CVD cardiovascular disease DHA dehydroascorbate DOPA dihydroxyphenylalanine dUCH Drosophila ubiquitin C-terminal hydrolase ET-1 endothelin-1 GABA gamma amino butyric acid GLUT glucose transporter ICAM-1 intercellular adhesion molecule 1 ICU intensive care unit IL-1 interleukin-1 IL-6 interleukin-6 IL-8 interleukin-8 IV intravenous LDL low-density lipoprotein NF-κB pathway nuclear factor kappa B pathway NMDA N-methyl-D-aspartic acid or N-methyl-D-aspartate NO nitric oxide RDA recommended dietary allowance ROS reactive oxygen species SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 SLC23A solute carrier family 23 m1 SNP single nucleotide polymorphism SVCT sodium ascorbate co-transporter TNF-α tumour necrosis factor alpha Authors’ contribution: All the authors contributed equally to prepare this article, read and approved the final manuscript. Consent for publication: All the authors have agreed to submit this paper for publication in Nutrition and Health. The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article. ORCID iD: Saritha R Shetty https://orcid.org/0000-0003-2440-5398 Supplemental material: Supplemental material for this article is available online. ==== Refs References Abobaker A Alzwi A Alraied AHA (2020) Overview of the possible role of vitamin C in management of COVID-19. Pharmacological Reports 72 (6 ): 1517–1528.33113146 Agus DB Gambhir SS Pardridge WM , et al. (1997) Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters. Journal of Clinical Investigation 100 (11 ): 2842–2848.9389750 Alvarado C Alvarez P Puerto M , et al. 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==== Front Am J Otolaryngol Am J Otolaryngol American Journal of Otolaryngology 0196-0709 1532-818X Saunders S0196-0709(22)00336-2 10.1016/S0196-0709(22)00336-2 103709 Article Table of Contents 1 12 2022 January-February 2023 1 12 2022 44 1 103709103709 2023 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmc	0	PMC9713689	NO-CC CODE	2022-12-02 23:23:07	no	Am J Otolaryngol. 2023 Dec 1 January-February; 44(1):103709	utf-8	Am J Otolaryngol	2,022	10.1016/S0196-0709(22)00336-2	oa_other
==== Front Early Child Educ J Early Child Educ J Early Childhood Education Journal 1082-3301 1573-1707 Springer Netherlands Dordrecht 1419 10.1007/s10643-022-01419-x Article Fostering Play Through Virtual Teaching: Challenges, Barriers, and Strategies http://orcid.org/0000-0002-5263-5206 Ethridge Elizabeth A. [email protected] 1 http://orcid.org/0000-0001-7224-3165 Malek-Lasater Adrien D. [email protected] 2 http://orcid.org/0000-0002-7771-4752 Kwon Kyong-Ah [email protected] 3 1 grid.266900.b 0000 0004 0447 0018 Instructional Leadership and Academic Curriculum, University of Oklahoma, Tulsa, OK USA 2 grid.266865.9 0000 0001 2109 4358 Department of Teaching, Learning and Curriculum, University of North Florida, Jacksonville, FL USA 3 grid.266900.b 0000 0004 0447 0018 Instructional Leadership and Academic Curriculum, University of Oklahoma, Norman, OK USA 1 12 2022 111 26 10 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Early childhood teachers routinely facilitate play-based learning experiences in their physical classrooms; however, the pivot to virtual teaching platforms created a barrier for providing age appropriate, play-based learning opportunities during the COVID-19 pandemic. There are few studies exploring how to promote play in the virtual classroom or what types of activities and learning experiences promote play in synchronous and asynchronous settings. Therefore, this study explored the barriers and challenges to fostering play through virtual teaching and the types of play-based instruction teachers were effectively able to implement in their virtual classroom. This study used content analysis along with descriptive analysis of an online survey with open-ended prompts that early childhood teachers completed (n = 76). Findings revealed two major themes related to challenges and barriers in teachers’ efforts to foster play-based learning through virtual formats. Even though teachers noted significant challenges and barriers they identified multiple play-based activities they were able to facilitate effectively through virtual formats. These activities were categorized through the theoretical framework of Piaget’s stages of the development of play with the addition of guided play. Implications for how play can be fostered through virtual teaching in early childhood classrooms were discussed. Keywords Play-based instruction Virtual teaching Guided play Virtual classroom Challenges and barriers ==== Body pmcDue to the impact of the COVID-19 global pandemic, many early childhood centers and schools transitioned to virtual (online) learning, which is an educational process that takes place over the internet (Merriam-Webster, n.d.); this shift changed the nature and scope of teaching and learning. Teachers who had to transition to teaching virtually adapted to the unprecedented situation and made significant efforts to provide virtual learning activities (Steed & Leech, 2021). Rooted in developmentally appropriate practice (Copple & Bredekamp, 2008), the National Association for the Education of Young Children and the Fred Rogers Center provided the position statement which noted that when used intentionally and appropriately, technology and interactive media can be effective tools to support children’s learning in 2012 (National Association for the Education of Young Children [NAEYC], 2012). Still, virtual teaching is not the norm for most teachers. Therefore, it poses significant challenges (Dhawan, 2020), especially for early childhood educators. For example, early childhood teachers may have trouble translating play-based learning experiences to the virtual setting. According to constructivist theory, the principal theory of learning in early childhood education (ECE), play is a central component in early learning. Play-based learning combined with some degree of adult guidance and scaffolding engages children to construct new knowledge (Van Hoorn et al., 2015; Weisberg et al., 2016) and has been linked with higher academic achievement (Marcon, 2002; Zosh et al., 2013) and executive functioning (Berk & Meyers, 2013). Children in child-centered, play-based environments have been shown to be engaged in more effective problem-solving behaviors compared to children in more teacher-directed environments (Gmitrová & Gmitrov, 2004; McInnes et al., 2009). Although ECE teachers routinely create play-based learning experiences in the classroom, the virtual setting can pose a barrier for providing interactive and engaging learning experiences. Even though there is emerging evidence on various opportunities and challenges for ECE teachers’ virtual teaching (e.g., Dayal & Tiko, 2020; O’Keeffe & McNally, 2021), information on how teachers incorporate play into virtual teaching to support children’s learning is scarce. Thus, the current study examined the challenges and barriers ECE teachers faced in their efforts to foster play through virtual teaching, focusing on the types of play-based instruction teachers were effectively able to facilitate virtually. Theoretical Framework The constructivist approach guides our study, which endorses play as child-directed, active, engaging, and involving a child’s social world and community (Mooney, 2013). For this paper, the understanding and organization of play in virtual settings are based on Piaget’s stages of the development of play (Van Hoorn, et al, 2015) and extended by including the category of guided play (Weisberg et al., 2016). Piaget’s stages of the development of play, also known as types of play, include the categories of functional play and symbolic play, which consist of constructive play, dramatic play, and games with rules (Van Hoorn et al., 2015). Functional play is predominantly a form of play for infants and toddlers in which children repeatedly practice interacting with objects, people, and language to develop schemes (Gestwicki, 1999). However, older children also engage in functional play such as riding bikes, moving their bodies to music, repeatedly putting the same puzzle together, along with several other repetitive activities they find engaging. Symbolic play involves mental representation and make-believe, also known as pretend play. It forms the foundation for abstract thinking and is critical to human development (Van Hoorn et al., 2015). Constructive play is a form of symbolic play because it involves children representing their experiences by utilizing objects to make other things (Gestwicki, 1995). For example, a child may construct a house out of blocks or draw a picture to illustrate a firetruck. Dramatic play is another form of symbolic play; children create imaginary roles and situations using representations of objects that are more abstract, utilizing language and gestures (Van Hoorn et al., 2015). Finally, games with rules are one more form of symbolic play that includes adherence to external governed play, such as Chutes and Ladders, or negotiated or agreed upon rules that the children can invent (Gestwicki, 1995; Van Hoorn et al., 2015). Another category of play is guided play. Weisberg et al. (2016) describe guided play as play-based activities integrating teacher engagement to support learning. This type of play is referred to as ‘intentional teaching’ (Epstein, 2007), ‘conceptual play’ (Fleer, 2011), ‘educational inquiry’ (Youngquist & Pataray-Ching, 2004), and ‘pedagogical play’ (Edwards & Cutter-Mackenzie, 2013). While play holds the characteristic of being child-centered, in an ECE classroom, the teacher remains key to the play centered-curriculum. Therefore, in guided play, the knowledgeable teacher offers scaffolding when appropriate and orchestrates the classroom environment or activity in a way that meets and fosters the developmental needs of the children in their play (Van Hoorn et al., 2015). Use of Technology and Virtual Teaching in Early Childhood Classrooms Virtual teaching has grown in the last two decades (Singh & Thurman, 2019). Research in the last decade has shown that virtual instruction in some early intervention and K-12 settings has been effective in improving students’ learning outcomes (Behl et al., 2017; Merchant et al., 2014; Olsen et al., 2012; Poole et al., 2020). Despite its growing popularity and potential benefits, research findings on the quality and impact of virtual teaching are mostly mixed, which has raised concern among practitioners, researchers, and the public about the effectiveness of such methods (Dong et al., 2020; Dorn et al., 2020; Duckworth et al., 2021; Means et al., 2009). Also, most studies on virtual teaching were conducted in K-12 or early intervention settings but not in early childhood settings. While these findings are informative, they may not apply to virtual teaching in ECE settings due to its inherent differences from early intervention (e.g., serving multiple children vs. one–one–one interaction). Evidence on virtual teaching and learning in ECE settings is emerging, but there is still a significant gap in the literature. In the field of ECE, integrating technology into classrooms for young children has not received positive views from teachers and parents (Chen & Chang, 2006; Mertala, 2019). Many ECE teachers believe that traditional methods (e.g., hands-on and in-person) are more effective and afford a tactile experience for the child which allows for a more active learning role (Mertala, 2019). While this belief is supported by educational theory, the fact that virtual teaching is becoming more of a permanent option for children and families suggests there is a need for a mindset that incorporates ways to teach children virtually using developmentally appropriate practices. Teachers' positive views and attitudes toward technology integration are critical as they affect teachers’ use of technology and virtual teaching practices and impact children's motivation and learning (Kara & Cagiltay, 2017; Mertala, 2019; Miranda & Russell, 2012). Another common concern is the lack of training and support for using technology and virtual teaching for ECE teachers (Trust & Whalen, 2020). This lack of training and support may make teachers feel unprepared and incompetent in choosing and implementing new technologies (Chen & Chang, 2006). During the onset of the COVID-19 pandemic, virtual teaching became mandatory in many ECE settings. Several teachers and administrators expect that the need for virtual teaching will continue in the future (Hu et al., 2021). With the shift to virtual teaching, early childhood teachers attempted to quickly create online lessons for children in various ways (Hodges et al., 2020), such as replicating their traditional classes via video conferencing platforms (Lederman, 2020) and modifying their traditional methods to engage children (Allen et al., 2020). In the past, early childhood teachers have used technology as a teaching tool (e.g., showing pictures or video, Kalogiannakis, 2010), but teaching virtually requires next-level knowledge and skills in technology integration. Teaching virtually as a primary teaching modality requires teachers to gain new skills and feel competent in providing meaningful learning experiences online (Chen & Chang, 2006; Dhawan, 2020; Trust & Whalen, 2020). Providing play-based experiences is vital in creating meaningful learning environments for children. However, some evidence suggests teachers are experiencing barriers to providing play virtually. As indicated by Ford et al. (2021), virtual teaching is inherently challenging for any age group, but it would be even more challenging for young children. The abrupt transition to virtual teaching does not allow much time to prepare for virtual teaching and has intensified job demands and stress (Allen et al., 2020). Many teachers reported that children do not have access to basic technological equipment and internet access to access learning content and participate in virtual classes (Dias et al., 2020; Ford et al., 2021). Teachers often reported difficulty engaging children and communicating with parents through virtual teaching (Dayal & Tiko, 2020; Dias et al., 2020; Ford et al., 2021; Hu et al., 2021; Szente, 2020; Tarrant & Nagasawa, 2020). As teachers experienced various challenges, many teachers experienced lack of resources and support, training, and consistent guidance for virtual teaching (Ford et al., 2021). According to Hu et al., (2021), most preschool teachers provided digital content (e.g., uploading videos, sharing virtual resources) and have limited teacher–child interactions to deliver teaching (e.g., real-time interaction in a synchronous environment). Despite myriad challenges and lack of resources and training, many ECE teachers actively sought resources, learned independently, and collaborated with others to engage children and provide them developmentally appropriate and play-based virtual learning experiences (Ford et al., 2021). While more research evidence on virtual teaching is emerging, there is still a lack of research on how teachers deliver content, which includes their ability to support play. As the effects and impacts of the COVID-19 pandemic may be long-lasting, play is even more necessary as it provides a natural medium to help reduce stress and build resilience in children, particularly in times of crisis (Anderson-McNamee & Bailey, 2010; Rao, 2018). In addition, play provides a valuable escape from uncertainty and stress, offering a sense of normalcy and safety (Yogman et al., 2018). Thus, it is crucial to study how ECE teachers provided learning opportunities through play in a virtual space during the COVID-19 pandemic. Current Study There are a few studies that indicated endorsement for play-based virtual teaching (O’Keeffe & McNally, 2021), but there is uncertainty regarding the capacity for ECE teachers to use this type of teaching during the COVID-19 pandemic (Steed & Leech, 2021). Currently, limited resources and research are available on how to apply developmentally appropriate practices (including play-based instruction) in an ECE virtual classroom. Further, understanding the challenges associated with implementing play-based learning through virtual teaching is an understudied area. More research is needed to understand teachers’ experiences and what types of activities and learning experiences promote play in a virtual classroom. Thus, the current study examined ECE teachers’ provision of virtual teaching, focusing on play-based learning activities and related challenges by seeking to answer the following research questions:What challenges and barriers to providing play-based instruction are more prevalent in virtual teaching? What type of play-based instruction did teachers effectively implement in their virtual classroom? Methods This qualitative study used content analysis of the open-ended responses along with descriptive analysis of an online survey to understand how ECE teachers implemented play-based instruction in a virtual setting and what challenges and barriers existed. Participants After obtaining IRB approval, ECE teachers (serving children from birth through age 8) who had taught any form of virtual instruction in the United States (hybrid, synchronous, asynchronous) during the 2020–2021 school year were invited through email and social media to participate in this study. Recruitment methods included emailing local schools, using social media such as Facebook, and reaching out through national organizations. The COVID-19 pandemic caused a shift in school functioning with closures beginning in March 2020 and various rearrangements of teaching options by January 2021, including in-person or virtual teaching (i.e., hybrid, synchronous, asynchronous) options. Therefore, recruitment efforts reached teachers with a range of experience teaching virtually. Between January and June 2021, an online consent form and survey were sent out to teachers who were currently teaching in ECE classrooms. To participate, teachers had to have utilized some form of virtual teaching in the 2020–2021 school year and have taught children between the ages of birth through third grade between the 2020–2021 school year. Initially, 86 teachers completed the survey however, ten were excluded because they either taught higher-ed or served in a non-teaching role (i.e., retired, principal). Therefore, 76 early childhood teachers from around the United States participated in this study (see Table 1).Table 1 Teacher demographics and background information (n = 76 teachers) Characteristics % Age (years) 18–24 7.9 25–34 22.4 35–44 32.9 45–54 26.3 55+ 10.5 Race White 77.3 Black 4.0 American Indian/Alaska Native 8.0 Asian 6.7 Other 4.0 Hispanic/Latino Origin (yes) 5.3 Gender Female 99.0 Other 1.0 Education level Graduate degree 30.3 Bachelor’s degree 64.5 Associate degree or lower 5.2 No experience teaching virtually prior to COVID-19 89.0 Total years teaching 1–5 years 26.3 6–10 years 26.3 11–15 years 15.8 15+ years 31.6 Age teaching (2020–2021 school year) Infant/Toddler (birth—2 years old) 5.3 Preschool/PreK (3–5 years old) 63.2 Kindergarten 7.9 First grade 7.9 Second grade 7.9 Mixed ages/other 7.9 Taught a different ECE age/grade in the previous year 21.1 Teaching Modality (2020–2021 school year) Fully in-person 22.7 Fully online 25.3 Hybrid (some in-person/some online) 32.0 Began in-person and moved online 6.7 Began online and moved to in-person 8.0 Other combination of modalities 5.3 Teaching Philosophy Constructivist 77.1 Behaviorist 4.3 Maturationist 5.7 Combination 12.9 Measures Teachers in the study completed an online survey with multiple choice and open-ended response questions. Multiple choice questions addressed demographic and background information (e.g., What grade do you currently teach?, Have you had previous experience teaching prior to the COVID-19 pandemic?), and questions about teaching experience, play-based instruction, and teaching beliefs and practices (e.g., How have you incorporated play-based instruction in the virtual classroom using pretend, imaginative, or dramatic play?, How would you describe your personal teaching philosophy?). The purpose of the teaching beliefs and practices questions was to better understand teachers' foundational beliefs related to play-based instructional practices, given that certain philosophies more clearly align with implementing play (i.e., constructivist and maturationist philosophies) (Kohlberg & Mayer, 1972). Open-ended response questions asked teachers their personal perspectives on:1) types of play-based activities that were effectively implemented in their online classroom, and 2) challenges and barriers they experienced when trying to implement play-based activities in their online classroom. Data Analysis Content analyses of open-ended responses and descriptive analysis were used for this study. Content analysis as a research technique allowed the researchers to determine the presence of words, concepts, and themes within given qualitative data of participant responses and analyze the meanings and relationships among those words, concepts, and themes (Berelson, 1954). This technique has been applied to the verbatim responses to code those responses into a meaningful set of categories that lend themselves to further analysis (Berelson, 1954). Descriptive statistics were used to analyze teacher and background information and the frequency of play categories teachers found effective in their virtual teaching. Qualitative analysis was used for the open-ended survey questions and included conducting level one (provisional coding) and level two (pattern) coding (Saldaña, 2021). After cleaning the data, the first two authors developed a list of provisional codes and then individually coded the qualitative data (i.e., teachers’ open-ended responses in the online survey). Once all responses were coded, the authors met to refine codes and categories and discuss any discrepancy until reaching consensus. Finally, the two authors conducted reliability checks with all responses to the open-ended question and established inter-coder reliability ranging from 90 to 100% agreement. Each open-ended question was analyzed separately using two cycles of coding. First, the open-ended question on challenges and barriers to implementing play-based virtual instruction was analyzed using provisional codes for level one (Saldaña, 2021) that were developed before the analysis. These codes (e.g., technical issue, connectivity issue, parent involvement, and student participation) were based on research related to challenges and barriers to virtual teaching (Dayal & Tiko, 2020; O’Keeffe & McNally, 2021) and play-based instruction (e.g., Kane, 2016). Pattern coding was used for level two coding (Saldaña, 2021) with emerging themes related to whether challenges and barriers were unique to virtual instruction (connection issues, home distractions, and lack of access to materials) or specific to fostering play virtually (lack of experience teaching virtually, lack of support, and teacher mindset). The second open-ended question on forms of play in the virtual classroom asked teachers to describe effective ways they implemented play-based activities in the virtual classroom. Level one provisional codes (Saldaña, 2021) were based on common but not mutally exclusive characteristics of play (e.g., child-directed, open-ended, scaffolding, hands-on, pretend play, games, inquiry, and physical movement) (Van Hoorn, et al., 2015; Weisberg et al., 2016). The process of level two pattern coding (Saldaña, 2021) organized the play-based activities into categories of play guided by the theoretical framework). Once all responses were coded and inter-coder reliability was established for this question, the frequency of occurrence of the activities in each type of category of play was calculated. Results Challenges and Barriers The survey data collected teachers’ open-ended responses that described challenges and barriers they encountered when trying to incorporate play-based instruction in their virtual classroom. Two major themes emerged from the analysis of the open-ended responses using the two-level coding process: (1) challenges related to virtual instruction and (2) specific barriers to fostering play-based instruction. Challenges Related to Virtual Instruction The first major theme of challenges related to virtual instruction included reported difficulties unique to teaching online. The greatest challenge reported regarding virtual instruction was that students did not consistently have access to materials at home that were needed for lessons. One teacher explained, “Not every student has the same resources at home” while another teacher listed, “Lack of knowing what is available at home” to be a significant challenge. The second greatest challenge reported was student engagement in the virtual setting. Teachers reported a range of student participation and interaction. For example, some teachers with asynchronous classrooms did not directly interact with the students and reported having inadequate information on whether they participated and how they engaged with the material and activities. Several teachers with synchronous classrooms also expressed difficulty fostering student engagement and interaction. A few teachers attributed this challenge to children’s age and comfort level. For instance, a teacher explained it is “hard for preschoolers to interact with peers or teachers in a play situation” in a synchronous virtual classroom. Another teacher noted, “Students are more shy on camera than in person and some refuse to interact.” External distractions such as background noise or siblings interfering and distracting the students were also often mentioned as interruptions to the level of engagement in a virtual classroom. Lastly, several teachers also listed common technology issues, such as bad internet connections and insufficient wi-fi, as being a virtual instruction challenge. Barriers to Fostering Play Virtually The second major theme that emerged was specific barriers to fostering play virtually. The most highly reported barrier was the lack of support. Survey responses showed that most teachers felt they had a higher degree of autonomy in their instructional practices (52.9% felt a great extent of autonomy) prior to the COVID-19 pandemic. Whereas, during the 2020–2021 school year 49.3% felt they had more autonomy. Teachers noted a lack of support from both parents and school administration. Regarding lack of support from parents, one teacher wrote, “Many parents don’t understand play-based instruction and prefer rote learning.” Another noted, “Lack of understanding of the importance of play from parents despite different ways of explaining it.” One teacher explained that in asynchronous classrooms,It’s hard to document what standard play is covering. It’s also hard to get parents to take a picture or record a video of the students playing for documentation purposes. Without me being right there, it is hard for me to justify putting that as a completed standard or assignment. In addition, a teacher shared that some parents’ lack of support for play-based activities was due to not wanting “a mess or a ton of things to clean-up.” This comment refers to the fact that hands-on and exploratory activities may utilize “messy” materials such as paint, mud, or water or require many materials that require extensive clean-up (i.e., small beads or pretend food). Teachers also referenced the lack of support from administration in implementing play-based teaching with statements such as, “Being forced to follow curriculum to fidelity,” “schedule is rigid,” and “everyone has different expectations.” A second barrier highly reported related to implementing play virtually was teachers’ lack of experience with virtual instruction. This lack of experience teaching virtually was woven throughout the teachers’ comments related to their lack of experience with the technology platform chosen by their school district, and lack of education on how to teach in a virtual classroom. Teachers’ lack of experience was also evident in comments like “[providing play virtually is] impossible.” These types of ‘absolute’ comments came from many teachers even though 77.1% of teachers in the study identified themselves as having a constructivist teaching philosophy (see Table 1). In addition, teachers expressed how difficult it was to not be able to teach in person, saying, “The time for extended free play and dramatic play that would happen in a classroom is not feasible online.” Another explained, “Young children do not have the attention span that would allow them to remain attentive through a virtual lesson.” Play-Based Lessons Facilitated Virtually Even with significant challenges and barriers to fostering play through virtual teaching, many teachers provided specific explanations for play-based lessons or activities they were able to facilitate virtually. Of the 76 participating teachers who completed the survey, 40 teachers provided a total of 57 examples of types of play that were implemented effectively in the virtual classroom (see Table 2). Some participants listed multiple examples of play-based activities which explains why there were more examples than the number of teachers’ responses. Findings were organized and guided by our theoretical framework of Piaget’s stages of the development of play (Van Hoorn et al., 2015) with the addition of guided play (Weisberg et al., 2016). It is important to note that some examples provided under each type of play could be interpreted to fit in another type of play, which demonstrates that play is not one-dimensional and is influenced by each child’s individuality and unique nature.Table 2 Frequency of reported effective play-based lessons or activities in virtual classroom (n = 40 teachers) Type of play Frequency % Sample comments Functional play 13 23 “Object play—everyone had same household object to explore together” Constructive play 8 14 “Using loose parts for building.” Dramatic play 11 19 “A favorite of the students was a dramatic play experience, where we either dressed up as a character, or used puppets or stuffed animals and pretended to be the puppet or stuffed animal. We would talk, ask them, think of games that the animal/character would enjoy, think about how they would respond, etc.” Games with rules 16 28 “We played Simon Says…, made up Bingo games…, played I Spy games as well as guessing sounds of animals and guessing songs.” Guided play 9 16 “Activities that asked children to go outside, look for things, create using those things.” Frequency calculations based on the total number of examples (57) provided by teachers aCategories of play are listed by the theoretical framework rather than by the highest frequency Play-based activities in the category of functional play were reported thirteen times (23%). Functional play was evident when teachers described lessons in which the children engaged in:Singing, fingerplays, Go Noodle, yoga, dance parties Sink or float activities Making shadows with flashlights Interacting with White Board features Constructive play was reported the least frequently with eight examples (14%). Constructive play was represented through:Open-ended play with blocks, Play-Doh, Legos Making caves or forts at home Pretending to go on adventures with pretend equipment such as rope and ladders. Dramatic play was reported 11 (19%) times as an effective play-based virtual activity. Dramatic play is when children create imaginary roles that often goes along with their constructive play, however it is more abstract utilizing language, gestures and role playing with others. Examples teachers noted in the survey include:Virtual “tea party” Acting out stories with household objects Pretend cooking Pretend adventures Dress-up as character in books Use puppets and stuffed animals and talk for them Use story starters and children role play their own endings Pretend play (bakery, grocery shopping) The most frequently reported virtual play-based activity category was games with rules (28%). Examples of games with rules teachers provided included:Scavenger hunts Simon Says Bingo I Spy Hide and Seek (with objects and families helping) Next, virtual play-based activities in the category of guided play were reported approximately 16% of the time. Examples of guided play included:Investigative questioning of concepts Open-ended science activities Science experiments Outdoor investigations Nature walk, collecting materials, journaling Patterning and counting with toys Looking for things outdoors and creating with those items The findings suggest each type of play can be facilitated virtually; however, some types of play were reported to be effectively implemented more often than others. Games with rules and functional play were the two most frequently reported types of play used in virtual teaching by the teachers in this study. Discussion This study sought to answer questions related to what challenges and barriers exist for ECE teachers when implementing play-based instruction virtually and how teachers implemented virtual play-based activities effectively during the COVID-19 pandemic. Our study adds to the growing body of literature that has come out of the questions that arose from the pandemic’s impact on education. Given the lack of literature on virtual teaching and how teachers deliver content to children, our study is unique in that it captured teachers’ perspectives of ways to implement play-based instruction virtually during the pandemic when virtual teaching in ECE was increasing in use. Most teachers (89%) participating in our study did not have any experience teaching virtually, with limited training in this area prior to the 2020 school year. With our focus on play-based instruction, our findings highlight the unique demands of the virtual learning environment in ECE settings. Given that the option for virtual teaching in one form or another is here to stay even after the pandemic, it is necessary to examine the needs specific to ECE settings to understand and inform how to best support teachers in providing play-based virtual instruction. The biggest challenge to teaching virtually for the ECE teachers in our study was children’s lack of access to needed materials. Developmentally appropriate practices for ECE are grounded in teaching through experiences that often involve the use of hands-on materials (NAEYC, 2022), including items such as counting manipulatives, art supplies, building materials and visual supports. In a physical, in-person classroom, teachers utilize these typical ECE classroom materials or may borrow materials from other teachers in their school. However, in a virtual classroom, teachers must rely on the materials students have access to in their homes. Navigating the range of access to materials their students can use may decrease instruction quality or force teachers to rely on their own personal finances to purchase individual supplies for students as reported by teachers in other studies (i.e., Atiles et al., 2021). Teachers also reported difficulty engaging children virtually and reducing distractions, which suggests the ECE virtual learning environment requires a different way of engaging children. Teachers understand that play is essential to children’s learning and development and have reported the desire to reach and engage their students in developmentally appropriate ways, even virtually, which is consistent with previous studies (Crawford et al., 2021; Ford et al., 2021; Kwon et al., 2022; Szente, 2020). This commitment has been evidenced by teachers’ ability to pivot their teaching to a virtual modality while still trying to incorporate play-based instruction. However, the highly reported challenge of engaging children mentioned in other recent research (e.g., Ford et al., 2021; Hu et al., 2021) suggests that teachers need to be better equipped to teach virtually in developmentally appropriate ways (Atiles et al., 2021; Crawford et al., 2021; Szente, 2020). Debates about supporting play in the in-person classroom are not new (Chien et al., 2010; Pyle & Danniels, 2017), and teachers in our study expressed a lack of support as a barrier to providing play-based instruction in their virtual classroom. While many of the teachers assumed a level of teacher autonomy, the lack of parent and administrative support in their fostering play-based instruction was prevalent. This could have been due to the unprecedented situation of the COVID-19 pandemic, where school administrators and teachers alike might have felt overwhelmed by the changes. However, as schools have adapted more to providing virtual instruction since the onset of the pandemic, another examination of teachers’ feelings of support in this context is needed (Ford et al., 2021). Many teachers also expressed that lack of support from parents was a significant barrier to fostering play-based instruction, which supports the findings of previous studies (Dias et al., 2020; Ford et al., 2021; Hu et al., 2021; Kwon et al., 2022). The most notable reason was due to parents’ lack of understanding of the role play has on children’s learning, which is often an issue for teachers even when they are teaching in person. Ultimately, parents want what is best for their children and they likely experienced behavioristic, traditional teaching practices throughout their own schooling and think that is how one learns. Play has often been considered frivolous or something children do to relax and entertain themselves, not academic or a way of learning. This indicates that educators must find ways to share with families how play is essential to children’s learning and the merits it has in children’s development. Since this has been a challenge for early childhood educators for decades, it might be wise to change our approach. Guided play was listed as a type of play in the research and in the data analysis for this study. Purposefully framed play or guided play can be connected to standards for learning and documented accordingly, which may help parents and even educators be more receptive to such instructional processes. As virtual teaching is becoming a more adopted modality, these findings provide insight into how to support teachers who offer play-based instruction virtually. Teachers also reported their lack of experience teaching virtually as a barrier to implementing play. This finding extends recent research findings that ECE teachers experience a lack of preparation for teaching online (Ford et al., 2021) by specifically focusing on implementing play. Interestingly, while some teachers tended to take an approach that was flexible, creative, and oriented to problem-solving, several teachers in our study were hesitant to try facilitating play virtually. Some comments inferred a strong resistance to fostering play through virtual teaching, indicating a fixed mindset. Teachers’ attitudes about integrating technology and teaching can have an influence on their ability to use it as an effective tool for teaching, even in the early childhood ages (NAEYC, 2012; Nuri & Cagiltay, 2017). O’Keeffe & McNally (2021) found that generally early childhood teachers believed play-based activities are an important pedagogical tool in supporting children’s learning, even in the context of the COVID-19 pandemic. Therefore, it could be that the stressful experiences reported by teachers in our study could explain some teachers reverting to traditional, behavioristic practices such as teaching mainly through direct instruction and worksheets or cause some teachers to adopt a mindset that it is not possible or feasible to foster play through virtual teaching. While the consideration of the challenges and barriers is important in knowing how to best support teachers who are teaching virtually, perhaps the equally important finding is that despite reported barriers and challenges, teachers found ways to foster play in the virtual setting in every category of play. The most frequently reported category of play was games with rules such as scavenger hunts, Bingo, and Hide-and-Seek to engage children with learning material available at home. This type of symbolic play may have been the most seamlessly translated to the online format, given that many versions of online games already exist, and games with rules allow for some teacher-direction and built-in structure. Teachers in our study reported that engaging children was a challenge to virtual teaching, and the use of games may have been a way that helped encourage children to participate and interact. Given the lack of research on what teachers do in their virtual classrooms (e.g., their use of developmentally appropriate and play-based teaching strategies to promote children's learning), it is promising that we found a variety of examples of play-based instruction provided in each category of play during the pandemic. Teachers were able to facilitate dramatic play, provoke children’s thinking through investigations, encourage physical activity, as well as provide opportunities for open-ended exploration. These findings offer hope that a mindset can be shifted away from the belief that providing play in a virtual setting is “impossible”. Teachers are motivated to best meet children’s learning needs by providing opportunities for play and exploration (O’Keefe & McNally, 2021). With additional support and training, those who may be assigned to teach virtually will be able to do so effectively and more confidently. Limitations This study has several limitations. First, the study is somewhat limited in that it primarily focused on which activities teachers used to foster play virtually and the challenges and barriers they faced. Much more information is needed regarding teaching children virtually, such as how teachers connect play experiences to state standards and how they assess children’s learning. More research documenting whether teachers are having success in scaffolding children’s play virtually and what strategies they are utilizing is needed. Second, the online survey data were collected between January and June 2021 during the pandemic. While data were collected for a six-month span, the COVID-19 cases and school responses to the pandemic (e.g., transitioning from one teaching modality to another repeatedly) have fluctuated since its onset. Our data only serves as a snapshot and does not capture how teachers adjusted and tailored their teaching to the ongoing changes and challenges during COVID-19. It would be ideal to collect longitudinal data to address this process. In addition, most teachers in our study were highly educated and taught PreK and children in public school settings. Thus, it may not fully represent the teachers in a range of ECE settings. More research is needed to understand the perspective of teachers serving other age groups in different ECE settings (e.g., childcare, Head Start programs). Notably, the perspectives of teachers who served in early care and education centers or family childcare homes (i.e., serving infant, toddler, and preschool-aged children) are needed, given that these teachers tend to have potential unique needs and experiences when related to virtual teaching. Implications for Practice and Policy This study has implications for practice. First, this study’s findings highlight the need to prepare teachers to provide play-based and developmentally appropriate instruction virtually. While various professional organizations (e.g., NAEYC, the Division for Early Childhood) provide valuable resources and information, teachers benefit from more resources and training on different strategies for engaging children virtually. Although, evidence from this study demonstrates that teachers are capable of creatively fostering play through virtual teaching that includes all types of play, teachers face various challenges and barriers when trying to implement play-based instruction virtually. Teachers in the field need to be provided with training that keeps them up to date with the virtual teaching and learning platforms as well as strategies that can elicit student engagement. Ideas and tips from experts in special education and early intervention, practitioners in telehealth, and faculty in higher education can be adopted for the ECE classroom such as chunking time and information into smaller parts, adding humor, adding an element of performance, and storytelling (Cavanagh, 2019; Olsen et al., 2012). Teacher educators also need to be knowledgeable in virtual teaching practices and online learning systems that are being used in schools and programs and teacher preparation programs need to incorporate elements of virtual instruction into their courses. Second, schools and programs need to equip families with the knowledge of how play and learning are connected in the early childhood years. Administrators must understand teacher challenges and barriers and support teachers in overcoming these difficulties. Families need to be educated on the importance of play, even in virtual settings. This can be achieved through family engagement activities, informational sessions, and school-family communication. This study also has implications for policy and research. As virtual instruction is more prevalent, policies need to be implemented that provide equitable access to materials including educational hands-on materials, as well as stable access to the internet and needed devices. For schools that offer the options for virtual instruction, it is vital for administrators to consider this need for students to access educational materials that are appropriate for early childhood ages. Schools can confront this challenge through suggestions such as allowing space in their budget to purchase materials for students to use at home or having community resource centers where virtual students can borrow materials they may not have at home. More research is needed on how to make virtual learning more equitable. In addition, more research is necessary to examine any changes in early childhood teachers’ ability to provide play-based instruction in a post-pandemic era. Given the known barriers and challenges that currently exist, research similar to this study will help to illuminate the struggles teachers face and allow for better approaches to meet the needs of teachers and children. Our ultimate responsibility as educators is to set the conditions for learning and development, which can include a virtual environment. This can be accomplished by supporting play in efforts to guide children’s learning related to academic standards in ways that honor children’s development (Van Hoorn et al., 2015). Our research is not making a case for virtual learning with young children; however, the study provides essential information for when virtual teaching is the modality being used and the importance of play-based teaching regardless of whether it is in person or virtual. It has been documented that play is the primary force in child development and learning and therefore, it is essential for early childhood teachers to incorporate different kinds of play in their teaching pedagogies whether they are in person or virtual. In this new era, schools will likely continue to utilize virtual teaching to some degree to make up for children's time away from school due to weather, illness, and a host of other reasons. Therefore, it is critical that the findings of such research are disseminated in the field of ECE. This research helps to expand notions of how play can be encouraged, supported, and scaffolded through both synchronous and asynchronous formats. The sharing of ideas and lessons learned regarding fostering play through virtual teaching is always a great way to build cognitive, social, and emotional connections that will influence teaching, research, and service. Declarations Conflict of interest The authors did not receive support from any organization for the submitted work. The authors have no relevant financial or non-financial interests to disclose. 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==== Front Early Child Educ J Early Child Educ J Early Childhood Education Journal 1082-3301 1573-1707 Springer Netherlands Dordrecht 1419 10.1007/s10643-022-01419-x Article Fostering Play Through Virtual Teaching: Challenges, Barriers, and Strategies http://orcid.org/0000-0002-5263-5206 Ethridge Elizabeth A. [email protected] 1 http://orcid.org/0000-0001-7224-3165 Malek-Lasater Adrien D. [email protected] 2 http://orcid.org/0000-0002-7771-4752 Kwon Kyong-Ah [email protected] 3 1 grid.266900.b 0000 0004 0447 0018 Instructional Leadership and Academic Curriculum, University of Oklahoma, Tulsa, OK USA 2 grid.266865.9 0000 0001 2109 4358 Department of Teaching, Learning and Curriculum, University of North Florida, Jacksonville, FL USA 3 grid.266900.b 0000 0004 0447 0018 Instructional Leadership and Academic Curriculum, University of Oklahoma, Norman, OK USA 1 12 2022 111 26 10 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Early childhood teachers routinely facilitate play-based learning experiences in their physical classrooms; however, the pivot to virtual teaching platforms created a barrier for providing age appropriate, play-based learning opportunities during the COVID-19 pandemic. There are few studies exploring how to promote play in the virtual classroom or what types of activities and learning experiences promote play in synchronous and asynchronous settings. Therefore, this study explored the barriers and challenges to fostering play through virtual teaching and the types of play-based instruction teachers were effectively able to implement in their virtual classroom. This study used content analysis along with descriptive analysis of an online survey with open-ended prompts that early childhood teachers completed (n = 76). Findings revealed two major themes related to challenges and barriers in teachers’ efforts to foster play-based learning through virtual formats. Even though teachers noted significant challenges and barriers they identified multiple play-based activities they were able to facilitate effectively through virtual formats. These activities were categorized through the theoretical framework of Piaget’s stages of the development of play with the addition of guided play. Implications for how play can be fostered through virtual teaching in early childhood classrooms were discussed. Keywords Play-based instruction Virtual teaching Guided play Virtual classroom Challenges and barriers ==== Body pmcDue to the impact of the COVID-19 global pandemic, many early childhood centers and schools transitioned to virtual (online) learning, which is an educational process that takes place over the internet (Merriam-Webster, n.d.); this shift changed the nature and scope of teaching and learning. Teachers who had to transition to teaching virtually adapted to the unprecedented situation and made significant efforts to provide virtual learning activities (Steed & Leech, 2021). Rooted in developmentally appropriate practice (Copple & Bredekamp, 2008), the National Association for the Education of Young Children and the Fred Rogers Center provided the position statement which noted that when used intentionally and appropriately, technology and interactive media can be effective tools to support children’s learning in 2012 (National Association for the Education of Young Children [NAEYC], 2012). Still, virtual teaching is not the norm for most teachers. Therefore, it poses significant challenges (Dhawan, 2020), especially for early childhood educators. For example, early childhood teachers may have trouble translating play-based learning experiences to the virtual setting. According to constructivist theory, the principal theory of learning in early childhood education (ECE), play is a central component in early learning. Play-based learning combined with some degree of adult guidance and scaffolding engages children to construct new knowledge (Van Hoorn et al., 2015; Weisberg et al., 2016) and has been linked with higher academic achievement (Marcon, 2002; Zosh et al., 2013) and executive functioning (Berk & Meyers, 2013). Children in child-centered, play-based environments have been shown to be engaged in more effective problem-solving behaviors compared to children in more teacher-directed environments (Gmitrová & Gmitrov, 2004; McInnes et al., 2009). Although ECE teachers routinely create play-based learning experiences in the classroom, the virtual setting can pose a barrier for providing interactive and engaging learning experiences. Even though there is emerging evidence on various opportunities and challenges for ECE teachers’ virtual teaching (e.g., Dayal & Tiko, 2020; O’Keeffe & McNally, 2021), information on how teachers incorporate play into virtual teaching to support children’s learning is scarce. Thus, the current study examined the challenges and barriers ECE teachers faced in their efforts to foster play through virtual teaching, focusing on the types of play-based instruction teachers were effectively able to facilitate virtually. Theoretical Framework The constructivist approach guides our study, which endorses play as child-directed, active, engaging, and involving a child’s social world and community (Mooney, 2013). For this paper, the understanding and organization of play in virtual settings are based on Piaget’s stages of the development of play (Van Hoorn, et al, 2015) and extended by including the category of guided play (Weisberg et al., 2016). Piaget’s stages of the development of play, also known as types of play, include the categories of functional play and symbolic play, which consist of constructive play, dramatic play, and games with rules (Van Hoorn et al., 2015). Functional play is predominantly a form of play for infants and toddlers in which children repeatedly practice interacting with objects, people, and language to develop schemes (Gestwicki, 1999). However, older children also engage in functional play such as riding bikes, moving their bodies to music, repeatedly putting the same puzzle together, along with several other repetitive activities they find engaging. Symbolic play involves mental representation and make-believe, also known as pretend play. It forms the foundation for abstract thinking and is critical to human development (Van Hoorn et al., 2015). Constructive play is a form of symbolic play because it involves children representing their experiences by utilizing objects to make other things (Gestwicki, 1995). For example, a child may construct a house out of blocks or draw a picture to illustrate a firetruck. Dramatic play is another form of symbolic play; children create imaginary roles and situations using representations of objects that are more abstract, utilizing language and gestures (Van Hoorn et al., 2015). Finally, games with rules are one more form of symbolic play that includes adherence to external governed play, such as Chutes and Ladders, or negotiated or agreed upon rules that the children can invent (Gestwicki, 1995; Van Hoorn et al., 2015). Another category of play is guided play. Weisberg et al. (2016) describe guided play as play-based activities integrating teacher engagement to support learning. This type of play is referred to as ‘intentional teaching’ (Epstein, 2007), ‘conceptual play’ (Fleer, 2011), ‘educational inquiry’ (Youngquist & Pataray-Ching, 2004), and ‘pedagogical play’ (Edwards & Cutter-Mackenzie, 2013). While play holds the characteristic of being child-centered, in an ECE classroom, the teacher remains key to the play centered-curriculum. Therefore, in guided play, the knowledgeable teacher offers scaffolding when appropriate and orchestrates the classroom environment or activity in a way that meets and fosters the developmental needs of the children in their play (Van Hoorn et al., 2015). Use of Technology and Virtual Teaching in Early Childhood Classrooms Virtual teaching has grown in the last two decades (Singh & Thurman, 2019). Research in the last decade has shown that virtual instruction in some early intervention and K-12 settings has been effective in improving students’ learning outcomes (Behl et al., 2017; Merchant et al., 2014; Olsen et al., 2012; Poole et al., 2020). Despite its growing popularity and potential benefits, research findings on the quality and impact of virtual teaching are mostly mixed, which has raised concern among practitioners, researchers, and the public about the effectiveness of such methods (Dong et al., 2020; Dorn et al., 2020; Duckworth et al., 2021; Means et al., 2009). Also, most studies on virtual teaching were conducted in K-12 or early intervention settings but not in early childhood settings. While these findings are informative, they may not apply to virtual teaching in ECE settings due to its inherent differences from early intervention (e.g., serving multiple children vs. one–one–one interaction). Evidence on virtual teaching and learning in ECE settings is emerging, but there is still a significant gap in the literature. In the field of ECE, integrating technology into classrooms for young children has not received positive views from teachers and parents (Chen & Chang, 2006; Mertala, 2019). Many ECE teachers believe that traditional methods (e.g., hands-on and in-person) are more effective and afford a tactile experience for the child which allows for a more active learning role (Mertala, 2019). While this belief is supported by educational theory, the fact that virtual teaching is becoming more of a permanent option for children and families suggests there is a need for a mindset that incorporates ways to teach children virtually using developmentally appropriate practices. Teachers' positive views and attitudes toward technology integration are critical as they affect teachers’ use of technology and virtual teaching practices and impact children's motivation and learning (Kara & Cagiltay, 2017; Mertala, 2019; Miranda & Russell, 2012). Another common concern is the lack of training and support for using technology and virtual teaching for ECE teachers (Trust & Whalen, 2020). This lack of training and support may make teachers feel unprepared and incompetent in choosing and implementing new technologies (Chen & Chang, 2006). During the onset of the COVID-19 pandemic, virtual teaching became mandatory in many ECE settings. Several teachers and administrators expect that the need for virtual teaching will continue in the future (Hu et al., 2021). With the shift to virtual teaching, early childhood teachers attempted to quickly create online lessons for children in various ways (Hodges et al., 2020), such as replicating their traditional classes via video conferencing platforms (Lederman, 2020) and modifying their traditional methods to engage children (Allen et al., 2020). In the past, early childhood teachers have used technology as a teaching tool (e.g., showing pictures or video, Kalogiannakis, 2010), but teaching virtually requires next-level knowledge and skills in technology integration. Teaching virtually as a primary teaching modality requires teachers to gain new skills and feel competent in providing meaningful learning experiences online (Chen & Chang, 2006; Dhawan, 2020; Trust & Whalen, 2020). Providing play-based experiences is vital in creating meaningful learning environments for children. However, some evidence suggests teachers are experiencing barriers to providing play virtually. As indicated by Ford et al. (2021), virtual teaching is inherently challenging for any age group, but it would be even more challenging for young children. The abrupt transition to virtual teaching does not allow much time to prepare for virtual teaching and has intensified job demands and stress (Allen et al., 2020). Many teachers reported that children do not have access to basic technological equipment and internet access to access learning content and participate in virtual classes (Dias et al., 2020; Ford et al., 2021). Teachers often reported difficulty engaging children and communicating with parents through virtual teaching (Dayal & Tiko, 2020; Dias et al., 2020; Ford et al., 2021; Hu et al., 2021; Szente, 2020; Tarrant & Nagasawa, 2020). As teachers experienced various challenges, many teachers experienced lack of resources and support, training, and consistent guidance for virtual teaching (Ford et al., 2021). According to Hu et al., (2021), most preschool teachers provided digital content (e.g., uploading videos, sharing virtual resources) and have limited teacher–child interactions to deliver teaching (e.g., real-time interaction in a synchronous environment). Despite myriad challenges and lack of resources and training, many ECE teachers actively sought resources, learned independently, and collaborated with others to engage children and provide them developmentally appropriate and play-based virtual learning experiences (Ford et al., 2021). While more research evidence on virtual teaching is emerging, there is still a lack of research on how teachers deliver content, which includes their ability to support play. As the effects and impacts of the COVID-19 pandemic may be long-lasting, play is even more necessary as it provides a natural medium to help reduce stress and build resilience in children, particularly in times of crisis (Anderson-McNamee & Bailey, 2010; Rao, 2018). In addition, play provides a valuable escape from uncertainty and stress, offering a sense of normalcy and safety (Yogman et al., 2018). Thus, it is crucial to study how ECE teachers provided learning opportunities through play in a virtual space during the COVID-19 pandemic. Current Study There are a few studies that indicated endorsement for play-based virtual teaching (O’Keeffe & McNally, 2021), but there is uncertainty regarding the capacity for ECE teachers to use this type of teaching during the COVID-19 pandemic (Steed & Leech, 2021). Currently, limited resources and research are available on how to apply developmentally appropriate practices (including play-based instruction) in an ECE virtual classroom. Further, understanding the challenges associated with implementing play-based learning through virtual teaching is an understudied area. More research is needed to understand teachers’ experiences and what types of activities and learning experiences promote play in a virtual classroom. Thus, the current study examined ECE teachers’ provision of virtual teaching, focusing on play-based learning activities and related challenges by seeking to answer the following research questions:What challenges and barriers to providing play-based instruction are more prevalent in virtual teaching? What type of play-based instruction did teachers effectively implement in their virtual classroom? Methods This qualitative study used content analysis of the open-ended responses along with descriptive analysis of an online survey to understand how ECE teachers implemented play-based instruction in a virtual setting and what challenges and barriers existed. Participants After obtaining IRB approval, ECE teachers (serving children from birth through age 8) who had taught any form of virtual instruction in the United States (hybrid, synchronous, asynchronous) during the 2020–2021 school year were invited through email and social media to participate in this study. Recruitment methods included emailing local schools, using social media such as Facebook, and reaching out through national organizations. The COVID-19 pandemic caused a shift in school functioning with closures beginning in March 2020 and various rearrangements of teaching options by January 2021, including in-person or virtual teaching (i.e., hybrid, synchronous, asynchronous) options. Therefore, recruitment efforts reached teachers with a range of experience teaching virtually. Between January and June 2021, an online consent form and survey were sent out to teachers who were currently teaching in ECE classrooms. To participate, teachers had to have utilized some form of virtual teaching in the 2020–2021 school year and have taught children between the ages of birth through third grade between the 2020–2021 school year. Initially, 86 teachers completed the survey however, ten were excluded because they either taught higher-ed or served in a non-teaching role (i.e., retired, principal). Therefore, 76 early childhood teachers from around the United States participated in this study (see Table 1).Table 1 Teacher demographics and background information (n = 76 teachers) Characteristics % Age (years) 18–24 7.9 25–34 22.4 35–44 32.9 45–54 26.3 55+ 10.5 Race White 77.3 Black 4.0 American Indian/Alaska Native 8.0 Asian 6.7 Other 4.0 Hispanic/Latino Origin (yes) 5.3 Gender Female 99.0 Other 1.0 Education level Graduate degree 30.3 Bachelor’s degree 64.5 Associate degree or lower 5.2 No experience teaching virtually prior to COVID-19 89.0 Total years teaching 1–5 years 26.3 6–10 years 26.3 11–15 years 15.8 15+ years 31.6 Age teaching (2020–2021 school year) Infant/Toddler (birth—2 years old) 5.3 Preschool/PreK (3–5 years old) 63.2 Kindergarten 7.9 First grade 7.9 Second grade 7.9 Mixed ages/other 7.9 Taught a different ECE age/grade in the previous year 21.1 Teaching Modality (2020–2021 school year) Fully in-person 22.7 Fully online 25.3 Hybrid (some in-person/some online) 32.0 Began in-person and moved online 6.7 Began online and moved to in-person 8.0 Other combination of modalities 5.3 Teaching Philosophy Constructivist 77.1 Behaviorist 4.3 Maturationist 5.7 Combination 12.9 Measures Teachers in the study completed an online survey with multiple choice and open-ended response questions. Multiple choice questions addressed demographic and background information (e.g., What grade do you currently teach?, Have you had previous experience teaching prior to the COVID-19 pandemic?), and questions about teaching experience, play-based instruction, and teaching beliefs and practices (e.g., How have you incorporated play-based instruction in the virtual classroom using pretend, imaginative, or dramatic play?, How would you describe your personal teaching philosophy?). The purpose of the teaching beliefs and practices questions was to better understand teachers' foundational beliefs related to play-based instructional practices, given that certain philosophies more clearly align with implementing play (i.e., constructivist and maturationist philosophies) (Kohlberg & Mayer, 1972). Open-ended response questions asked teachers their personal perspectives on:1) types of play-based activities that were effectively implemented in their online classroom, and 2) challenges and barriers they experienced when trying to implement play-based activities in their online classroom. Data Analysis Content analyses of open-ended responses and descriptive analysis were used for this study. Content analysis as a research technique allowed the researchers to determine the presence of words, concepts, and themes within given qualitative data of participant responses and analyze the meanings and relationships among those words, concepts, and themes (Berelson, 1954). This technique has been applied to the verbatim responses to code those responses into a meaningful set of categories that lend themselves to further analysis (Berelson, 1954). Descriptive statistics were used to analyze teacher and background information and the frequency of play categories teachers found effective in their virtual teaching. Qualitative analysis was used for the open-ended survey questions and included conducting level one (provisional coding) and level two (pattern) coding (Saldaña, 2021). After cleaning the data, the first two authors developed a list of provisional codes and then individually coded the qualitative data (i.e., teachers’ open-ended responses in the online survey). Once all responses were coded, the authors met to refine codes and categories and discuss any discrepancy until reaching consensus. Finally, the two authors conducted reliability checks with all responses to the open-ended question and established inter-coder reliability ranging from 90 to 100% agreement. Each open-ended question was analyzed separately using two cycles of coding. First, the open-ended question on challenges and barriers to implementing play-based virtual instruction was analyzed using provisional codes for level one (Saldaña, 2021) that were developed before the analysis. These codes (e.g., technical issue, connectivity issue, parent involvement, and student participation) were based on research related to challenges and barriers to virtual teaching (Dayal & Tiko, 2020; O’Keeffe & McNally, 2021) and play-based instruction (e.g., Kane, 2016). Pattern coding was used for level two coding (Saldaña, 2021) with emerging themes related to whether challenges and barriers were unique to virtual instruction (connection issues, home distractions, and lack of access to materials) or specific to fostering play virtually (lack of experience teaching virtually, lack of support, and teacher mindset). The second open-ended question on forms of play in the virtual classroom asked teachers to describe effective ways they implemented play-based activities in the virtual classroom. Level one provisional codes (Saldaña, 2021) were based on common but not mutally exclusive characteristics of play (e.g., child-directed, open-ended, scaffolding, hands-on, pretend play, games, inquiry, and physical movement) (Van Hoorn, et al., 2015; Weisberg et al., 2016). The process of level two pattern coding (Saldaña, 2021) organized the play-based activities into categories of play guided by the theoretical framework). Once all responses were coded and inter-coder reliability was established for this question, the frequency of occurrence of the activities in each type of category of play was calculated. Results Challenges and Barriers The survey data collected teachers’ open-ended responses that described challenges and barriers they encountered when trying to incorporate play-based instruction in their virtual classroom. Two major themes emerged from the analysis of the open-ended responses using the two-level coding process: (1) challenges related to virtual instruction and (2) specific barriers to fostering play-based instruction. Challenges Related to Virtual Instruction The first major theme of challenges related to virtual instruction included reported difficulties unique to teaching online. The greatest challenge reported regarding virtual instruction was that students did not consistently have access to materials at home that were needed for lessons. One teacher explained, “Not every student has the same resources at home” while another teacher listed, “Lack of knowing what is available at home” to be a significant challenge. The second greatest challenge reported was student engagement in the virtual setting. Teachers reported a range of student participation and interaction. For example, some teachers with asynchronous classrooms did not directly interact with the students and reported having inadequate information on whether they participated and how they engaged with the material and activities. Several teachers with synchronous classrooms also expressed difficulty fostering student engagement and interaction. A few teachers attributed this challenge to children’s age and comfort level. For instance, a teacher explained it is “hard for preschoolers to interact with peers or teachers in a play situation” in a synchronous virtual classroom. Another teacher noted, “Students are more shy on camera than in person and some refuse to interact.” External distractions such as background noise or siblings interfering and distracting the students were also often mentioned as interruptions to the level of engagement in a virtual classroom. Lastly, several teachers also listed common technology issues, such as bad internet connections and insufficient wi-fi, as being a virtual instruction challenge. Barriers to Fostering Play Virtually The second major theme that emerged was specific barriers to fostering play virtually. The most highly reported barrier was the lack of support. Survey responses showed that most teachers felt they had a higher degree of autonomy in their instructional practices (52.9% felt a great extent of autonomy) prior to the COVID-19 pandemic. Whereas, during the 2020–2021 school year 49.3% felt they had more autonomy. Teachers noted a lack of support from both parents and school administration. Regarding lack of support from parents, one teacher wrote, “Many parents don’t understand play-based instruction and prefer rote learning.” Another noted, “Lack of understanding of the importance of play from parents despite different ways of explaining it.” One teacher explained that in asynchronous classrooms,It’s hard to document what standard play is covering. It’s also hard to get parents to take a picture or record a video of the students playing for documentation purposes. Without me being right there, it is hard for me to justify putting that as a completed standard or assignment. In addition, a teacher shared that some parents’ lack of support for play-based activities was due to not wanting “a mess or a ton of things to clean-up.” This comment refers to the fact that hands-on and exploratory activities may utilize “messy” materials such as paint, mud, or water or require many materials that require extensive clean-up (i.e., small beads or pretend food). Teachers also referenced the lack of support from administration in implementing play-based teaching with statements such as, “Being forced to follow curriculum to fidelity,” “schedule is rigid,” and “everyone has different expectations.” A second barrier highly reported related to implementing play virtually was teachers’ lack of experience with virtual instruction. This lack of experience teaching virtually was woven throughout the teachers’ comments related to their lack of experience with the technology platform chosen by their school district, and lack of education on how to teach in a virtual classroom. Teachers’ lack of experience was also evident in comments like “[providing play virtually is] impossible.” These types of ‘absolute’ comments came from many teachers even though 77.1% of teachers in the study identified themselves as having a constructivist teaching philosophy (see Table 1). In addition, teachers expressed how difficult it was to not be able to teach in person, saying, “The time for extended free play and dramatic play that would happen in a classroom is not feasible online.” Another explained, “Young children do not have the attention span that would allow them to remain attentive through a virtual lesson.” Play-Based Lessons Facilitated Virtually Even with significant challenges and barriers to fostering play through virtual teaching, many teachers provided specific explanations for play-based lessons or activities they were able to facilitate virtually. Of the 76 participating teachers who completed the survey, 40 teachers provided a total of 57 examples of types of play that were implemented effectively in the virtual classroom (see Table 2). Some participants listed multiple examples of play-based activities which explains why there were more examples than the number of teachers’ responses. Findings were organized and guided by our theoretical framework of Piaget’s stages of the development of play (Van Hoorn et al., 2015) with the addition of guided play (Weisberg et al., 2016). It is important to note that some examples provided under each type of play could be interpreted to fit in another type of play, which demonstrates that play is not one-dimensional and is influenced by each child’s individuality and unique nature.Table 2 Frequency of reported effective play-based lessons or activities in virtual classroom (n = 40 teachers) Type of play Frequency % Sample comments Functional play 13 23 “Object play—everyone had same household object to explore together” Constructive play 8 14 “Using loose parts for building.” Dramatic play 11 19 “A favorite of the students was a dramatic play experience, where we either dressed up as a character, or used puppets or stuffed animals and pretended to be the puppet or stuffed animal. We would talk, ask them, think of games that the animal/character would enjoy, think about how they would respond, etc.” Games with rules 16 28 “We played Simon Says…, made up Bingo games…, played I Spy games as well as guessing sounds of animals and guessing songs.” Guided play 9 16 “Activities that asked children to go outside, look for things, create using those things.” Frequency calculations based on the total number of examples (57) provided by teachers aCategories of play are listed by the theoretical framework rather than by the highest frequency Play-based activities in the category of functional play were reported thirteen times (23%). Functional play was evident when teachers described lessons in which the children engaged in:Singing, fingerplays, Go Noodle, yoga, dance parties Sink or float activities Making shadows with flashlights Interacting with White Board features Constructive play was reported the least frequently with eight examples (14%). Constructive play was represented through:Open-ended play with blocks, Play-Doh, Legos Making caves or forts at home Pretending to go on adventures with pretend equipment such as rope and ladders. Dramatic play was reported 11 (19%) times as an effective play-based virtual activity. Dramatic play is when children create imaginary roles that often goes along with their constructive play, however it is more abstract utilizing language, gestures and role playing with others. Examples teachers noted in the survey include:Virtual “tea party” Acting out stories with household objects Pretend cooking Pretend adventures Dress-up as character in books Use puppets and stuffed animals and talk for them Use story starters and children role play their own endings Pretend play (bakery, grocery shopping) The most frequently reported virtual play-based activity category was games with rules (28%). Examples of games with rules teachers provided included:Scavenger hunts Simon Says Bingo I Spy Hide and Seek (with objects and families helping) Next, virtual play-based activities in the category of guided play were reported approximately 16% of the time. Examples of guided play included:Investigative questioning of concepts Open-ended science activities Science experiments Outdoor investigations Nature walk, collecting materials, journaling Patterning and counting with toys Looking for things outdoors and creating with those items The findings suggest each type of play can be facilitated virtually; however, some types of play were reported to be effectively implemented more often than others. Games with rules and functional play were the two most frequently reported types of play used in virtual teaching by the teachers in this study. Discussion This study sought to answer questions related to what challenges and barriers exist for ECE teachers when implementing play-based instruction virtually and how teachers implemented virtual play-based activities effectively during the COVID-19 pandemic. Our study adds to the growing body of literature that has come out of the questions that arose from the pandemic’s impact on education. Given the lack of literature on virtual teaching and how teachers deliver content to children, our study is unique in that it captured teachers’ perspectives of ways to implement play-based instruction virtually during the pandemic when virtual teaching in ECE was increasing in use. Most teachers (89%) participating in our study did not have any experience teaching virtually, with limited training in this area prior to the 2020 school year. With our focus on play-based instruction, our findings highlight the unique demands of the virtual learning environment in ECE settings. Given that the option for virtual teaching in one form or another is here to stay even after the pandemic, it is necessary to examine the needs specific to ECE settings to understand and inform how to best support teachers in providing play-based virtual instruction. The biggest challenge to teaching virtually for the ECE teachers in our study was children’s lack of access to needed materials. Developmentally appropriate practices for ECE are grounded in teaching through experiences that often involve the use of hands-on materials (NAEYC, 2022), including items such as counting manipulatives, art supplies, building materials and visual supports. In a physical, in-person classroom, teachers utilize these typical ECE classroom materials or may borrow materials from other teachers in their school. However, in a virtual classroom, teachers must rely on the materials students have access to in their homes. Navigating the range of access to materials their students can use may decrease instruction quality or force teachers to rely on their own personal finances to purchase individual supplies for students as reported by teachers in other studies (i.e., Atiles et al., 2021). Teachers also reported difficulty engaging children virtually and reducing distractions, which suggests the ECE virtual learning environment requires a different way of engaging children. Teachers understand that play is essential to children’s learning and development and have reported the desire to reach and engage their students in developmentally appropriate ways, even virtually, which is consistent with previous studies (Crawford et al., 2021; Ford et al., 2021; Kwon et al., 2022; Szente, 2020). This commitment has been evidenced by teachers’ ability to pivot their teaching to a virtual modality while still trying to incorporate play-based instruction. However, the highly reported challenge of engaging children mentioned in other recent research (e.g., Ford et al., 2021; Hu et al., 2021) suggests that teachers need to be better equipped to teach virtually in developmentally appropriate ways (Atiles et al., 2021; Crawford et al., 2021; Szente, 2020). Debates about supporting play in the in-person classroom are not new (Chien et al., 2010; Pyle & Danniels, 2017), and teachers in our study expressed a lack of support as a barrier to providing play-based instruction in their virtual classroom. While many of the teachers assumed a level of teacher autonomy, the lack of parent and administrative support in their fostering play-based instruction was prevalent. This could have been due to the unprecedented situation of the COVID-19 pandemic, where school administrators and teachers alike might have felt overwhelmed by the changes. However, as schools have adapted more to providing virtual instruction since the onset of the pandemic, another examination of teachers’ feelings of support in this context is needed (Ford et al., 2021). Many teachers also expressed that lack of support from parents was a significant barrier to fostering play-based instruction, which supports the findings of previous studies (Dias et al., 2020; Ford et al., 2021; Hu et al., 2021; Kwon et al., 2022). The most notable reason was due to parents’ lack of understanding of the role play has on children’s learning, which is often an issue for teachers even when they are teaching in person. Ultimately, parents want what is best for their children and they likely experienced behavioristic, traditional teaching practices throughout their own schooling and think that is how one learns. Play has often been considered frivolous or something children do to relax and entertain themselves, not academic or a way of learning. This indicates that educators must find ways to share with families how play is essential to children’s learning and the merits it has in children’s development. Since this has been a challenge for early childhood educators for decades, it might be wise to change our approach. Guided play was listed as a type of play in the research and in the data analysis for this study. Purposefully framed play or guided play can be connected to standards for learning and documented accordingly, which may help parents and even educators be more receptive to such instructional processes. As virtual teaching is becoming a more adopted modality, these findings provide insight into how to support teachers who offer play-based instruction virtually. Teachers also reported their lack of experience teaching virtually as a barrier to implementing play. This finding extends recent research findings that ECE teachers experience a lack of preparation for teaching online (Ford et al., 2021) by specifically focusing on implementing play. Interestingly, while some teachers tended to take an approach that was flexible, creative, and oriented to problem-solving, several teachers in our study were hesitant to try facilitating play virtually. Some comments inferred a strong resistance to fostering play through virtual teaching, indicating a fixed mindset. Teachers’ attitudes about integrating technology and teaching can have an influence on their ability to use it as an effective tool for teaching, even in the early childhood ages (NAEYC, 2012; Nuri & Cagiltay, 2017). O’Keeffe & McNally (2021) found that generally early childhood teachers believed play-based activities are an important pedagogical tool in supporting children’s learning, even in the context of the COVID-19 pandemic. Therefore, it could be that the stressful experiences reported by teachers in our study could explain some teachers reverting to traditional, behavioristic practices such as teaching mainly through direct instruction and worksheets or cause some teachers to adopt a mindset that it is not possible or feasible to foster play through virtual teaching. While the consideration of the challenges and barriers is important in knowing how to best support teachers who are teaching virtually, perhaps the equally important finding is that despite reported barriers and challenges, teachers found ways to foster play in the virtual setting in every category of play. The most frequently reported category of play was games with rules such as scavenger hunts, Bingo, and Hide-and-Seek to engage children with learning material available at home. This type of symbolic play may have been the most seamlessly translated to the online format, given that many versions of online games already exist, and games with rules allow for some teacher-direction and built-in structure. Teachers in our study reported that engaging children was a challenge to virtual teaching, and the use of games may have been a way that helped encourage children to participate and interact. Given the lack of research on what teachers do in their virtual classrooms (e.g., their use of developmentally appropriate and play-based teaching strategies to promote children's learning), it is promising that we found a variety of examples of play-based instruction provided in each category of play during the pandemic. Teachers were able to facilitate dramatic play, provoke children’s thinking through investigations, encourage physical activity, as well as provide opportunities for open-ended exploration. These findings offer hope that a mindset can be shifted away from the belief that providing play in a virtual setting is “impossible”. Teachers are motivated to best meet children’s learning needs by providing opportunities for play and exploration (O’Keefe & McNally, 2021). With additional support and training, those who may be assigned to teach virtually will be able to do so effectively and more confidently. Limitations This study has several limitations. First, the study is somewhat limited in that it primarily focused on which activities teachers used to foster play virtually and the challenges and barriers they faced. Much more information is needed regarding teaching children virtually, such as how teachers connect play experiences to state standards and how they assess children’s learning. More research documenting whether teachers are having success in scaffolding children’s play virtually and what strategies they are utilizing is needed. Second, the online survey data were collected between January and June 2021 during the pandemic. While data were collected for a six-month span, the COVID-19 cases and school responses to the pandemic (e.g., transitioning from one teaching modality to another repeatedly) have fluctuated since its onset. Our data only serves as a snapshot and does not capture how teachers adjusted and tailored their teaching to the ongoing changes and challenges during COVID-19. It would be ideal to collect longitudinal data to address this process. In addition, most teachers in our study were highly educated and taught PreK and children in public school settings. Thus, it may not fully represent the teachers in a range of ECE settings. More research is needed to understand the perspective of teachers serving other age groups in different ECE settings (e.g., childcare, Head Start programs). Notably, the perspectives of teachers who served in early care and education centers or family childcare homes (i.e., serving infant, toddler, and preschool-aged children) are needed, given that these teachers tend to have potential unique needs and experiences when related to virtual teaching. Implications for Practice and Policy This study has implications for practice. First, this study’s findings highlight the need to prepare teachers to provide play-based and developmentally appropriate instruction virtually. While various professional organizations (e.g., NAEYC, the Division for Early Childhood) provide valuable resources and information, teachers benefit from more resources and training on different strategies for engaging children virtually. Although, evidence from this study demonstrates that teachers are capable of creatively fostering play through virtual teaching that includes all types of play, teachers face various challenges and barriers when trying to implement play-based instruction virtually. Teachers in the field need to be provided with training that keeps them up to date with the virtual teaching and learning platforms as well as strategies that can elicit student engagement. Ideas and tips from experts in special education and early intervention, practitioners in telehealth, and faculty in higher education can be adopted for the ECE classroom such as chunking time and information into smaller parts, adding humor, adding an element of performance, and storytelling (Cavanagh, 2019; Olsen et al., 2012). Teacher educators also need to be knowledgeable in virtual teaching practices and online learning systems that are being used in schools and programs and teacher preparation programs need to incorporate elements of virtual instruction into their courses. Second, schools and programs need to equip families with the knowledge of how play and learning are connected in the early childhood years. Administrators must understand teacher challenges and barriers and support teachers in overcoming these difficulties. Families need to be educated on the importance of play, even in virtual settings. This can be achieved through family engagement activities, informational sessions, and school-family communication. This study also has implications for policy and research. As virtual instruction is more prevalent, policies need to be implemented that provide equitable access to materials including educational hands-on materials, as well as stable access to the internet and needed devices. For schools that offer the options for virtual instruction, it is vital for administrators to consider this need for students to access educational materials that are appropriate for early childhood ages. Schools can confront this challenge through suggestions such as allowing space in their budget to purchase materials for students to use at home or having community resource centers where virtual students can borrow materials they may not have at home. More research is needed on how to make virtual learning more equitable. In addition, more research is necessary to examine any changes in early childhood teachers’ ability to provide play-based instruction in a post-pandemic era. Given the known barriers and challenges that currently exist, research similar to this study will help to illuminate the struggles teachers face and allow for better approaches to meet the needs of teachers and children. Our ultimate responsibility as educators is to set the conditions for learning and development, which can include a virtual environment. This can be accomplished by supporting play in efforts to guide children’s learning related to academic standards in ways that honor children’s development (Van Hoorn et al., 2015). Our research is not making a case for virtual learning with young children; however, the study provides essential information for when virtual teaching is the modality being used and the importance of play-based teaching regardless of whether it is in person or virtual. It has been documented that play is the primary force in child development and learning and therefore, it is essential for early childhood teachers to incorporate different kinds of play in their teaching pedagogies whether they are in person or virtual. In this new era, schools will likely continue to utilize virtual teaching to some degree to make up for children's time away from school due to weather, illness, and a host of other reasons. Therefore, it is critical that the findings of such research are disseminated in the field of ECE. This research helps to expand notions of how play can be encouraged, supported, and scaffolded through both synchronous and asynchronous formats. The sharing of ideas and lessons learned regarding fostering play through virtual teaching is always a great way to build cognitive, social, and emotional connections that will influence teaching, research, and service. Declarations Conflict of interest The authors did not receive support from any organization for the submitted work. The authors have no relevant financial or non-financial interests to disclose. 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==== Front J Child Adolesc Trauma J Child Adolesc Trauma Journal of Child & Adolescent Trauma 1936-1521 1936-153X Springer International Publishing Cham 496 10.1007/s40653-022-00496-9 Exploratory Study Mitigating Rural Adolescent Trauma: Remote Delivery of a Trauma-Informed Yoga Intervention During COVID-19 http://orcid.org/0000-0003-1535-6643 Davis Lauren [email protected] Aylward Alexandra [email protected] grid.41891.35 0000 0001 2156 6108 Montana State University, 115 Reid Hall, Bozeman, MT 59717 USA 1 12 2022 114 28 10 2022 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Given the prevalence of childhood trauma in rural Montana, this project is intended to help mitigate stressors that may contribute to poor behavioral and mental health in high school-aged children, which may be exacerbated by the collective trauma of the COVID-19 pandemic. The immediate goal was to measure physical and mental health outcomes in adolescents resulting from a remotely delivered trauma-informed yoga intervention designed to foster positive youth development. Our study builds on the successes from an initial feasibility pilot study one year prior in order to evaluate a more robust intervention comparing experimental and control group outcomes. Students at a small, rural high school in Montana volunteered to participate in a 6-week, twice-weekly trauma-informed yoga intervention in their physical education class. Validated survey measures, including the PHQ-A, GAD-7, and ACE-Q instruments, were utilized to measure mental health outcomes pre- vs. post-intervention. Salivary cortisol levels were also measured pre-, mid-, and post-intervention. Statistically significant declines in cortisol levels and improvements in sleep duration were noted when comparing experimental vs. control groups. Noteworthy declines in depression and anxiety levels were also seen when comparing the treatment to control groups. Descriptive differences between the control and experimental groups illustrate the mental health benefits of reduced anxiety and depressive symptoms in rural adolescents resulting from a remotely delivered trauma-informed yoga intervention. Our study holds the potential for a long-term public health impact in reducing adolescent rates of anxiety and depression while mitigating trauma in geographically isolated settings. Trial Registration: ClinicalTrials.gov identifier: NCT04664855. Keywords Adolescents Trauma Depression Anxiety Yoga COVID-19 http://dx.doi.org/10.13039/100000002 National Institutes of Health P20GM104417 Davis Lauren ==== Body pmcIntroduction “…it is easier to build strong children than repair broken men.” (Frederick Douglass) It is no secret that adolescence can be one of the most emotionally difficult phases in one’s life, even in the best of times; adding the collective trauma of a global pandemic compounds these challenges. Recent evidence indicates that mental health crises are on the rise for both adolescents and adults, and an early study indicates that increases in adolescent depression and anxiety may be linked to the coronavirus pandemic (Chattergee, 2021; Courtney et al., 2020; Lee, 2020). Further, school scheduling changes, like remote learning, shatter routines necessary for students, which are especially crucial coping mechanisms for those with mental health conditions (Lee, 2020). This study sought to mitigate these, and other mental health difficulties, through a novel, remotely delivered trauma-informed yoga intervention to rural high school students in Montana during the COVID-19 pandemic. Significance Given the prevalence of suicide and mental health issues in rural Montana (Montana Department of Health and Human Services, 2016), which will be elucidated below, this efficacy study was designed to help mitigate the impact of contributing factors by providing coping strategies through breathing techniques and somatic experiencing for rural high school adolescents; these coping strategies have been linked to improvements in overall mental health and behavioral outcomes, which may ultimately lead to reduced suicide rates (Khalsa et al., 2012a). Our study builds on the successes from an initial feasibility pilot study one year prior (Authors, manuscript in preparation) in order to evaluate a more robust intervention comparing experimental and control group outcomes. Ultimately, this project aimed to promote positive youth development and student success in school-aged children concurrently with providing rural access to novel interventions during a worldwide pandemic crisis. Further, it aligns with multi-tiered systems of support (MTSS) conceptual frameworks that are currently implemented in most public schools nationally (including the district involved in this study) and provides a tiered support system for students in a trauma-informed educational setting (Dorado, 2020). Context: Rural Montana and the Mental Health Crisis The Rocky Mountain region is frequently referred to as the nation’s “suicide belt;” both empirical evidence, as well as observed behaviors and anecdotes, point to high altitude, lower oxygen levels, and prolonged cold and darkness in the winter as possible contributing factors (Shukitt-Hale & Lieberman, 1996; Siegler, 2018). Recent findings from the Centers for Disease Control and Prevention (2020) indicate that Montana consistently ranks in the top 5 states with highest suicide rates in the nation. The town in which this study took place typically exceeds three times the national average in suicide rates and, unfortunately, these often involve youth suicides (KULR 8, 2013). Adolescent Mental Health in Montana A 2010–2014 longitudinal study showed that 39.1% of Montana adolescents aged 12–17 sought treatment for a major depressive episode (Montana Department of Health and Human Services, 2016). Depression likely contributes to Montana’s high rate of youth (ages 11–17) suicides, which, as a state as whole, more than double the U.S. youth suicide rates; 74% of those suicides presented with warning signs (American Foundation for Suicide Prevention, 2017). In the county in which this study took place, the 2019 Youth Risk Behavior Survey indicated that 39% of high schoolers experienced severe depression, 20% made a suicide plan, and 16% attempted suicide (Montana Office of Public Instruction, 2019). Tragically, one high school suicide took place (of a student not involved in the study) during the study implementation period. Barriers to Mental Health Care Geographic challenges unique to this area due to rugged terrain lead to significant barriers to receiving many services; germane to this study, accessing mental health care services is exceedingly difficult in the rural mountain west, but especially in Montana (Kaiser Family Foundation, 2016). As nearly half of the state is classified as rural or frontier, rural Montana has a starkly deficient quantity of mental health care providers to meet rural community members’ mental health care needs and faces lengthy travel distances and poor travel conditions to reach quality providers (National Network of Libraries of Medicine, 2020; National Center for Frontier Communities, 2018). While this study is unique to the state of Montana, similar geographic barriers and access to care difficulties exist nationally amongst other rural locales. Thus, new treatment and prevention delivery modalities are needed to increase access to evidence-based mental health treatments for rural citizens–especially by intervening adolescents in the school setting. Indication for a Trauma-Informed Intervention In children, specific traumas are considered an “adverse childhood experience,” or an “ACE;” the seminal study by Felitti et al. (1998) categorize ten forms of trauma into emotional, physical, and household challenges and link these experiences to a myriad of mental and physical health complications in adulthood. In addition to these primary traumas, the coronavirus pandemic is also considered to be a collective stressor, which can cause resulting (and potentially collective) traumatic events (Kaysen, 2021). Further, Porges (2020), recently highlighted the need for the mitigation of trauma during and following the coronavirus pandemic with his assertion that “We are a traumatized species…. [and] trauma will be the second pandemic.” COVID-19 has challenged our perceptions of safety, which is a necessity for a regulated autonomic nervous system (and key for handling stressors and traumatic events); Porges postulates that feelings of safety not only promotes mental and physical health but quarantining and isolation limited opportunities to co-regulate with others, which is especially true for children who have been unable to attend school during the pandemic (2020). While it is true that not everyone has the same genetic vulnerability toward maladaptation from stressors, trauma can shift the autonomic nervous state, disrupting neurophysiological regulation (such as sleep, digestion, etc.) (Heim & Teicher, 2020; Porges, 2020). Additionally, this shift caused by stressors in the autonomic nervous system can release hormones, including the corticotropin releasing factor (CRF), which triggers the release of cortisol, a stress hormone; the release of CRF can cause a permanent upregulation of the autonomic nervous system, which can lead to autoimmune diseases, depression, stress, and anxiety (Heim & Teicher, 2020). This dysregulation of the nervous system, caused by stressors and trauma, underscores the need for a new paradigm of treatment of trauma focusing on the autonomic state as the intervening variable (Porges, 2020). The intervention employed in this study, trauma-informed yoga, utilizes physical sequences and breathing strategies that have proven useful in mediating physiological trauma responses, including regulating the nervous system (Cook-Cottone et al., 2017). Intervention Design and Framework: Trauma-Informed Yoga Yoga has been proven to benefit individuals’ mental and physical health consistently; studies on yoga has indicated its use as a therapeutic intervention nearly 50 times in the last 15 years, reporting positive mental health outcomes in measures of resilience, anger, anxiety, stress, depression and fatigue as well as physical health improvements such as flexibility, strength, and weight loss (Khalsa et al., 2012b). For youth, yoga can be particularly beneficial in improving cortisol levels, stress, anxiety, depression, academic performance, and sleep (Davis & Buchanan, 2020a, b; Butzer et al., 2015; Cook-Cottone et al., 2017; Emerson & Hopper, 2011). Due to high rates of adverse childhood experiences in the county in which this study was completed (of over 20% of its adults reporting having an ACE score of 4 or more), a trauma-informed yoga intervention is indicated; in alignment with trauma-informed multi-tiered systems of support frameworks (MTSS), this intervention addresses Tier 1 (80% of the student population) by providing strategies for coping with stress for students (Dorado, 2020; Montana Office of Public Instruction, 2019; PRC Custom Research, 2020). A trauma-informed yoga session differs from traditional yoga practice. A trauma-informed yoga instructor can make intentional, trauma-informed choices regarding avoidance of certain props, like use of straps, which may emulate restraints that could trigger the yoga practitioners or avoiding certain poses that could be triggering in positionality (Emerson & Hopper, 2011). It is also prudent for the trauma-informed yoga instructor to educate the participants on how yoga can help heal the dysregulated stress response/nervous system for their own understanding of the practice (Emerson & Hopper, 2011; Cook-Cottone et al., 2017). As referenced earlier, empirical evidence supports that trauma-informed yoga can calm the nervous system; rhythmic, self-soothing somatosensory input (which is fostered in a yoga practice) leads to physical senses of safety (which is key in healing trauma) and pleasure, leading to the release of “pleasure” hormones and regulation of stress-related neural systems (Malchiodi & Perry, 2020; Porges, 2020). Further, an indicator of autonomic nervous system health, yoga has proven improvements in heart rate variability in trauma survivors (van der Kolk, 2014). Additionally, trauma can cause difficulties in perceiving one’s physical body and personal boundaries; the loss of the “somatic sense of self” has led to those who have experienced trauma as sharing comments like “I feel detached from my body” and “I feel like my body does not belong to me” as well as experiencing physical symptoms like rapid heart rate, shortness of breath, and pain (Lanius et al., 2020). These sensations result in a condition known as alexithymia, which is difficulty in identifying one’s feelings and distinguishing between those feelings and bodily sensations from emotional arousal (and communicating these feelings with words) (Lanius, 2021). Moreover, trauma may impact “peri-personal space” in that traumatized individuals will frequently misperceive how far away objects and other people are due to a lack of spatial awareness (Rabellino, 2021). Yoga, which focuses on both interoception (understanding one’s own interior landscape) and proprioception (understanding one’s relationship to others in an environment), can help individuals regain their sense of physical space (Malchiodi & Perry, 2020). To regain this somatic sense of self and heal one’s trauma, trauma experts assert that a traumatized individual must regain a sense of embodiment by integrating interoceptive and exteroceptive sensations, which leads to the “agentive embodied self” (Rabellino, 2021). Trauma-informed yoga works to reintegrate these sensations through intentional teaching of mindfulness practices, stress reduction, and an embodied practice, as illustrated by Table 1 below and utilized as the conceptual framework for this study (Cook-Cottone et al., 2017). This conceptual framework was adapted for use for our 6 week program, as shown in Fig. 1 below.Table 1 Conceptual Framework for Trauma-Informed Yoga Intervention(Cook-Cottone et al., 2017) Symptoms Related to Trauma Exposure Elements of YIS-TIY Hyperarousal and Dissociation Embodied Practice (Physicality and Interoceptive Awareness) Avoidance and Re-experiencing Engagement in the Present Moment (Yoga and Mindfulness Practices) Alterations in Cognitions Intentional, Empowered Thinking (12 Cognitive Intentions) Relational Disconnection Yoga Teacher Presence and Responsiveness (Relational Attunement) Fig. 1 Conceptual Framework for Trauma-Informed Yoga Intervention (adapted fromCook-Cottone et al., 2017) Implications for School-Based Mental Health Initiatives. ACEs not only impact one’s adult health outcomes, but they also cause multiple mental health and academic complications during youth. Brackett (2019) asserts the following:One in five American children is experiencing a mental health issue, such as depression or anxiety, and over half of all seventeen-year-olds report having either experienced trauma directly, ranging from neglect to abuse, or witnessed it at least once as a child. By failing to recognize trauma’s effects on learning, educators risk compounding the trauma and jeopardizing students’ prospects in school…. depending on our emotional state, our chemical and hormonal profiles change dramatically, and our brains function differently. The three most important aspects of learning--attention, focus, and memory--are all controlled by our emotions, not by cognition (p. 192; p. 195). Because most ACEs tend to originate from the household and/or caregivers, schools become the low-hanging fruit as a logical setting for providing widespread support for traumatized youth. Brackett further posits that by “failing to recognize trauma’s effects on learning, educators risk compounding the trauma and jeopardizing students’ prospects in school” (2019, p. 192). Porges (2020) also notes that trauma disrupts primary learning functions, including but not limited to: difficulties in listening, following verbal commands, and speech-language delays, prosody, and behavioral regulation. Moreover, The Aspen Institute's National Commission on Social, Emotional, & Academic Development also argues that the promotion of social, emotional, and academic learning is “the substance of education itself” (2019, p. 6). Therefore, a school-based intervention is indicated in the alleviation of childhood trauma. Theoretical Framework: Community-Based Participatory Research To successfully integrate a school-based intervention into the curriculum, as well as understand the environment and demographics of the study, the research team must be embedded within the context; therefore, a community-based participatory research framework (CBPR) is necessary in carrying out this important work. Israel et al. (2001) define community-based participatory research as “focusing on social, structural, and physical environmental inequities through active involvement of community members, organizational representatives, and researchers in all aspects of the research process” (p. 182). A community advisory board (CAB) had already been established in previous iterations of the research to represent key community and school stakeholders impacted by the project. For this study, the principal investigator strengthened the existing school-community-academic partnership through strategic involvement in multiple community action and school district committees, including a community-focused suicide prevention and resilience steering committee, a school district-focused “Suicide, Intervention, and Refer to Treatment” steering committee, and CAB meetings. Informal feedback from the CAB was gathered at the conclusion of the previous study and throughout the implementation of this study to continuously assess local relevance, collaborative engagement, disseminate results, and make programmatic adjustments for future studies. Research Aims As a limited feasibility and efficacy study for a remotely delivered, rural intervention, our study had two primary objectives:Aim 1: Pilot test a limited feasibility and efficacy study of a remotely delivered (via Zoom) trauma-informed yoga intervention for 15–18 year-old male and female high school students. To prepare for this study, feedback was gathered from the school district, teachers, and students via focus groups and email communications to assess the burden from the various assessment instruments and participation in the pilot. Assessment measures listed later in this manuscript comport with this feedback. Retention and satisfaction of participants, as measured by survey instrumentation from the previous iteration of the research indicated the need for an ongoing partnership with the school district and an expansion to additional high school physical education classes for a 6-week intervention while also including a new control group. The intervention’s efficacy for youth mental and physical health was assessed with validated surveys measuring depressive and anxiety symptomology, as well as resilience, and cortisol testing to measure physiological changes. Participants were given Fitbit Inspire HR wearable fitness trackers as both an incentive and as an additional physiological measure; however, participant engagement with fitness trackers (both in wearing and syncing devices) was inconsistent at best and thus, data from these trackers were not included as a measure in this study. Participation rates and assessment outcomes were considered to determine feasibility for future remotely delivered interventions as well as to explore scalability to rural communities without access to yoga instructors while providing a safe, socially-distanced contingency for COVID-19 school closures and schedule changes. Aim 2: Evaluate the study’s school-community-academic partnership at the conclusion of the program using a qualitative survey. The results were compared with data from the same survey, collected at the end of the previous year’s study. The PI shared results from these surveys, along with study outcomes, with the CAB to determine areas for program refinement and assess the need for a study continuation and expansion within the district. Methods Sample Forty-five students at a small, rural high school in Montana volunteered to participate in this study. The analytical sample includes 23 students who completed both the pre and post measures in the treatment group and 22 students who completed the same assets in the control group. The majority of participants identified as white (n = 41) and male (n = 25 males). The number of sessions attended ranged between three and twelve, with a median of nine sessions. The majority of students (47.8%) attended at least 10 sessions and 1 student attended every session. Among our sample, 23.5% of our participants had a moderate ACE score at the onset of the study (an ACE score of 2–3), while 38.2% had a high ACE score (of 4 or more ACEs). Descriptive statistics for the full sample on the outcome measures are provided in Table 2.Table 2 Summary Statistics on Outcome Measures Overall (n = 45) Control (n = 22) Percent Change Control Group Treatment (n = 23) Percent Change Treatment Group Variable Mean (SD) Mean (SD) Mean (SD) Pre-GAD7 6.07 (5.19) 5.27 (5.13) 6.83 (5.25) Post-GAD7 4.96 (5.28) 4.55 (4.54) -13.66% 5.35 (5.97) -21.67% Pre-PHQA 6.71 (6.00) 5.36 (4.89) 8.00 (6.75) Post-PHQA 5.31 (6.09) 4.18 (5.24) -22.01% 6.39 (6.73) -20.13% Pre-CDRISC 6.82 (7.35) 28.32 (7.11) 25.39 (7.45) Post-CDRISC 27.60 (8.66) 30.45 (6.44) 7.52% 24.87 (9.71) 2.05% Pre- Cortisol 0.53 (0.40) 0.71 (0.44) 0.30 (0.19) Post- Cortisol 0.36 (0.25) 0.49 (0.27) -30.99% 0.22 (0.12) -26.67% Measures Adverse Childhood Experiences The Center for Youth Wellness ACE-Q Teen self-report is a 19-item survey that is broken into two subscales; the first subscale measures traditional adverse childhood experiences (on a scale of 0–10) while the second quantifies events that are hypothesized to be correlated to a dysregulated stress response on a scale of 0–9 (i.e., toxic stress caused by discrimination, bullying, unsafe housing, etc.) (Bucci et al., 2015). For the purposes of this study, only the first subscale for adverse childhood experiences was utilized in calculating ACE scores; other circumstances perpetuating toxic stress (i.e., bullying, homelessness, food insecurity, etc.) were excluded. ACE scores pre intervention ranged from 0 to 10 with a mean of 3.09 and a standard deviation of 2.95 and post mean of 2.22 with a standard deviation of 2.51. Thirty-eight percent of students were categorized as having a low ACE score (a score of 0–1), 23.5% had a moderate ACE score (a score of 2–3), and 38.2% were categorized as having a high ACE score (a score of 4–10). Adolescent Anxiety The Generalized Anxiety Disorder Scale (GAD-7) (Spitzer et al., 2006) is a 7-item practical self-report anxiety questionnaire where participants are asked how often, during the last 2 weeks, they have been bothered by each of the 7 core symptoms of generalized anxiety disorder. Response options are "not at all," "several days," "more than half the days," and "nearly every day,” scored as 0, 1, 2, and 3 respectively. Therefore, GAD-7 scores range from 0 to 21, with scores of > 5, > 10, and > 15 representing mild, moderate, and severe anxiety symptom levels. In this study, the GAD-7 demonstrated high internal consistency (Cronbach’s alpha of 0.90) for the sample in this study. The GAD-7 has also shown strong reliability (0.85) and validity (73.3%) in prior research (Rutter & Brown, 2017). Adolescent Depressive Symptoms The Patient Health Questionnaire for Depressive Symptomology for Adolescents (PHQ-A) (Johnson, 2002) is a self-report 9-item instrument to assess symptoms of depression among adolescents at study onset. Participants were asked to indicate how often they have been bothered by eight possible problems or symptoms over the last 2 weeks (e.g., “feeling down, depressed, or hopeless,” “feeling tired or having little energy,” and “feeling bad about yourself, or that you are a failure, or have let yourself or your family down”). Each item was rated 0 (not at all), 1 (several days), 2 (more than half the days), or 3 (nearly every day), and items were summed to obtain scale scores. Internal consistency for this scale was high (Cronbach’s alpha = 0.90) in the sample. The PHQ-A has also demonstrated strong reliability (0.875) (Bian et al., 2011). Resilience The Connor-Davidson Resilience Scale (CD-RISC) (Connor & Davidson, 2003) is a self-report 10-item instrument to assess one’s perceptions of self-resilience and agency. Each question is scored on a Likert scale of 0 to 4, where 0 indicates “not at all,” 1 indicates “rarely true,” 2 indicates “sometimes true,” and 4 indicates “true nearly all the time.” Scores can thus range from 0–40, with higher scores indicating higher resilience. Scores are broken into four quartiles; scores from 0–29 exemplify a low resilience score, 30–32 is low-intermediate resilience, 33–36 is high intermediate resilience, and a score of 37–40 exemplifies high resilience. Internal consistency for this study’s scale was high with a Cronbach’s alpha of 0.90 in the sample. The 10-item CD-RISC measure has also shown adequate internal reliability (0.85) in other studies (Gonzales et al., 2016). Below is a table of the various survey measures, and their corresponding validity and reliability, utilized in this study; additionally, all students were all screened pre- and post-intervention by the Columbia Suicidality Screener, per the school district’s request, but these results were not included in the findings from this study Table 3.Table 3 Assessment Measures Description of Measure Source Timeline for Dissemination Cronbach’s Alpha/Validity & Reliability from Prior Research Center for Youth Wellness ACE-Q Self-Reporting Screener for Teens Bucci et al., 2015 Pre-intervention (in order to determine ACE scores of participants) Longitudinal testing currently underway to measure content and construct validity/reliability Generalized Anxiety Disorder Scale (GAD-7) Spitzer et al., 2006 Pre- and post-intervention Cronbach's alpha: .79-.91 Reliability = .85 Validity = 73.3% Patient Health Questionnaire for Depressive Symptomology for Adolescents (PHQ-A) Johnson, 2002 Pre- and post-intervention Chronbach’s alpha = .835 Reliability = .875 Validity = 89.5% Connor-Davidson Resilience Scale (CD-RISC) Connor & Davidson, 2003 Pre- and post-intervention Reliability = .85 Intervention Design District leaders wished to hold the study during the Jan-March 2021 window, as this timeframe has shown a historical spike in inappropriate student behaviors and absenteeism. This study aimed to have at least 30 students in each group (control and experimental); while the project initially had a sufficient sample size for statistical significance (n > 30 in each group), due to attrition, COVID-19 quarantining students, and other difficulties beyond the research team’s control, the final data set included 23 students in the treatment group and 22 in the control group. Students in both groups were from three different physical education classes taught by the same physical education teacher; the experimental group was composed solely of freshmen students, and the control group was a variety of sophomore, junior, and senior students. Students in the experimental group were asked to provide qualitative feedback after each yoga session (according to prompts provided by the yoga instructors) as well as at the conclusion of the study to provide overall feedback regarding their perceptions of the intervention. During the 2020–2021 academic year, the high school involved in this study was administered in an A/B Cohort model; that is, students were split in half into two separate groups (A and B). Students in Cohort A attended school face-to-face twice weekly while students in Cohort B attended school remotely from home while Cohort A students were in school, and the reverse happened for Cohort B students to attend school while Cohort A students worked from home. To test the remote delivery of this intervention, sessions were held for each cohort during their regularly scheduled physical education class time while they worked remotely from home; this not only protected the physical education teacher’s in-person instructional time for each cohort, but it also promoted healthy activity and wellness for students as they learned from home. Students in the control group did not receive any yoga during the intervention, but they were provided with recordings from the intervention sessions after the study’s conclusion. Sessions for the experimental group were twice weekly for six weeks for each cohort, and each session lasted 45 min via Zoom and were led by two certified trauma-informed yoga instructors; one benefit to remote delivery was that it created the opportunity for instructors outside of rural Montana to be a part of the yoga instruction. Thus, one instructor taught virtually from within the rural Montana community, and the other instructor taught remotely from Madison, Wisconsin. Considerations for the remote delivery of this intervention primarily revolved around student safety. Given that trauma-informed yoga can bring up strong emotions (related to previous adverse experiences), as well as potential physical injury, students had brief “check-ins” with the instructors at the beginning of each session. At the conclusion of each session, yoga instructors provided journaling prompts, via the Zoom Chat feature, to help participants process and reflect upon how the session impacted their physical and mental states. Participants entered these journal entries into a confidential Google Form survey, which was then reviewed by the researchers and yoga instructors. Wraparound support services were offered to participants who reported any kinds of difficulties resulting from the class, whether physical or emotional. Students who shared physical difficulties were given modifications for following classes by the instructors, and those who reported emotional distress were referred to school-based therapists and counselors. Any student with mental health/safety concerns were also immediately referred to school administration, who then initiated a parent/guardian contact for student safety. Analytical Methods Because these classes were pulled from the same physical education teacher, this allowed for computer-generated randomization. Instead of individuals being randomly assigned, gym class clusters of individuals were randomly allocated to intervention groups. This approach is necessary because randomization at the individual level is impractical and we sought to avoid contamination between treatment groups. For all participants, baseline surveys (see Table 2 below), were conducted prior to exposure to Yoga (T0). Follow-up surveys were collected at 6 weeks (T1). Analyses were conducted using R Version 3.6. Baseline differences between intervention and control group were examined using independent t test or χ2 test. All data was analyzed according to the treatment assignment. Cortisol Analysis Students in both the experimental and control groups were administered salivary cortisol testing at the beginning of weeks 1 and 3 and the conclusion of week 6, each time on an “in-person” day at school, prior to their physical education class. Specifically, students provided a saliva sample to PI, which was deidentified (using a code key system) before analysis by the Center for American Indian and Rural Health Equity’s Translational Biomarkers Core Lab at Montana State University. Testing used the Abcam (ab154996) cortisol in vitro competitive ELISA (Enzyme-Linked Immunosorbent Assay) kit designed for accurate quantitative measurement of cortisol in saliva (sensitivity 0.12 ng/ml). Deidentified cortisol data was returned to the PI for re-identification using the code key, and comparisons using paired t tests assessed trends to determine usefulness of this measure. Quantitative Results Analyses The study investigated the impact of participating in yoga on various outcomes among a sample of 45 high school students randomly assigned to either a treatment (n = 23) or control group (n = 22). Of the 45 students, 25 were male and 20 were female. Randomization eliminates selection bias definitionally, leaving a role only to chance differences. This reduces the plausibility of other threats to internal validity. Because groups were randomly formed, any initial group differences in maturational rates, in the experience of simultaneous historical events, and in regression artifacts, ought to be due to chance. For all three outcomes, the first regression model, or the unadjusted model, included the treatment group variable only. The second model adjusted for baseline levels of the measures, and the third and final regression model includes gender as a covariate. The data were analyzed as a treatment on the treated, as surveys were only completed by participants. Additionally, dosage effects were measured by the number of sessions attended. The number of sessions attended ranged between three and twelve, with a median of nine sessions. The dose was examined as a predictor variable added to the third model, but there were no significant relationships detected between attendance and the outcome measures (depression, anxiety, or resilience). We further investigated if there were interactions between baseline scores on measures and treatment group; based on our limited statistical power, no interaction was significant. Model 1 YPost = β0 + β1Treatment. Model 2: YPost = β0 + β1Treatment + β2Pre. Model 3: YPost + β1Treatment + β2Pre + β3Gender. Based on the summary statistics earlier in Table 2, the treatment group saw a greater decline in anxiety versus the control group (21.67% vs. 13.66%). The decline in depression was similar between the two groups (20.13% vs. 22.01%). Interestingly, the control group had a greater increase in resilience than the treatment group. However, there were no significant differences in any measure between the groups. Further, a sensitivity analysis was also conducted to assess the dose–response relationship between number of treatments received and post-study outcomes. Using dosage, or number of sessions as the outcome, as opposed to the treatment dummy of treatment versus control, did not result in different results for anxiety, depression, or resilience. Again, we did not detect statistical significance. Additionally, no significant differences were observed between males and females in any of the study outcomes. Our null findings may reflect the fact that we had low power due to small sample sizes, resulting in a reduced probability of rejecting the null hypothesis when it is indeed false. Implications and possible reasons explaining this will be discussed in the Discussion Section; tables illustrating these outcomes are provided below Tables 4, 5, 6, 7 and 8.Table 4 Model Results for GAD-7 (Anxiety) Model 1 Model 2 Model 3 GAD-7 Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 4.546* 1.135 [2.256, 6.834] 0.271 0.869 [-1.482, 2.024 0.314 0.872 [-1.446, 2.074] Treatment 0.802 1.588 [-2.399, 4.004] -0.457 0.995 [-2.464, 1.550] -1.010 1.162 [-3.356, 1.337] Baseline 0.811* 0.097 [0.615, 1.006] 0.760*** 0.111 [0.535, 0.985] Gender (Male as ref) 1.228 1.328 [-1.455, 3.911] Adj. R2 -0.017 0.610 0.609 The Cohen’s d effect size for the treatment is -0.19 and represents the average intervention effect on the treated * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 5 Model Results for PHQ-A (Depression) Model 1 Model 2 Model 3 PHQ-A Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 4.182 1.290 [1.580, 6.784] -0.271 0.909 [-2.105, 1.563] -0.273 0.915 [-2.121, 1.575] Treatment 2.209 1.805 [-1.431, 5.849] 0.021 1.094 [-2.187, 2.229] -0.378 1.256 [-2.915, 2.158] Baseline 0.830* 0.092 [0.644, 1.016] 0.799*** 0.104 [0.588, 1.009[ Gender (Male as ref) 0.939 1.419 [-1.926, 3.803] Adj. R2 0.011 0.655 0.650 The Cohen’s d effect size for the treatment is -0.06 and represents the average intervention effect on the treated * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 6 Model Results for CD-RISC (Resilience) Model 1 Model 2 Model 3 CD-RISC Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 30.455*** 1.765 [26.896, 34.013] 9.765* 4.043 [1.605, 17.924] 8.575 4.378 [-0.265, 17.416] Treatment -5.585* 2.468 [-10.563, -0.607] -3.447 1.953 [-7.388, 0.495] -4.248 2.248 [-8.788, 0.292] Baseline 0.731* 0.134 [0.459, 1.002] 0.762* 0.142 [0.476, 1.047] Gender (Male as ref) 1.736 2.368 [–3.047, 6.518] Adj. R2 0.086 0.451 0.445 The Cohen’s d effect size for the treatment is -0.49 and represents the average intervention effect on the treated * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 7 Association Between Number of Treatments Received and Pre- and Post-Intervention: GAD-7 Model 1 Model 2 Model 3 GAD7 Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 4.684* 1.103 [2.459, 6.909] 0.165 0.871 [-1.593, 1.923 0.205 0.877 [-1.565, 1.976] Treatment Count 0.061 0.171 [-0.285, 0.406] -0.022 0.107 [-0.237, 0.193] -0.073 0.126 [-0.326, 0.181] Baseline 0.806* 0.096 [0.611, 1.000] 0.760*** 0.113 [0.532, 0.989] Gender (Male as ref) 1.041 1.342 [-1.670, 3.751] Adj. R2 -0.020 0.608 0.605 * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 8 Association Between Number of Treatments Received Pre- and Post-Intervention: PHQ-A Model 1 Model 2 Model 3 PHQ-A Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 4.544* 1.263 [1.998, 7.090] -0.445 0.918 [-2.298, 1.408] -0.433 0.927 [-2.306, 1.440] Treatment Count 0.172 0.196 [-0.2241, 0.567] 0.047 0.115 [-0.186, 0.280] 0.017 0.136 [-0.257, 0.290] Baseline 0.826* 0.090 [0.644, 1.009] 0.802*** 0.106 [0.589, 1.016] Gender (Male as ref) 0.643 1.447 [-2.280, 3.566] Adj. R2 -0.005 0.656 0.649 * indicates p < .05.; indicates p < .01.; * indicates p < .001 Cortisol Analysis Although descriptive statistics indicated a greater downward trend in PHQ-A and GAD-7 scores for the treatment group, we did not detect significant differences in depression, anxiety, or resilience among students who received the yoga treatment in comparison to the control group. However, a significant difference in cortisol level was observed between students assigned to the treatment and control group. Students in the treatment group had significantly lower cortisol levels at post-intervention; this finding remained significant when controlling for differences in baseline cortisol levels. No significant differences were observed between males and females in cortisol levels. A sensitivity analysis was then conducted to assess the dose–response relationship between number of treatments received and post-study cortisol levels. An unadjusted association was observed between the number of treatments and post-study cortisol levels, with an average cortisol reduction of 0.026 ug/dl for every additional treatment received by participants (p < 0.001). This association remained when controlling for baseline cortisol levels, with a 0.016 ug/dl reduction in cortisol, on average per treatment (< 0.01), and average reduction of 0.025 ug/dl (p < 0.01) when controlling for baseline cortisol levels and gender. In sum, there was a statistically significant association based upon the number of sessions completed; the more sessions a participant engaged in, the greater the reduction in cortisol levels Tables 9, 10, 11 and 12.Table 9 Model Results for Cortisol Levels Model 1 Model 2 Model 3 CORTISOL (ug/dl) Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 0.492* 0.044 [0.407, 0.577] 0.319* 0.073 [0.177, 0.462] 0.333* 0.070 [0.196, 0.470] Treatment -0.271* 0.062 [-0.392, -0.151] -0.167* 0.070 [-0.303, -0.030] -0.263 0.084 [-0.428, -0.099] Baseline 0.246 0.085 [0.078, 0.413] 0.199* 0.085 [0.032, 0.367] Gender (Male as ref) 0.142 0.074 [-0.004, 0.287] Adj. R2 0.290 0.377 0.412 The Cohen’s d effect size for the treatment is 1.30 and represents the average intervention effect on the treated * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 10 Association Between Number of Treatments Received Pre- and Post-Intervention: Cortisol Levels Model 1 Model 2 Model 3 CORTISOL (ug/dl) Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 0.472* 0.044 [0.386, 0.559] 0.306* 0.071 [0.166, 0.446] 0.315* 0.069 [0.180, 0.450] Treatment Count -0.026* 0.007 [-0.040, -0.013] -0.016* 0.007 [-0.030, -0.002] -0.025 0.009 [-0.042, -0.008] Baseline 0.257 0.085 [0.090, 0.424] 0.217* 0.085 [0.050, 0.383] Gender (Male as ref) 0.132 0.075 [-0.015, 0.279] Adj. R2 0.232 0.367 0.396 * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 11 Mean Cortisol Levels among Males and Females at Pre-, Mid-, and Post-Intervention Cortisol Levels Females Males p-valuea Mean (SD) Mean (SD) Cortisol- Time 1 0.51 (0.50) 0.54 (0.33) 0.83 Cortisol- Time 2 0.43 (0.29) 0.49 (0.29) 0.52 Cortisol- Time 3 0.33 (0.28) 0.38 (0.23) 0.54 Change in Cortisol (Time 3—Time 1) -0.12 (0.42) -0.16 (0.28) 0.69 aP-values derived from pooled T-Test Table 12 Linear Association between Number of Treatments Received and Change in Cortisol Levels between Pre- and Post-Intervention Period Predictor Variable Geometric Mean 95% Confidence Interval p-value Per each additional treatment received Unadjusted 1 0.01 (-0.01, 0.04) 0.17 Adjusted for baseline cortisol levels2 -0.02 (-0.03, -0.01) 0.03 Adjusted for baseline cortisol levels and gender3 -0.03 (-0.04, -0.01) < 0.01 Additionally, a sleep analysis was performed to determine whether the yoga intervention had any impacts on participants’ self-reported sleep quality and duration; no significant linear association was observed between sleep duration (n hours) and cortisol level. However, when expressed as a binary variable (less than 8 h vs. 8 or more hours), a significant difference in means was observed (p = 0.05), with a mean cortisol level of 0.49 ug/dl among those sleeping less than 8 h compared to 0.38 ug/dl among those sleeping 8 or more hours. When comparing mean cortisol levels according to self-reported sleep quality, no statistically significant difference was observed between those who “slept well” and those who “did not sleep well” the prior night (p = 0.18). Additionally, no association was observed between number of treatments and sleep duration, and no association was observed between number of treatments and perceived sleep quality (comparing those who slept well to those who didn’t sleep well) (as evidenced by a linear regression to model the association between number of treatments and hours of sleep and a logistic regression to model the odds of sleeping well for every additional treatment received) Tables 13, 14, 15 and 16.Table 13 Linear Association between Sleep Duration and Cortisol Levels Predictor Variable Geometric Mean 95% Confidence Interval p-value Hours Slept -0.01 (-0.05, 0.02) 0.44 Table 14 Difference in Cortisol Levels by Sleep Quality and Duration Variable Mean Cortisol Level SD N p-value Perceived quality of prior night's sleep Didn't sleep well 0.48 0.31 48 0.18 Slept well 0.4 0.32 76 Sleep Duration Less than 8 h 0.49 0.37 81 0.05 8 or more hours 0.38 0.23 48 Table 15 Association between Number of Treatments Received and Post-Intervention Sleep Duration Model 1 Model 2 Model 3 Hours Slept Est SE 95% Conf Int Est SE 95% Conf Int Est SE 95% Conf Int Intercept 7.762* 0.329 [7.117, 8.406] 3.938 1.218 [1.551, 6.325] 4.166 1.303 [1.613, 6.720] Treatment Count -0.039 0.051 [-0.138, 0.061] -0.003 0.044 [-0.090, 0.083] 0.01 0.052 [-0.092, 0.112] Baseline 0.514 0.157 [0.206, 0.821] 0.489** 0.165 [0.166, 0.811] Gender (Male as ref) -0.258 0.534 [-1.304, 0.788] Adj. R2 0.172 0.155 * indicates p < .05.; indicates p < .01.; * indicates p < .001 Table 16 Association between Number of Treatments Received and Post-Intervention Sleep Quality Model 1 Model 2 Model 3 Sleep Quality Est SE Odds Ratio 95% Conf Int Est SE Odds Ratio 95% Conf Int Est SE Odds Ratio 95% Conf Int Intercept 0.865 0.479 -3.135 2.262 -3.504 2.497 Number of Treatments -0.013 0.071 0.987 [.859, 1.134] 0.028 0.079 1.029 [.882, 1.200] 0.189 0.141 1.208 [.917, 1.593] Baseline 0.545 0.302 1.724 [.953, 3.116] 0.483 0.326 1.62 [.856, 3.066] Gender (Male as ref) -1.044 0.649 0.124 [.010, 1.580] AIC 55.94 51.808 50.618 * indicates p < .05.; indicates p < .01.; * indicates p < .001 Discussion While the study indicates descriptively that the intervention of trauma-informed yoga was beneficial to experimental group participants (when comparing outcomes to the control group), insufficient sample sizes limited our statistical power to detect significance. Furthermore, it is highly likely that the external stressors caused by the pandemic may have artificially deflated post-intervention survey outcomes and/or underestimated improvements. During the six week intervention period, multiple traumatic events occurred within the local and school community that directly impacted all participants in this study; as mentioned earlier, one high school student (not involved in this study) took their life (and many of the experimental group participants knew and/or were friends with this student). Another crisis related to school/student safety occurred mid-intervention period which led to a two-day school closure, heavy police activity, and evacuation of some students from their homes. Therefore, both the safety concerns that could have caused excessive stress and/or trauma responses in participants–as well as the need for a trauma-informed intervention in this sample–are important to highlight here. Further research is needed to explore possible differences in resilience levels between the control and treatment groups (i.e., age may be a factor, etc.). Additionally, the research team has concerns regarding potential survey bias for students who indicated suicidal ideation on the Columbia Suicidality Screener, which was not included in the results of this study but was requested to be completed by the school district. These students, per medical/suicide intervention guidance, were pulled the same day of the pre-survey to assess immediate risk by the school nurse to assess imminent risk to their one safety (and determine a potential need for referral to mental health services); this action, however, may have influenced those students to not answer in such a way that they would be “called out” (or parents notified) on their PHQ-A post-surveys, which also included a question on suicidal ideation. Study Limitations As alluded to previously, there were limitations to this study, both in design and also extenuating circumstances related to COVID-19. While the research team hoped to have enough of a sample size to analyze statistical significance, due to attrition rates and participants quarantined for COVID-19 exposure and/or infection, our sample size had limited power to detect statistical significance, although descriptives indicate the strong potential of this intervention. Additionally, this sample was ethnically homogenous (all but four students were white); further research needs to explore this intervention with a more diverse group of participants, both in age and ethnicity. Attendance for the intervention also was inconsistent among several participants, which inevitably impacted study results, but also makes our findings more conservative; poor school attendance and engagement are often related to the incidence of adverse childhood experiences (Blodgett & Lanigan, 2018). Moreover, regarding the intervention itself, the research team was unable to secure a single yoga teacher for every session of the intervention, so two teachers were used each week of the intervention, which introduces the possibility of variability of results based on different teachers’ styles. While this research study also intended to utilize additional, reliable physiological data with sleep from wearable fitness trackers worn by all participants across both groups, participant engagement with the devices was inconsistent at best. Many participants did not wear their devices regularly, nor did they sync them at the requested intervals (and several lost the device altogether); therefore, self-reports of sleep quality and duration used in this study may be considered less reliable than what could have potentially been gathered from these wearable health devices. Finally, it is worth noting that self-reporting by adolescents is inherently less reliable than physiological data. For example, when asked on a qualitative, open-ended survey how “stressed out” students felt about COVID-19, most students replied that they “were not stressed” about the pandemic; however, physiological evidence suggests the inverse is true. While baseline levels of cortisol were much lower in the experimental group compared to the control group, (baseline level = 0.31 ug/dl in the experimental group and 0.71 ug/dl in the control group), a normal/healthy range is 0.10–0.20 ug/dl, so the participants all had notably high baseline cortisol levels. This indicates a potential mismatch in how students perceive their own anxiety levels compared to their actual physiological response to stress. The research team postulates that, among other possibilities, there are two distinct ones that rise above the others to explain this misalignment. First, as mentioned above, this community suffered multiple crises not only during the intervention period, but also during the entire course of the pandemic; one theory is that students’ positioning on the hierarchy of needs (in terms of immediate survival and safety) may have superseded those of the threat of COVID-19 (Maslow, 1970). An additional possibility relates to alexithymia, which is often attributed to trauma survivors. As mentioned earlier in this manuscript, alexithymia is defined as difficulty in identifying one’s feelings and communicating these feelings with words; this leads to the distinct possibility that the students’ self-reports were inaccurate (as compared to the physiological findings) because they were incapable of identifying their own feelings and/or describing them on the survey, thus leading to further potential survey bias (Lanius, 2021). Conclusions and Implications Given the prevalence of suicide and mental health issues in rural Montana, and especially in the community in which this study took place, this project was intended to help mitigate stressors that may contribute to poor behavioral and mental health in high school-aged children, which may be exacerbated by the collective trauma of the COVID-19 pandemic. Twenty percent of American adolescents experience mental health difficulties, and over 50% of all seventeen-year-olds have reported experiencing some form of trauma (Brackett, 2019). Further, “anxiety disorders are the most common mental illness in the U.S., affecting 25% of children between 13 and 18 years old….[and] Depression is the leading cause of disability worldwide” (Brackett, 2019, p. 3). Despite limitations of statistical power (based on insufficient sample sizes in each group) to indicate significance in self-reported survey measures in this study, the descriptive differences between the control and experimental groups illustrate the mental health benefits of reduced anxiety and depressive symptoms in rural adolescents resulting from a remotely delivered trauma-informed yoga intervention. Physiologically, statistically significant differences of post-intervention cortisol levels between the control and treatment groups exemplify the promise of this intervention in the reduction of stress hormone levels. Further exploration and research are needed to include a larger and more diverse sample to determine generalizability to both rural and urban contexts; however, our study holds the potential for a long-term public health impact in reducing adolescent rates of anxiety and depression while improving stress-related physical health and mitigating trauma in geographically isolated settings. Funding The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: The research highlighted in this article was supported by a grant from the National Institutes of Health, award no. P20GM104417 (grant year 2020). Data Availability Due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data are not available. Declarations Ethical Approval The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: The research highlighted in this article was supported by a grant from the National Institutes of Health, award no. P20GM104417 (grant year 2020). Consent to Participate Parent Consent/Student Assent was obtained for all study participants. This study involving human participants was in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standard and was approved by the Institutional Review Board at Montana State University. Conflicts of Interest/Competing Interests There are no conflicts of interest/competing interests to disclose for this study. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References American Foundation for Suicide Prevention. (2017). Suicide: Montana 2017 facts & figures. Aspen Institute National Commission on Social, Emotional, and Academic Development. (2019). From a nation at risk to a nation at hope. https://nationathope.org/wp-content/uploads/2018_aspen_final-report_full_webversion.pdf Brackett M Permission to feel 2019 Celadon Books Bucci M Gutiérrez W Koita K Purewal S Silvério MB Harris N Center for youth wellness ACE-questionnaire user guide 2015 Center for Youth Wellness Butzer, B., van Over, M., Noggle Taylor, J., & Khalsa, S. (2015). Yoga may mitigate decreases in high school grades. 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NPR. https://www.npr.org/sections/health-shots/2018/10/23/658834805/how-one-colorado-town-is-tackling-suicide-prevention-starting-with-the-kids Shukitt-Hale, B. Lieberman, H. (1996). The Effect of altitude on cognitive performance and mood states. Available from: https://www.ncbi.nlm.nih.gov/books/NBK232882 Spitzer R Kroenke K Williams J A brief measure for assessing generalized anxiety disorder: The GAD-7 Archives of Internal Medicine 2006 166 1092 1097 10.1001/archinte.166.10.1092 16717171 van der Kolk BA The body keeps the score: Brain, mind, and body in the healing of trauma 2014 Viking Suicide rates in Livingston triple national average. (2013, August 20). Retrieved from https://www.kulr8.com/connections/suicide-rates-in-livingston-triple-national-average/article_c4f6f24d-9ef7-53ffaf5c-3d113d5631fb.html	36471891	PMC9713729	NO-CC CODE	2022-12-02 23:23:08	no	J Child Adolesc Trauma. 2022 Dec 1;:1-14	utf-8	J Child Adolesc Trauma	2,022	10.1007/s40653-022-00496-9	oa_other
==== Front J Hum Hypertens J Hum Hypertens Journal of Human Hypertension 0950-9240 1476-5527 Nature Publishing Group UK London 784 10.1038/s41371-022-00784-9 Article A new and specific automated blood pressure device for exercise stress testing http://orcid.org/0000-0002-1491-6576 McLaurin Natalie N. http://orcid.org/0000-0001-8602-8749 Wang Tianyu Chen Lin-Sheng http://orcid.org/0000-0002-1780-7471 Tanaka Hirofumi [email protected] grid.89336.37 0000 0004 1936 9924 Cardiovascular Aging Research Laboratory, Department of Kinesiology and Health Education, The University of Texas at Austin, Austin, TX 78712 USA 1 12 2022 15 9 5 2022 14 11 2022 22 11 2022 © The Author(s), under exclusive licence to Springer Nature Limited 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Blood pressure (BP) measurement plays a critical role in cardiac stress testing and is most commonly assessed manually. The emphasis of social distancing during the COVID-19 pandemic has renewed the interest in and the need for an automated BP device for incremental exercise stress testing. We assessed the accuracy of a new automated blood pressure device specifically manufactured for cardiac stress testing. Thirty-five adults aged 35 ± 16 years were studied during an incremental stress test on the cycle ergometer. Three observers measured BP simultaneously, two listening to Korotkoff sounds using a dual-headed stethoscope and one using headphones to listen to sounds generated by an automated BP device. With increasing workload, systolic BP increased progressively without significant differences in BP readings between any observer compared with the automated monitor at any stage during exercise. Systolic BP obtained with the BP machine was strongly correlated with those obtained by the stethoscope observers (r = 0.96) and the observer with headphones (r = 0.95). Diastolic BP obtained with the BP machine was moderately and significantly associated with those obtained by the stethoscope observers (r = 0.75) and the observer with headphones (r = 0.75). The automated BP monitor specifically made for cardiac stress testing accurately measured both systolic and diastolic blood pressure during exercise. Subject terms Diagnosis Hypertension ==== Body pmcIntroduction Blood pressure (BP) is an essential measurement during exercise testing as it is a relative contraindication to terminate exercise [1]. Typically, manual BP measurements by a single technician are used during exercise testing in clinical settings. However, using a manual sphygmomanometer to measure BP during exercise has limitations as noise and motion artifact from the exerciser make these measurements difficult to take [2, 3]. This reduces the accuracy of BP measurements during exercise and could alter their clinical interpretation [4–9]. As many world organizations have expressed the importance of proper social distancing during the COVID-19 pandemic, the interest in an accurate and reliable automated BP monitor that can be used during exercise has been renewed. A new automated BP monitor has recently been developed for the purpose of exercise stress testing. This monitor uses R-wave gating from an electrocardiogram paired with acoustic transduction via a microphone placed over the brachial artery within the BP cuff in order to measure BP. The purpose of the present study is to evaluate the accuracy of this new automated blood pressure device at rest, during exercise, and recovery. These automated measurements were compared to manual blood pressure measurements which served as the reference standard. We hypothesized that the measurements taken by the automated device would not be different from the reference measurements. Methods Participants A total of 35 apparently healthy adults (20 men and 15 women) varying widely in age (19–66 years) were studied (Table 1). Due to limitations placed during the COVID-19 pandemic, we were instructed by the Institutional Review Board to avoid recruiting hypertensive participants as they fell into the “high-risk” category for COVID-19. As such, the International Organization for Standardization (ISO) requirement that 10% of participants must have a resting BP ≥ 140 mmHg was not met. The health status of each participant was determined using a questionnaire. The 2020 PAR-Q + questionnaire was used to assess the safety of exercise. Informed consent was obtained from all participants before involvement in the study. This study was conducted in accordance with the Declaration of Helsinki, and all procedures were approved by the Institutional Review Board of The University of Texas at Austin.Table 1 Selected participant characteristics (n = 35). Variable Means (SD) or n Male/Female 20/15 Age (years) 35 (16) Height (cm) 173 (10) Body Weight (kg) 72.8 (14.6) Heart Rate at Rest (bpm) 69 (14) Maximal Heart Rate (bpm) 183 (12) Race White/Caucasian 19 Black/African American 2 Asian 13 Multiracial 1 Ethnicity Hispanic/Latino 2 Procedures Participants came into the laboratory on two separate occasions separated by at least 2 days and at the same time of day. All participants refrained from caffeine on the day of the test, food intake >4 h prior to testing, and alcohol intake and strenuous exercise for >24 h prior to testing. Height and body weight were measured using an electronic balance scale (Seca #769, Vogel & Halke, Hamburg, Germany). Upon entry into the laboratory, each participant was equipped with a 5-lead ECG, and the circumference of the upper arm was measured to ensure that a properly-sized blood pressure cuff was used. A new automated BP monitor (FBX-1000, Fukuda Denshi, Tokyo, Japan) specifically developed for exercise testing was evaluated during the study. Prior to testing, an aneroid sphygmomanometer was calibrated using a pressure gauge and was found to be within ±2 mmHg. The automated BP monitor was calibrated against the aneroid sphygmomanometer and was within ±2 mmHg. Each participant was then seated in an armchair with their back, elbows, and forearms supported. All participants were instructed to rest their feet flat on the ground. A BP cuff from the automated BP monitor was placed over the brachial artery of the designated arm, chosen at random. The opposite arm was used during the second day of testing. Following 10 min of seated rest, resting BP was simultaneously measured using three different methods: (1) by two experienced observers using a stethoscope with dual earpieces (one set for each observer) (reference), (2) by the Fukuda automated BP monitor, and (3) by an observer using headphones to listen to sounds generated by the microphone of the Fukuda BP monitor. The armrest of the chair was utilized so the measurement arm could remain relaxed and supported throughout the BP measurements. In addition, the height of the armrest allowed the BP cuff to be positioned at heart level. Participants were asked to keep their arm relaxed and not to tense their shoulder throughout the duration of the BP measurement. Each observer measured BP using the same method throughout the duration of the study and for each participant. The automated BP monitor was connected to a standard aneroid manometer using a Y-tube connector allowing each observer to be blinded to the BP readings of the automated monitor. The appearance of Korotkoff sounds (phase I) defined systolic BP and the disappearance of Korotkoff sounds (phase V) defined diastolic BP. Each observer was blinded to the BP measurements taken by each other. Heart rate was also obtained from the automated BP monitor. An incremental exercise test was performed on a cycle ergometer (Excalibur, Lode B.V., Groningen, The Netherlands). Exercise began at 25 W and increased by 25 W every 2 min until a rating of perceived exertion (RPE) of 16 (Borg’s original scale) or 80% of age-predicted maximal heart rate [10] was reached. BP was measured one minute into each exercise stage. The measurement arm of the participant was extended to the side so the BP cuff was positioned at heart level and supported by an observer to avoid excessive movement. All participants were asked to keep their arm relaxed during the BP measurement. Heart rate and RPE were measured within the final 15 s of each exercise stage. Immediately upon completion of exercise, the participant was quickly moved from the cycle ergometer and seated into an armchair with their back, elbow, and forearms supported. Heart rate and BP were measured every 2 min until BP returned to resting values ±5 mmHg. Data analyses Data analyses were performed using SPSS version 22 (IBM Corp., Armonk, NY). Shapiro–Wilk tests indicated a normal (Gaussian) distribution for all continuous variables. Differences between BP values obtained with the different methods were evaluated using two-factor analysis of variance (ANOVA) with repeated measures (BP methodologies × exercise stage). The LSD post-hoc analysis was used to determine specific differences between the groups. Pearson correlation analyses were conducted to observe relationships between the automated BP devices and the reference manual BP measurements. The Bland-Altman analyses of agreement were then conducted to observe the relative differences between two different methods. P < 0.05 was considered significant for all analyses. Sample size calculation was conducted using a power calculation software (GPower 3.0.10, Dusseldorf, Germany) with power = 0.8, alpha level = 0.05, and effect size = 0.33. Results Selected participant characteristics are shown in Table 1. The participants were relatively diverse in age, sex/gender, and racial distributions. Pearson correlation coefficients for the two observers using a stethoscope were 0.98 for systolic BP and 0.91 for diastolic BP. BP measurements between the two observers using a stethoscope were not significantly different at any stage. Due to the similarity in BP values, the measurements of the two stethoscope observers were averaged to provide one set of reference data in accordance with ISO guidelines. Systolic and diastolic BP at rest were not different between the reference, the automated BP monitor, and the observer using headphones (Fig. 1 and Table 2). Systolic BP showed gradual and significant increases throughout the incremental exercise protocol (p < 0.05, Fig. 1 and Table 2). Diastolic BP increased slightly and significantly during the incremental exercise protocol. There were no significant differences in systolic or diastolic BP between the methods at any individual stage during the incremental exercise protocol.Fig. 1 Systolic and diastolic blood pressure (BP) at rest, during exercise (left panel) and recovery from exercise (right panel). Data are means (SD). p < 0.05 vs. Rest, †p < 0.05 vs. Immediately preceding stage, ‡p < 0.05 vs. Reference, §p < 0.05 vs. All other methods at the same stage. Table 2 Systolic (SBP) and diastolic blood pressure (DBP) measurements at rest and during incremental exercise. Stage Reference (A) Fukuda monitor (B) Fukuda monitor with headphones B – A Heart rate SBP DBP SBP DBP SBP DBP SBP DBP Rest 116 (9) 74 (8) 114 (9) 73 (9) 115 (9) 74 (8) −2 (5) −1 (4) 69 (14) 25W 123 (14)* 75 (11) 125 (13)* 76 (13)* 123 (13)* 76 (11) 2 (6) 1 (7) 95 (14)* 50W 131 (13)† 74 (10) 134 (15)† 75 (11)* 131 (14)† 77 (10) 3 (7) 1 (6) 106 (18)† 75W 141 (15)† 76 (10) 142 (14)† 77 (12)† 142 (14)† 77 (11) 1 (5) 1 (6) 119 (20)† 100W 152 (15)† 77 (11) 152 (15)† 78 (12) 153 (15)† 79 (11) 0 (7) 1 (8) 129 (20)† 125W 157 (13)† 75 (9) 158 (14)† 78 (11) 158 (14)† 77 (8) 1 (6) 3 (8) 129 (14)* 150W 161 (13)† 74 (8) 161 (15)† 74 (9)† 163 (13)† 75 (9) 0 (7) 0 (6) 134 (11)† 175W 175 (8)† 77 (5)† 175 (8)† 78 (7)† 175 (9)† 77 (6)† 0 (5) 1 (7) 125 (7)† Data are means (SD). p < 0.05 vs. Rest, †p < 0.05 vs. Immediately preceding stage. Systolic BP decreased progressively for the first 4 min of recovery (Fig. 1 and Table 3). Immediately after exercise, the automated BP monitor recorded a higher systolic and diastolic BP than the other two methods (p < 0.05, Fig. 1 and Table 2).Table 3 Systolic (SBP) and diastolic blood pressure (DBP) measurements during recovery from exercise. Recovery (min) Reference (A) Fukuda monitor (B) Fukuda monitor with headphones B – A Heart rate SBP DBP SBP DBP SBP DBP SBP DBP 0 138 (13) 69 (10) 143 (15)† 75 (12)‡ 139 (13) 70 (11) 5 (7) 6 (10) 92 (17) 2 126 (11)* 70 (10) 128 (12)* 73 (11)* 127 (11)* 71 (10) 2 (7) 3 (7) 86 (15)* 4 120 (9)* 71 (9) 123 (10)* 73 (11) 121 (9)* 72 (10) 3 (7) 2 (5) 80 (11)* 6 122 (6) 73 (4)* 122 (6) 74 (4) 122 (7) 74 (4) 0 (5) 1 (3) 75 (7)* Data are means (SD). p < 0.05 vs. Immediately preceding stage, †p < 0.05 vs. Reference, ‡p < 0.05 vs. All other methods at the same stage. As shown in Fig. 2, systolic BP obtained with the reference was strongly associated with the Fukuda automated BP monitor (p < 0.001) as evidenced by a strong Pearson correlation coefficient of 0.96. Similar significant associations were obtained with diastolic BP although the strength of the association was slightly weaker at 0.75. These agreements are consistent with the Bland-Altman analysis. Comparable results were observed between the observer using headphones and the automated BP monitor (Supplemental Figs. 1 and 2). Systolic and diastolic BP with the reference showed significant associations with the observer listening via headphones as shown by strong Pearson correlation coefficients of 0.97 and 0.90 and Bland-Altman analysis.Fig. 2 Association of systolic and diastolic blood pressure (BP) between the Fukuda monitor and the reference (the observer using a stethoscope). Pearson correlation analysis of systolic and diastolic blood pressure are shown in (A and B). Bland-Altman analysis of agreement plots with mean difference ±2 standard deviations are shown in (C and D). During exercise, all participants obtained a heart rate at least 30% greater than their heart rate at rest. Heart rate increased progressively and significantly throughout the incremental exercise protocol (p < 0.05, Table 2). Heart rate significantly decreased throughout recovery from exercise (p < 0.05). Discussion In the present study, the accuracy of a newly-developed automated BP device was compared with standard BP measurements at rest, during exercise, and recovery. The automated BP monitor accurately measured both systolic and diastolic BP at rest and during exercise. Both correlational analyses and Bland-Altman plots indicated that the BP obtained with the automated BP device were strongly associated with those obtained with the reference methods. The Fukuda automated BP device may be a suitable alternative to manual BP measurement especially when the proper social distance is needed. Previous studies have assessed the accuracy of automated BP devices during exercise [2, 4, 11–14]. These studies produced highly mixed findings. In one of the earlier studies that evaluated four different BP monitors during cycle exercise at 100 W [14], the two BP devices (Colin 630 and Accutracker II BP monitors) were found to accurately measure BP at 100 W with minimal arm movement. However, it was stated that these devices would be unlikely to produce accurate and reliable measurement at higher intensities [14]. The other two devices (Del Mar P-IV and SpaceLabs 90202 BP devices) could not accurately measure BP until the participant had completely stopped movement [14]. Colin later developed a new automated BP monitor (Colin 680) specifically for the purpose of exercise stress testing which was evaluated during high-intensity cycle and treadmill exercise [11]. The device produced significantly higher systolic BP during cycle ergometer and treadmill exercise compared with the reference (stethoscope) measurements. In contrast, in a follow-up study conducted by a different group [2], the monitor accurately measured systolic BP during low, moderate, and high-intensity cycle exercise but did not accurately measure diastolic BP at moderate and high-intensity exercise. The Colin BP monitor is no longer manufactured or sold in the market. The Tango M2 stress test monitor was also specifically developed for exercise stress testing [15]. Compared with manual BP measurements during incremental cycle exercise, the device produced reliable BP values with absolute BP differences within an acceptable clinical range [15]. Similar findings have been reported in individuals with diabetes [16] and other populations [17–21]. The present study found the newly-developed Fukuda automated BP monitor accurately measured both systolic and diastolic BP during exercise indicating that this is a suitable device which can be used during exercise stress testing. It should be noted that the precise ISO guidelines were not followed strictly due to the design of our full experiment. We evaluated the Fukuda automated BP monitor as well as a new wristwatch that measures BP during incremental exercise. It was not feasible to measure brachial BP in both arms simultaneously as the arm with the wristwatch was required to be in a flexed position in order for the wristwatch to be positioned at heart level. The wristwatch was not able to accurately measure BP during exercise due to excessive movement artifact and was therefore removed from the paper. In addition, the use of the “same arm sequential” method of measuring BP as described in the ISO guidelines was not feasible during an incremental exercise protocol as BP tends to change even at the same exercise stage as exercise intensity progressed. Although ISO standard guidelines were constructed for resting BP and were not meant to be applied to exercise BP, the automated BP monitor has passed the validation criteria for non-invasive sphygmomanometers intended for use during exercise stress testing. Overall, the automated brachial BP monitor accurately measured both systolic and diastolic BP at rest and during incremental cycle ergometer exercise. The accuracy and simplicity of this BP monitor make it a suitable option for use in laboratory and clinical settings and can aid proper social distancing in situations such as the COVID-19 pandemic. Summary What is known about the topic Exercise stress testing is conducted to predict the risk of developing hypertension as well as diagnose and evaluate the treatment of cardiovascular disease. Manual blood pressure measurements during exercise are difficult to perform as motion and sound artifact from exercising participants often impede the ability of a technician to record accurate blood pressure. Blood pressure (BP) is a vital measurement during exercise stress testing as it is an indicator to terminate exercise. The onset of the COVID-19 pandemic has led international organizations to recommend social distancing to reduce the spread of the coronavirus. What this study adds The validation of a new automated blood pressure device which was specifically developed for exercise stress testing and can be utilized in environments where social distancing is recommended. Supplementary information Supplemental figures Supplementary information The online version contains supplementary material available at 10.1038/s41371-022-00784-9. Acknowledgements The authors would like to thank the volunteers that donated their time for the success of this study. Author contributions NM, and HT designed the experiments, analyzed, and interpreted the data. NM, TW, and LC performed the data collection. NM wrote the initial draft of the paper and prepared figures. All authors edited, revised, and approved the final version of the paper. Funding This study was supported by The University of Texas at Austin. Data availability The datasets generated and analyzed for the present study are available from the corresponding author upon reasonable request. Competing interests Fukuda Denshi provided the automated BP device evaluated in the present study. Ethics approval This study was approved by Institutional Review Board at The University of Texas at Austin. 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==== Front Urolithiasis Urolithiasis Urolithiasis 2194-7228 2194-7236 Springer Berlin Heidelberg Berlin/Heidelberg 36454280 1390 10.1007/s00240-022-01390-7 Research The effect of COVID-19 outbreak on urological procedures for urinary stones: data from three high-volumes centers in China Mazzon Giorgio 1 Zhang Xin 1 Yang Xingguo 2 Cheng Fan 3 Liu Yongda 1 Zeng Guohua [email protected] 1 1 grid.470124.4 Department of Urology and Guangdong Key Laboratory of Urology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 2 Department of Urology, Yiling Hospital, Yiling District, Yichang City, China 3 grid.412632.0 0000 0004 1758 2270 Department of Urology, Renmin Hospital of Wuhan University, Wuhan, China 1 12 2022 2023 51 1 512 10 2022 27 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. China has been the first country to be affected by the COVID-19 outbreak. The pandemic resulted in significant disruption of Health Care Services worldwide, and this effect on treatments for urinary stones is currently unclear. This is the first retrospective study involving three tertiary referral centers for urolithiasis across China. We evaluated surgical volumes and peri-operative outcomes of procedures delivered for upper urinary tract stones. We compared trimester prior to restrictions for COVID-19 (October 1st, 2019 to December 31st, 2019, period A), during restrictions (February 1st, 2020 to March 31st, 2020, period B), and afterword (April 1st, 2020 to June 31st, 2020, period C). A total of 2,543 procedures have been carried out. We observed a loss of 743 cases during period B (−29.2%) and 201 during period C (−7.9%). Percutaneous surgery showed the worst reduction, with 507 mini-PCNLs delivered in period A, 168 in period B (−60.8%), and 389 (−18.3%) in period C (p = 0.001). A worst trend was shown for standard PCNLs with 84 procedures carried out in period A, 5 in period B (−95.2%), and 9 (−89.2%) in period C (p = 0.001). Retrograde surgery also decreased, from 420 cases in period A to 190 cases in period B (−54.8%). An increment was however seen in period C when 468 cases have been carried out (+ 11.4%, p = 0.008). In term of SFRs, a difference was noticed for RIRSs, being 69.2%, 80.5%, and 69.3% during three periods (p = 0.045) and semirigid ureteroscopies (90.3%, 97.1%, and 84.8%, p = 0.013). Charlson’s Comorbidity Score could not show any difference between groups as well as no differences in term of post-operative complications have been noticed. Supplementary Information The online version contains supplementary material available at 10.1007/s00240-022-01390-7. Keywords COVID-19 outbreak Urinary stones PCNL RIRS ESWL Ureteroscopy issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2023 ==== Body pmcIntroduction The novel coronavirus 2019 (COVID-19) has been responsible of a new rapidly spreading viral infection classified as pandemic on March 11, 2020 by World Health Organization (WHO) [1]. In July 2020, more than 13,340,000 cases have been registered worldwide including 578,000 deaths. By the end of January 2020, all provinces in mainland of China reported cases of COVID-19. By the beginning of February 2020, 44,672 cases were reported in all 31 provinces of mainland of China, with the peak reached on February 12th, 2020. The Hubei province was the first to report absence of new confirmed cases on March 18th 2020, followed by other 23 provinces. By March 23rd 2020, a total of 81.773 cases were reported in 31 provinces of mainland of China, with 8 provinces with no new cases and vast majority of new diagnoses made on patients coming from abroad [2]. This exponentially growing number of cases, has rapidly overwhelmed Health Care Systems and saturated hospital services across the world, causing a significant disruption of elective and emergency activities. In fact, there has been a dramatic change in clinical priorities, both medical and surgical, with urology services affected in several ways. Non-urgent operations have been postponed, emergency cases were advised to be managed differently, aiming to reduce the number of patients requiring hospitalization and access to operating rooms and intensive care units [3]. Additionally, in many cases, urologists had been redeployed in the emergency services to support the care of COVID-19; elective out-patients clinics were also stopped because of the risk of gathering people within the hospital implementing services of telemedicine [4]. Additionally, due to restrictions and uncertainties related to the pandemic, several patients may have decided to not report to hospital for assistance, with a potential risk of clinical deterioration of their conditions. Endourological procedures were also dramatically affected, even though urinary stones are considered a benign condition, they can lead to severe complications if not treated timely such as urosepsis, renal function deterioration, prolonged pain, need of repeated access to Emergency Care Services, etc. This may represent a risk for patients and, at the same time, an overload for Health Care Systems struggling already to deliver an adequate care during the outbreak [5]. A delayed treatment may also lead to a poor quality of life for patients with urinary stones, for example because of prolonged stent-related irritative symptoms, anxiety, stone growth requiring a more invasive procedure at a later stage. In this study, we describe the effect of COVID-19 pandemic on urological procedures for upper urinary tract stones in three highly specialized Chinese Institutions during the first outbreak. Materials and methods We conducted a retrospective study involving three tertiary referral high-volume Centers specialized in the treatments of urinary stones. We compared surgical volumes and peri-operative outcomes during three periods: trimester prior to COVID-19 outbreak (period A), during COVID-19 (period B), and after COVID-19 outbreak (period C). All Centers involved in the study were asked to collect clinical, surgical, and peri-operative data of adult patients (> 18-year-old) undergoing elective and emergency surgeries for upper tract urinary stones and completing at least a 3-month follow-up. Lengths of study periods were decided taking in consideration the duration of first COVID-19 outbreak in China. In fact, the government notified it on January 24th 2020, during Chinese New Year celebrations. Period B was, therefore, defined as the time interval between the beginning of restrictions and their release, happened on April 30th, 2020. Surgical activities have been compared to analogue periods prior and after the emergency. Following this principle, period A started on October 1st, 2019, and finished on December 31st, 2019. Period B was from February 1st, 2020, to April 30th, 2020. Period C was from May 1st, 2020, to July 30th, 2020. These three study periods have been also identified as all involved hospitals were subjected to analogue situations and limitations, aiming to obtain uniform data. We decided to exclude cases carried out in January 2020 as this period contained many national holidays with the potential effect of biasing results. From beginning of period B to date, as per national regulations, patients must have been rigorously screened for COVID-19 before receiving any surgical treatment. If negative, they were admitted to a COVID-19-free department for either elective or urgent surgical treatment. If positive, they would have been admitted to a dedicated ward with specific precautions measurements and only non-deferrable conditions would have been treated. According to national and local hospital regulations, there were no formal limitations or prioritization rules on which type of surgeries should have been delivered across China during COVID-19 first wave of pandemic and afterward, after confirmation that patients were COVID-19 free. In fact, surgeons were left free to decide which types of conditions had to be treated based on their patient’s conditions. For the same reason, patients with urinary stones may have delayed their access to hospitals due to social distancing measurements and/or fear to report to hospitals. In this study, we investigated procedures with intention to treat ureteric and renal stones or complications related to their presence, including extracorporeal shock wave Llthotripsy (SWL), ureterolithotripsy (URS), retrograde intra-renal surgery (RIRS), percutaneous nephrolithotomy (PCNL), ureterolithotomy, and pyelolithotomy. Data have been retrospectively extrapolated from hospital electronic databases. PCNLs were defined as “mini” in case of utilization of an equal or smaller than 22 Fr Sheath. Standard PCNL was defined as > 22 Fr sheath. Procedures were further categorized as elective or emergency cases. Post-operative complications were reported at 30 days post-op using Clavien–Dindo classification [6]. Stone free status (SFR) was evaluated at 1–3 month post-op and cutoff for clinically insignificant residual fragments was fixed at 2 mm. Due to the nature of the study, Centers adopted different imaging modalities during follow-ups, including unenhanced CT scan (NCCT), ultrasound scan of the urinary tract (US), and X-ray of kidney–ureter and bladder (KUB). The study has been approved in each Center by respective ethic committees. Peri-operative data Patient’s demographic data included sex and age. Age-adjusted Charlson’s Comorbidities Index [7] was utilized to assess and quantify comorbidities. Stone characteristics included laterality, stone location, cumulative stone diameter (mm), hydronephrosis (none, mild, moderate, severe). Stone complexity has been reported using the Guy’s Stone Score (GSS) [8]. We also reported the number of patients who preliminarily received a ureteric stent or percutaneous nephrostomy insertion. Peri-operative data included the rate of patients which received a stent insertion at the end of treatment, 30-days complications rates, mean post-operative length of stay (days from completion of treatment to decision to discharge), SFR. In case of persistence of significant fragments, we also collected the subsequent planned treatment strategy. Usually, after admission, patients receive full assistance from the pre-operative surgical preparation to the final stone treatment. In case of patients requiring immediate renal drainage, a stent/nephrostomy was placed and generally the stone clearance was completed during same admission as soon as the clinical conditions are favorable. For this reason, procedures have been divided in emergencies if delivered on first day of admission or elective if carried out after more days and adequate pre-operative work-up. Therefore, emergency procedures include cases operated for different reasons at day-1, including deterioration of renal function, hydronephrosis, infectious complications, pain. Due to the characteristics of the electronic database, we could not record separately ureteric stents and percutaneous nephrostomy placements, as happening during same admission episode. Statistical analysis Descriptive statistics were obtained reporting means and standard deviations for continuous variables. Frequencies and proportions were used for categorical variables. Continuous variables were compared using the Student t or the Mann–Whitney U test, based on normal or not-normal distribution. Categorical variables were tested with Chi-square test. All tests were two-sided, and significancy was set at p < 0.05. Statistical analyses were performed using SPSS v.24 (IBM SPSS Statistics for Mac, Armonk, NY, IBM Corp). Results Surgical volumes A total of 2,543 procedures have been delivered across three sites, including 2,062 elective cases and 481 emergency cases (Table 1). During period B, we observed a loss of 457 elective cases (−53.3%) and 286 emergency cases (−93.5%). A more modest decrease was noticed also in period C, including 55 elective cases (−6.4%) and 151 emergency cases (−49.3%). These differences resulted statistically significant (p < 0.001). The most significant reduction was due to PCNLs, with mini-PCNLs reduced by 60.8% in period B and 18.29% in period C. Standard PCNLs showed a worst decrease (−95.2% and −89.2% in period B and C, respectively). Among elective cases, we observed a significant increase of retrograde procedures (+ 31.1% for URSs and + 38.1% for RIRSs) during period C. Same effect was not noticed for emergency cases. Open surgeries included a total of eight procedures. During period B, these procedures have not been carried out, ureterolithotomies were reduced in period C (−25%) but pyelolithotomies increased by 75%. In Table 1, we also reported relative variations of different types of surgery during three periods. In Supplementary Table 1, we reported variations of expenses related to stone surgeries. Compared to period A, costs were reduced by 64.8% and 17.1% during period B and C, respectively.Table 1 Procedures delivered during trimesters, subdivided in elective and urgent and variations of overall surgical volumes Type of surgery Pre-COVID COVID (difference compared to pre-COVID in %) Post-COVID (difference compared to pre-COVID in %) Significancy Elective procedures ESWLs 56 46 (−17.9%) 49 (−12.5%) p < 0.001 URSs 135 65 (−51.9%) 177 (+ 31.1%) p < 0.001 RIRSs 155 121 (−21.9%) 214 (+ 38.1%) p < 0.001 Mini-PCNLs 421 165 (−60.8%) 344 (−18.3%) p < 0.001 Standard PCNLs 83 4 (−95.2%) 9 (−89.2%) p < 0.001 Ureterolithotomies 4 0 (−100%) 3 (−25.0%) p < 0.001 Pyelolithotomies 4 0 (−100%) 7 (+ 75.0%) p < 0.001 Total 858 401 (−53.3%) 803 (−6.4%) p < 0.001 Emergency procedures ESWLs 89 12 (−86.5%) 33 (−62.9%) p < 0.001 URSs 71 2 (−97.2%) 34 (−52.1%) p < 0.001 RIRSs 59 2 (−96.6) 43 (−27.1) p < 0.001 Mini-PCNLs 86 3 (−96.5%) 45 (−47.7) p < 0.001 Standard PCNLs 1 1 (no difference) 0 (−100%) p < 0.001 Ureterolithotomies 0 0 0 Non applicable Pyelolithotomies 0 0 0 Non applicable Total 306 20 (−93.5%) 155 (−49.3%) p < 0.001 Overall procedures —rates of different surgical techniques within periods ESWLs 145 (12.4%) 58 (13.7%) 82 (8.5%) p < 0.001 URSs 206 (17.7%) 67 (15.9%) 211 (22.0%) p < 0.001 RIRSs 214 (18.3) 123 (29.2%) 257 (26.8%) p < 0.001 Mini-PCNLs 507 (43.5%) 168 (39.9%) 389 (40.6%) p = 0.036 Standard PCNLs 84 (7.2%) 5 (1.2%) 9 (0.9%) p < 0.001 Ureterolithotomies 4 (0.3%) 0 (0%) 3 (0.3%) p = 0.059 Pyelolithotomies 4 (0.3%) 0 (0%) 7 (0.7%) p = 0.062 Total 1164 (100%) 421 (100%) 958 (100%) p < 0.001 In the bottom part of the table, variations of rates of different types of surgery within study periods are reported Patients and stone characteristics In Tables 2, 3, 4, and 5, most relevant peri-operative data for PCNLs, RIRSs, URSs, and ESWLs, respectively are reported Additional data are reported in Supplementary Table 2. Patients’ complexity did not change during three periods, with CCI having a mean value between 1 and 2 (p = 0.062 for PCNLs, p = 0.851 for RIRSs, p = 0.763 for URSs and p = 0.397 for ESWLs). During period B, we observed a different level of stone complexity compared to period A. For PCNLs, absence or mild hydronephrosis was observed in 53.5% during period A compared to 64.9% in period B and 58.8% in period C (p = 0.04). Regarding RIRSs, none or mild hydronephrosis was noticed in 72.8% of cases during period A, 81.7% in period B, and 78.3% in period C (p = 0.025). Similar trend was noticed also for URSs, showing 62.2% during period A, 66.7% in period B, and 70.4% in period C (p:0.07). ESWLs did not show the same trend with 97.9%, 96.5%, and 95.7% in periods A, B, and C, respectively (p = 0.189).Table 2 Peri-operative data on PCNLs PCNL Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.464 Male (%) 327 (55.3%) 95 (54.9%) 235 (59%) Female (%) 264 (44.7%) 78 (45.1%) 163 (41%) Mean age (SD) 53.42 (11.9) 51.71 (11.8) 53 (13.1) 0.274 Stone site 0.001 Ureter (%) 65 (11.1%) 18 (10.5%) 53 (13.3%) Kidney (%) 479 (82%) 124 (72.5%) 299 (75.1%) Ureter + kidney (%) 40 (6.8%) 29 (17%) 46 (11.6%) Stone location 0.003 Single (%) 157 (31.2%) 28 (18.2%) 84 (24.3%) Multiple/Staghorn (%) 347 (68.8%) 126 (81.8%) 261 (75.7%) Mean Charlson’s Comorbidity Index (SD) 1.55 (1.34) 1.31 (1.12) 1.51 (1.23) 0.063 Mean cumulative stone diameter (SD) 26.78 (16.3) 28.42 (16.9) 25.18 (16.9) 0.009 Hydronephrosis (%) 0.004 None 39 (6.9%) 28 (16.4%) 37 (9.5%) Mild 262 (46.6%) 83 (48.5%) 193 (49.4%) Moderate 144 (25.6%) 31 (18.1%) 97 (24.8%) Severe 117 (20.8%) 29 (17%) 64 (16.4%) Guy’s Stone Score 0.01 1 64 (12.8%) 19 (13.5%) 28 (8.4%) 2 309 (61.7%) 78 (55.3%) 206 (61.5%) 3 84 (16.8%) 22 (15.6%) 76 (22.7%) 4 44 (8.8%) 22 (15.6%) 25 (1.5%) Pre-operatively stented (%) 0.001 No 507 (89.6%) 144 (83.2%) 263 (66.8%) Yes 59 (10.4%) 29 (16.8%) 131 (33.2%) Pre-operatively nephrostomized (%) 0.001 No 378 (66.8%) 159 (91.9%) 269 (68.4%) Yes 188 (33.2%) 14 (8.1%) 124 (31.6%) Mean post-op length of stay (SD) 4.0 (3) 4.9 (2.94) 3.57 (2.68) 0.001 Clavien–Dindo score for complications (%) 0.047 0 491 (83.1%) 152 (87.9%) 331 (81.7%) 1 86 (14.6%) 13 (7.5%) 57 (14.1%) 2 14 (2.4%) 8 (4.6%) 16 (4%) 3a 0 0 1 (0.2%) Stone free patients (%) 0.913 No 157 (26.4%) 46 (26.6%) 110 (27.6%) Yes 434 (73.6%) 127 (73.4%) 288 (72.4%) Table 3 Peri-operative data on RIRSs RIRS Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.561 Male (%) 131 (61.2%) 81 (65.9%) 169 (65.5%) Female (%) 83 (38.8%) 42 (34.1%) 89 (34.5%) Mean age (SD) 48.88 (12.9) 49.96 (11.9) 50.25 (13.5) 0.500 Stone site 0.01 Ureter (%) 50 (23.5%) 45 (36.6%) 92 (35.9%) Kidney (%) 151 (70.9%) 55 (44.7%) 135 (52.7%) Ureter + kidney (%) 12 (5.6%) 23 (18.7%) 29 (11.3%) Stone location 0.041 Single (%) 77 (47.8%) 24 (31.2%) 64 (39.3%) Multiple/Staghorn (%) 84 (52.2%) 53 (68.8%) 99 (60.7%) Median Charlson’s Comorbidity Index (SD) 1.3 (1.3) 1.2 (1.3) 1.4 (1.4) 0.851 Mean cumulative stone diameter (SD) 17.1 (9.3) 14.5 (7.0) 13.9 (7.1) 0.0001 Hydronephrosis (%) 0.025 None 18 (8.5%) 18 (15%) 46 (18.5%) Mild 137 (64.3%) 80 (66.7%) 149 (59.8%) Moderate 43 (20.2%) 16 (13.3%) 46 (18.5%) Severe 15 (7%) 6 (5%) 8 (3.2%) Pre-operatively stented (%) 0.001 No 170 (80.2%) 98 (79.7%) 147 (57.4%) Yes 42 (19.8%) 25 (20.3%) 109 (42.6%) Pre-operatively nephrostomized (%) 0.052 No 201 (96.6%) 122 (99.2%) 241 (94.1%) Yes 7 (3.4%) 1 (0.8%) 15 (5.9%) Clavien–Dindo Score for complications (%) 0.978 0 202 (95.7%) 119 (96.7%) 244 (95.9%) 1 6 (2.8%) 3 (2.4%) 9 (3.5%) 2 3 (1.4%) 1 (0.8%) 4 (1.6%) Stone free patients (%) 0.048 No 66 (30.8%) 24 (19.5%) 80 (30.7%) Yes 148 (69.2%) 99 (80.5%) 178 (69.3%) Table 4 Peri-operative data on URSs URS Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.194 Male (%) 112 (54.1%) 44 (64.7%) 129 (61.1%) Female (%) 95 (45.9%) 24 (35.3%) 82 (38.9%) Mean age (SD) 51.4 (13.3) 49.1 (14.4) 50.9 (13.4) 0.482 Stone site 0.783 Proximal ureter (%) 50 (28.4%) 24 (36.4%) 48 (30.4%) Mid ureter (%) 37 (21%) 15 (22.7%) 34 (21.5%) Distal ureter (%) 84 (47.7%) 26 (39.4%) 69 (43.7%) Multiple sites (%) 5 (2.8%) 1 (1.5%) 7 (4.4%) Mean Charlson’s Comorbidity Index (SD) 1.4 (1.6) 1.3 (1.1) 1.4 (1.4) 0.763 Mean cumulative stone diameter (SD) 12.1 (7.6) 10.8 (5.5) 11.6 (7.2) 0.588 Hydronephrosis (%) 0.007 None 14 (7%) 5 (7.6%) 39 (19.9%) Mild 111 (55.2%) 39 (59.1%) 99 (50.5%) Moderate 57 (28.4%) 16 (24.2%) 42 (21.4%) Severe 19 (9.5%) 6 (9.1%) 16 (8.2%) Pre-operatively stented (%) 0.001 No 174 (89.7%) 57 (86.4%) 126 (60.3%) Yes 20 (10.3%) 9 (13.6%) 83 (39.7%) Pre-operatively nephrostomized (%) 0.087 No 187 (96.9%) 65 (98.5%) 195 (93.3%) Yes 6 (3.1%) 1 (1.5%) 14 (6.7%) Mean post-op length of stay (SD) 1.83 (2.69) 1.89 (2.09) 1.79 (1.85) 0.117 Clavien Dindo Score for complications (%) 0.638 0 195 (94.2%) 63 (92.6%) 200 (94.8%) 1 9 (4.3%) 5 (7.4%) 10 (4.7%) 2 3 (1.4%) 0 1 (0.5%) Stone free patients (%) 0.013 No 20 (9.7%) 2 (2.9%) 32 (15.2%) Yes 187 (90.3%) 66 (97.1%) 179 (84.8%) Table 5 Peri-operative data on ESWLs ESWL Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.003 Male (%) 93 (64.1%) 51 (87.9%) 60 (73.2%) Female (%) 52 (5.9%) 7 (12.1%) 22 (26.8%) Mean age (SD) 48.9 (13.9) 45.4 (12.1) 50.0 (14.5) 0.127 Stone site 0.019 Ureter (%) 101 (70.1%) 51 (87.9%) 64 (78%) Kidney (%) 43 (29.9%) 6 (10.3%) 17 (20.7%) Ureter + kidney (%) 0 1 (1.7%) 1 (1.2%) Mean Charlson’s Comorbidity Index (SD) 1.0 (1.2) 0.9 (1.1) 1.3 (1.4) 0.397 Mean cumulative stone diameter (SD) 8.6 (3.4) 9.1 (3.6) 7.8 (3.4) 0.096 Hydronephrosis (%) 0.189 None 8 (8.5%) 7 (12.3%) 14 (20.3%) Mild 84 (89.4%) 48 (84.2%) 52 (75.4%) Moderate 2 (2.1%) 2 (3.5%) 3 (4.3%) Severe 0 (0.0%) 0 (0.0%) 0 (0.0%) Pre-operatively stented (%) 0.501 No 120 (94.5%) 57 (98.3%) 63 (94%) Yes 7 (5.5%) 1 (1.7%) 4 (6%) Pre-operatively nephrostomized (%) 0.947 No 0 0 0 Yes 0 0 0 Mean post-op length of stay (SD) 1.90 (3.31) 4.41 (3.82) 3.49 (4.1) 0.001 Stone free patients (%) 0.578 No 16 (11.0%) 7 (12.1%) 13 (15.9%) Yes 129 (89.0%) 51 (87.9%) 69 (84.1%) GSS was also calculated for PCNLs. In this case, data suggest that more complex procedures have been carried out during the pandemic, with GSS 3 and 4 representing 25.6% of cases during period A, 31.2% in period B, and 24.2% in period C (p = 0.01). For PCNL, 68.8% of patients in period A had multiple/staghorn stones, 81.8% in period B, and 75.7% in period C. For RIRS, 52.2% of patients in stage A had multiple/staghorn stones, 68.8% in period B, and 60.7% in period C. For PCNL, bilateral stones were observed in 5.4% of cases in period A, 13.3% in period B, and 25.4% in period C (p = 0.001). In period A, 6.8% of cases had both kidney and ureteral stones, 17% in period B, and 11.6% in period C (p = 0.001). For RIRS, bilateral stones were observed in 4.2% of cases in period A, 5.7% in period B, and 25% in period C (p = 0.004). In period A, 5.6% of cases had both kidney and ureteral stones, 18.7% in period B, and 11.3% in period C (p = 0.01). Regarding ESWLs, after COVID-19 appearance, the proportion of ureteric stones increased significantly, representing 70.1% in period A, 87.9% in period B, and 78% in period C (p = 0.019). Using stone diameter data, we also found that post-COVID-19 stone burdens changed in stages B and C. For PCNL, the data showed a greater stone burden in stages B and C, 26.8 in stage A, 28.42 in stage B, and 25.2 in stage C (p = 0.009). For RIRS, the data showed a lower stone burden in stages B and C, 17.15 in stage A, 14.5 in stage B, and 13.9 in stage C (p = 0.0001). The number of patients pre-operatively stented also increased. For PCNLs, the rates of pre-stented patients were 10.4%, 16.8%, and 33.2% for period A, B, and C, respectively (p = 0.01). For RIRSs, rates were 19.8%, 20.3%, and 42.6% (p = 0.001). For URSs, they were 10.3%, 13.6%, and 39.7% (p = 0.001). This difference was not noticed for ESWLs (p = 0.501). Intra-operative data and follow-ups SFRs did not show any relevant difference for PCNLs and ESWLs (p = 0.913 and p = 0.578, respectively) while a difference was noticed for RIRSs and URSs (p = 0.048 and p = 0.013, respectively). For RIRS, SFR in stage A was 69.2%, stage B was 80.5%, and stage C was 69.3% (p = 0.048). For URS, SFR in stage A was 90.3%, stage B was 97.1%, and stage C was 84.8% (p = 0.013). Patients requiring ureteric stenting after treatment increased after period A. In fact, for RIRSs, the rates were 84.4%, 93.1%, and 98.2% in period A, B, and C, respectively (p = 0.001). URSs showed analogue trends, with rates of stented patients of 84.1%, 99.2%, and 97.3%, respectively (p = 0.01). For PCNLs, the rate of patients requiring nephrostomy and/or ureteric stent was 84.4%, 93.1%, 98.2%, respectively (p = 0.001). The Clavien–Dindo Score for complications differed only for PCNLs, with 2.4% of patients in stage A having a score of 2, 2.6% in stage B, and 4% in stage C (p = 0.047). Among patients not stone free, the post-operative strategy did not differ between groups for all types of procedures throughout three study periods. Discussion COVID-19 pandemic has caused a significant disruption of Health Care Services and its effects are still evident. The main result was the partial or total interruption of routine clinical and surgical activities. Naspro et al. documented a reduction of urological surgical volumes by 30% within 15 days from the beginning of the outbreak. Same authors reported a total shutdown on March 19th, 2020. This fact was determined by utilization of hospital beds for COVID-19 patients. Additionally, the capacity of delivering urgent urological operations was significantly affected due to the lack of anesthetists, operating rooms, and ventilators, required for critically ill COVID-19 patients [9]. Furthermore, Novara G et al. reported a 55% decrease of urgent consultations in Italy, with a peak of 64% in areas more severely involved by the COVID-19 pandemic [10]. Similar effects were reported by Bin Hamri et al. [11] in Saudi Arabia. Overall, authors observed a decrease of 78% of out-patients clinic appointments (with 90.8% of them delivered as telephone consultations) and 34% of all elective procedures. Additionally, emergency procedures were reduced by 9.3%. In China, although preventive measurements have been promptly adopted, we observed a significant reduction of surgical and clinical activities. This effect has been caused by different phenomena which, we believe, have been challenging to control and prevent. On one side, clinicians may have decided to carry out only urgent operations and postpone all non-urgent complex procedures. On the other hand, the isolation protocols introduced in different cities may have limited the possibility of seeking medical assistance. Additionally, we cannot properly quantify the psychological stress and fear of citizens, possibly stopping them from reporting to hospitals. The sum of these events is difficult to clearly quantify but, as shown by our data, has likely determined a temporary significant disruption of quality of Health Services which continued partially even after the complete release of restrictions. In fact, we reported a loss of 743 procedures during period B and 206 procedures during period C. The impact of the inappropriate management of urinary stones can have a dramatic impact on patients’ safety. As reported by Galiabovitch et al. [12], approximately 10% of all surgical deaths are secondary to stones. Also, urinary sepsis are demonstrated to be responsible for 49.5% of all deaths. Same authors revealed how delayed/suboptimal management of infections was responsible of 39% of these events. Postponed access to medical care and adoption of non-ideal treatments are phenomena widely reported during the pandemic. These data underline how deep the effect of COVID-19 on patients with urinary stones could have been. The real impact of delayed treatments for urolithiasis is difficult to balance but we can expect an increased risk of renal function loss, ureteric strictures, and infectious complications. In literature, the delayed management of complications secondary to renal obstruction seems to have caused more dangerous events. Before the pandemic, the mean time between onset of symptoms for obstructing urosepsis and the appropriate intervention was a 2–3 days, with an associated positive correlation between a longer time and occurrence of septic shock [13, 14]. Furthermore, Meller et al. [15] were able to show how during COVID-19 pandemic, patients with obstructive pyelonephritis received delayed assistance (mean of 7.8 days versus 4.3 days of the pre-COVID period) and experiencing more severe clinical conditions including SIRS (57% versus 25%), perirenal abscesses (13% versus 0%), and longer hospitalization (mean 7.6 days versus 3.8 days). In this study, the authors attributed the delay of treatment to strict measures adopted in Brazil and the concomitant reluctance of the population to seek hospital assistance. In our study, we observed a reduction of emergency treatments too, although our data do not demonstrate an increased incidence of life-threatening events. This fact is likely to be attributed to different factors which have limited access to hospitals. We might speculate that many patients preferred to be managed locally or avoid medical care completely, particularly those with less severe urgent conditions (pain, low-degree fever, asymptomatic renal obstruction, etc.). To the best of our knowledge, this study represents the first report of COVID-19 effects on the treatment of urinary stones in China. In literature, reports on changes of surgical volumes during the outbreak are sparse and not evaluating patient’s complexity and surgical results. In this study, we analyzed surgical volumes in three highly Specialized Centers including also peri-operative data, documenting how COVID-19 affected treatment decisions. For PCNLs, our data showed that the number of patients pre-operatively stented was higher in period B compared to period A, whereas the number of nephrostomies has reduced. Additionally, during period B, stone complexity seemed to be higher (31.2% of stones resulted to be GSS 3 or 4 compared to 25.6% in period A and 24.2% in period C, p = 0.01). In period B, we observed varying degrees of hydronephrosis and stone burden compared to period A. We believe that more patients with complex stones in period B reported to hospitals for non-deferrable reasons (pain, infections, etc.), while those who were generally well and with no obvious symptoms preferred to stay at home and delay treatments. Additionally, for safety reasons, surgeons seemed to be more inclined to stents patients before definitive surgery, which greatly reduces the degree of hydronephrosis during period B. Besides, in our opinion, the more complex scenarios lead to an increased incidence of post-operative complications after PCNL. However, all Centers applied treatment plans to avoid complications through rational surgical selection and good surgeon decision-making. In our opinion, the reduced number of nephrostomies in favor of the utilization of ureteric stents has been determined by different factors. In fact, percutaneous drainages may come along with risks such as bleeding and dislodgements which may lead to a prolonged length of stay. For this reason, we believe that surgeons have favored ureteric stents to minimize this risk. Additionally, the rate of “non-tubeless” patients increased during period B. In our opinion, this was due to a higher stone complexity and the need of minimizing the risk of post-operative complications and patients’ re-access to hospitals, which would have been particularly problematic especially during period B. Similarly, the longer post-operative hospital stay has been determined by more cautious strategies. Regarding RIRS/URSs, data show that the stone burden was smaller during period B and C, coupled with an increased rate of post-operative stent placement and a longer post-operative hospital stay. From an overview of all these elements, it appears evident that, during the pandemic, surgeons preferred treating undelayable large and complex stones with PCNLs and offer RIRS/URS for smaller stones with low risk of post-operative complications. It seems also evident that peri-operative strategies varied, favoring solutions which would have guaranteed lower chances of complications and hospital re-admissions. These phenomena may have determined a deviation from standard and ideal clinical practice alongside increased costs. In fact, such a strategy has obvious effects, greatly improving the SFR of RIRS and URS without affecting the SFR of PCNL. Data presented in this document, seem to have some discordant trends if compared to other geographic regions. In fact, Byrne et al. analyzed treatment modalities for ureteric stones in United Kingdom highlighting a reduced number of patients managed with surgical interventions (from 57.2% to 47.5%) and an increased number of ESWL (from 22.7% to 34.1%). Also, adoption of medical expulsive therapy increased (from 17.4% to 25.4%). In our opinion, these differences have been determined by different factors [16]. Certainly, China is categorized by a very vast territory, and with a less diffused health care network. In this scenario, and with less possibilities of routine monitoring, surgeons in China had to guarantee a more certain resolution of the problem during pandemic period, then favoring more invasive strategies. In the creation of this study, we certainly expected a drastic reduction of surgical volume during pandemic. However, certain unexpected results emerged. For example, SWL treatments did not increase compared to retrograde surgeries in period B, although they would have required no anesthesia and hospital admissions. The reason is probably multifactorial and determined by specific case characteristics. We believe that the need to clear the stone in a single treatment was favored; therefore, many surgeons did not opt for SWL. Although rigid safety protocols have been introduced to treat COVID-19 positive stone patients, none of three hospitals reported such cases and all procedures have been carried out in patients free from COVID-19. This also represents an unexpected results. This study has some limitations. Data have been collected retrospectively. In fact, due to the characteristics of electronic databases and the retrospective nature of the study, we cannot extrapolate the reasons for urgent operations. For the same reasons, data on accesses to Accident & Emergency Departments for stones were unavailable and have not been investigated in this document. In this research, all involved Urology Units are centralized tertiary referral Centers and frequently accepting patients after a diagnosis already obtained locally. In patients requiring treatments, preliminary investigations are usually carried out during same admission and appropriate surgical treatment is usually delivered without the patient returning to hospital at a later stage. In this context, it has not been possible evaluating eventual delays in patients’ treatment as a consequence of the pandemic. Furthermore, we cannot state whether patients acquired COVID-19 as a consequence of the hospitalization; this was determined by the fact that patients were managed with early discharges when possible. We also need considering that patients could have been located in areas very distant from hospitals, and therefore these elements were not available. Additionally, all procedures have been delivered safely. In fact, all indicators of surgical adequacy (SFR, complication rates and need of subsequent auxiliary procedures) remained similar. However, we acknowledge that a bias may be involved in the comparison of SFR as post-operative imaging modalities adopted for follow-up have been rather heterogeneous, including NCCT, ultrasound, and plain X-rays. Finally, we provided an estimation of costs for treatments of urinary stones (Supplementary Table 2). These data have to be considered indicative as they cannot take in consideration cost related to eventual complications, auxiliary procedure, different medical treatments, etc. Conclusion COVID-19 caused a significant disruption of endourological services for urinary stones in terms of both surgical volumes and cases complexity. Alongside a reduced number of treated patients, a deviation from the pre-COVID standard practice has been observed. These effects were still evident after the end of the outbreak and the release of government restrictions. Although the effects of these phenomena in the long term are still uncertain and will need further investigation, in our experience endourological procedures can be adequately and safely carried out. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 18 KB) Acknowledgements Authors would like to thank all patients participating in the study. Author contributions Study design: GM, GZ, XZ. Data collection: XZ, XY, FC, YL. Statistical analysis: GM, XZ. Tables preparation: FC, YL. All authors reviewed and agreed with the paper. Data availability The data that support the findings of this study are available request from the corresponding author (GZ). They are not publicly available due to informations that could compromise the privacy of research participants. Declarations Conflict of interest Authors have nothing to disclose. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Giorgio Mazzon, Xin Zhang authors equally contributed to the paper. ==== Refs References 1. Cucinotta D Vanelli M WHO declares COVID-19 a pandemic Acta Biomed 2020 91 157 160 10.23750/ABM.V91I1.9397 32191675 2. Jin Y Yang H Ji W Virology, epidemiology, pathogenesis, and control of COVID-19 Viruses 2020 12 372 10.3390/V12040372 32230900 3. Ficarra V Novara G Abrate A Urology practice during the COViD-19 pandemic Minerva Urol Nefrol 2020 72 369 375 10.23736/S0393-2249.20.03846-1 32202401 4. Hollander JE Carr BG Virtually perfect? Telemedicine for Covid-19 N Engl J Med 2020 382 1679 1681 10.1056/NEJMP2003539 32160451 5. 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Silva AB Freschi G Carrera RV COVID-19 pandemic impact on clinical outcomes of patients with obstructive pyelonephritis Int Urol Nephrol 2021 53 627 633 10.1007/S11255-020-02708-3 33219920 16. Byrne MHV Georgiades F Light A Impact of COVID-19 on the management and outcomes of ureteric stones in the UK: a multicentre retrospective study BJU Int 2022 10.1111/BJU.15882 36083711	36454280	PMC9713732	NO-CC CODE	2022-12-02 23:24:45	no	Urolithiasis. 2023 Dec 1; 51(1):5	utf-8	Urolithiasis	2,022	10.1007/s00240-022-01390-7	oa_other
==== Front Urolithiasis Urolithiasis Urolithiasis 2194-7228 2194-7236 Springer Berlin Heidelberg Berlin/Heidelberg 36454280 1390 10.1007/s00240-022-01390-7 Research The effect of COVID-19 outbreak on urological procedures for urinary stones: data from three high-volumes centers in China Mazzon Giorgio 1 Zhang Xin 1 Yang Xingguo 2 Cheng Fan 3 Liu Yongda 1 Zeng Guohua [email protected] 1 1 grid.470124.4 Department of Urology and Guangdong Key Laboratory of Urology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 2 Department of Urology, Yiling Hospital, Yiling District, Yichang City, China 3 grid.412632.0 0000 0004 1758 2270 Department of Urology, Renmin Hospital of Wuhan University, Wuhan, China 1 12 2022 2023 51 1 512 10 2022 27 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. China has been the first country to be affected by the COVID-19 outbreak. The pandemic resulted in significant disruption of Health Care Services worldwide, and this effect on treatments for urinary stones is currently unclear. This is the first retrospective study involving three tertiary referral centers for urolithiasis across China. We evaluated surgical volumes and peri-operative outcomes of procedures delivered for upper urinary tract stones. We compared trimester prior to restrictions for COVID-19 (October 1st, 2019 to December 31st, 2019, period A), during restrictions (February 1st, 2020 to March 31st, 2020, period B), and afterword (April 1st, 2020 to June 31st, 2020, period C). A total of 2,543 procedures have been carried out. We observed a loss of 743 cases during period B (−29.2%) and 201 during period C (−7.9%). Percutaneous surgery showed the worst reduction, with 507 mini-PCNLs delivered in period A, 168 in period B (−60.8%), and 389 (−18.3%) in period C (p = 0.001). A worst trend was shown for standard PCNLs with 84 procedures carried out in period A, 5 in period B (−95.2%), and 9 (−89.2%) in period C (p = 0.001). Retrograde surgery also decreased, from 420 cases in period A to 190 cases in period B (−54.8%). An increment was however seen in period C when 468 cases have been carried out (+ 11.4%, p = 0.008). In term of SFRs, a difference was noticed for RIRSs, being 69.2%, 80.5%, and 69.3% during three periods (p = 0.045) and semirigid ureteroscopies (90.3%, 97.1%, and 84.8%, p = 0.013). Charlson’s Comorbidity Score could not show any difference between groups as well as no differences in term of post-operative complications have been noticed. Supplementary Information The online version contains supplementary material available at 10.1007/s00240-022-01390-7. Keywords COVID-19 outbreak Urinary stones PCNL RIRS ESWL Ureteroscopy issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2023 ==== Body pmcIntroduction The novel coronavirus 2019 (COVID-19) has been responsible of a new rapidly spreading viral infection classified as pandemic on March 11, 2020 by World Health Organization (WHO) [1]. In July 2020, more than 13,340,000 cases have been registered worldwide including 578,000 deaths. By the end of January 2020, all provinces in mainland of China reported cases of COVID-19. By the beginning of February 2020, 44,672 cases were reported in all 31 provinces of mainland of China, with the peak reached on February 12th, 2020. The Hubei province was the first to report absence of new confirmed cases on March 18th 2020, followed by other 23 provinces. By March 23rd 2020, a total of 81.773 cases were reported in 31 provinces of mainland of China, with 8 provinces with no new cases and vast majority of new diagnoses made on patients coming from abroad [2]. This exponentially growing number of cases, has rapidly overwhelmed Health Care Systems and saturated hospital services across the world, causing a significant disruption of elective and emergency activities. In fact, there has been a dramatic change in clinical priorities, both medical and surgical, with urology services affected in several ways. Non-urgent operations have been postponed, emergency cases were advised to be managed differently, aiming to reduce the number of patients requiring hospitalization and access to operating rooms and intensive care units [3]. Additionally, in many cases, urologists had been redeployed in the emergency services to support the care of COVID-19; elective out-patients clinics were also stopped because of the risk of gathering people within the hospital implementing services of telemedicine [4]. Additionally, due to restrictions and uncertainties related to the pandemic, several patients may have decided to not report to hospital for assistance, with a potential risk of clinical deterioration of their conditions. Endourological procedures were also dramatically affected, even though urinary stones are considered a benign condition, they can lead to severe complications if not treated timely such as urosepsis, renal function deterioration, prolonged pain, need of repeated access to Emergency Care Services, etc. This may represent a risk for patients and, at the same time, an overload for Health Care Systems struggling already to deliver an adequate care during the outbreak [5]. A delayed treatment may also lead to a poor quality of life for patients with urinary stones, for example because of prolonged stent-related irritative symptoms, anxiety, stone growth requiring a more invasive procedure at a later stage. In this study, we describe the effect of COVID-19 pandemic on urological procedures for upper urinary tract stones in three highly specialized Chinese Institutions during the first outbreak. Materials and methods We conducted a retrospective study involving three tertiary referral high-volume Centers specialized in the treatments of urinary stones. We compared surgical volumes and peri-operative outcomes during three periods: trimester prior to COVID-19 outbreak (period A), during COVID-19 (period B), and after COVID-19 outbreak (period C). All Centers involved in the study were asked to collect clinical, surgical, and peri-operative data of adult patients (> 18-year-old) undergoing elective and emergency surgeries for upper tract urinary stones and completing at least a 3-month follow-up. Lengths of study periods were decided taking in consideration the duration of first COVID-19 outbreak in China. In fact, the government notified it on January 24th 2020, during Chinese New Year celebrations. Period B was, therefore, defined as the time interval between the beginning of restrictions and their release, happened on April 30th, 2020. Surgical activities have been compared to analogue periods prior and after the emergency. Following this principle, period A started on October 1st, 2019, and finished on December 31st, 2019. Period B was from February 1st, 2020, to April 30th, 2020. Period C was from May 1st, 2020, to July 30th, 2020. These three study periods have been also identified as all involved hospitals were subjected to analogue situations and limitations, aiming to obtain uniform data. We decided to exclude cases carried out in January 2020 as this period contained many national holidays with the potential effect of biasing results. From beginning of period B to date, as per national regulations, patients must have been rigorously screened for COVID-19 before receiving any surgical treatment. If negative, they were admitted to a COVID-19-free department for either elective or urgent surgical treatment. If positive, they would have been admitted to a dedicated ward with specific precautions measurements and only non-deferrable conditions would have been treated. According to national and local hospital regulations, there were no formal limitations or prioritization rules on which type of surgeries should have been delivered across China during COVID-19 first wave of pandemic and afterward, after confirmation that patients were COVID-19 free. In fact, surgeons were left free to decide which types of conditions had to be treated based on their patient’s conditions. For the same reason, patients with urinary stones may have delayed their access to hospitals due to social distancing measurements and/or fear to report to hospitals. In this study, we investigated procedures with intention to treat ureteric and renal stones or complications related to their presence, including extracorporeal shock wave Llthotripsy (SWL), ureterolithotripsy (URS), retrograde intra-renal surgery (RIRS), percutaneous nephrolithotomy (PCNL), ureterolithotomy, and pyelolithotomy. Data have been retrospectively extrapolated from hospital electronic databases. PCNLs were defined as “mini” in case of utilization of an equal or smaller than 22 Fr Sheath. Standard PCNL was defined as > 22 Fr sheath. Procedures were further categorized as elective or emergency cases. Post-operative complications were reported at 30 days post-op using Clavien–Dindo classification [6]. Stone free status (SFR) was evaluated at 1–3 month post-op and cutoff for clinically insignificant residual fragments was fixed at 2 mm. Due to the nature of the study, Centers adopted different imaging modalities during follow-ups, including unenhanced CT scan (NCCT), ultrasound scan of the urinary tract (US), and X-ray of kidney–ureter and bladder (KUB). The study has been approved in each Center by respective ethic committees. Peri-operative data Patient’s demographic data included sex and age. Age-adjusted Charlson’s Comorbidities Index [7] was utilized to assess and quantify comorbidities. Stone characteristics included laterality, stone location, cumulative stone diameter (mm), hydronephrosis (none, mild, moderate, severe). Stone complexity has been reported using the Guy’s Stone Score (GSS) [8]. We also reported the number of patients who preliminarily received a ureteric stent or percutaneous nephrostomy insertion. Peri-operative data included the rate of patients which received a stent insertion at the end of treatment, 30-days complications rates, mean post-operative length of stay (days from completion of treatment to decision to discharge), SFR. In case of persistence of significant fragments, we also collected the subsequent planned treatment strategy. Usually, after admission, patients receive full assistance from the pre-operative surgical preparation to the final stone treatment. In case of patients requiring immediate renal drainage, a stent/nephrostomy was placed and generally the stone clearance was completed during same admission as soon as the clinical conditions are favorable. For this reason, procedures have been divided in emergencies if delivered on first day of admission or elective if carried out after more days and adequate pre-operative work-up. Therefore, emergency procedures include cases operated for different reasons at day-1, including deterioration of renal function, hydronephrosis, infectious complications, pain. Due to the characteristics of the electronic database, we could not record separately ureteric stents and percutaneous nephrostomy placements, as happening during same admission episode. Statistical analysis Descriptive statistics were obtained reporting means and standard deviations for continuous variables. Frequencies and proportions were used for categorical variables. Continuous variables were compared using the Student t or the Mann–Whitney U test, based on normal or not-normal distribution. Categorical variables were tested with Chi-square test. All tests were two-sided, and significancy was set at p < 0.05. Statistical analyses were performed using SPSS v.24 (IBM SPSS Statistics for Mac, Armonk, NY, IBM Corp). Results Surgical volumes A total of 2,543 procedures have been delivered across three sites, including 2,062 elective cases and 481 emergency cases (Table 1). During period B, we observed a loss of 457 elective cases (−53.3%) and 286 emergency cases (−93.5%). A more modest decrease was noticed also in period C, including 55 elective cases (−6.4%) and 151 emergency cases (−49.3%). These differences resulted statistically significant (p < 0.001). The most significant reduction was due to PCNLs, with mini-PCNLs reduced by 60.8% in period B and 18.29% in period C. Standard PCNLs showed a worst decrease (−95.2% and −89.2% in period B and C, respectively). Among elective cases, we observed a significant increase of retrograde procedures (+ 31.1% for URSs and + 38.1% for RIRSs) during period C. Same effect was not noticed for emergency cases. Open surgeries included a total of eight procedures. During period B, these procedures have not been carried out, ureterolithotomies were reduced in period C (−25%) but pyelolithotomies increased by 75%. In Table 1, we also reported relative variations of different types of surgery during three periods. In Supplementary Table 1, we reported variations of expenses related to stone surgeries. Compared to period A, costs were reduced by 64.8% and 17.1% during period B and C, respectively.Table 1 Procedures delivered during trimesters, subdivided in elective and urgent and variations of overall surgical volumes Type of surgery Pre-COVID COVID (difference compared to pre-COVID in %) Post-COVID (difference compared to pre-COVID in %) Significancy Elective procedures ESWLs 56 46 (−17.9%) 49 (−12.5%) p < 0.001 URSs 135 65 (−51.9%) 177 (+ 31.1%) p < 0.001 RIRSs 155 121 (−21.9%) 214 (+ 38.1%) p < 0.001 Mini-PCNLs 421 165 (−60.8%) 344 (−18.3%) p < 0.001 Standard PCNLs 83 4 (−95.2%) 9 (−89.2%) p < 0.001 Ureterolithotomies 4 0 (−100%) 3 (−25.0%) p < 0.001 Pyelolithotomies 4 0 (−100%) 7 (+ 75.0%) p < 0.001 Total 858 401 (−53.3%) 803 (−6.4%) p < 0.001 Emergency procedures ESWLs 89 12 (−86.5%) 33 (−62.9%) p < 0.001 URSs 71 2 (−97.2%) 34 (−52.1%) p < 0.001 RIRSs 59 2 (−96.6) 43 (−27.1) p < 0.001 Mini-PCNLs 86 3 (−96.5%) 45 (−47.7) p < 0.001 Standard PCNLs 1 1 (no difference) 0 (−100%) p < 0.001 Ureterolithotomies 0 0 0 Non applicable Pyelolithotomies 0 0 0 Non applicable Total 306 20 (−93.5%) 155 (−49.3%) p < 0.001 Overall procedures —rates of different surgical techniques within periods ESWLs 145 (12.4%) 58 (13.7%) 82 (8.5%) p < 0.001 URSs 206 (17.7%) 67 (15.9%) 211 (22.0%) p < 0.001 RIRSs 214 (18.3) 123 (29.2%) 257 (26.8%) p < 0.001 Mini-PCNLs 507 (43.5%) 168 (39.9%) 389 (40.6%) p = 0.036 Standard PCNLs 84 (7.2%) 5 (1.2%) 9 (0.9%) p < 0.001 Ureterolithotomies 4 (0.3%) 0 (0%) 3 (0.3%) p = 0.059 Pyelolithotomies 4 (0.3%) 0 (0%) 7 (0.7%) p = 0.062 Total 1164 (100%) 421 (100%) 958 (100%) p < 0.001 In the bottom part of the table, variations of rates of different types of surgery within study periods are reported Patients and stone characteristics In Tables 2, 3, 4, and 5, most relevant peri-operative data for PCNLs, RIRSs, URSs, and ESWLs, respectively are reported Additional data are reported in Supplementary Table 2. Patients’ complexity did not change during three periods, with CCI having a mean value between 1 and 2 (p = 0.062 for PCNLs, p = 0.851 for RIRSs, p = 0.763 for URSs and p = 0.397 for ESWLs). During period B, we observed a different level of stone complexity compared to period A. For PCNLs, absence or mild hydronephrosis was observed in 53.5% during period A compared to 64.9% in period B and 58.8% in period C (p = 0.04). Regarding RIRSs, none or mild hydronephrosis was noticed in 72.8% of cases during period A, 81.7% in period B, and 78.3% in period C (p = 0.025). Similar trend was noticed also for URSs, showing 62.2% during period A, 66.7% in period B, and 70.4% in period C (p:0.07). ESWLs did not show the same trend with 97.9%, 96.5%, and 95.7% in periods A, B, and C, respectively (p = 0.189).Table 2 Peri-operative data on PCNLs PCNL Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.464 Male (%) 327 (55.3%) 95 (54.9%) 235 (59%) Female (%) 264 (44.7%) 78 (45.1%) 163 (41%) Mean age (SD) 53.42 (11.9) 51.71 (11.8) 53 (13.1) 0.274 Stone site 0.001 Ureter (%) 65 (11.1%) 18 (10.5%) 53 (13.3%) Kidney (%) 479 (82%) 124 (72.5%) 299 (75.1%) Ureter + kidney (%) 40 (6.8%) 29 (17%) 46 (11.6%) Stone location 0.003 Single (%) 157 (31.2%) 28 (18.2%) 84 (24.3%) Multiple/Staghorn (%) 347 (68.8%) 126 (81.8%) 261 (75.7%) Mean Charlson’s Comorbidity Index (SD) 1.55 (1.34) 1.31 (1.12) 1.51 (1.23) 0.063 Mean cumulative stone diameter (SD) 26.78 (16.3) 28.42 (16.9) 25.18 (16.9) 0.009 Hydronephrosis (%) 0.004 None 39 (6.9%) 28 (16.4%) 37 (9.5%) Mild 262 (46.6%) 83 (48.5%) 193 (49.4%) Moderate 144 (25.6%) 31 (18.1%) 97 (24.8%) Severe 117 (20.8%) 29 (17%) 64 (16.4%) Guy’s Stone Score 0.01 1 64 (12.8%) 19 (13.5%) 28 (8.4%) 2 309 (61.7%) 78 (55.3%) 206 (61.5%) 3 84 (16.8%) 22 (15.6%) 76 (22.7%) 4 44 (8.8%) 22 (15.6%) 25 (1.5%) Pre-operatively stented (%) 0.001 No 507 (89.6%) 144 (83.2%) 263 (66.8%) Yes 59 (10.4%) 29 (16.8%) 131 (33.2%) Pre-operatively nephrostomized (%) 0.001 No 378 (66.8%) 159 (91.9%) 269 (68.4%) Yes 188 (33.2%) 14 (8.1%) 124 (31.6%) Mean post-op length of stay (SD) 4.0 (3) 4.9 (2.94) 3.57 (2.68) 0.001 Clavien–Dindo score for complications (%) 0.047 0 491 (83.1%) 152 (87.9%) 331 (81.7%) 1 86 (14.6%) 13 (7.5%) 57 (14.1%) 2 14 (2.4%) 8 (4.6%) 16 (4%) 3a 0 0 1 (0.2%) Stone free patients (%) 0.913 No 157 (26.4%) 46 (26.6%) 110 (27.6%) Yes 434 (73.6%) 127 (73.4%) 288 (72.4%) Table 3 Peri-operative data on RIRSs RIRS Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.561 Male (%) 131 (61.2%) 81 (65.9%) 169 (65.5%) Female (%) 83 (38.8%) 42 (34.1%) 89 (34.5%) Mean age (SD) 48.88 (12.9) 49.96 (11.9) 50.25 (13.5) 0.500 Stone site 0.01 Ureter (%) 50 (23.5%) 45 (36.6%) 92 (35.9%) Kidney (%) 151 (70.9%) 55 (44.7%) 135 (52.7%) Ureter + kidney (%) 12 (5.6%) 23 (18.7%) 29 (11.3%) Stone location 0.041 Single (%) 77 (47.8%) 24 (31.2%) 64 (39.3%) Multiple/Staghorn (%) 84 (52.2%) 53 (68.8%) 99 (60.7%) Median Charlson’s Comorbidity Index (SD) 1.3 (1.3) 1.2 (1.3) 1.4 (1.4) 0.851 Mean cumulative stone diameter (SD) 17.1 (9.3) 14.5 (7.0) 13.9 (7.1) 0.0001 Hydronephrosis (%) 0.025 None 18 (8.5%) 18 (15%) 46 (18.5%) Mild 137 (64.3%) 80 (66.7%) 149 (59.8%) Moderate 43 (20.2%) 16 (13.3%) 46 (18.5%) Severe 15 (7%) 6 (5%) 8 (3.2%) Pre-operatively stented (%) 0.001 No 170 (80.2%) 98 (79.7%) 147 (57.4%) Yes 42 (19.8%) 25 (20.3%) 109 (42.6%) Pre-operatively nephrostomized (%) 0.052 No 201 (96.6%) 122 (99.2%) 241 (94.1%) Yes 7 (3.4%) 1 (0.8%) 15 (5.9%) Clavien–Dindo Score for complications (%) 0.978 0 202 (95.7%) 119 (96.7%) 244 (95.9%) 1 6 (2.8%) 3 (2.4%) 9 (3.5%) 2 3 (1.4%) 1 (0.8%) 4 (1.6%) Stone free patients (%) 0.048 No 66 (30.8%) 24 (19.5%) 80 (30.7%) Yes 148 (69.2%) 99 (80.5%) 178 (69.3%) Table 4 Peri-operative data on URSs URS Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.194 Male (%) 112 (54.1%) 44 (64.7%) 129 (61.1%) Female (%) 95 (45.9%) 24 (35.3%) 82 (38.9%) Mean age (SD) 51.4 (13.3) 49.1 (14.4) 50.9 (13.4) 0.482 Stone site 0.783 Proximal ureter (%) 50 (28.4%) 24 (36.4%) 48 (30.4%) Mid ureter (%) 37 (21%) 15 (22.7%) 34 (21.5%) Distal ureter (%) 84 (47.7%) 26 (39.4%) 69 (43.7%) Multiple sites (%) 5 (2.8%) 1 (1.5%) 7 (4.4%) Mean Charlson’s Comorbidity Index (SD) 1.4 (1.6) 1.3 (1.1) 1.4 (1.4) 0.763 Mean cumulative stone diameter (SD) 12.1 (7.6) 10.8 (5.5) 11.6 (7.2) 0.588 Hydronephrosis (%) 0.007 None 14 (7%) 5 (7.6%) 39 (19.9%) Mild 111 (55.2%) 39 (59.1%) 99 (50.5%) Moderate 57 (28.4%) 16 (24.2%) 42 (21.4%) Severe 19 (9.5%) 6 (9.1%) 16 (8.2%) Pre-operatively stented (%) 0.001 No 174 (89.7%) 57 (86.4%) 126 (60.3%) Yes 20 (10.3%) 9 (13.6%) 83 (39.7%) Pre-operatively nephrostomized (%) 0.087 No 187 (96.9%) 65 (98.5%) 195 (93.3%) Yes 6 (3.1%) 1 (1.5%) 14 (6.7%) Mean post-op length of stay (SD) 1.83 (2.69) 1.89 (2.09) 1.79 (1.85) 0.117 Clavien Dindo Score for complications (%) 0.638 0 195 (94.2%) 63 (92.6%) 200 (94.8%) 1 9 (4.3%) 5 (7.4%) 10 (4.7%) 2 3 (1.4%) 0 1 (0.5%) Stone free patients (%) 0.013 No 20 (9.7%) 2 (2.9%) 32 (15.2%) Yes 187 (90.3%) 66 (97.1%) 179 (84.8%) Table 5 Peri-operative data on ESWLs ESWL Parameter Pre-COVID COVID Post-COVID Significancy Sex 0.003 Male (%) 93 (64.1%) 51 (87.9%) 60 (73.2%) Female (%) 52 (5.9%) 7 (12.1%) 22 (26.8%) Mean age (SD) 48.9 (13.9) 45.4 (12.1) 50.0 (14.5) 0.127 Stone site 0.019 Ureter (%) 101 (70.1%) 51 (87.9%) 64 (78%) Kidney (%) 43 (29.9%) 6 (10.3%) 17 (20.7%) Ureter + kidney (%) 0 1 (1.7%) 1 (1.2%) Mean Charlson’s Comorbidity Index (SD) 1.0 (1.2) 0.9 (1.1) 1.3 (1.4) 0.397 Mean cumulative stone diameter (SD) 8.6 (3.4) 9.1 (3.6) 7.8 (3.4) 0.096 Hydronephrosis (%) 0.189 None 8 (8.5%) 7 (12.3%) 14 (20.3%) Mild 84 (89.4%) 48 (84.2%) 52 (75.4%) Moderate 2 (2.1%) 2 (3.5%) 3 (4.3%) Severe 0 (0.0%) 0 (0.0%) 0 (0.0%) Pre-operatively stented (%) 0.501 No 120 (94.5%) 57 (98.3%) 63 (94%) Yes 7 (5.5%) 1 (1.7%) 4 (6%) Pre-operatively nephrostomized (%) 0.947 No 0 0 0 Yes 0 0 0 Mean post-op length of stay (SD) 1.90 (3.31) 4.41 (3.82) 3.49 (4.1) 0.001 Stone free patients (%) 0.578 No 16 (11.0%) 7 (12.1%) 13 (15.9%) Yes 129 (89.0%) 51 (87.9%) 69 (84.1%) GSS was also calculated for PCNLs. In this case, data suggest that more complex procedures have been carried out during the pandemic, with GSS 3 and 4 representing 25.6% of cases during period A, 31.2% in period B, and 24.2% in period C (p = 0.01). For PCNL, 68.8% of patients in period A had multiple/staghorn stones, 81.8% in period B, and 75.7% in period C. For RIRS, 52.2% of patients in stage A had multiple/staghorn stones, 68.8% in period B, and 60.7% in period C. For PCNL, bilateral stones were observed in 5.4% of cases in period A, 13.3% in period B, and 25.4% in period C (p = 0.001). In period A, 6.8% of cases had both kidney and ureteral stones, 17% in period B, and 11.6% in period C (p = 0.001). For RIRS, bilateral stones were observed in 4.2% of cases in period A, 5.7% in period B, and 25% in period C (p = 0.004). In period A, 5.6% of cases had both kidney and ureteral stones, 18.7% in period B, and 11.3% in period C (p = 0.01). Regarding ESWLs, after COVID-19 appearance, the proportion of ureteric stones increased significantly, representing 70.1% in period A, 87.9% in period B, and 78% in period C (p = 0.019). Using stone diameter data, we also found that post-COVID-19 stone burdens changed in stages B and C. For PCNL, the data showed a greater stone burden in stages B and C, 26.8 in stage A, 28.42 in stage B, and 25.2 in stage C (p = 0.009). For RIRS, the data showed a lower stone burden in stages B and C, 17.15 in stage A, 14.5 in stage B, and 13.9 in stage C (p = 0.0001). The number of patients pre-operatively stented also increased. For PCNLs, the rates of pre-stented patients were 10.4%, 16.8%, and 33.2% for period A, B, and C, respectively (p = 0.01). For RIRSs, rates were 19.8%, 20.3%, and 42.6% (p = 0.001). For URSs, they were 10.3%, 13.6%, and 39.7% (p = 0.001). This difference was not noticed for ESWLs (p = 0.501). Intra-operative data and follow-ups SFRs did not show any relevant difference for PCNLs and ESWLs (p = 0.913 and p = 0.578, respectively) while a difference was noticed for RIRSs and URSs (p = 0.048 and p = 0.013, respectively). For RIRS, SFR in stage A was 69.2%, stage B was 80.5%, and stage C was 69.3% (p = 0.048). For URS, SFR in stage A was 90.3%, stage B was 97.1%, and stage C was 84.8% (p = 0.013). Patients requiring ureteric stenting after treatment increased after period A. In fact, for RIRSs, the rates were 84.4%, 93.1%, and 98.2% in period A, B, and C, respectively (p = 0.001). URSs showed analogue trends, with rates of stented patients of 84.1%, 99.2%, and 97.3%, respectively (p = 0.01). For PCNLs, the rate of patients requiring nephrostomy and/or ureteric stent was 84.4%, 93.1%, 98.2%, respectively (p = 0.001). The Clavien–Dindo Score for complications differed only for PCNLs, with 2.4% of patients in stage A having a score of 2, 2.6% in stage B, and 4% in stage C (p = 0.047). Among patients not stone free, the post-operative strategy did not differ between groups for all types of procedures throughout three study periods. Discussion COVID-19 pandemic has caused a significant disruption of Health Care Services and its effects are still evident. The main result was the partial or total interruption of routine clinical and surgical activities. Naspro et al. documented a reduction of urological surgical volumes by 30% within 15 days from the beginning of the outbreak. Same authors reported a total shutdown on March 19th, 2020. This fact was determined by utilization of hospital beds for COVID-19 patients. Additionally, the capacity of delivering urgent urological operations was significantly affected due to the lack of anesthetists, operating rooms, and ventilators, required for critically ill COVID-19 patients [9]. Furthermore, Novara G et al. reported a 55% decrease of urgent consultations in Italy, with a peak of 64% in areas more severely involved by the COVID-19 pandemic [10]. Similar effects were reported by Bin Hamri et al. [11] in Saudi Arabia. Overall, authors observed a decrease of 78% of out-patients clinic appointments (with 90.8% of them delivered as telephone consultations) and 34% of all elective procedures. Additionally, emergency procedures were reduced by 9.3%. In China, although preventive measurements have been promptly adopted, we observed a significant reduction of surgical and clinical activities. This effect has been caused by different phenomena which, we believe, have been challenging to control and prevent. On one side, clinicians may have decided to carry out only urgent operations and postpone all non-urgent complex procedures. On the other hand, the isolation protocols introduced in different cities may have limited the possibility of seeking medical assistance. Additionally, we cannot properly quantify the psychological stress and fear of citizens, possibly stopping them from reporting to hospitals. The sum of these events is difficult to clearly quantify but, as shown by our data, has likely determined a temporary significant disruption of quality of Health Services which continued partially even after the complete release of restrictions. In fact, we reported a loss of 743 procedures during period B and 206 procedures during period C. The impact of the inappropriate management of urinary stones can have a dramatic impact on patients’ safety. As reported by Galiabovitch et al. [12], approximately 10% of all surgical deaths are secondary to stones. Also, urinary sepsis are demonstrated to be responsible for 49.5% of all deaths. Same authors revealed how delayed/suboptimal management of infections was responsible of 39% of these events. Postponed access to medical care and adoption of non-ideal treatments are phenomena widely reported during the pandemic. These data underline how deep the effect of COVID-19 on patients with urinary stones could have been. The real impact of delayed treatments for urolithiasis is difficult to balance but we can expect an increased risk of renal function loss, ureteric strictures, and infectious complications. In literature, the delayed management of complications secondary to renal obstruction seems to have caused more dangerous events. Before the pandemic, the mean time between onset of symptoms for obstructing urosepsis and the appropriate intervention was a 2–3 days, with an associated positive correlation between a longer time and occurrence of septic shock [13, 14]. Furthermore, Meller et al. [15] were able to show how during COVID-19 pandemic, patients with obstructive pyelonephritis received delayed assistance (mean of 7.8 days versus 4.3 days of the pre-COVID period) and experiencing more severe clinical conditions including SIRS (57% versus 25%), perirenal abscesses (13% versus 0%), and longer hospitalization (mean 7.6 days versus 3.8 days). In this study, the authors attributed the delay of treatment to strict measures adopted in Brazil and the concomitant reluctance of the population to seek hospital assistance. In our study, we observed a reduction of emergency treatments too, although our data do not demonstrate an increased incidence of life-threatening events. This fact is likely to be attributed to different factors which have limited access to hospitals. We might speculate that many patients preferred to be managed locally or avoid medical care completely, particularly those with less severe urgent conditions (pain, low-degree fever, asymptomatic renal obstruction, etc.). To the best of our knowledge, this study represents the first report of COVID-19 effects on the treatment of urinary stones in China. In literature, reports on changes of surgical volumes during the outbreak are sparse and not evaluating patient’s complexity and surgical results. In this study, we analyzed surgical volumes in three highly Specialized Centers including also peri-operative data, documenting how COVID-19 affected treatment decisions. For PCNLs, our data showed that the number of patients pre-operatively stented was higher in period B compared to period A, whereas the number of nephrostomies has reduced. Additionally, during period B, stone complexity seemed to be higher (31.2% of stones resulted to be GSS 3 or 4 compared to 25.6% in period A and 24.2% in period C, p = 0.01). In period B, we observed varying degrees of hydronephrosis and stone burden compared to period A. We believe that more patients with complex stones in period B reported to hospitals for non-deferrable reasons (pain, infections, etc.), while those who were generally well and with no obvious symptoms preferred to stay at home and delay treatments. Additionally, for safety reasons, surgeons seemed to be more inclined to stents patients before definitive surgery, which greatly reduces the degree of hydronephrosis during period B. Besides, in our opinion, the more complex scenarios lead to an increased incidence of post-operative complications after PCNL. However, all Centers applied treatment plans to avoid complications through rational surgical selection and good surgeon decision-making. In our opinion, the reduced number of nephrostomies in favor of the utilization of ureteric stents has been determined by different factors. In fact, percutaneous drainages may come along with risks such as bleeding and dislodgements which may lead to a prolonged length of stay. For this reason, we believe that surgeons have favored ureteric stents to minimize this risk. Additionally, the rate of “non-tubeless” patients increased during period B. In our opinion, this was due to a higher stone complexity and the need of minimizing the risk of post-operative complications and patients’ re-access to hospitals, which would have been particularly problematic especially during period B. Similarly, the longer post-operative hospital stay has been determined by more cautious strategies. Regarding RIRS/URSs, data show that the stone burden was smaller during period B and C, coupled with an increased rate of post-operative stent placement and a longer post-operative hospital stay. From an overview of all these elements, it appears evident that, during the pandemic, surgeons preferred treating undelayable large and complex stones with PCNLs and offer RIRS/URS for smaller stones with low risk of post-operative complications. It seems also evident that peri-operative strategies varied, favoring solutions which would have guaranteed lower chances of complications and hospital re-admissions. These phenomena may have determined a deviation from standard and ideal clinical practice alongside increased costs. In fact, such a strategy has obvious effects, greatly improving the SFR of RIRS and URS without affecting the SFR of PCNL. Data presented in this document, seem to have some discordant trends if compared to other geographic regions. In fact, Byrne et al. analyzed treatment modalities for ureteric stones in United Kingdom highlighting a reduced number of patients managed with surgical interventions (from 57.2% to 47.5%) and an increased number of ESWL (from 22.7% to 34.1%). Also, adoption of medical expulsive therapy increased (from 17.4% to 25.4%). In our opinion, these differences have been determined by different factors [16]. Certainly, China is categorized by a very vast territory, and with a less diffused health care network. In this scenario, and with less possibilities of routine monitoring, surgeons in China had to guarantee a more certain resolution of the problem during pandemic period, then favoring more invasive strategies. In the creation of this study, we certainly expected a drastic reduction of surgical volume during pandemic. However, certain unexpected results emerged. For example, SWL treatments did not increase compared to retrograde surgeries in period B, although they would have required no anesthesia and hospital admissions. The reason is probably multifactorial and determined by specific case characteristics. We believe that the need to clear the stone in a single treatment was favored; therefore, many surgeons did not opt for SWL. Although rigid safety protocols have been introduced to treat COVID-19 positive stone patients, none of three hospitals reported such cases and all procedures have been carried out in patients free from COVID-19. This also represents an unexpected results. This study has some limitations. Data have been collected retrospectively. In fact, due to the characteristics of electronic databases and the retrospective nature of the study, we cannot extrapolate the reasons for urgent operations. For the same reasons, data on accesses to Accident & Emergency Departments for stones were unavailable and have not been investigated in this document. In this research, all involved Urology Units are centralized tertiary referral Centers and frequently accepting patients after a diagnosis already obtained locally. In patients requiring treatments, preliminary investigations are usually carried out during same admission and appropriate surgical treatment is usually delivered without the patient returning to hospital at a later stage. In this context, it has not been possible evaluating eventual delays in patients’ treatment as a consequence of the pandemic. Furthermore, we cannot state whether patients acquired COVID-19 as a consequence of the hospitalization; this was determined by the fact that patients were managed with early discharges when possible. We also need considering that patients could have been located in areas very distant from hospitals, and therefore these elements were not available. Additionally, all procedures have been delivered safely. In fact, all indicators of surgical adequacy (SFR, complication rates and need of subsequent auxiliary procedures) remained similar. However, we acknowledge that a bias may be involved in the comparison of SFR as post-operative imaging modalities adopted for follow-up have been rather heterogeneous, including NCCT, ultrasound, and plain X-rays. Finally, we provided an estimation of costs for treatments of urinary stones (Supplementary Table 2). These data have to be considered indicative as they cannot take in consideration cost related to eventual complications, auxiliary procedure, different medical treatments, etc. Conclusion COVID-19 caused a significant disruption of endourological services for urinary stones in terms of both surgical volumes and cases complexity. Alongside a reduced number of treated patients, a deviation from the pre-COVID standard practice has been observed. These effects were still evident after the end of the outbreak and the release of government restrictions. Although the effects of these phenomena in the long term are still uncertain and will need further investigation, in our experience endourological procedures can be adequately and safely carried out. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 18 KB) Acknowledgements Authors would like to thank all patients participating in the study. Author contributions Study design: GM, GZ, XZ. Data collection: XZ, XY, FC, YL. Statistical analysis: GM, XZ. Tables preparation: FC, YL. All authors reviewed and agreed with the paper. Data availability The data that support the findings of this study are available request from the corresponding author (GZ). They are not publicly available due to informations that could compromise the privacy of research participants. Declarations Conflict of interest Authors have nothing to disclose. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Giorgio Mazzon, Xin Zhang authors equally contributed to the paper. ==== Refs References 1. Cucinotta D Vanelli M WHO declares COVID-19 a pandemic Acta Biomed 2020 91 157 160 10.23750/ABM.V91I1.9397 32191675 2. Jin Y Yang H Ji W Virology, epidemiology, pathogenesis, and control of COVID-19 Viruses 2020 12 372 10.3390/V12040372 32230900 3. Ficarra V Novara G Abrate A Urology practice during the COViD-19 pandemic Minerva Urol Nefrol 2020 72 369 375 10.23736/S0393-2249.20.03846-1 32202401 4. Hollander JE Carr BG Virtually perfect? Telemedicine for Covid-19 N Engl J Med 2020 382 1679 1681 10.1056/NEJMP2003539 32160451 5. Fukushima H Kobayashi M Kawano K Morimoto S Performance of quick sequential (sepsis related) and sequential (sepsis related) organ failure assessment to predict mortality in patients with acute pyelonephritis associated with upper urinary tract calculi J Urol 2018 199 1526 1533 10.1016/J.JURO.2017.12.052 29291417 6. de La Rosette JJMCH Opondo D Daels FPJ Categorisation of complications and validation of the Clavien score for percutaneous nephrolithotomy Eur Urol 2012 62 246 255 10.1016/J.EURURO.2012.03.055 22487016 7. Brusselaers N Lagergren J The Charlson comorbidity index in registry-based research Methods Inf Med 2017 56 401 406 10.3414/ME17-01-0051 29582935 8. Thomas K Smith NC Hegarty N Glass JM The Guy’s stone score—grading the complexity of percutaneous nephrolithotomy procedures Urology 2011 78 277 281 10.1016/J.UROLOGY.2010.12.026 21333334 9. Naspro R da Pozzo LF Urology in the time of corona Nat Rev Urol 2020 17 251 253 10.1038/s41585-020-0312-1 32203310 10. Novara G Bartoletti R Crestani A Impact of the COVID-19 pandemic on urological practice in emergency departments in Italy BJU Int 2020 126 245 247 10.1111/BJU.15107 32407585 11. Raheem Ali A Ghazwani Y Alowidah I Impact of COVID-19 on endourology surgical practice in Saudi Arabia: a national multicenter study Asian J Urol 2021 8 416 423 10.1016/J.AJUR.2021.03.006 34765449 12. Galiabovitch E Hansen D Retegan C McCahy P Urinary tract stone deaths: data from the Australian and New Zealand audits of surgical mortality BJU Int 2020 126 604 609 10.1111/BJU.15171 32654379 13. Yamamoto Y Fujita K Nakazawa S Clinical characteristics and risk factors for septic shock in patients receiving emergency drainage for acute pyelonephritis with upper urinary tract calculi BMC Urol 2012 12 4 10.1186/1471-2490-12-4 22413829 14. Tambo M Okegawa T Shishido T Predictors of septic shock in obstructive acute pyelonephritis World J Urol 2014 32 803 811 10.1007/S00345-013-1166-4 24037335 15. Silva AB Freschi G Carrera RV COVID-19 pandemic impact on clinical outcomes of patients with obstructive pyelonephritis Int Urol Nephrol 2021 53 627 633 10.1007/S11255-020-02708-3 33219920 16. Byrne MHV Georgiades F Light A Impact of COVID-19 on the management and outcomes of ureteric stones in the UK: a multicentre retrospective study BJU Int 2022 10.1111/BJU.15882 36083711	36471787	PMC9713733	NO-CC CODE	2022-12-02 23:23:08	no	Heilberufe. 2022 Dec 1; 74(12):36-37	latin-1	Heilberufe	2,022	10.1007/s00058-022-2973-z	oa_other
==== Front J Community Health J Community Health Journal of Community Health 0094-5145 1573-3610 Springer US New York 36454479 1175 10.1007/s10900-022-01175-4 Original Paper Remote Consultations for Mental Health: Patient Experiences http://orcid.org/0000-0002-8798-0448 Crunelle Cleo L. [email protected] 1 Van Daele Tom 2 Van Laere Sven 3 Blancke Stefaan 4 Vanderbruggen Nathalie 1 Matthys Frieda 1 1 grid.8767.e 0000 0001 2290 8069 Departement of Psychiatry, Vrije Universiteit Brussel (VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium 2 Expertise Unit Psychology, Technology & Society, Thomas More University of Applied Sciences, Molenstraat 8, 2018 Antwerp, Belgium 3 grid.8767.e 0000 0001 2290 8069 Support for Quantitative and Qualitative Research (SQUARE), Vrije Universiteit Brussel, Core facility, Laarbeeklaan 103, 1090 Brussels, Belgium 4 CGG Andante, Bredabaan 579, 2170 Antwerp, Belgium 1 12 2022 16 16 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Objective An exponential implementation of remote mental health care has been observed, but little data is available on experiences and barriers of remote health from a patient’s perspective. This study investigated experiences associated with several forms of remote consultations (both telephone and online video) for mental health care during the COVID-19 coronavirus pandemic with a particular focus on patients’ experiences. Methods This study includes results of an online web-based survey filled in by 512 patients on the use and experiences of remote mental health consultations and circulating between March and October 2021. Results Psychiatric consultations were initiated by the health care provider in 47.0% of cases, while psychological consultations were most often initiated in shared decision with the patient (54.9%). Only 28.8% of participants mentioned advantages regarding teleconsultations over face-to-face, compared to 39.3% for online video consultations. Moreover, 49.3% saw clear disadvantages for teleconsultations and 32.7% for video consultations. Positive factors associated with remote mental health care included when faced with transportation problems, followed by consultations primarily focusing on medication (for telephone consultations) or on more practical aspects (for video consultations). 25.0% of patients deemed conversations when being angry or sad to be feasible by telephone, and 33.0% considered these feasibly using video consultations. Conclusion Remote consultations were deemed feasible, but the positive factors did not seem to outweigh the face-to-face contacts from a patient’s perspective. Remote consultations will probably remain present in the following decades, although care must be taken when providing the possibility of remote mental health care. Supplementary Information The online version contains supplementary material available at 10.1007/s10900-022-01175-4. Keywords COVID-19 mental health patient experience remote care ==== Body pmcIntroduction With the COVID-19 pandemic, there has been an important growth in the use and implementation of remote (telephone and online video) consultations in mental health [1]. This growth is reflected in the almost four-fold increase in literature around remote mental health care between 2018 (n = 201) and 2021 (n = 749). Many studies indicated the negative effects of the pandemic on mental well-being [2–5]. In this regard, digital technologies may play a key role in the care of psychological difficulties and for providing necessary support, most specifically in a period of quarantine and social distancing. The initiation and promotion of remote (mental) health was considered one way of optimally residing patient contacts during the pandemic, but more studies on the use of remote (mental) health are deemed necessary. For many mental health care practitioners, the COVID-19 pandemic has been an opportunity towards remote care, being able to implement and evaluate remote mental health in practice. In general, implementation of online video consultations was particularly slow to start but intensified with the increasing length of the pandemic [6]. In a recent study amongst patients and practitioners, online video consultations were deemed overall well-accepted, especially for clinicians working from home [7]. Others indicated rather mixed experiences with the changes of service activity towards remote care, with patients addressing the lack of a therapeutic ‘safe space’ and concerns regarding those with limited technical options and socio-demographic inequalities [3, 8]. Other barriers mentioned by health practitioners included changing workflow routines and schedules, a lack of training, privacy considerations, increased provider and staff acceptance, and reimbursement possibilities for health care providers (6, 9–10). From a health care provider point of view, remote care was deemed satisfactory for quite some respondents (59%), of which the majority indicated being open to the further use of remote care after the pandemic [11]. While remote mental health has been deemed cost-effective (or at least as effective as face-to-face care), studies are generally of poor quality and several barriers of implementation have been noted towards generalizability of findings [12]. In addition, few patient-centered studies were available such that identifying the acceptance and difficulties associated with remote mental health from a patient perspective proves to be challenging. This study aims to describe and understand the experiences associated with several forms of remote consultations (both telephone and online video) for mental health care during the COVID-19 coronavirus pandemic with psychiatrist and psychologists, focusing in depth on the experiences of patients. Methods This study was a web-based survey, presented in Dutch and circulated online from 11 March to 12 October 2021. The study was approved by the Ethical Committee of the University Hospital Brussels (UZ Brussel), number B1432020000152. The custom-designed survey included demographic questions regarding age, gender, housing and education. It was followed by questions regarding their ongoing mental health consultations: regarding the profession of their therapist (i.e., psychologist or psychiatrist), the nature of the consultations (telephone, online video, face-to-face or combinations) and by who any remote consultations were initiated and whether this was by choice. This section was followed by 13 exploratory multiple-choice questions asking participants to compare previous offline experiences with their experiences with telephone consultations and /or online video consultations, depending on the channel(s) they had reported using. These questions were based on results of a previous study exploring patients’ attitudes using focus groups (Blancke et al., in preparation). Questions were answered on a 5-point Likert-scale, ranging from ‘highly disagree’ to ‘highly agree’. Afterwards, these 13 questions were clustered into four categories. The first and second category focused on advantages for respectively telephone and online video consultations (6 items, αtele = 0.72; αvideo = 0.77) and included statements such as ‘Video consultations made it easier for me to discuss difficult topics’. The third and fourth category focused on disadvantages for respectively telephone and video consultations (7 items, αtele = 0.85; αvideo = 0.86) and included statements such as ‘I found my therapist to be less involved’. A final multiple-choice question (with the possibility to provide multiple answers) probed for patient’s opinions regarding the potential use of online video and/or telephone consultations in a number of situations, e.g., when the patient faces transportation problems or when the patient might feel sad, angry or anxious. Statistics Descriptive statistics were reported in terms of absolute and relative frequencies for categorical outcomes, and in terms of mean (M), standard deviation (SD) and range for continuous outcomes. To compare the nature of initiation of the consultations between psychiatrists and psychologists, the chi squared test of independence was used, corrected for multiple testing using the Benjamini-Hochberg (False Discovery Rate (FDR)) correction. To interpret the general stance of the participants for both telephone consultation and online video consultation separately, two-sided one-sample t-tests were conducted, comparing category means with the value ‘3’, which corresponded with a neutral position towards that statement. To compare participants’ feelings towards possible advantages and disadvantages towards telephone and online video consultations, two-sided independent sample t-tests were conducted. To compare telephone and online video consultations for a subsample of participants who reported experiences with both, two-sided paired sample t-tests were conducted. For both analyses, the obtained p values were again corrected for multiple testing using the Benjamini-Hochberg FDR correction. Patient preferences about whether or not they approved the use of telephone or online video consultations in certain situations were described in terms of absolute and relative frequencies. For testing differences between telephone and online video consultations, the chi square test of independence was used again complemented with a correction for multiple testing (Benjamini-Hochberg). All data were analyzed using SPPS (version 26.0). Results Participants A total of 521 patients participated in the survey. Participants who did not report any experience with telephone or online video consultation (N = 142) or were not residing in Belgium (N = 4) were excluded, resulting in 375 participants to be included for data analysis (see Fig. 1). Participants were mainly female (80.0%). The mean age was 40.4 (SD 11.6, range 11–80). Of the 375 respondents, 164 (43.9%) lived with a partner, 78 (20.7%) lived with others than a partner, and 133 (35.4%) lived alone. Of respondents, 2.9% had completed primary education, 26.4% completed secondary education and 70.7% had completed higher education or university. Fig. 1 Flowchart with overview of included study participants Nature of Consultations Respectively, 202 participants (53.9%) and 308 participants (82.1%) indicated having visited the psychiatrist or the psychologist during the last year. Furthermore, 145 participants (38.7%) indicated to have visited both a psychiatrist and a psychologist during the last year. In total, 88 participants reported only having had remote consultations with a psychiatrist, 220 participants only with a psychologist, and 67 participants had remote consultations with both. Regarding psychiatric consultations that were kept by video and/or telephone (N = 155), 105 participants (67.7%) reported on their experiences regarding video-consultations, 36 participants (23.2%) reported on only telephone consultations and 14 participants (9.0%) reported on a combination of online video and telephone consultations. Regarding psychological consultations that were kept by video and/or telephone (N = 287), 241 (84.0%) consulted by means of online video, 30 (10.5%) by telephone and 16 (5.6%) had a combination of online video- and telephone consultations (see Fig. 1). The nature of consultations between psychiatrists and psychologists was statistically different (p < .001) with more use of online video consultations and less face-to-face or telephone consultations by the psychologist. Psychiatric remote consultations were initiated on request of the treating psychiatrist and psychologist without possibility of choice for 47.0% and 29.1% of respondents, respectively N = 95 and N = 89. Psychological consultations were initiated more often together with the participants’ agreement compared to psychiatrist consultations (54.9% compared to 38.6%, p = .008). Remote consultations initiated solely on the behalf of participants were present in 14.4% (consultation with a psychiatrist) and 16.0% (consultation with a psychologist) of cases. Also here, the person and manner of initiating the consultation was statistically different between psychiatrist and psychologist (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\chi }_{2}^{2}=17.701$$\end{document}, p<.001). Patient Experience To explore the patient experience, results were analyzed for the sample as a whole, irrespective of the professional (i.e., psychiatrist or psychologist) providing the services. Of all the participants, 28.8% of participants saw advantages (average score > 3) regarding telephone consultations, compared to 39.3% for remote online video consultations. Moreover, 49.3% saw clear disadvantages (average score < 3) for telephone consultations and 32.7% for online video consultations. More specifically, for telephone consultations, participants tended to disagree with potential advantages over face-to-face (M = 2.57, SD = 0.76), t(71) = -4.80, p < .001) and took a neutral stance towards potential disadvantages (M = 2.96, SD = 0.95), t(71) = − 0.32, p = .74. For online video consultations, participants again tended to disagree with potential advantages (M = 2.85, SD = 0.79), t(301) = -3.26, p < .002, but also disagreed with potential disadvantages (M = 2.58, SD = 0.91), t(301) = -8.05, p < .001. When comparing the experiences of the subsample of participants who reported experience with both telephone and video consultations (N = 30), the advantages of video consultations (M = 2.76, SD = 0.72) were considered slightly stronger compared to telephone consultations (M = 2.58, SD = 0.70), t(29) = -2.59, p = .023. No difference in disadvantages was found between online video consultations (M = 2.71, SD = 0.81) compared to telephone consultations (M = 2.93, SD = 0.87), t(29) = 2.12, p = .051. Patient Preference When participants were asked regarding the appropriate use of remote consultations, less than 10% of respondents argued that these would never be appropriate. Transportation problems were most mentioned, followed by consultations primarily focusing on medication (for telephone consultations) and for consultations primarily focusing on practical aspects (for online video consultations). Conversations where participants were angry or sad were deemed feasible to be held remotely using telephone for close to a quarter of participants, whereas over one third considered these to be feasibly using online video consultations. Introspective consultations via video were considered feasible by twice as much participants compared to consultations over the phone. A detailed overview about the observed appropriateness according to participants can be found in Table 1. Patients’ perceptions about the possible advantages and disadvantages of telephone and online video consultations are listed in Table 2. Online video consultations were more accepted in comparison to telephone consultations, as the patient did not have to move around to go to the therapist and as it gave a better insight into the home situation. Furthermore, telephone conversations made the interaction shallower, made the participant feel more isolated, made the participant feel more as if the therapist did not understand and sense the participant well, and left the participant with a less involved feeling about the therapist in comparison to online video consultations (p < .05). Table 1 Appropriate use of remote consultations, according to the personal experience of patients having used telephone consultations (N = 72) and online video consultations (N = 302) Telephone consultation Video consultation Adjusted P-value N % N % For someone having transportation problems. 55 76.4 262 86.8 0.056 For a consultation primarily focussing on medication.a 30 60.0 78 65.5 0.021 For a consultation primarily focussing on practical aspects, symptoms and events. 37 51.4 208 68.9 0.020 As preparation for a live conversation 30 41.7 100 33.1 0.228 For a conversation in which I am sad, angry or anxious. 17 23.6 109 36.1 0.070 For a conversation where I introspectively explore myself to get to know myself better. 12 16.7 104 34.4 0.020 As a first conversation. 13 18.1 60 19.9 0.727 Not at all. 7 9.7 20 6.6 0.413 a Only taking into account participants having had a psychiatric consultation either over telephone (N = 50) or over video (N = 119) Table 2 Patients’ perceptions of possible advantages and disadvantages of telephone and online video consultations Telephone consultations (N = 72) Video consultations (N = 302) Adjusted P-value M SD M SD Possible advantages … are more comfortable, as I do not have to move around to go to my therapist 3.00 1.34 3.52 1.31 0.017 … make my therapist more easily available 2.88 1.21 3.17 1.18 0.106 … get to the heart of the conversation faster, as there is less lead time for the consultation 2.63 1.08 2.77 1.07 0.392 … can give the therapist more insight into my home situation 2.39 1.14 2.74 1.02 0.038 … are easier for me because it provides less stimuli 2.32 1.15 2.46 1.19 0.398 … make it easier for me to discuss difficult topics 2.19 1.13 2.46 1.14 0.106 Possible disadvantages … make the conversations more shallow 3.18 1.38 2.57 1.35 0.013 … make me feel more isolated 3.15 1.31 2.75 1.27 0.038 … trouble me, as there is no run-up and run-down time for consultation 3.14 1.13 2.69 1.17 0.017 … make it harder to remain focused 3.07 1.14 2.89 1.31 0.392 … feel like my practitioner did not understand and sense me as well 2.92 1.30 2.45 1.25 0.018 … make me experience my therapist as less involved 2.75 1.43 2.25 1.21 0.018 … result in less silence; I felt more pressure to talk 2.54 1.07 2.44 1.06 0.487 To analyze the differences in feelings towards possible advantages and disadvantages the categorical data were transformed to numeric data where 1 = ‘highly disagree’ and 5 = ‘highly agree’ and were analyzed as such Discussion This study described specific patients’ experiences associated with remote mental health care consultations (both telephone and online video). In comparison to earlier studies addressing the health practitioners’ perspective regarding remote care as being satisfactory for approximately 59% of health practitioners [11], our study showed a more tempered acceptance towards remote consultations from a patient perspective. However, several situations were deemed positive, for which remote consultations should remain present, including when being faced with transportation issues of when discussing more practical aspects of care or medication. This was in line with earlier reports from practitioners providing them with the opportunity to work from home [7], but also addressing difficulties related to privacy considerations which made remote consultations less ‘attractive’ [6]. The results of this study must be approached with care. There may be a certain response bias as to receiving primarily the more ‘positive’ and ‘negative’ responses, leaving out the more ‘neutral’ ones. In addition, remote consultations may have been appreciated more during the COVID-19 pandemic as they would have been outside of a pandemic, primarily out of fear for contamination and a way of retaining contact for patients residing in loneliness. Also, remote consultations may have been more imposed upon the patients, leaving fewer room for choice by the patient him/herself in terms of the ongoing pandemic. Finally, patients may have been reluctant about sharing personal issues over modalities that provided little or no guarantees of privacy. Our study, however, did not focus on privacy-related disadvantages, which is an item that should be further investigated. Overall, whether the here presented results reflect an overall satisfaction surrounding remote consultation that remains present upon the ending of the pandemic, will need further evaluation in the near future. One of the challenges for remote mental health care will remain to reach the more vulnerable patient populations, including patients with limited remote access [13]. If these barriers can be countered, remote consultations become a true possibility for integration within standard care [6, 14], going rather towards a hybrid model of patient-centered care integrating both face-to-face and remote care depending on the patient’s needs. Conclusion Compared to face-to-face consultations, video consultations were better received by health-care patients compared to telephone consultations, especially when the patient was restricted to home or when the consultation aim included sharing rather straightforward information such as medication. Remote consultations were less well received and offered more barriers when there was a need for a more therapeutic consultation and when discussing emotions. In conclusion, online video consultations compared to telephone consultations performed significantly better, especially in terms of sharing emotions of anger and sadness, although it must be mentioned that the majority of patients tended to disagree with potential advantage over face-to-face contacts and approximately 10% of respondents argued that neither form of remote consultations would ever be appropriate. Electronic Supplementary Material Below is the link to the electronic supplementary material. There is no supplementarry material. Authors’ Contributions FM, TVD, NV, SB and CC initiated the project. SVL and TVD analysed the data. CC wrote the first draft. All authors contributed to the writing of the final manuscript. Funding There was no funding for this study. Data Availability The data that support the findings of this study are available from the corresponding author upon request. Code Availability N/A. Declarations Conflicts of Interest All authors declare no conflict of interest. Ethics Approval The study was approved by the Ethical Committee of the University Hospital Brussels (UZ Brussel) under number B1432020000152. Consent for Publication N/A. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Abraham A Jithesh A Doraiswamy S Al-Khawaga N Mamtani R Cheema S Telemental Health Use in the COVID-19 pandemic: a scoping review and evidence gap mapping Frontiers Psychiatry 2021 12 748069 10.3389/fpsyt.2021.748069 2. Evanoff, B. A., Strickland, J. R., Dale, A. M., et al. (2021). Work-related and personal factors Associated with Mental Well-Being during the COVID-19 response: survey of Health Care and other workers.Journal of Medical Internet Research, 22, e21366. 3. Rains LS Johnson S Barnett P Early impacts of the COVID-19 pandemic on mental health care and on people with mental health conditions: framework synthesis of international experiences and responses Social Psychiatry and Psychiatric Epidemiology 2020 56 1 13 24 10.1007/s00127-020-01924-7 32804258 4. Vanderbruggen N Matthys F Van Laere S Self-reported Alcohol, Tobacco, and Cannabis Use during COVID-19 Lockdown Measures: results from a web-based survey European Addiction Research 2020 26 6 309 315 10.1159/000510822 32961535 5. Mansfield KE Mathur R Tazare J Indirect acute effects of the COVID-19 pandemic on physical and mental health in the UK: a population-based study Lancet Digital Health 2021 3 4 217 230 10.1016/S2589-7500(21)00017-0 6. Budhwani, S., Fujioka, J. K., Chu, C., et al. (2021). Delivering Mental Health Care virtually during the COVID-19 pandemic: qualitative evaluation of provider experiences in a scaled context.JMIR Formative Research, 5(9), e30280. 7. Johns, G., Burhouse, A., Tan, J., et al. (2021). Remote mental health services: a mixed-methods survey and interview study on the use, value, benefits and challenges of a national video consulting service in NHS Wales, UK.BMJ Open, 11(9), e053014. 8. Liberati, E., Richards, N., Parker, J., et al. (2021). Remote care for mental health: qualitative study with service users, carers and staff during the COVID-19 pandemic.BMJ Open, 11(4), e049210. 9. De Beurs E Blankers M Goudriaan AE de Koning M Dekker JJM Mental health care in times of corona: experiences of professionals with eHealth Tijdschrift Psychiatrie 2021 63 4 242 249 10. De Witte NA Carlbring P Etzelmueller A Online consultations in mental healthcare during the Covid-19 outbreak: an international survey study on uptake and experiences Internet Interventions 2021 25 100405 10.1016/j.invent.2021.100405 34401365 11. Ceniti AK Abdelmoemin WR Ho K One degree of separation: a mixed-methods evaluation of canadian Mental Health Care user and provider experiences with Remote Care during COVID-19 Canadian Journal of Psychiatry 2022 67 9 712 722 10.1177/07067437211070656 34986035 12. Eze, N. D., Mateus, C., & Hashiguchi, T. C. O. (2020). Telemedicine in the OECD: an umbrella review of clinical and cost-effectiveness, patient experience and implementation.PLoS One, 15(8), e0237585. 13. Johnson S Dalton-Locke C San Juan NV Impact on mental health care and on mental health service users of the COVID-19 pandemic: a mixed methods survey of UK mental health care staff Social Psychiatry and Psychiatric Epidemiology 2021 56 1 25 37 10.1007/s00127-020-01927-4 32857218 14. Khanna A Jones GB Envisioning post-pandemic Digital Neurological, Psychiatric and Mental Health Care Frontiers Digital Health 2021 3 803315 10.3389/fdgth.2021.803315	36454479	PMC9713736	NO-CC CODE	2022-12-02 23:24:45	no	J Community Health. 2022 Dec 1;:1-6	utf-8	J Community Health	2,022	10.1007/s10900-022-01175-4	oa_other
==== Front Z Bild Forsch Z Bild Forsch Zeitschrift Fur Bildungsforschung 2190-6890 2190-6904 Springer Fachmedien Wiesbaden Wiesbaden 371 10.1007/s35834-022-00371-5 Editorial Editorial Eder Ferdinand [email protected] 1 Brauckmann-Sajkiewicz Stefan 2 Paseka Angelika 3 1 grid.7039.d 0000000110156330 Present Address: Fachbereich Erziehungswissenschaft, Paris Lodron Universität, Salzburg, Österreich 2 grid.7520.0 0000 0001 2196 3349 Present Address: Institut für Unterrichts- und Schulentwicklung, Alpen-Adria-Universität, Klagenfurt, Österreich 3 grid.9026.d 0000 0001 2287 2617 Present Address: Fakultät für Erziehungswissenschaft, Universität Hamburg, Hamburg, Deutschland 1 12 2022 15 10 11 2022 © Springer Fachmedien Wiesbaden GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcDie Beiträge für Heft 3/2022 liefern ein Abbild laufender Trends und Entwicklungen im Bildungswesen: Neue Anforderungen an Schulleitungen und Ausbildungseinrichtungen durch fachfremd eingesetzte Lehrpersonen oder Noch-Studierende, die wegen Lehrermangels bereits unterrichten; Fragen der Unterstützung von Lehrpersonen beim Einstieg in die Praxis, aber auch bei der Weiterentwicklung des Unterrichts; Herausforderungen des Systems „Schule“ vor dem Hintergrund von Schulschließungen, digitalem Lernen und Flucht aus der öffentlichen Schule ins Homeschooling, oder durch Begleiterscheinungen der „asozialen“ Nutzung „sozialer“ Medien. Das Phänomen des fachfremd erteilten Unterrichts wurde von der Schulleitungsforschung bislang eher selten behandelt. Dieser Umstand verwundert, zeigen sich doch Schulleitungen im Rahmen Neuer Steuerungskonzepte neben stark mediatisierten Fragen der Unterrichtsversorgung auch für die Gewährleistung qualitätsvollen Unterrichts sowie die Führung des pädagogischen Personals verantwortlich (Brauckmann und Eder 2019; Thillmann, Brauckmann, Herrmann und Thiel 2015). Der Beitrag von Raphaela Porsch und Fabian Gräsel („Fachfremdes Unterrichten und Schulleitungen: Einstellungen und Maßnahmen zur Unterstützung fachfremd tätiger Lehrkräfte“) greift somit auf der Basis einer mit 11 deutschen Schulleitungen an Grundschulen und weiterführenden, nichtgymnasialen Schulen durchgeführten Interviewstudie ein Forschungsdesiderat der empirischen Schulleitungsforschung auf. Ziel dieser Interviewstudie ist es, die Einstellungen von Schulleitungen sowie Maßnahmen der Personalentwicklung anhand von Interviewaussagen zu extrapolieren und überdies zu systematisieren. Die Befragungen der Schulleitungen erfolgten mithilfe eines teilstandardisierten Interviewleitfadens, der auf die Biographie der (stellvertretenden) Schulleitung, die schulischen Ausgangsbedingungen und ferner die Einstellungen und Praxis des fachfremd erteilten Unterrichts und der damit verbundenen Unterstützungsmaßnahmen fokussierte. Es zeigt sich, dass die Schulleitungen dem fachfremd erteilten Unterricht grundsätzlich kritisch gegenüberstehen, ihre anfängliche defizitorientierte Perspektive auf selbiges Phänomen im Verlauf der Interviews jedoch auch zu relativieren vermögen. Der Beitrag schließt mit weiterführenden Überlegungen zu gebotenen Anschlussarbeiten im Bereich der Schulleitungsforschung. Falk Scheidig und Monika Holmeier vergleichen in ihrem Beitrag „Unterrichten neben dem Studium“ Studierende, die bereits während des Studiums an einer Schule unterrichten, mit Studierenden, die dies nicht tun. Die Zahl der Studierenden, die bereits während des Studiums an einer Schule arbeiten, ist in den vergangenen Jahren aufgrund des Mangels an Lehrpersonal deutlich gestiegen; vor allem im Bereich der Sekundarschulen werden viele Studierende angefragt, um Lücken im System zu füllen. Auf Basis einer schriftlichen Befragung von 929 Lehramtsstudierenden konnten interessante Ergebnisse herausgearbeitet werden: Das Ausmaß dieser Unterrichtstätigkeit variiert deutlich, ebenso wie die Art der dabei gemachten Praxiserfahrungen. Studierende, die einer Unterrichtstätigkeit nachgehen, unterscheiden sich jedoch über weite Strecken kaum von jenen Studierenden, die das nicht tun. Es überrascht, dass die Beurteilung der Berufsrelevanz des Studiums und der Bedeutung der Auseinandersetzung mit wissenschaftlichem Wissen von beiden Gruppen in gleicher Weise eingeschätzt wird. Die bereits unterrichtenden Studierenden erfahren jedoch einen geringeren Kompetenzzuwachs durch das Studium, was darauf hindeutet, dass die erlebte Praxis als großer Zugewinn für die selbst eingeschätzte Kompetenz wahrgenommen wird. Studierende, die neben dem Studium nicht unterrichten, wünschen sich hingegen mehr praxisrelevante Lehrveranstaltungen und solche, die ihnen ein differenzierteres Bild der auf sie zukommenden beruflichen Herausforderungen ermöglichen. Annika Braun, Sabine Weiß, Susanne Langenohl, Clemens M. Schlegel und Ewald Kiel untersuchen in ihrem Beitrag über „Feedbackprozesse in Mentor/in-Mentee-Beziehungen im Praktikum“ die Art und Weise, wie Feedbackgespräche zwischen Praktikumslehrkräften und Lehramtsstudierenden ablaufen. Aus der Auswertung von 21 Gruppendiskussionen rekonstruieren sie als dominierenden Zugang eine auf Wertschätzung und Offenheit basierende und an einem konstruktivistischen Verständnis von Mentoring orientierte Beratungspraxis, die auf eine gemeinsame Gestaltung von Lernen, Unterrichten und Reflektieren ausgerichtet ist. Daraus erarbeiten die Autor:innen dann Impulse und Ideen für die Gestaltung von Beratungsgesprächen. Margaretha Florin, Rita Stebler, Christine Pauli und Kurt Reusser untersuchen die Bedeutung der „Selbstwirksamkeit von Lehrpersonen bei der Entwicklung einer personalisierten Lehr-Lernkultur“. Die Autorinnen verstehen darunter einen individualisierenden Zugang, der „den Lernenden durch eine optimale, partizipativ erzielte Passung zwischen Lernangeboten und individuellen Nutzungsbereitschaften einen maßgeschneiderten Kompetenzaufbau ermöglichen und zu einer umfassenderen und fortan sozial gerechteren Realisierung von Bildungspotenzialen beitragen“ will. Die Auswertung von längsschnittlich erhobenen Fragebogendaten von 188 Lehrpersonen aus 42 Schweizer Primar- und Sekundarschulen, die sich für die Teilnahme an einem einschlägigen Projekt zur Förderung personalisierten Lernens entschieden hatten, zeigte die Bedeutung der Selbstwirksamkeit für die Umsetzung des Projekts, zugleich aber auch, dass überwiegend individuelle Faktoren (Enthusiasmus, Engagement für das Projekt) sowie bisherige positive Erfahrungen für die Entstehung einer hohen Selbstwirksamkeit Ausschlag gebend waren. Covid-19 hat sich in vieler Hinsicht als Katalysator für Prozesse im Bildungswesen erwiesen, einerseits als Motor für – von manchen als längst fällig eingeschätzte – Umsetzungen von Digitalisierung, andererseits aber auch für manche als Anstoß, ihre Kinder einem als übergriffig oder chaotisch empfundenen System zu entziehen, das seine vermeintliche Entbehrlichkeit ohnehin durch Schulschließungen belegt hatte. Diese zuletzt genannte Gruppe thematisiert der Beitrag von Veronika Hofinger, Werner Reisinger und Rebecca Walter über „Homeschooling in Österreich in problematischen Kontexten“. Die Autorinnen richten den Focus auf die Tendenz, Kinder aus der Schule zu nehmen und zu Hause zu unterrichten (was in Österreich möglich und während der Covid-19 verstärkt aufgetreten ist). Während diese Tendenz früher eher mit Skepsis gegenüber einem als problematisch empfundenen Schulsystem bzw. dem Wunsch nach alternativen Lern- und Beschulungsmethoden verbunden war, diagnostizieren die Autorinnen während der Covid-19-Krise verstärkt die Nähe zu einem rechtsesoterischen Spektrum mit massiven staats- und wissenschaftsfeindlichen sowie verschwörungsideologischen Zügen und plädieren – im Interesse der Kinder – für eine stärkere staatliche Kontrolle dieses Bereichs. Im Kontext einer Zunahme digital gestützter Lernformate lässt sich der Beitrag von Uwe Maier und Christian Klotz („Misserfolge beim digitalen Lernen verhindern: ,Predictive learning analytics‘ am Beispiel einer Web-App für Grammatik und Rechtschreibung“) einordnen. Es geht um die Frage, inwieweit sich aus der Art und Weise, wie Schülerinnen ein elektronisches Tool zum Erlernen von Grammatik bearbeiten, Vorhersagen ableiten lassen, ob sie letzten Endes lernerfolgreich sein werden oder nicht. Dazu erheben sie in einem experimentellen Design von den Schülerinnen, die mit dieser App arbeiten, fortlaufend Kennwerte für die Art der Bearbeitung (z. B. Bearbeitungszeit, Ausmaß, in dem Erläuterungen oder Rückmeldungen gelesen werden, u.Ä.) und analysieren, ob bereits zu einem frühen Zeitpunkt der Nutzung Vorhersagen eines späteren (Miss‑)Erfolgs möglich sind, sodass Lehrpersonen gegebenenfalls unterstützend eingreifen könnten. Im Gegensatz zu Erfahrungen beim Einsatz elektronischer Lern-Apps im akademischen Bereich erweisen sich die Vorhersagemöglichkeiten im schulischen Bereich jedoch als weniger verlässlich. Zwei Beiträge beschäftigen sich mit dem Thema „Hatespeech“, jeweils aus unterschiedlichen Perspektiven. Cindy Ballaschk, Friederike Schule-Reichheit, Sebastian Wachs, Norman Krause, Alexander Wettstein, Julia Kansok-Dusche, Ludwig Bilz und Wilfried Schubarth erkunden das Verständnis von pädagogischem Schulpersonal über Hatespeech in ihrem Beitrag „Ist das (schon) Hatespeech?“. Dazu befragten sie in einer Interview Studie 18 Lehrpersonen und 16 Sozialpädagogen und Sozialpädagoginnen. Den Befragten fällt es nicht immer leicht, Hatespeech als solche wahrzunehmen, vor allem wenn subtile Formen genutzt werden. Abwertungen lassen sich schwerer erkennen, wenn sie in humoristischer Form auftauchen oder in einem spaßigen Spiel mit Stereotypen. Es konnten zwei Muster beim Erkennen von Hatespeech rekonstruiert werden, die davon abhängen, ob die Rolle der Adressierten eingenommen wird oder die Rolle der Ausübenden von Hatespeech. In einem zweiten Beitrag der Autor:innengruppe Lissanne Seeman-Herz, Julia Kansok-Dusche, Alexandra Dix, Sebastian Wachs, Norman Krause, Cindy Ballaschk, Friederike Schulze-Reichelt und Ludwig Bilz werden „Schulbezogene Programme zum Umgang mit Hatespeech“ mithilfe von ausgewählten Kriterien untersucht: Art der Definition des Phänomens Hatespeech, wissenschaftliche Fundierung der präsentierten Präventions- und Interventionsansätze, Verknüpfung dieser beiden Ansätze, Ausmaß der Aktivierung von teilnehmenden Schülerinnen und Schülern sowie das Vorhandensein einer Evaluation des Programms. 14 deutschsprachige Programme zur Prävention von und Intervention bei Hatespeech wurden in die Analyse einbezogen. Das Ergebnis ist jedoch ernüchternd: es gibt kaum Programme, die diesen Kriterien gerecht werden. In einem bildungstheoretisch orientierten Beitrag zu „Digitalisierung in der Schule: Vorschlag eines systematisierenden Rahmenmodells aus schulpädagogischer Perspektive“ versuchen Marcus Syring, Thorsten Bohl und Andreas Lachner die heterogenen Zugänge zum Thema Digitalisierung in einem Rahmenmodell zusammen zu fassen. Ihre Analyse fußt auf einer als „narrativer Review“ bezeichneten Zusammenschau verschiedener Ansätze und will eine Antwort auf die Frage, „welche Aspekte von Digitalisierung(sprozessen) aus einer dezidiert schulpädagogischen Perspektive in der Literatur existieren“. Digitalisierung verstehen die Autoren im Anschluss an die Literatur einerseits technologisch als Prozess, in dem digitale Medien bisherige analoge Prozesse des Unterrichtens ergänzen und erweitern, andererseits gesellschaftlich als eine Herausforderung, deren Bewältigung „Medienbildung“ erfordert. Insgesamt handelt es sich um eine verdienstvolle Synopse der unterschiedlichen Zugänge, die eine Einordnung der laufenden Entwicklungen unter einer pädagogischen Perspektive erleichtert. Direkt im Kontext von Covid-19 steht ein think piece von Dagmar Schulze Heuling und Christoph Helm: „Schulschließungen als ethische Herausforderung“. Schulschließungen sind unbestritten mit erheblichen Nachteilen und Beeinträchtigungen für die Schülerinnen verbunden, sodass sich die Frage aufdrängt, inwieweit eine solche Maßnahme überhaupt zu rechtfertigen ist. Die Autor*innen diskutieren daher vor dem Hintergrund deontischer und konsequentialistischer Zugänge, wie Schulschließungen ethisch zu bewerten sind – mit ambivalentem Ergebnis! Friedhelm Käpnik rezensiert das Buch „Begabungsüberzeugungen und ihr Einfluss auf kognitiv herausfordernden Unterricht“ von Silke Rogl. In den Nachrichten aus der ÖFEB werden aktuelle Entwicklungen innerhalb der Forschungsgesellschaft berichtet und abschließend, wie immer, die neuesten Buchpublikationen von ÖFEB-Mitgliedern vorgestellt. ==== Refs Literatur Brauckmann S Eder F Führungsforschung im Bildungsbereich: Schulleitung im Spannungsfeld erweiterter Rechte und Pflichten Zeitschrift für Bildungsforschung 2019 9 5 15 10.1007/s35834-019-00242-6 Thillmann K Brauckmann S Herrmann C Thiel F Abs H Brüsemeister T Schemmann M Wissinger J Praxis schulischer Personalentwicklung unter den Bedingungen der Neuen Steuerung Governance im Bildungssystem. Educational Governance 2015 Wiesbaden Springer VS	36471725	PMC9713738	NO-CC CODE	2022-12-02 23:23:08	no	Z Bild Forsch. 2022 Dec 1;:1-5	utf-8	Z Bild Forsch	2,022	10.1007/s35834-022-00371-5	oa_other
==== Front Datenbank Spektrum Datenbank Spektrum Datenbank-Spektrum 1618-2162 1610-1995 Springer Berlin Heidelberg Berlin/Heidelberg 430 10.1007/s13222-022-00430-1 Community Reviving the Workshop Series on Testing Database Systems – DBTest Binnig Carsten [email protected] 1 Böhm Alexander [email protected] 2 Rabl Tilmann [email protected] 3 Tözün Pınar [email protected] 4 1 Darmstadt, Germany 2 Google, ABC-Str. 19, Hamburg, Germany 3 grid.11348.3f 0000 0001 0942 1117 Hasso Plattner Institute, Uni Potsdam, Potsdam, Germany 4 grid.32190.39 0000 0004 0620 5453 IT University of Copenhagen, Copenhagen, Denmark 1 12 2022 14 © Gesellschaft für Informatik e.V. and Springer-Verlag GmbH Germany, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. With the ever increasing complexity of database systems and their pervasive use in industry, testing them has been an important issue for a long time. Recognizing this relevance, researchers and industry have started the Workshop Series on Testing Database Systems in 2008 collocated with ACM SIGMOD. Six instances of the workshop were successfully run until 2013. Five years later, in 2018, we revived the workshop in a new, biannual format. Today, the DBTest workshop consistently has high-quality submissions, expert presenters, and active participants across both academia and industry. Going forward, we plan to open the workshop up to an even more diverse audience, especially the research communities that focus on software testing and debugging in general, and not only on database systems. Keywords DBTest SIGMOD ==== Body pmcIntroduction Software testing has been an essential topic in computer systems in general. Database systems are complex software systems with large-scale adoption. They have been evolving at a fast pace following the diverse needs of emerging data-intensive applications. The correctness of their behavior and the efficiency and stability of their performance is therefore at high importance. Recognizing this importance, researchers in database community as well as practitioners from industry started the Workshop Series on Testing Database Systems in 2008 collocated with ACM SIGMOD Conference. The workshop ran very successfully every year collocated with SIGMOD from 2008 until 2013. After a five year hiatus, in 2018, we revived the workshop in a new, biannual format. Today, the DBTest workshop1 is a venue for industry and academia to come together and exchange best practices from real-world applications and latest research insights covering the broad topics of testing, debugging, and benchmarking of data-related systems. In addition, the workshop often offers stories of how an academic research prototype can grow into a full-fledged real-world product (e.g., HyPer [7, 8], DuckDB [11]) from the people who build such systems. This way, DBTest also serves as an educational venue for especially junior researchers and developers. Focus of the Workshop As for the topics to focus on during the workshop, we believe that there is the need and interest for discussing topics that are related to not only testing database management systems but also data-intensive systems in general. Specifically, emerging new technologies such as non-volatile memory impose new challenges (e.g., avoiding persistent memory leaks and partial writes), and novel system designs including FPGAs, GPUs, and RDMA call for additional attention and sophistication. Moreover, there are new classes of data-driven applications (i.e., machine learning and big-data scenarios) that need to be considered. Another dimension includes completely new system designs – such as crowdsourcing applications, where testing imposes various monetary and functional challenges (i.e., result set quality and verification), scalable machine-learning systems that combine distributed computing with modern hardware, or even cloud-based data management systems with high release cadence and extreme global reach. Finally, there is an ever-increasing popularity and proliferation of NoSQL systems. These systems are taking new approaches and design decisions (e.g., by replacing the traditional ACID guarantees with relaxed consistency models such as BASE) to increase performance and scalability. Consequentially, they require special testing efforts and rigor to ensure classical database strengths such as reliability, integrity, and performance can be successfully carried over to these novel system architectures and designs. Following these focus areas, the workshop accepts submissions and presentations on the set of topics listed below:Testing of database systems, storage services, and database applications Testing of database systems using novel hardware and software technology (non-volatile memory, hardware transactional memory, …) Testing heterogeneous systems with hardware accelerators (GPUs, FPGAs, ASICs, …) Testing distributed and big-data systems Testing machine learning systems Specific challenges of testing and quality assurance for cloud-based systems War stories and lessons learned Performance and scalability testing Testing the reliability and availability of database systems Algorithms and techniques for automatic program verification Maximizing code coverage when testing database systems and applications Generation of synthetic data for test databases Testing the effectiveness of adaptive policies and components Tools for analyzing database management systems (profilers, debuggers, …) Workload characterization with respect to performance metrics and engine components Metrics for test quality, robustness, efficiency, and effectiveness Operational aspects such as continuous integration and delivery pipelines Security and vulnerability testing In the rest of this paper, we first cover the highlights from the different instances of DBTest. Then, we summarize the plans for the future of the DBTest workshop. From 2008 to 2013 The first DBTest workshop was collocated with SIGMOD 2008 in Vancouver, Canada [5]. It was organized by Leo Giakoumakis and Donald Kossmann. DBTest 2008 had a clear focus on testing with a significant number of contributions from academia and industry. DBTest 2009, which was collocated with SIGMOD 2009 in Providence, USA, was organized by Carsten Binnig and Benoit Dageville [3]. Again, the workshop featured many contributions by academia and industry, mostly on the topic of testing, but also workload modelling and benchmarking. In 2010, DBtest was organized by Shivnath Babu and Glenn Paulley, collocated with SIGMOD in Indianapolis, USA [1]. For the first time, DBTest also included a panel with experts from industry and academia, discussing the grand challenges in database system testing. DBTest 2011, which was collocated with SIGMOD in Athens, Greece, was organized by Goetz Graefe and Kenneth Salem [6]. The 2011 instance of DBTest saw a stronger shift towards tooling and benchmarks. The trend of tooling continued in DBTest 2012, which was held in Scottsdale, USA, and organized by Eric Lo and Florian Waas [9]. DBTest 2012 introduced auto-tuning into the program and saw submissions on the topic of big data. The 2013 issue of DBTest, collocated with SIGMOD in New York, USA, was focused on data generation and tooling for database analysis [10]. DBTest 2013 was organized by Vivek Narasayya and Neoklis Polyzotis. DBTest 2013 was the last issue after a series of six yearly workshops. From 2018 to 2022 After four years of absence, DBTest was successfully revived at SIGMOD 2018 in Houston, USA, with over 40 participants, 15 paper submissions (out of which 8 were accepted), and three industry sponsors [2]. DBTest 2018 was organized by Alexander Böhm and Tilmann Rabl as chairs together with Carsten Binnig who was involved in the early iterations of the workshop2. In the reincarnation of 2018, there was a lot of positive feedback and we were encouraged by the audience to re-establish DBTest in a yearly cadence. Hence, we decided to only run with a biannual cadence to prevent from running out of topics and high-quality submissions. DBTest 2020 was then organized by Pınar Tözün and Alexander Böhm. With the global challenges caused by the COVID-19 pandemic, both SIGMOD and DBTest moved to an online format in 2020 [14]. Despite these challenges, the interest in the DBTest workshop increased further, with now more than 150 registrations, and over 70 online participants during sessions. With seven accepted papers, three keynotes and four industry sponsors and a diverse set of topics ranging from debugging query compilers, to automated performance testing, to correctness tests for big-data systems, the workshop was well received by the audience. The last instance of DBTest took place in hybrid mode co-located with SIGMOD 2022 conference in Philadelphia, USA and was organized by Manuel Rigger and Pınar Tözün [13]. The program consisted of one panel, two keynotes, and four paper presentations with two industry sponsors. It covered topics ranging from software testing practices in large-scale projects, automatic bug finding, benchmark generation for systems with different characteristics. 15–35 people participated in-person during sessions, while 15–25 participated online. The sessions with most participation was, as in previous years, the keynote sessions. Both the in-person and virtual audience actively participated in all Q&A sessions highlighting the overall interest in the workshop topics. One of the remarkable aspects of each DBTest instance has been the high number of industry participants and submissions creating an interesting mix of both academic work and industry perspectives. The workshop has always created interesting lively discussions among the academic and industry participants. Such exchanges enrich DBTest to also be an educational platform for everyone when it comes to building realistic and practical benchmarking and testings infrastructures. Looking into the Future DBTest is a workshop at the intersection of database and software engineering research. Therefore, we are currently reaching out to the software engineering community to see if an alternating collocation with a software engineering conference equivalent to SIGMOD would be sensible and possible. Techniques like fuzzing have been a topic in DBTest from the start (e.g., Garcia [4]) and continue to be relevant (e.g., Rehman et al. [12]). These have their roots in the software engineering community and debugging and testing database systems is a relevant application. Conclusion In the article, we briefly summarized the history of the International Workshop on Database Testing (DBTest), which currently is collocated with SIGMOD in a biyearly cadence. DBTest is a venue for research in testing, debugging, and benchmarking of database systems and related technologies. All papers are available through the ACM digital library and listed in DBLP3. Declarations Conflict of interest The authors do not have any financial or non-financial interests that are directly or indirectly related to the work submitted for publication. 1 https://dbtest.io/. 2 Today, Alexander Böhm, Tilmann Rabl and Carsten Binnig form the steering committee of the workshop. 3 DBLP page – International Workshop on Testing Database Systems – https://dblp.uni-trier.de/db/conf/dbtest-ws/index.html. ==== Refs References 1. Babu S Paulley GN Proceedings of the third international workshop on testing database systems 2010 New York ACM 2. Böhm A Rabl T Proceedings of the 7th international workshop on testing database systems 2018 New York ACM 3. Dageville B Binnig C Proceedings of the 2nd international workshop on testing database systems 2009 New York ACM 4. Garcia R Dageville B Binnig C Case study: experiences on SQL language fuzz testing Proceedings of the 2nd International Workshop on Testing Database Systems 2009 New York ACM 5. Giakoumakis L Kossmann D Proceedings of the 1st international workshop on testing database systems 2008 New York ACM 6. Graefe G Salem K Proceedings of the fourth international workshop on testing database systems 2011 New York ACM 7. Kemper A Neumann T Funke F Leis V Mühe H Hyper: adapting columnar main-memory data management for transactional AND query processing IEEE Data Eng Bull 2012 35 1 46 51 8. Kersten T Neumann T Tözün P Böhm A On another level: how to debug compiling query engines Proceedings of the 8th international workshop on testing database systems 2020 New York ACM 1 2 9. Lo E Waas F Proceedings of the fifth international workshop on testing database systems 2012 New York ACM 10. Narasayya VR Polyzotis N Proceedings of the sixth international workshop on testing database systems 2013 New York, NY, USA ACM 11. Raasveldt M Mühleisen H Boncz PA Manegold S Ailamaki A Deshpande A Kraska T Duckdb: an embeddable analytical database Proceedings of the 2019 International Conference on Management of Data 2019 New York ACM 1981 1984 12. Rehman MS Elmore AJ Rigger M Tözün P Fuzzydata: a scalable workload generator for testing dataframe workflow systems DBTest@SIGMOD ’22: Proceedings of the 9th International Workshop of Testing Database Systems 2022 New York ACM 17 24 13. Rigger M Tözün P DBtest@SIGMOD ’22: proceedings of the 9th international workshop of testing database systems 2022 New York ACM 14. Tözün P Böhm A Proceedings of the 8th international workshop on testing database systems 2020 New York ACM	36471748	PMC9713739	NO-CC CODE	2022-12-02 23:23:08	no	Datenbank Spektrum. 2022 Dec 1;:1-4	utf-8	Datenbank Spektrum	2,022	10.1007/s13222-022-00430-1	oa_other
==== Front Z Gerontol Geriatr Z Gerontol Geriatr Zeitschrift Fur Gerontologie Und Geriatrie 0948-6704 1435-1269 Springer Medizin Heidelberg 36454314 2135 10.1007/s00391-022-02135-2 Case Reports Depression as a major component of a gait disorder—Successful multimodal treatment including electroconvulsive therapy A case report Depression als Hauptkomponente einer Gangstörung – Erfolgreiche multimodale Behandlung inklusive ElektrokonvulsionstherapieEin Fallbericht Hobert Markus A. [email protected] 12 Bruhn Daniel 1 Koch Jakob 1 Studt Simone 1 1 grid.9764.c 0000 0001 2153 9986 Zentrum für Integrative Psychiatrie Campus Kiel, Christian-Albrechts-University zu Kiel, Kiel, Germany 2 grid.9764.c 0000 0001 2153 9986 UKSH Campus Kiel, Klinik für Neurologie, Christian-Albrechts-University zu Kiel, Arnold-Heller-Str. 3, 24105 Kiel, Germany 1 12 2022 15 26 4 2022 29 9 2022 © The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. This case report describes an 82-year-old patient who was treated in a gerontological psychiatric ward due to a multifactorial gait disorder with falls. The main component of the gait disorder was depression, which was accompanied by a pronounced fear of falling. Other factors were polyneuropathy, gonarthrosis and an exercise deficit after previous inactivity. An important part of the multimodal treatment was electroconvulsive therapy (ECT). A total of nine sessions of ECT in right unilateral stimulation were conducted. The treatment resulted in a significant improvement of the depression and the gait disorder, which was impressively reflected in the geriatric assessment. The presented case shows that depression and fear of falling should not be underestimated as (main) components of a gait disorder. Here, a multimodal treatment including treatment of the depression by ECT was successful. Supplementary Information The online version of this article (10.1007/s00391-022-02135-2) contains supplementary material, which is available to authorized users. Dieser Fallbericht beschreibt einen 82-jährigen Patienten, der aufgrund einer multifaktoriellen Gangstörung mit Stürzen auf einer gerontopsychiatrischen Station behandelt wurde. Die Hauptkomponente der Gangstörung war eine Depression, die mit einer ausgeprägten Sturzangst einherging. Die weiteren Anteile waren eine Polyneuropathie, eine Gonarthrose und ein Trainingsdefizit im Rahmen einer vorhergegangenen Inaktivität. Die Behandlung erfolgte in einem multimodalen Therapiekonzept. Ein wichtiger Teil der Behandlung war die Elektrokonvulsionstherapie (EKT). Es erfolgten insgesamt neun Sitzungen der EKT in rechts unilateraler Stimulation. Durch die Behandlung zeigten sich die Depression und die Gangstörung deutlich gebessert, was sich eindrücklich im geriatrischen Assessment widerspiegelte. Der dargestellte Fall zeigte, dass Depressionen und Sturzangst als (Haupt‑)Komponenten einer Gangstörung nicht unterschätzt werden sollten und, dass eine multimodale Behandlung mit Behandlung der Depression durch die EKT in diesem Fall zum Erfolg führte. Keywords Walking Falls Geriatric assessment Electroconvulsive therapy Fear of falling Schlüsselwörter Gehen Stürze Geriatrisches Assessment Elektrokonvulsionstherapie Sturzangst ==== Body pmcMethods Description of a case report and discussion considering psychiatric and geriatric perspectives. Anamnesis An 82-year-old male patient was brought to the interdisciplinary emergency room after two falls. After exclusion of severe trauma sequelae, a gait disorder and depression became apparent. The patient was transferred to a gerontological psychiatric ward of the Department for Psychiatry of the University of Kiel. The patient reported that he had been feeling lonely and increasingly socially isolated since the death of his wife. He suspected that his inactivity had led to his gait insecurity and blames himself for it. Due to the falls, he had been very insecure and described his biggest worry as not being able to live in his house anymore but having to go to a nursing home. He described himself as joyless and listless and expressed a wish to be able to care for himself again as he used to. The medication consisted of venlafaxine sustained release 150 mg and olanzapine 5 mg per day. Psychiatric history The patient suffered from recurrent depressive episodes practically all his life. During his professional training the patient had developed obsessive thoughts for the first time. The patient had been in inpatient psychiatric treatment several times and had had six series of treatment with electroconvulsive therapy (ECT). These took place at the ages of 72, 73, 74, 75, 77 and 78 years. The most recent series of ECT was about 3 ½ years ago. In total, 56 ECT sessions (6–10 per series) were carried out cumulatively. Details are given in Table 1 of the web appendix. Diagnostic findings Mental status examination using the Manual for the Assessment and Documentation of Psychopathology in Psychiatry (AMDP system) Alert and clear, fully oriented, concentration and retentiveness moderately disturbed, short-term memory slightly disturbed, long-term memory not disturbed, no confabulation. Sleep undisturbed. Formal thinking slowed down, circumstantial, preoccupied, constricted. Thought content undisturbed. No disorders of the self. No perceptual disturbance. Depressed mood. Affect sad, blunted, perplexed, anxious. Fears of doing something wrong, of having to go to a nursing home. Fear of falling. Obsessive thoughts and compulsive actions, e.g. regarding toilet procedures, impoverished drive. Feeling of illness and insight into illness present. Reduced appetite with weight loss. Unobtrusive appearance, polite and friendly in contact. No endangerment to self and others. Physical examination Reduced general and slightly underweight nutritional status (53 kg, 168 cm, BMI 18.8 kg/m2). Generalized muscular atrophy without paresis. Unsteady gait with slightly bent knee joints and with phobic component. Step length symmetrically reduced, step width fluctuating. Turning with slightly increased number of steps and too small step width. Romberg test unsteady with undirected falling tendency. Pallhypesthesia (1–2/8) of the legs, ankle jerk reflexes extinguished. Genua vara with pain in the right knee joint. Further general medical and neurological examinations were unremarkable. Diagnostic investigations The cranial CT scan revealed no trauma sequelae. It showed a generalised brain atrophy and a mild microangiopathy. ECG and EEG were unremarkable. Comprehensive geriatric assessment In the Geriatrie-Check, the patient scored 1 point in part A and 2 points in part B and was classified as a probable geriatric patient [1, 2]. In the short physical performance battery (SPPB), the patient achieved 7 points as an expression of the risk of falling. In the DemTect the patient scored 6/18 and in the mini-mental status test 26/30 points. In the Barthel index, the patient scored 75/100 points. The falls efficacy scale international (FES-I) revealed 45/64 as an expression of a pronounced fear of falling [3]. In the visual analogue scale of the EQ5D (standardised measure of health-related quality of life), the patient rated his quality of life with 20%. In the geriatric depression scale (GDS), the patient scored 9/15 points indicating mild to moderate depression. The patient was not able to answer the Beck’s depression inventory (BDI) II due to obsessive thoughts to give a wrong answer. The first assessment of the BDI II was after the third ECT with 10 points, reflecting a normal score. The course of the results of the geriatric assessment is shown in Fig. 1.Fig. 1 Overview of course and therapy effects of treatment in geriatric assessment. Assessment of depressiveness with Beck’s depression inventory (BDI) and geriatric depression scale (GDS), fear of falling with falls efficacy scale (FES‑I), mobility with short physical performance battery (SPPB) and quality of life with EQ5D. The blue arrows indicate the individual treatments using electroconvulsive therapy (ECT) Clinical course and therapy At the beginning of the inpatient stay, the patient appeared very anxious, was ruminating, and very constricted to his gait disorder. Compulsive actions also occurred, especially in personal hygiene. He reported that his own rules would protect him from doing something wrong. As a result, he repeatedly found himself in situations that made him very insecure when something deviated from the usual pattern. In the activities of daily living, the patient demanded support especially in personal hygiene but could usually be encouraged to carry this on his own. He stayed mainly in his room at the beginning and reported a great fear of falling. He used a walker when walking. We classified the gait disorder as multifactorial and planned further treatment accordingly. Besides the pre-existing polyneuropathy, a known gonarthrosis on the right side and an exercise deficit due to the previous inactivity, fear of falling and depression were the main components. The patient explicitly wanted ECT treatment because ECTs had shown good effects in the past and nine right unilateral ECTs were performed with a stimulation intensity of 60–110%. Anesthesia was performed with propofol and succinylcholine. Convulsion durations of up to 55 s were achieved. The detailed parameters of ECT are listed in Table 1. Initially, two treatments were given per week, after the fifth ECT one per week. After the third ECT, an effect could be observed. After the fifth ECT, also the patient noticed an improvement. The improvement was also reflected in the geriatric assessment (see Fig. 1).Table 1 Overview of the parameters of the electroconvulsive therapy sessions Nr. Position of the stimulation electrodes Dose of propofol (in mg) Doss of succinylcholine (in mg) Energy set (in %) Stimulation strength (in mC) Seizure duration in EEG (in s) BIS 1 Right unilateral 90/30 50 60/80 299.7/400.7 13/20 32 2 Right unilateral 90 80 80 400.1 34 53 3 Right unilateral 120 50 90 450.4 52 32 4 Right unilateral 140 60 90 449.9 55 33 5 Right unilateral 100 60 90 450.0 46 30 6 Right unilateral 100 60 90 449.9 41 31 7 Right unilateral 120 60 90/110 554.6 20/13 36/41 8 Right unilateral 100 60 110 551.6 54 54 9 Right unilateral 100 60 110 553.3 40 40 BIS Bispectral Index, EEG Electroencephalography Before ECT was performed, the medication venlafaxine sustained release was reduced to 75 mg per day, olanzapine was taken unchanged. After the last ECT, venlafaxine sustained release was increased again to the original dose of 150 mg per day. The patient regularly participated in group therapies for occupational therapy, physiotherapy and music therapy. He received also individual physiotherapy with the aim of improving mobility and preventing falls. In addition to gait training, he received training on the ergometer to improve leg strength, and stair training. In weekly individual therapy sessions, the cycle of “falls → fear of falling → inactivity → lack of training → new falls” was worked out and behavior therapy with elements from acceptance and commitment therapy (ACT) and exposure exercises were carried out. The patient received high-calorie drinks and gained weight from 53 to 59 kg during the inpatient stay. The treatment resulted in a significant improvement in mobility, depression, anxiety, and compulsions. Three successful stress tests at home were carried out, so that the patient could be discharged home after 10 weeks of inpatient treatment. At discharge, the patient was able to walk safely without a walker. Further treatment The further treatment after the inpatient stay was carried out with medication and psychotherapy in the psychiatric outpatient department of the clinic. No maintenance ECT was considered because the patient had been stable for 3.5 years after the last ECT series and because the local structural conditions during the COVID-19 pandemic did not permit this. Diagnoses and ICD-10 classification Psychiatric diagnoses. Recurrent depressive disorder, current episode severe without psychotic symptoms F33.2. Predominantly obsessional thoughts or ruminations F42.0. Geriatric diagnoses. Multifactorial gait disorder consisting of:Gait disorder in the context of depression and fear of falling R26.8. Right gonarthrosis with genu varum M17.1. Polyneuropathy, unspecified G62.9. Unspecified protein-energy malnutrition E46. Sarcopenia M62.50. Discussion In geriatric patients, gait and balance disorders are very common and often multifactorial. In the case presented here, the leading contribution of depression and anxiety is particularly impressive. In a recently published model, important factors in the relationship between fear of falling and falls are discussed. Experienced and observed falls lead to fear of falling, which in combination with fall efficacy leads to avoidance behavior [4]. In this model, depression influences both fear of falling and fall efficacy, but is also directly associated with falls. Avoidance behavior (of situations that can lead to falls) plays a central role, as it leads to inactivity, lack of exercise and muscle atrophy. These factors are themselves risk factors for falls. In the described case the multilayered treatment led to success. The depression was mainly treated by ECT. As the depression improved, so did the anxiety, compulsions, gait and balance disturbance. Individual therapies included exposure exercises and walking. In physiotherapy, gait and stair training and improvement of leg strength took place. Activation took place throughout the entire therapy concept. In the geriatric assessment, the improvement of depression, gait and balance disturbance, fear of falling and resulting quality of life is reflected (see Fig. 1). In the literature, the effect of ECT on gait and balance disorders is described only in few studies. In one study, a gait and balance assessment was carried out after ECT. The aim was to investigate the association of falls as a side effect after ECT. No (short-term) deterioration of gait and balance in the assessment was found [5]. A study with wearable sensors showed an improvement of mobility after ECT [6]. Studies analyzing quantitative parameters to describe gait quality (i.e. with an instrumented assessment [7]) after ECT have not yet been published to our knowledge. In principle, it can be assumed that the effect of ECT is not directly on the gait disorder but is a consequence of the improvement of the depression. Under effective drug therapy, the same result would probably have been achieved, but possibly later [8]. In the case described here, treatment with ECT was chosen for various reasons without first trying another medication: treatment with ECT was wanted by the patient. In the past, ECT had shown a faster and better effect than treatment with tranylcypromine, olanzapine and flupentixol. From a geriatric point of view, a rapid improvement of the depression is preferred to treat the main component of the gait disorder and to break the cycle of immobilization, muscle atrophy, worsening of the gait disorder and falls. This patient showed a relevant improvement after the third ECT, i.e. in the fourth week of the inpatient stay. ECT is an important therapy option in old age for therapy-refractory depression. The response rate is even somewhat higher compared to younger patients [9]. It is discussed that older age may increase the risk of intervention due to somatic, especially cardiovascular, disease [12, 14]. Cognitive side effects of ECT are discussed [10]. In some cases, an improvement in cognition after ECT, at least in the medium term, is described [11, 12]. An increased risk of dementia due to ECT has not been confirmed [13]. In the case described, no cognitive side effects occurred, and cognition improved (DemTect 6/18 points before the first ECT and 12/18 points after the last ECT) due to the improvement of depression. Practical conclusion The case presented shows that depression and anxiety should not be underestimated as (main) components of a gait disorder. Multimodal treatment, including treatment of the depression by ECT, led to success here. The use of the geriatric assessment of motor function and fear of falling is also useful in the gerontological psychiatry. Effects of therapy can also be reflected in the geriatric assessment, as described in the case presented. Supplementary Information Appendix Table 1: Overview of all ECT series Declarations Conflict of interest M.A. Hobert, D. Bruhn, J. Koch and S. Studt declare that they have no competing interests. The patient provided consent to publish his medical history as a case report. A written declaration of consent is available. Scan QR code & read article online ==== Refs References 1. Gerhard T Mayer K Braisch U Validierung des Geriatrie-Checks zur Identifikation geriatrischer Patienten in der Notaufnahme Z Gerontol Geriatr 2021 54 106 112 10.1007/s00391-020-01699-1 32112273 2. Hobert MA Bernhard FP Bettecken K Validierung des Geriatrie-Checks in einer Kohorte von stationären neurologischen Patienten Z Gerontol Geriatr 2019 52 172 178 10.1007/s00391-018-1441-5 30206742 3. Dias N Kempen GI Todd CJ The German version of the Falls Efficacy Scale-International Version (FES-I) Z Gerontol Geriatr 2006 39 297 300 10.1007/s00391-006-0400-8 16900450 4. Peeters G Bennett M Donoghue OA Understanding the aetiology of fear of falling from the perspective of a fear-avoidance model—A narrative review Clin Psychol Rev 2020 79 101862 10.1016/j.cpr.2020.101862 32442854 5. Plakiotis C Barson F Vengadasalam B Balance and gait in older electroconvulsive therapy recipients: a pilot study Neuropsychiatr Dis Treat 2013 9 805 812 10.2147/NDT.S42628 23766650 6. Winkler D Pjrek E Lanzenberger R Actigraphy in patients with treatment-resistant depression undergoing electroconvulsive therapy J Psychiatr Res 2014 57 96 100 10.1016/j.jpsychires.2014.06.006 24998016 7. Hobert MA Jamour M Assessment von Mobilität – geriatrisches Assessment zur Erfassung lokomotorischer Mobilitätseinschränkungen und Perspektiven der Instrumentierung Z Gerontol Geriatr 2022 10.1007/s00391-022-02040-8 35181808 8. Gálvez V Ho K-A Alonzo A Neuromodulation Therapies for Geriatric Depression Curr Psychiatry Rep 2015 17 59 10.1007/s11920-015-0592-y 25995098 9. Haq AU Sitzmann AF Goldman ML Response of depression to electroconvulsive therapy: a meta-analysis of clinical predictors J Clin Psychiatry 2015 76 1374 1384 10.4088/JCP.14r09528 26528644 10. Gardner BK O’Connor DW A review of the cognitive effects of electroconvulsive therapy in older adults J Ect 2008 24 68 80 10.1097/YCT.0b013e318165c7b0 18379338 11. Verwijk E Comijs HC Kok RM Short- and long-term neurocognitive functioning after electroconvulsive therapy in depressed elderly: a prospective naturalistic study Int Psychogeriatrics 2014 26 315 324 10.1017/S1041610213001932 12. Dominiak M Antosik-Wójcińska AZ Wojnar M Mierzejewski P Electroconvulsive therapy and age: effectiveness, safety and tolerability in the treatment of major depression among patients under and over 65 years of age Pharm (basel) 2021 10.3390/ph14060582 13. Osler M Rozing MP Christensen GT Electroconvulsive therapy and risk of dementia in patients with affective disorders: a cohort study Lancet Psychiatry 2018 5 348 356 10.1016/S2215-0366(18)30056-7 29523431 14. Cattan RA Barry PP Mead G Electroconvulsive therapy in octogenarians J Am Geriatr Soc 1990 38 753 758 10.1111/j.1532-5415.1990.tb01465.x 2370395	36454314	PMC9713740	NO-CC CODE	2022-12-02 23:24:43	no	Z Gerontol Geriatr. 2022 Dec 1;:1-5	utf-8	Z Gerontol Geriatr	2,022	10.1007/s00391-022-02135-2	oa_other
==== Front Ophthalmologie Ophthalmologie Die Ophthalmologie 2731-720X 2731-7218 Springer Medizin Heidelberg 36454264 1759 10.1007/s00347-022-01759-4 Leitlinien, Stellungnahmen und Empfehlungen Empfehlungen bei progredienter Myopie im Kindes- und Jugendalter. Stellungnahme von DOG, BVA und der Bielschowsky Gesellschaft für Schielforschung und Neuroophthalmologie Stand Juni 2022 Recommendations for progressive myopia in childhood and adolescence. Statement of the DOG, BVA and the Bielschowsky Society for Strabismus Research and NeuroophthalmologyStatus June 2022 Deutsche Ophthalmologische Gesellschaft (DOG)[email protected] 1 Bielschowsky Gesellschaft für Schielforschung und Neuroophthalmologie2 Berufsverband der Augenärzte Deutschlands e. V. (BVA)Lagrèze Wolf Bertram Bernd Ehrt Oliver Friedburg Dieter Reck Barbara Schaeffel Frank Schittkowski Michael Ziemssen Focke 3 1 grid.470768.a 0000 0001 0532 5937 Deutsche Ophthalmologische Gesellschaft, Platenstr. 1, 80336 München, Deutschland 2 Bielschowsky Gesellschaft für Schielforschung und Neuroophthalmologie e. V., Wiesbaden, Deutschland 3 Berufsverband der Augenärzte Deutschlands e. V., Düsseldorf, Deutschland 1 12 2022 19 19 10 2022 © The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Seit der letzten und gleichzeitig ersten Stellungnahme der deutschen ophthalmologischen Fachgesellschaften zu den Möglichkeiten der Minderung von Myopieprogression im Kindes- und Jugendalter haben sich in der klinischen Forschung viele neue Details und Aspekte ergeben. Die nun vorliegende, zweite Stellungnahme aktualisiert das bisherige Dokuments und konkretisiert die Empfehlungen sowohl zum Seh- und Leseverhalten als auch zu den pharmakologischen und optischen Therapieoptionen, die in der Zwischenzeit sowohl verfeinert als auch neu entwickelt wurden. Since the last and at the same time first statement of the German ophthalmological societies on the possibilities of reducing myopia progression in childhood and adolescence, many new details and aspects have emerged in clinical research. This second statement updates the previous document and specifies the recommendations on visual and reading behavior as well as on pharmacological and optical therapy options, which have been both refined and newly developed in the meantime. Schlüsselwörter Kinder Jugendliche Myopie Risikofaktor Atropin Kontaktlinsen Multisegmentgläser Keywords Children Adolescents Myopia Risk factor Atropine Contact lenses Multisegment spectacles ==== Body pmc1. Präambel Die folgenden Empfehlungen beziehen sich auf die sog. „Schulmyopie“, die häufigste Form der Kurzsichtigkeit, die ab dem 6. Lebensjahr auftritt und bis zum späten Teenageralter progredient ist. Die Empfehlungen gelten NICHT für die seltenen Sonderformen der Myopie z. B. aufgrund von Syndromen, Linsenerkrankungen, genetischen Varianten oder auf eine Myopie im Vorschulalter, da für diese Fälle keine Studien zur Progressionsminderung vorliegen. 2. Epidemiologie und Folgen der Myopie Weltweit hat die Myopie insgesamt über die letzten Dekaden zugenommen. Für eine differenzierte Bewertung ist es sinnvoll, die regionalen Unterschiede und verwendeten Definitionen zu berücksichtigen. Insbesondere aus asiatischen Ländern wird berichtet, dass im jüngeren Teil der Bevölkerung die Raten der Kurzsichtigkeit (geringer und hoher) zugenommen haben [1–3]. Bereits im jungen Alter ist eine deutliche Zunahme über die letzten 40 Jahre zu sehen (Prävalenz ab −0,25 dpt 7‑Jährige: 5 % auf 25 %, 12-Jährige: 31 % auf 77 %; Prävalenz ab −6 dpt 12-Jährige: 1,4 % auf 4,3 %) [3]. In der Altersgruppe zwischen 25 und 29 Jahren wurde in Europa eine Myopieprävalenz (ab −0,75 dpt) von knapp unter 50 % berichtet [4]. Aktuell gibt es keine sicheren Belege dafür, dass der Anteil kurzsichtiger Menschen in den letzten ca. 20 Jahren in Deutschland zugenommen hat [5, 6]. In der KIGGS-Studie lag die Prävalenz der Myopie im Alter von 3 bis 17 Jahren (basierend auf Angabe der Eltern) bei 13,3 % und zeigte in den Erhebungszeiträumen (2003 bis 2006 vs. 2014 bis 2017) keinen Unterschied [7]. In einer Leipziger Stichprobe wurde eine Prävalenz von 10,8 % (ab −0,75 dpt, ohne Zykloplegie gemessen) gefunden, wobei der Anteil von 3,5 % (6 Jahre) auf 27 % (16 Jahre) zunahm [8]. Basierend auf Daten der Rotterdam Eye Study wurde aus der Zunahme der mittleren Achslänge in Abhängigkeit des Geburtsjahres eine Zunahme des kumulativen Lebenszeitrisikos einer Sehbehinderung extrapoliert [9]: Myopie von weniger als −6 dpt birgt kein relevantes Risiko, bei −6 bis −10 dpt ist ab dem 65. Lebensjahr eine geringe Risikozunahme zu beobachten. Myope zwischen −10 und −15 dpt zeigen ab der Mitte der sechsten Dekade eine eindeutige Risikozunahme. Sehr deutlich steigt das Risiko bereits ab 50 Jahren Lebensalter an, wenn jemand kurzsichtiger als −15 dpt ist. In diesem Fall beträgt das Lebenszeitrisiko für myopiebedingte Sehbehinderung fast 80 %. Bereits 1970 wiesen Curtin und Karlin darauf hin, dass sekundäre Fundusveränderungen bereits ab einer mittleren Myopie beobachtet werden [10]. Curtin beschrieb das erhöhte Risiko einer Netzhautablösung (geringe Myopie: 4 ×, hohe Myopie: 22 ×). Registerdaten zufolge ist das Risiko einer Netzhautablösung mit einer größeren Achslänge insbesondere für Patienten mit Pseudophakie im berufstätigen Alter relevant [11]. In der Gutenberg Health Study nahm die Odds Ratio (OR) für eine rhegmatogene Ablösung mit jeder weiteren Dioptrie um 1,3 zu [12]. Das Risiko einer myopen Makuladegeneration steigt mit Ausprägung der Myopie. Die OR steigt von niedriger Myopie (−1 bis −3 dpt) mit 14, über die mittlere Myopie (−3 bis −6 dpt) mit 73 auf 845 bei hoher Myopie (über −6 dpt) [3]. Auch das Risiko für Glaukom und Strabismus nimmt mit zunehmender Myopie zu. Fazit. Myopie hat in erster Linie in Asien zugenommen. In den letzten knapp 20 Jahren ist die Prävalenz der Myopie im deutschsprachigen Raum nicht nennenswert gestiegen. Das Risiko von Sekundärerkrankungen mit zunehmender Myopie ist gut dokumentiert. Die Gefahr einer Erkrankung mit bleibender Sehbehinderung steigt spätestens ab einer Schwelle von −6 dpt deutlich an. Patienten mit einer hohen Myopie sollten über Risiken, Warnsymptome einer Makuladegeneration und Netzhautablösung und die Notwendigkeit von Kontrollen des zentralen und peripheren Augenhintergrunds bei entsprechenden Beschwerden (s. Leitlinie Risikofaktoren und Prophylaxe der rhegmatogenen Netzhautablösung bei Erwachsenen, www.awmf.org/leitlinien/detail/ll/045-025.html) informiert werden. 3. Risikofaktoren für Myopie Aus vielen Studien ist zwar bekannt, dass die elterliche Refraktion einen deutlichen Hinweis auf die spätere Refraktionsentwicklung des Kindes gibt [13, 14]. In einer US-amerikanischen Studie betrug das Myopierisiko bei Emmetropie der Eltern 10 %, bei Myopie eines Elternteils 30 und 60 %, wenn beide Eltern myop waren [3]. Verhalten und Umweltfaktoren haben aber einen noch größeren Einfluss auf die Entwicklung der Myopie als die genetische Veranlagung [7, 15]. Die Zeit der Aktivität in der Nähe und der Leseabstand korrelierten mit Myopie und ihrer Progression. Körperliche Aktivität war mit einer geringeren Ausprägung der Kurzsichtigkeit assoziiert, während Schule und Ausbildungsdauer mit einer ausgeprägten Zunahme zusammenhingen. Mit jeder Stunde Naharbeit pro Woche wurde eine Zunahme des Myopierisikos um 2 % gesehen [3]. Der Aufenthalt im Freien von mindestens 2 h/Tag war mit einer Abnahme des Myopierisikos und einer langsameren Progression um 0,13 dpt/Jahr verbunden [3]. Der Einfluss der Beleuchtung stellt sich auch in der saisonal stärkeren Progression im Winter dar und unterstreicht die Bedeutung von Aktivitäten im Freien [16]. In einigen Ländern wurde eine Zunahme der Myopie während der COVID-19-Pandemie beobachtet [17, 18]. Fazit. Die günstigen Auswirkungen von Aufenthaltszeit im Freien und die gegenteiligen Effekte von Naharbeit und kurzer Lesedistanz wurden eindeutig belegt. Dem sollte in der Ausgestaltung des Alltags und Verhaltensempfehlungen hohe Priorität eingeräumt werden. 4. Beratung zu Verhalten und präventiven Maßnahmen Grundsätzlich ist eine gezielte Information sinnvoll, welche Verhaltensmaßnahmen zum Auftreten einer Myopie und zur Abnahme einer Progression beitragen können:Lichtexposition Eine ausreichende Aufenthaltszeit im Freien zeigte schon mit relativ geringen Lichtintensitäten (1000 lx) positive Auswirkungen [19]. Die tägliche Exposition sollte besonders auch im Winter beachtet werden bzw. die Aufenthaltsbereiche der Kinder in Schule und zu Hause einschließen. Die Empfehlung von 2 h täglicher Aufenthaltszeit im Freien ist sinnvoll. In Taiwan hat der zwangsweise Aufenthalt im Freien während der Schulpause schon zu einer Abnahme der Myopieprävalenz geführt. Nahsicht In Untersuchungen stellte sich die kontinuierliche Beschäftigung in der Nähe wie z. B. beim Lesen als relevanter Einfluss dar [20]. Obwohl zuverlässige Daten zu Bildschirmzeit oder verwendeten Abständen für große Kohorten fehlen, sollte neben Abständen auch an regelmäßige Pausenzeiten erinnert werden. Möglicherweise handelt es sich bei der akkommodationsassoziierten Progression um einen Mechanismus, der insbesondere bei einer bereits bestehenden Myopie zum Tragen kommt [21]. Lesezeiten von über 30 min bei weniger als 30 cm Leseabstand sollten durch 10 min Blick in die Ferne unterbrochen werden. Für die Maßnahmen und Verhaltensempfehlungen könnte sich eine frühe Berücksichtigung ab dem Kindergarten auszahlen [22]. Der Nachweis eines Nutzens präventiver Maßnahmen bezieht sich daher v. a. auf Kinder, die im Schulalter eine Zunahme der Myopie zeigen [23]. Nach wie vor reichen die Daten nicht aus, um – abgesehen von Lichtexposition und Reduzierung von Nahsichtaktivitäten – eine generelle Prävention, d. h. z. B. die Therapie emmetroper Kinder zu empfehlen. 5. Praktisches Vorgehen zum Bestimmen der Progression Die Refraktionsbestimmung sollte speziell bei jüngeren Kindern, bei denen eine unbewusste Akkommodation nicht ausgeschlossen werden kann, in Zykloplegie erfolgen. Ferner ist eine Orientierung an der individuellen Änderung der Achslänge für die Verlaufskontrolle sinnvoll [8]. Eine sichere Beurteilung der Myopieentwicklung ist in der Rückschau auf längere Zeiträume von mindestens 12 Monaten möglich. Die Orientierung an Perzentilen kann hilfreich sein. Es gibt Hinweise, dass ein Zusammenhang mit dem Körperwachstum oder dem Einfluss von Wachstumshormonen besteht. 6. Interventionen zur Progressionsminderung Neben den erwähnten Hinweisen zu alltäglichen Umweltfaktoren stehen Maßnahmen zur Verringerung der Progression zur Verfügung [3, 24]. Die Evidenz der unterschiedlichen Ansätze und die Qualität der berichteten Studien wurden in einem Cochrane Review zuletzt 2020 kritisch bewertet [25]. 6.1. Atropin Die Mehrheit der publizierten randomisierten kontrollierten Studien zur Sicherheit und Wirkung von niedrig dosiertem Atropin stammen bis auf wenige Ausnahmen [26] aus Asien [3]. Beispielsweise sind für große Studien aus Australien und England erst die Protokolle bzw. Baseline-Faktoren publiziert [27, 28]. Nach der ATOM-II-Studie aus Singapur, welche den weltweiten Einsatz von niedrig dosiertem Atropin angestoßen hat [29], wurden in einer weiteren Studie die Konzentrationen 0,05 %, 0,025 % und 0,01 % Atropin gegen Placebo verglichen [30]. Nach 1 Jahr Therapie betrugen die entsprechenden Progressionsraten −0,27 dpt, −0,46 dpt, −0,59 dpt und −0,81 dpt. Die Nebenwirkungen waren gering und unterschieden sich bei diesen 3 Konzentrationen nicht wesentlich und wurden auch fast gleichhäufig in der Placebogruppe berichtet. Im zweiten Jahr dieser Studie wurde der Placeboarm auf 0,05 % umgestellt und die Zweijahresergebnisse bestätigen die Wirksamkeit [31]. Inzwischen liegen die Dreijahresergebnisse vor. Im dritten Jahr führte die eine Hälfte die Therapie weiter, die andere hatte die Therapie beendet. Der Rebound nach Therapieende war gering und war statistisch nicht signifikant [3]. Eine weitere Studie aus China randomisierte 220 Kinder auf 0,01 % Atropin oder Placebo [32]. Nach 1 Jahr betrug die Progression in der Placebogruppe 0,76 dpt, in der Atropin-Gruppe 0,49 dpt. Die Achslänge nahm in der Placebogruppe um 0,41 mm und in der Atropin-Gruppe um 0,32 mm zu. Vor dem Hintergrund dieser Daten erscheint eine Verordnung von Atropin in der Konzentration von 0,5 % und der damit einhergehenden Blendungsempfindlichkeit nicht sinnvoll oder erforderlich [33, 34]. Allerdings kann auch die Konzentration von 0,05 % mit einer Pupillenerweiterung von 3 mm einhergehen [35]. Konservierungsmittel haben keinen wesentlichen Einfluss auf die Penetration der Tropfen ins Auge [36]. Eine asiatische Studie hat keinen Unterschied bezüglich reduzierter Akkommodation und vergrößerten Pupillendurchmessers bei Konzentrationen zwischen 0,01 und 0,02 % gefunden [37]. Grundsätzlich sprechen die Daten für eine mehrjährige Therapiedauer [3]. Die Progression der Myopie ist gegen Ende des Grundschulalters oft höher als in den Folgejahren, sodass eine Intervention hier größeren Nutzen verspricht. Mit 14 bis 16 Jahren ist die Zunahme der Achslänge im Regelfall so gering, dass eine Therapie nur in Ausnahmefällen sinnvoll erscheint. Die Fortsetzung einer begonnenen Therapie mit niedrig dosiertem Atropin ist spätestens nach einer Dauer von 2 Jahren oder Erreichen des 15. Lebensjahrs zu prüfen. Eine anhaltende Zunahme der Kurzsichtigkeit spricht eher für eine Verlängerung der Therapie. Ansonsten sollte der Verlauf nach einer Pause beobachtet und beim Vorliegen von einer Progression von über 0,5 dpt/Jahr eine Wiederaufnahme evaluiert werden. Somit kann grundsätzlich myopen Kindern im Alter zwischen ca. 6 und 14 Jahren und bei einer beobachteten Progression von ≥ 0,5 dpt/Jahr Atropin 0,01 % angeboten werden. Dabei handelt es sich um ein Rezepturarzneimittel (s. Anhang) bei fehlender Medikamentenzulassung für diese Dosierung und diese Indikation. Diese Tropfen werden 1‑mal abends vor dem zu Bett gehen in beide Augen getropft und sollen konform mit den Vorgaben konservierungsmittelfrei zubereitet werden. Eine Kontrolle zur Verträglichkeit kann in der frühen Phase z. B. 2 bis 6 Wochen nach Beginn der Atropin-Therapie sinnvoll sein. Eltern und Kinder sollten gerade zu Beginn der Therapie über eine mögliche geringe Pupillenerweiterung und Blendempfindlichkeit informiert werden. Mit einer relevant verminderten Akkommodation oder einem reduzierten Nahvisus ist in der Regel nicht zu rechnen [38, 39]. Sollte dies im Einzelfall doch störend werden, kann eine temporäre Gleitsichtbrille angeboten werden. Kombinationstherapien mit den in folgenden optischen Interventionen sind im Einzelfall sinnvoll und können dann erwogen werden. Das Studiendesign und die Erfassung möglicher Nebenwirkungen sind zu heterogen, um eine abschließende Beurteilung der Dosierung in dem Bereich von 0,01–0,05 % mit dem besten Verhältnis von Wirkung und Nebenwirkungen zu erlauben [40]. Die bisherigen Metaanalysen können als Beleg für die Sicherheit herangezogen werden, ohne die Verträglichkeit im Einzelfall zu garantieren [41]. Eine aktuelle Recherche in ClinicalTrials.gov mit den Suchstichworten „Atropine AND Myopia“ erbrachte insgesamt 26 Studien, von denen sich 10 mit kurzfristigen Nebenwirkungen und Änderungen der Aderhautdicke befassten. Die anderen 16 Studien vergleichen fast immer 0,01 % entweder gegen Placebo oder andere Atropin-Konzentrationen. Entsprechend ist in den kommenden Jahren mit einer Vielzahl weiterer Studienergebnisse zu diesem Thema auch zur Wirksamkeit bei Nicht-Asiaten zu rechnen. Fazit. Die Prophylaxe mit niedrig dosiertem Atropin, meist in der Konzentration von 0,01 %, hat sich inzwischen weltweit verbreitet und kann im Alter von 6 bis14 Jahren angeboten werden, wenn eine jährliche Myopieprogression von mindestens 0,5 dpt vorliegt. Die Datenlage hierzu ist solide und das Verhältnis von Nutzen und Nebenwirkungen günstig. Laufende Studien dienen unter anderem der Dosisoptimierung. Gleiche Konzentrationen werden bei verschiedenen Ethnien vermutlich verschiedene Effektgrößen zeigen. Aktuell werden Therapie und Kontrolle in Deutschland im Rahmen klinischer Studien angeboten: In der multizentrischen AIM-Studie (EUDRACT 2020-001575-33) werden 300 Kinder im Alter zwischen 8 und 12 Jahren (Refraktion im Bereich von −1 bis −6 dpt) eingeschlossen, wenn eine Progression von ≥ 0,5 dpt pro Jahr erwartet wird (www.aim-studie.de). Im ersten Jahr wird 0,02 % Atropin gegen Placebo verglichen. Im zweiten Studienjahr erhält die Placebogruppe ebenfalls Atropin in der Konzentration 0,01 %. In der 3‑jährigen Beobachtungszeit erhalten alle Kinder mindestens 2 Jahre niedrig dosiertes Atropin. In einer weiteren Studie (EUDRACT 2021-004884-29) wird die Wirksamkeit 4 verschiedener Dosierungen (0,05 %, 0,025 %, 0,01 %, 0,005 %) über 12 Monate verglichen. Rezeptur Atropin: Atropinsulfat 0,01 % AT 0,5 ml Rezeptur: Atropinsulfat 0,05 mg, Natriumchlorid 4,45 mg, HCI-Lösung 0,1 % q.s., Aqua ad injectabilia ad 0,5 g in Einzeldosisbehältnis Haltbarkeit: 1 Jahr, Reste verwerfen xx Packungen (xx Stk. pro Packung) Anwendung am Auge: 1 × 1 Tropfen abends in beide Augen 6.2 Optische Methoden Es ist eine Gemeinsamkeit der optischen Methoden, dass entweder neben der ersten auf die Fovea fokussierten Bildschale eine zweite vor die Netzhaut fokussierte Bildschale (bei einigen Kontaktlinsen und bei Brillengläsern mit zentraler Aussparung) generiert wird oder die Bildschale so gebogen wird, dass sie in der Peripherie vor der Netzhaut liegt, was beides ein Stoppsignal für das Augenwachstum darstellt. 6.2.1 Multifokale und Orthokeratologiekontaktlinsen Für multifokale und Orthokeratologiekontaktlinsen existieren viele Studien mit gutem Evidenzniveau und Metaanalysen [42]. Multifokale Kontaktlinsen, die zur Myopieprogressionskontrolle eingesetzt werden, haben eine zentrale Fernzone und entweder konzentrisch aufgebaute ringförmige Zonen mit einer Plusaddition oder eine graduell ansteigende Plusaddition zur Peripherie hin. Dadurch entsteht eine zweite Bildschale vor der Netzhaut. In der sehr hochrangig publizierten, randomisiert kontrollierten BLINK-Studie beispielsweise wurden 294 Kinder auf Monatslinsen randomisiert, die entweder monofokal oder multifokal mit einer Addition von +1,5 dpt oder +2,5 dpt waren [3]. Die Studiendauer betrug 3 Jahre, und es zeigt sich ein progressionsmindernder Effekt von 43 % für die Linse mit der stärkeren Nahaddition und 15 % mit der schwächeren Nahaddition im Vergleich zu der monofokalen Kontaktlinse. Eine zweiarmige Studie mit multifokalen Tageslinsen an 253 Kindern und einer Dauer von 3 Jahren fand eine Progressionsminderung von 59 % im Vergleich zu monofokalen Linsen [43]. Orthokeratologische Linsen verfolgen das gleiche optische Prinzip und sind ähnlich wirksam [3]. Sie führen zu einer Abflachung der zentralen Hornhaut mit Aufsteilung der Hornhaut in der mittleren Peripherie. Durch die damit einhergehende Erhöhung der Brechkraft entsteht in der mittleren Peripherie eine zweite Bildschale vor der Netzhaut [44]. Der Effekt der Verlangsamung des Achslängenwachstums liegt bei ca. 40 % [45]. Eine Metaanalyse berichtet, dass der Bulbuslängenunterschied nach 2 Jahren Behandlung im Vergleich zu Kontrollgruppen 0,7 mm betrug [3]. Daneben gibt es weitere, speziell zur Myopieprogressionshemmung entwickelte Kontaktlinsendesigns, die unterschiedliche optische Prinzipien nutzen. Erste Studien zeigen auch für diese Designs eine Verlangsamung des Bulbuslängenwachstums von ca. 25 % [46]. Generell ist beim Einsatz von Kontaktlinsen im Kindesalter zu bedenken, dass eine Gefahr der Infektion mit zwar seltener, aber potenziell auch erheblicher Schädigung des Auges besteht. Deswegen sind eine gute Kooperation und die Einhaltung der Anforderungen an die Hygiene, die mit der Zeit nachlassen können, zwingend erforderlich. Daher sollen Eltern und Kind über das Risiko von Komplikationen wie eine Keratitis und Warnsymptome informiert werden. Dazu gehört, dass bei Beschwerden die Kontaktlinsen nicht getragen werden dürfen und eine augenärztliche Untersuchung erforderlich ist. Orthokeratologische Linsen sind bei guter Hygiene ähnlich sicher wie Tageslinsen, erfordern aber eine hohe Compliance, da andernfalls eine schwankende Refraktion resultiert. Die Quantifizierung der Myopieprogression kann nur über die Messung der Bulbuslänge erfolgen. Bei dem Vergleich von multifokalen Linsen und Ortho‑K sind ferner verschiedene Faktoren und die individuelle Verträglichkeit zu berücksichtigen. Weiche multifokale Linsen sind mit einem größeren Defokus und schlechterem Kontrastsehen verbunden [47]. Die über Nacht getragenen Linsen können dagegen neben einer nachlassenden Myopiekorrektur und stärkeren Induktion von Aberrationen höherer Ordnung mit unterschiedlichen Refraktionsschwankungen über den Tag einhergehen und verursachen ein schlechteres Sehen in den ersten Tagen nach dem Absetzen der Linsen. Obwohl eine Dezentrierung der Orthokeratologielinsen grundsätzlich zu vermeiden ist, zeigte sie zumindest keine wesentliche Auswirkung auf die Hemmung der Myopieprogression [48]. Fazit. Für die optische Therapie können multifokale oder orthokeratologische Kontaktlinsen angeboten werden, wenn eine Myopieprogression von mindestens 0,5 dpt pro Jahr besteht und bei den Kontaktlinsen ein korrektes Tragen und eine adäquate Hygiene garantiert sind. Das infrage kommende Altersspektrum entspricht dem, bei dem auch eine Atropin-Therapie indiziert ist. 6.2.2 Multisegmentgläser Ein vergleichbares optisches Konzept wurde mit speziellen Brillengläsern realisiert. Hierbei ist in die Brillengläser in der mittleren Peripherie eine Vielzahl kleiner Pluslentikel eingeschliffen, welche einen zweiten Fokus vor der peripheren Netzhaut generieren. Diese Gläser mit den integrierten Segmentlinsen werden von verschiedenen Herstellern angeboten. Die Ergebnisse prospektiver Studien wurden unter Koautorenschaft der Hersteller publiziert. Randomisierte, kontrollierte Studien mit einer Beobachtungsdauer von 2 bis 3 Jahren [49, 50] zeigten eine Halbierung der Myopieprogression. Das Tragen der Gläser verursachte keine langfristig negativen Konsequenzen auf andere Sehfunktionen wie Nahsehschärfe, Phorieruhelage oder die Stereosehschwelle [51]. Bei Seitblick erzeugen die Multisegmentgläser eine Bildunschärfe, die im Alltag von vielen Kindern nicht als störend empfunden wird. Aktuell ist noch unklar, welche Relevanz bzw. welche Mechanismen asphärische gegenüber sphärischen Segmenten haben [50]. Eine aktuelle, deutschsprachige Übersichtsarbeit stellt die Erfahrungen mit den Gläsern eines Herstellers zusammen [52]. Ansonsten existieren keine Studien, die unabhängig von den jeweiligen Herstellern Wirksamkeit und Nebenwirkungen untersucht haben. Gleitsicht- oder Bifokalgläser, wie sie z. B. vor vielen Jahren in der COMET-Studie [3] evaluiert wurden, konnten nur eine geringe Hemmung der Myopieprogression erreichen, sodass sie nicht empfohlen werden. Fazit. Erste Studien sprechen für die progressionsmindernde Wirkung von Multisegmentbrillengläsern, die parallel eine Myopie- bzw. Visuskorrektion erlauben. Für eine allgemeine Empfehlung sind zu diesen Brillengläsern weitere Studien wünschenswert. 6.2.3 Kombinationstherapie Einzelne Studien belegen, dass sich die Wirkung der Orthokeratologie und Atropin synergistisch ergänzen [3, 53]. Daher ist es nicht ausgeschlossen, dass dies auch für multifokale Kontaktlinsen oder Multisegmentgläser und die Atropin-Therapie gilt. Bisher gibt es hierzu jedoch keine Publikationen. Anhang Im Folgenden sind die Interessenerklärungen als tabellarische Zusammenfassung dargestellt sowie die Ergebnisse der Interessenkonfliktbewertung und Maßnahmen, die nach Diskussion der Sachverhalte von der der LL-Gruppe beschlossen und im Rahmen der Konsensuskonferenz umgesetzt wurden. Tätigkeit als Beraterin und/oder Gutachterin Mitarbeit in einem Wissenschaftlichen Beirat (advisory board) Bezahlte Vortrags-/oder Schulungstätigkeit Bezahlte Autorinnen-/oder Coautorinnenschaft Forschungsvorhaben/Durchführung klinischer Studien Eigentümerinneninteressen (Patent, Urheberinnenrecht, Aktienbesitz) Indirekte Interessen Von COI betroffene Themen der Leitlinie, Einstufung bzgl. der Relevanz, Konsequenz Prof. Dr. Bertram, Bernd Gutachter für Versorgungsverwaltung in NRW Gutachterkommission der Ärztekammer Nordrhein Nein Nein Nein Nein Nein Mitglied: Ärztekammer Nordrhein: Mitglied des Vorstandes, Mitglied in den Ausschüssen Berufsordnung, Strukturen ärztlicher Versorgung, Kooperation der Gesundheitsberufe und der Versorgungssektoren, Delegierter beim Dt. Ärztetag, Mitglied des Beratenden Fachausschusses Fachärzte der KBV, Ressortleiter Leitlinien/Stellungnahmen des Berufsverbands der Augenärzte Deutschland e. V., Mitglied des Gesamtpräsidium der Deutschen Ophthalmologischen Gesellschaft, Sprecher der Leitlinienkommission von DOG und BVA, Sprecher Kommission Ophthalmologische Rehabilitation von DOG und BVA, Sprecher der Makulakommission von DOG und BVA, Sprecher der Kommission für Weiter- und Fortbildung von DOG und BVA, Sachverständiger in AG des Gemeinsamen Bundesausschusses: Sehhilfen, wissenschaftliche Tätigkeit: Versorgungsforschung, wissenschaftliche Tätigkeit: nichtoperative Augenheilkunde; Lasertherapie, Beteiligung an Fort‑/Ausbildung: Augenärztliche Akademie Deutschlands (AAD) Kein Thema (keine), keine Prof. Dr. med. Dieter, Friedburg Nein Nein Leitung des Schoberkurses des Berufsverbandes der Augenärzte Deutschlands Nein entfällt entfällt Mitglied: Berufsverband der Augenärzte Deutschlands (BVA), Ressortleiter Ophthalmologische Optik, wissenschaftliche Tätigkeit: im Ruhestand seit Oktober 2000. Publikationen zu klinischen augenärztlichen Themen, zur Biochemie der Augenlinse, zu Fragen der Refraktion und Refraktionsbestimmung, wissenschaftliche Tätigkeit: Leitung der Augenklinik des Klinikums Krefeld, Beteiligung an Fort‑/Ausbildung: Augenklinik des Klinikums Krefeld, Lehrkrankenhaus Universität Düsseldorf, persönliche Beziehung: keine Kein Thema (keine), keine Prof. Dr. med. Ehrt, Oliver Nein Wiss. Beirats der Studiengänge AO, HA, DHM, HTM, OE, APH und MBD der Hochschule Aalen Amtsarztlehrgang bei Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), Strabologietage, Wackerkurse, AAD, DOG, ESA, EPOS, Kinderärztekongresse, – Fortbildungen, DOG update, Ärztlicher Leiter Lehrer an Orhtoptikschule München Thieme Verlag Nein Nein Mitglied: Bielschowsky Gesellschaft, Vorstandsmitglied, Vorsitzender seit 2021, Mitglied: Europäische Strabologische Gesellschaft, Vorstand, Mitglied: BVA, DOG, DHV, wissenschaftliche Tätigkeit: Klinische Forschung Kein Thema (keine), keine Prof. Lagrèze, Wolf Boehringer Ingelheim, Santhera, GenSight, InfectoPharm, Novartis AG Forschung der DOG, AG Ethik der DOG Santhera Nein Nein Nein Mitglied: DOG, BVA, ARVO, EUNOS, wissenschaftliche Tätigkeit: Neuroprotektion, Neuroophthalmologie, Kinderaugenheilkunde, Strabismus, Orbitaerkrankungen, wissenschaftliche Tätigkeit: Neuroophthalmologie, Kinderaugenheilkunde, Strabismus, Orbitaerkrankungen, Beteiligung an Fort‑/Ausbildung: Ophthalmo-Update Kein Thema (keine), keine Dr. med. Reck, Barbara Nein Nein BVA Vereinigung Kontaktlinsen-anpassender Augenärzte Österreich Nein Nein Nein Mitglied: Stellvertretende Ressortleiterin Kontaktlinsen im BVA Kein Thema (keine), keine Prof. Dr. rer. nat. Dr. h.c Schaeffel, Frank Zeiss Vision Nein Nein Nein Nein Nein Nein Kein Thema (keine), keine Prof. Schittkowski, Michael Nein Bielschowsky Gesellschaft Bayer, Novartis KliMo/Thieme Nein Nein Mitglied: Bielschowsky Gesellschaft, BVA, DOG, ESOPRS, DGPW, wissenschaftliche Tätigkeit: EO, Orbita, Anophthalmus, wissenschaftliche Tätigkeit: Strabismus, Neuroophthalmologie Okuloplastik, Beteiligung an Fort‑/Ausbildung: DOG, AAD, Norddeutsche Gesellschaft, Persönliche Beziehung: nein Kein Thema (keine), keine Prof. Dr. Ziemssen, Focke Allergan/AbbVie, Boehringer-Ingelheim, Novo Nordisk, MSD, Oxurion, Cochrane Institut Freiburg/IQWIG, Bayer, Novartis, Roche, MSD, MSD Novartis, Bayer, Roche, Thieme Verlag, Thieme Verlag, Bayer, Novartis, Roche, Roche Allergan/AbbVie, Allergan/AbbVie, Bayer, CME-Verlag, Diaplan, Alimera, Gerling, Dr. Pliquett Mehring Symposium, Novartis, Optimed, Roche, OD-OS Bonifatius Verlag, Thieme Verlag, DOG DFG, Bayer, Novartis, Novartis, Regeneron Nein Mitglied: Berufsverband der Augenärzte, Mitglied: Deutsche Ophthalmologische Gesellschaft, Mitglied: American Academy of Ophthalmology, Mitglied: Retinologische Gesellschaft, Mitglied: Verein Rheinisch-Westfälische Augenärzte, wissenschaftliche Tätigkeit: diabetische Retinopathie, Versorgungsforschung, Sehen im Alter, Beteiligung an Fort‑/Ausbildung: Universitätsaugenklinik Leipzig Keiner (keine), keine Redaktionskomitee Prof. Dr. Wolf Lagrèze (Freiburg; federführend); Prof. Dr. Bernd Bertram (Aachen); Prof. Dr. Oliver Ehrt (München); Prof. Dr. Dieter Friedburg (Krefeld); Dr. Barbara Reck (Stuttgart); Prof. Dr. Frank Schaeffel (Tübingen); Prof. Dr. Michael Schittkowski (Göttingen); Prof. Dr. Focke Ziemssen (Leipzig) Einhaltung ethischer Richtlinien Interessenkonflikt Siehe Tab. 1 im Anhang. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. Diese Leitlinie erscheint ebenfalls in der Zeitschrift Klinische Monatsblätter für Augenheilkunde, Georg Thieme Verlag, Stuttgart. Das Redaktionskomitee dieser Stellungnahme wird am Beitragsende gelistet. QR-Code scannen & Beitrag online lesen ==== Refs Literatur 1. Wang CY Hsu NW Yang YC Chen YL Shyong MP Tsai DC Premyopia at preschool age: population-based evidence of prevalence and risk factors from a serial survey in Taiwan Ophthalmology 2022 129 8 880 889 10.1016/j.ophtha.2022.03.017 35331752 2. Li SM Wei S Atchison DA Kang MT Liu L Li H Annual incidences and progressions of myopia and high myopia in Chinese schoolchildren based on a 5-year cohort study Invest Ophthalmol Vis Sci 2022 63 8 10.1167/iovs.63.1.8 34989760 3. 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==== Front Socioecol Pract Res Socioecol Pract Res Socio-Ecological Practice Research 2524-5279 2524-5287 Springer Nature Singapore Singapore 134 10.1007/s42532-022-00134-6 Research Article Assessing indigenous community’s perspectives and attitudes toward tourism development impacts in the northwestern Himalayas, India http://orcid.org/0000-0002-9811-9610 Wani Mohd Saleem [email protected] Mohd Saleem Wani is a Doctoral Fellow at the Department of Geography and Disaster Management, University of Kashmir, Jammu and Kashmir, India. He has received his master's in Geography and Disaster Management and is currently pursuing his Ph.D. in the environmental sustainability of Tourism. His research interests include sustainable development, Sustainable Tourism, and the Application of Geospatial techniques in Tourism. Bhat M. Sultan Dr. M. Sultan Bhat is presently working as a Professor with a teaching experience of over 28 years at the Department of Geography and Disaster Management, University of Kashmir, Jammu and Kashmir, India. He has authored more than a hundred research papers in national and internationally acclaimed peer-reviewed journals. He is a NABET-accredited functional area expert (FAE) in Socio-Economic consultant and has been associated with EIA & EMP and SIA & SMP of several hydroelectric projects, roadways, industrial complexes, and tourism township developments of Jammu & Kashmir state and has also completed many research projects 17 sponsored by ICSSR, UGC, Ministry of Earth Sciences (MoES), Government of India and DST. He has availed Baden Wurttemberg Visiting Professor Fellowship at South Asia Institute, Heidelberg University Germany, and has been also selected for the Leadership for Academician Programme (LEAP) by the UGC in March 2019. His research interests include Urban geography, Watershed Development, Climate Change, and Environmental studies. Alam Akhtar Dr. Akhtar Alam is working as an Assistant Professor at the Department of Geography and Disaster Management, University of Kashmir, Jammu and Kashmir, India. He has worked as a Physical Science Research Fellow in Conflict, Disaster, and Migration at the Institute for Risk and Disaster Reduction (IRDR), University College London (UCL), UK, for a period of 2 years, 2019–2020 as a postdoctoral fellow. Besides, he has completed several projects sponsored by various agencies (Ministry of Earth Sciences (MoES), Government of India; Indian Council of Social Science Research (ICSSR), Government of India; University Grants Commission (UGC), Government of India. He has authored more than thirty papers in national and international peer-reviewed journals. His areas of research interest include environmental change, geomorphology, natural hazards, and disaster risk. Mir Sajad Ahmad Sajad Ahmad Mir is a Doctoral research fellow at the Department of Geography and Disaster Management, University of Kashmir, Jammu and Kashmir, India. He is currently working on dissecting the interface between health and groundwater contamination in the western Himalayan region. He has several publications to his name. His research interest includes spatial analysis, water resource management, and groundwater quality zonation. grid.412997.0 0000 0001 2294 5433 Department of Geography and Disaster Management, University of Kashmir, Srinagar, 190006 India 1 12 2022 116 26 6 2022 17 11 2022 21 11 2022 © The Author(s), under exclusive licence to Springer Nature Singapore Pte Ltd. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. An assessment and monitoring of tourism impacts coupled with community perception have emerged as a vital tool for ensuring the sustainability of mountain tourism destinations in recent years. The present study aims to explore the indigenous community’s perspectives on tourism impacts and their participation in the process of tourism development at Doodhpathri, an emerging tourist resort in Jammu and Kashmir, India. A non-probability convenience sampling method based on 344 questionnaires has been used to accomplish the research objectives. Inferential statistics and factor analysis were employed to analyze the collected data. Our assessment reveals that in general, tourism is viewed as a development industry. Its positives are better perceived than its negatives, given that it generates employment prospects, boosts household income, improves the image of the area, and raises the indigenous community’s standard of living. However, a substantial portion of the population living in the area perceives tourism activities as the cause of multiple environmental and biophysical issues, such as increased waste generation leading to pollution and water quality deterioration. On the whole, most of the residents were positive about future tourism development and optimistic about tourism management practices. However, the area has recently observed a voluminous influx of both local and foreign tourists, which necessitates the formulation of a sustainable tourism planning strategy. Keywords Mountain tourism development Indigenous population Community perception Sustainable development http://dx.doi.org/10.13039/501100001839 University Grants Committee MANF-2018-19-JAM-98997 Wani Mohd Saleem ==== Body pmcIntroduction The tourism industry has seen tremendous growth over the years in concert with the local community’s perceptions and attitudes toward sustainable tourism management practices. Recognizing the consequences of tourism on local people has undergone a radical shift, and it is now a significant issue in constructing long-term and sustainable tourist initiatives (Abdollahzadeh and Sharifzadeh 2014, p. 127; Diedrich and Garca-Buades 2009, p. 512). Tourism development is an essential array of financial activities for mountain economies to develop and revitalize. Mountain landscapes, on the other hand, are ecologically susceptible to alteration and tourism-induced impacts (ICIMOD 2010). In the vast corpus of tourism research, the term ‘impacts of tourism’ has gained prominence. Since the 1970s, there has been a significant increase in interest in residents’ perspectives on tourism, prompting academics and professionals to examine local attitudes closely (Sharpley 2014). This phenomenon could be understood explicitly through an appraisal of residents' attitudes and perception-based assessment. Tourism is fundamentally an integrative transactional process, including a direct and indirect exchange between visitors and locals (Brida et al. 2010, p. 359). The essence of such shared values is the sole experiential takeaway that visitors receive in conjunction with the perception and feel of the place, forming a comprehensive set of overall auditing of the tourist destination. Multiple researchers have examined locals’ perceptions and interpretations of the consequences of tourism activities in specific neighborhoods in recent times (Ko and Stewart 2002, p. 552; Lankford and Howard 1994, p. 123; Alves et al. 2010, p. 23; Choi 2013, p. 76; Cardoso and Silva 2018, p. 688; Cheng et al. 2019, p. 3; Gidebo 2019, p. 651; Zhu et al. 2017, p. 2; Latip et al. 2018, section I; Peters et al. 2018, p. 2). Doodhpathri, a tourist destination of the union territory of J and K in the Union of India, is a nature-based tourist destination located in the northwestern Himalayas. Being a perennial tourist destination, it has diverse tourist attractions ranging from forests, gushing waters, glaciers, lush green meadows surrounded by snow-clad mountains, rural landscapes, and local traditional culture attracting a massive rush of tourists. Tourism activity at Doodhpathri is placed at an emergent level of development, thus augmenting its scope for being a prominent destination for tourist engagement. As a result, tourism is emerging as one of the main economic activities in the region. Even though tourism forms the mainstay of the local economy, not much is known about how people feel about tourism development and subsequent impacts. Moreover, no research has been conducted to investigate the residents’ perceptions of the impacts of tourism. Understanding local communities’ perceptions regarding various aspects of tourism and how they vary may be critical to managing tourism activity, especially in the mountain ecosystems, which are fragile to any tourism-induced impacts. A better understanding of residents’ knowledge of tourism development, their awareness of its benefits, and its perceived impact on their welfare are fundamental to developing and implementing management strategies that are both sustainable in the long term and sensitive to existing local needs. This paper investigates residents’ perceptions of the impact of tourism and their support for future tourism development at Doodhpathri, an emerging tourist destination in the Kashmir Valley, India. There is a lot of evidence in the literature that the local community is at the center of tourist growth, their participation being key to the long-term success of the industry (Lee 2013, p. 38; Gonzalez et al. 2018, p.1). Furthermore, the research demonstrates that the locals' perspectives regarding tourism’s effects are vital to the expansion of tourism in the region. It demonstrates that communities' support for tourism depends on their perceptions of its impacts on their community (Bimonte and Punzo 2016, p. 129). How much support tourism development receives from the locals is a big part of how long it will last. Such a level of support, in turn, reflects how local citizens view tourism’s influence on their community (Rasoolimanesh and Jaffer 2017, p. 2; Jackson 2008, p. 242). Several theories have been used to identify the factors influencing residents’ perceptions of tourism. These theories include the Social Exchange theory (Gursoy et al. 2002, pp. 81–82; Gursoy and Kendall 2006, p. 606; Jurowski et al. 1997, pp. 3–4; Nunkoo and So 2016, p. 848; Peters et al 2018, p. 3; Ko and Stewart 2002; Látková and Vogt 2012, pp. 52–53; Rasoolimanesh et al. 2015, p. 336; Wang and Pfister 2008, p. 86; Zhu et al. 2017, p. 3); Stakeholder theory (Byrd 2007). Among these, it seems that SET (Social Exchange Theory) is the most prominent theory employed to explain local’s perspectives (Sharply 2014, p. 45). The core tenet of social exchange theory is that people form opinions about something by weighing the advantages against the costs. This idea is interdisciplinary, with its origins in fields as diverse as economics, anthropology, sociology, and social psychology (Liu 2012, p. 62). Consequently, it was initially applied in the discipline of economics by Homans (1958) to interpret human behavior. Using an expanded social exchange theory, Emerson (1962) and Blau (1964) examined how inhabitants and institutions work together to optimize benefits and minimize costs. This was accompanied by the realization that social exchange theory could be used in different situations to help people better understand how they see each other in a relationship or interaction. When put into the context of tourism, this theory claims that locals would encourage the expansion of the industry if they perceive its positives would exceed its negatives (Rasoolimanesh et al. 2015, p. 336). Alternatively, locals will typically be hostile to tourism expansion if they feel the costs outweigh the advantages. Analyzing and evaluating resident perspectives on tourist development would fill the gap in residents’ assessment research. Most research on how locals feel about tourism effects has been done in the developed world. Minimal research has been conducted on this theme in developing nations (Liu and Li 2018, p. 1). The fragile mountain ecosystem regions like the Kashmir valley, prone to conflict, have received little consideration in most research conducted in developing countries. In the Indian context, few studies have examined residents’ perceptions of tourism development. It is often assumed that attracting tourists to mountainous regions will help boost the local economy, especially when many locals abandon the mountainous areas in favor of the cities in search of better livelihood prospects. In this context, the present study investigates residents’ perceptions of tourism’s economic, environmental, and socio-cultural implications in Doodhpathri, NW Himalayas, and their support for future tourism development using the social exchange theory (SET) as a theoretical framework. Furthermore, in the context of Kashmir valley, no empirical study of the resident's perception of tourism development and their support for future development in an emerging destination has been undertaken. The social exchange hypothesis also makes sense and is intuitively attractive as a sound theoretical basis for assessing tourism implications and how they might foster development in an emerging destination, in the case of Doodhpathri. The present study infers that it is a prerequisite to conduct further research on this theme in other geographical settings and socio-economic backgrounds to fill a research gap and add to the existing tourism literature, which could be applied to other mountain destinations across the globe. Literature review The growth of tourism is a “two-edged sword” as it encompasses both constructive and detrimental facets for the local inhabitants (Wang and Pfister 2007, p. 411). The intensity of indigenous inhabitants’ inclusion in tourism is dynamically linked to tourism impacts on public perception (Eshliki and Kaboudi 2012, p. 340). Individual benefits accrued from tourism are associated with local community involvement in tourism management (Hanafiah et al. 2013, p. 792; Zhu et al. 2017, p. 10). If costs exceed profits gained, residents will negatively perceive tourism activity. However, if residents benefited from this exercise without considerable cost, there would be a positively driven perception (Lee 2013, p.38). An integral part of the “visitor experience” is the behavior of the host community's residents (Murphy 1985, p. 16). Residents’ perspectives and goodwill toward tourists directly evoke a pleasant experience, influencing the likelihood of tourists' repeated visits to the destination and exuding a healthy experience of the place (Carmichael 2006, p. 117). There is a lot of evidence in the literature that the local community is at the center of tourist growth and that their participation is key to the long-term success of the industry (Lee 2013, p. 38; Gonzalez et al. 2018, p. 1). Thus, assessing residents' insights about tourism development prompts tourism and government functionaries to devise an actionable policy for community outreach. This motivates host communities to provide proactive support (Murphy et al. 2000, p. 50). The generosity of the locals of a destination largely determines the positioning of the tourism business and how tourist spots should be planned following an area-specific approach (Andriotis 2005, p. 83). Unlike most other sectors, tourism comprises visitors going places, which generates friction between tourists and inhabitants. It is crucial to examine and document communities’ impressions of tourist effects. This aids in outlining an ideal tourist development strategy while minimizing negative effects on residents. The local community's support and involvement in the decision-making process guarantee long-term sustainability in tourism development, which is imperative in local tourist attractions. Researchers advocate that it is challenging to sustain tourism in any region that does not value and acknowledge the local inhabitants' inclusion in the active tourism function (McCool et al. 2001, p. 130; Twining-Ward and Butler 2002, p. 372). According to Brida et al. (2010), destination planners must recognize residents' opinions of tourism effects, as they are the primary stakeholders in perceiving the cumulative bearings throughout the process of multiplier effects of tourism development. While tourism's importance to economic and social development as well as a multiplicity of benefits to local people are well documented, even though it has culminated in many effects on which the local people are likely to suffer from waste generation, change in their traditional culture, traffic jams, crimes, and the cost of living seems to be mounting (Nunkoo and Ramkissoon 2010, pp. 52–53). The future viability of the travel and leisure economy depends on several issues, particularly, with particular attention paid to tourism’s impacts on the host community (Chandralal 2010, p. 42). Taking the host community into account when developing tourism policies ensures positive effects on environmental, economic, social, and cultural aspects (Puiu and Ovidiu 2008, p. 44). Community attitudes to tourism Over the last three decades, researchers have looked into how host communities perceive tourism's effects. The host residents' outlook is central to productive and sustainable development because valuing people’s perceptions and attitudes and how they are evinced regarding tourism development might attract indispensable observance from policymakers. Involving the local community in the implementation of community outreach programs increases their likelihood of success and ensures positive effects on the environment, economy, culture, and society. (Jeelani et al. 2022, p. 7). Diverse channels of revenues and expenses affect host communities’ perspectives and, as observed from the studies, can be categorized into three parts; socio-economic, cultural, and environmental implications (Gursoy et al. 2010, p. 381; Murphy 1985, p. 16). A comprehensive visitor impact study may help regional planners and local decision-makers simultaneously flag actual problems and challenges so that appropriate policies and responses can be devised (Allen et al. 1988, p. 20; Belisle and Hoy 1980, p. 85). In every social setup, the variables that determine inhabitants' viewpoints, as well as the type and intensity of the consequences, are highly varying in nature. The social fabric of an immediate society substantially impacts its ability to constructively assimilate the many shared values and conventions that visitors bring with them (Mansfield 1992, p. 379). Since most tourism is an exchange between cultural and environmental expenses and economic benefits, people handle it by underplaying the adverse consequences and stressing financial revenues to uphold satisfaction with their community (Cavus and Tanrisevdi 2003, p. 267; Faulkner and Tideswell 1997, p. 24). Tourism development in any area can contribute positively to local development but also tends to cause environmental deterioration and erosion of local identity and traditional values if its growth is uncontrolled and unplanned (Syamlal 2008, p. 2). The indigenous society's engagement in tourism planning in emerging economies is generally insufficient, limited, or neglected (Dola and Mijan 2006, p. 2). Cater (1994) underscores the significance of local community engagement in responsible tourism, mainly in developing nations. Because of the emphasis on local collaboration and a community-based strategy for tourism planning, host communities are widely ignored in the planning process, administrative auditing, and decision-making. In emerging nations with centralized development cultures, marginalization is evident (Teye et al. 2002, p. 670). Drake (1991) believed that the involvement of local people is widely viewed as critical to the effectiveness of sustainable tourism management and planning. Social inclusion is not only at the heart of the policy-making and growth of tourism, but it is also an essential component of long-term tourism viability (Murphy 1988, pp. 97–98). Understanding the host community’s familiarity with mountain tourism development, its beliefs, and its responsiveness to its visible impacts is imperative for policymakers in developing and executing long-term sustainable tourism strategies. Tourism in the study area The northwestern Himalayas have long been a famous tourist destination attracting people worldwide. Since ancient times, sages and travelers from all over India and surrounding nations have come to the valley for knowledge and spiritual purposes, attracting sages and prodigies from all over India and surrounding nations (Malik and Bhat 2015, p. 11). Tourism accounts for almost 7% of the total GDP of Jammu and Kashmir, employing directly and indirectly about 2 million people. A flourishing sector transcending inter-regional boundaries, tourism can have either favorable or unfavorable effects on the region with economic, environmental, social, and cultural magnitudes (Arrow et al. 1993, p. 4608). However, the tourism impacts and non-tourism activities on socio-cultural and environmental issues are hard to separate and evaluate (Briassoulis 2002). Mountain ecosystems like the Himalayas are especially susceptible to climate change owing to limited adaptive ability, resources, and cascade effects on downstream environments (Ahsan et al. 2021, p 0.2, Ahsan et al. 2022, p. 1665). The increased pressure on the population, commercial forestry, tourism activities, and economic prosperity are the leading causes of the destruction of the environment in the Himalayan ecosystem (Rawat and Sharma 1997). Mountain tourism is essential as it is often promoted as a source of community outreach that may give substitute income options, encourage population growth, expand local economies, and address poverty problems (Sinclair and Ham 2000). However, this exercise is often fraught with perceived risks of environmental and cultural decadence, affecting the complete identity of bio-physical and the composite image of the area. The valley of Kashmir, tucked between the breathtaking Himalayas, offers a wide variety of natural delights such as serene meadows, gorgeous pine woods, captivating treks, streams, snow-clad mountains, freshwater lakes, and an appealing climate, which has made this valley a sought-after tourist destination (Malik and Bhat 2015, p. 11). As an outcome of these naturally gifted traits, the Kashmir Valley has been globally euphemized as “Heaven on Earth." Doodhpathri is the perfect example of a place that is filled with all the bounties of nature and adds another jewel to the glorious beauty of Kashmir. Tucked away among the magnificent hills and the lovely fir forests, the place presents a beauty so untouched and raw that it leaves one enchanted and mesmerized. Increasing tourism activity and a rising population have indeed been attributed to a large volume of solid trash creation in Srinagar. Most of Srinagar's municipal solid waste is deposited on land without any control. When the trash is thrown away haphazardly, it causes problems that directly impact human and animal health, leading to economic, ecological, and biological losses (Suhaib and Jyoti 2017, p. 2, Ahmad et al. 2015, p. 3661). In Kashmir, unplanned tourism has contributed to the deterioration of water resources. The picturesque Dal Lake has been severely impacted due to mismanagement and unregulated tourism, resulting in eutrophication and water quality deterioration (Mushtaq et al. 2013, p. 633). Residents perceive positive perceptions regarding economic impacts. However, the Kashmir valley has mixed views on ecological and socio-cultural impacts (Charag et al. 2020, pp. 750–752). The rising temperature at Gulmarg and Pahalgam tourist hill stations is a grave concern for the viability of tourism, specifically winter tourism in the area (Dar et al. 2014, p. 2560). So far, the influence of tourism on the host population at Doodhpathri has not been documented, as the region falls under the category of a "virgin and emerging tourist destination." The region’s economic base is primarily subsistence agriculture, like cattle rearing, wood collection, etc. With the development of tourism in the area, the agrarian economy may shift toward a market-oriented economy, which can boost the region’s economy. The development of tourism is expected to provide a significant economic platform that will not only be effective in terms of the growth of the economy but will also result in significant structural changes in the workforce of the region, which in turn will also result in the conversion of marginal workers into primary workers. The growth of tourism can enhance the living standard of local and indigenous people by providing them with new sources of revenue, more job openings, better public services, and more richness in their cultural traditions. Accommodation and other types of visitor lodging may offer a profitable market for local items, encouraging indigenous people to keep up their traditional arts and crafts as tourism grows. Tourism can also negatively influence land prices, the cost of living, frequent traffic jams, crowding, increased crime, drug use, and competition for resources and facilities. Although some studies have been conducted at different hill stations in the northwestern Himalayas mainly focusing on the environmental viewpoints of tourism effects, mismanagement and free flow of tourism have led to a sudden shift in land use, generation of solid waste, reduction in forest cover (Malik et al. 2011, p. 7), and deterioration in water quality (Rashid and Romshoo 2013, p. 4717–4718) and associated challenges in the Lidder catchment of Kashmir. Doodhpathri's growth as a tourist destination is in its early stage, but it is fastly turning out to be one of Kashmir's most popular tourist locations. Figure 1 shows that Doodhpathri is an emerging tourist destination in the Kashmir valley, with more than 1 million tourists having visited over the last decade. Figure 1 also reveals how the number of tourists changed over the years. Many factors, like COVID-19 and political turmoil, have affected the magnitude and flow of tourists to the destination. Therefore, destination planners must assess how residents interpret the effects of tourism during the initial stage of development, as they are the main stakeholders in ensuring the long-term sustainability of tourism. Therefore, the main aim of the current research is to look at the consequences of tourism on the local indigenous population and assess the influence of these factors on public support for future tourist development in Doodhpathri tourist hill station, in the Kashmir valley. This study will be the first attempt to examine the effects of tourism on this destination in conjunction with the perception-driven impact analysis. This would help to generate a valuable database for future tourism development strategies in similar tourist destinations worldwide.Fig. 1 Tourist arrivals at Doodhpathri (2011–2020) Study area Doodhpathri is popularly known as the "Valley of Milk" owing to its frothing milky waters formed due to the rapids from the stream (Shaliganga) flowing through the region. This place is emerging as the region’s new destination for tourist activities. It is located at an altitude of 2730 m (ASL) in a bowl-shaped valley in the middle range of the northwestern Himalayas. It is located in the Kashmir valley's Budgam district, about 45 km from the summer capital of the UT and 22 km from the district headquarters. Located within the geographical coordinates of 33° 57′ 60–33° 45′ 30 N latitude and 74°35′ 36–74° 21′ 20 E longitude (Fig. 2). The area is directly accessible from Srinagar via Khan Sahib, Beeru, Budgam, etc. Although the region can be reached via several routes, the current condition of roads is dilapidated, often acting as a bottleneck for tourism growth. As such, public transport service to the region is very inadequate and, as such, private taxis and personal cars are being used as a means of transportation. The sub-tropical climate of Doodhpathri is quite pleasing, and its temperature ranges between − 4 and 31 °C. An alpine valley surrounded by snow-capped mountains, lush green pastures carpeted with flowers in spring and summer, magnificent hills, evergreen fir forests, the sloping landscape dotted with shepherd huts, and the milky white flowing water of the Shaliganga stream through the meadows provides a rare view of juxtaposing both the natural and adventurous streams of tourist typologies in the area. The resort provides an opportunity for trekking, horse riding, angling, and river rafting beside lakes, glaciers, and religious shrines, adding tourist options. Doodhpathri, with its alluring beauty, attracts an increasing flow of tourists every year and is becoming one of the most sought-after places in Kashmir among tourists. The population of Doodhpathri is primarily migrant (Gujjars), though, in the surrounding sub-region, several hamlets and villages are found with a population of about 14,563 (2734 households), literacy rate of just 47%, and a sex ratio of 987 as per census 2011. The region's economy is mostly based on farming. More than 60% of the population is involved in subsistence farming, such as raising animals, collecting firewood, mining coal, which shows that the region has a fragile economic base.Fig. 2 Study area map (Doodhpathri, Kashmir, India) The survey instruments and data analysis The indigenous community's attitude and perspectives regarding tourism development effects and their enthusiasm for future tourism development were assessed using a quantitative research approach based on a self-reported questionnaire. As recommended by earlier studies, the investigation was conducted using a non-probability convenience sampling technique with 344 residents (Harun et al. 2018, p. 4; Charag et al. 2020, Section. iv; Korca 1998, p. 198; Teye et al. 2002, p. 673). The survey was developed in response to a thorough literature analysis of locals' attitudes and opinions on the impacts of tourism development, (Akis et al. 1996, pp. 486–487; Dyer et al. 2007, p. 413; Gursoy et al. 2002, p. 91; Gursoy and Rutherford 2004, p. 505; Johnson et al. 1994, pp. 636–635; Ko and Stewart 2002, pp. 524–525; Korça 1996, p. 696; Kuvan and Akan 2005, pp. 698–699; Liu and Var 1986, p. 200; Vargas-Sánchez et al. 2009, p. 378). As depicted in Fig. 2, the study was conducted at the tourist destination of Doodhpathri. It was based on personal interactions involving locals who were asked to participate by the researcher between May and June 2021. A pilot survey with 30 participants was conducted to determine the viability of the survey procedure. Some questions were dropped due to the pilot study's findings (e.g., tourism enhances soil erosion, exclusion of locals from natural resources, reduction in vegetation cover). The sample respondents interacted through a door-to-door survey, and a single respondent was considered from each household, thus making it more representative of the host indigenous community. According to the 2011 census, the area has 2734 households spread across 29 villages and hamlets. The optimum sample size for the present study by applying the formula (Yamane 1973) at a 95% confidence level was 349. However, only 344 valid responses (98.5%) were found valid for further analysis. The final survey instrument was categorized into three components. The first segment asked about residents’ demographic information, but no identities were revealed, preserving the interviewees' anonymity. The second section covers statements about tourism's environmental, socio-economic, and cultural components. Residents' viewpoints on the implications of tourism expansion were elicited through the use of 23 impact indicators. Participants were interviewed to rate these 23 impact statements on a 4-point Likert scale (1 = Strongly Disagree; 2 = Disagree; 3 = Agree; 4 = Strongly Agree). The third component of the survey focuses on people's willingness to participate actively in potential tourist development programs at Doodhpathri and comprises nine items. A descriptive statistical examination was performed to establish the indigenous population’s demographics and to compute the mean and standard deviation of all variables to reveal how the locals see and support potential tourism growth. The data were analyzed using SPSS version 16.0 (Statistical Package for Social Sciences). Exploratory factor analysis was used to evaluate the component structure of the items that represent the inhabitants' perspective of tourist development. To determine the underlying constructs, the 23 items were factor-analyzed using principal component analysis (PCA) and the varimax rotation method (Dolnicar and Grun 2008, p. 19; Hair et al. 2010). The Kaiser–Meyer–Olkin (KMO) sample appropriateness test and Bartlett's test of sphericity were used to determine the data fitness. The factor loading matrix was analyzed, and items with a factor loading of 0.45 or less and cross-loading on two or more items were removed from further analysis (Hair et al. 2010). Additionally, the Eigenvalues obtained for each factor were employed to support their retention for additional study. For each item, Cronbach's alpha reliability coefficient was determined to assess the scale’s internal consistency. Using descriptive statistics, which included the mean scores of each variable, the basic properties of the data set were described (Heung et al. 2010, p 0.248) as depicted in Table 2. Factor extraction is done using the Eigenvalue criteria, which must be greater than 1. Factor analysis extracted five components with factor loadings greater than 0.45, accounting for 60.48 percent of the total variance, as shown in Table 3. Four items in the exploratory factor analysis demonstrated considerable cross-loading with several factors and were eliminated from further analysis (Hair et al. 2016). So, as shown in Table 3, 19 of the 23 measurement items were loaded onto the five factors. Results Sample profile of the respondents (344) As shown in Table 1, most of the respondents comprised males (67.73%), whereas females made up (32.26%) of the whole sample. Most of the respondents (50.87%) were in the 18–35 age group; (35.17%) were in the 36–55 age group; and the rest (13.95%) were over 55. So far, the level of education is concerned, (8.13%) were postgraduates, (18.60%) were graduates, (46.22%) had up to secondary level education, and the rest (27.03%) had no formal education. Most of the respondents (60.17%) were in the income group of less than INR 5000 per month; (31.10%) of respondents were in the income group of INR 5000–20,000, and the rest (8.73%) had more than 20,000 per month. Thus, more than 92% of the respondents were in the low-income group.Table 1 Demographic profile of respondents (N = 344). Source: Primary Survey, 2021 Demographic characteristics Category N Percentage Sex Male 233 67.73 Female 111 32.26 Age 18–35 175 50.87 36–55 121 35.17 55 Above 48 13.95 Marital status Married 208 60.46 Unmarried 136 39.53 Education Postgraduate 28 8.13 Graduate 64 18.60 Up to secondary 159 46.22 No formal education 93 27.03 Income ≤ 5000 207 60.17 5000–20,000 107 31.10 ≥ 20,000 30 8.72 Descriptive statistics findings The outcome of the descriptive analysis for the 19 items is provided in Table 2. The outcome of the mean findings shows that certain products have a more significant impact on the community than others, while others have both favorable and detrimental effects. The advantages of tourism, for example, boost job opportunities (2.59), Meeting tourist is a valuable experience (2.88), tourism enhances the image of the area (2.76), generation of waste (2.77), changes in traditional culture (2.64), tourism increases the standard of living (2.89), tourism causes water quality deterioration (2.53), tourism increases recreational facilities (2.57). The impacts of tourism on waste generation, changing traditional cultures, and water quality deterioration are causes for concern. The relatively high standard deviation values manifest the non-uniformity of perceptions in the local community. Since the area is an emerging destination, the fruits of tourism development have not trickled down to the whole area. For such destinations, the disagreement is evident because only a tiny portion of the local community currently benefits from tourism and sees it as a boon. However, a significant portion of the population is yet to be exposed to the impacts of tourism. Overall, the mean values show that tourism is seen as a growing industry because most people in the area are more interested in its positive effects.Table 2 Descriptive analysis of Items. Source: Primary Survey, 2021 Variables/items Descriptive statistics Mean SD Tourism increases the Demand for labor 2.39 1.31 Tourism boosts job opportunities for local people 2.59 1.27 The expense of living rises as a result of tourism 2.11 1.19 Tourism increases household income 2.26 1.21 Prices are increasing because of tourism 2.61 1.28 Tourism interrupts the quiet life of the area 1.91 1.17 Tourism causes a change in traditional culture 2.64 1.33 Crime and social issues in the community are aggravated by tourism 1.87 1.19 Tourism enhances the area's image 2.76 1.30 Tourism development results in the destruction of agricultural lands 2.22 1.22 Tourism has led to water quality deterioration 2.53 1.30 Tourism led to the generation of waste products 2.77 1.32 Tourism enhances the well-being of the local community 2.50 1.30 Meeting tourists is a valuable experience 2.88 1.28 The Standard of living is increasing due to tourism 2.89 1.21 Tourism improves the quality of public services 1.85 1.17 Tourism increases recreational facilities in the area 2.57 1.36 Tourism increases traffic problems during peak season 2.03 1.20 Tourism increases congestion 1.61 1.08 Results of factor analysis To assess the dimensionality of 23 impact variables, the residents' attitudes toward tourism activities were assessed using principal component analysis (PCA). Upon applying the varimax rotation method, five factors were found to have eigenvalues greater than 1, explaining 60.48% of the overall variation. All components with an eigenvalue of > 1 were included. All components with a factor loading of more than 0.45 were considered for further testing. Hair et al. (2010) found that if the representative sample is higher than 150, a factor loading of 0.45 is sufficient. The results of Bartlett's sphericity test were noteworthy (Chi-square 2415.210, 0.0001). The Kaiser–Meyer–Olkin (KMO) sample adequacy score was 0.876, indicating that the data can be factored (Tabachnick and Fidell 1989, p. 614). The 19 variables' Cronbach alpha consistency is at 0.88, which is greater than 0.6, indicating that the data is eligible for further research (Kaiser 1974, p. 35). The higher the internal reliability of the variables in the scale, the closer the alpha coefficient is to 1.00 (Tabachnick and Fidell 1989). Tourism’s "economic effects" on the indigenous community are the first aspect to consider. It explains 34.63% (Table 3) of the overall variance and has a mean of 2.39; SD = 1.252, which holds an overall positive view. The results indicate that the demand for labor (factor loading 0.702), increasing job opportunities (factor loading 0.696), and increasing household income (factor loading 0.657) has a favorable effect on the host community. Most residents viewed it as a developmental opportunity for their community to improve their livelihood; besides the favorable economic impacts, most residents (Mean = 2.61, SD = 1.28) showed a negative attitude toward increasing prices because of tourism (Factor loading 0.500).Table 3 Factor analysis of tourism evaluation (Extraction method: PCA; Rotation method: varimax with Kaiser Normalization; Rotation convergence in 14 iterations). Source: Primary Survey, 2021 Eigenvalue Variance% Factor Items Factor loading Communalities 6.58 34.63 Economic Impacts α = 0.80 Tourism increases the demand for labor 0.702 0.631 Tourism boosts job opportunities for local people 0.696 0.683 The expense of living rises as a result of tourism 0.688 0.570 Tourism increases household income 0.657 0.549 Prices are increasing because of tourism 0.500 0.511 1.63 8.60 Social and Cultural impacts α = 0.77 Tourism interrupts the quiet life of the area 0.761 0.636 Tourism causes a change in traditional culture 0.617 0.636 Crime and social issues in the community are aggravated by tourism 0.587 0.573 Tourism enhances the area's image 0.455 0.532 1.15 6.05 Environmental impacts α = 0.71 Tourism development results in the destruction of agricultural lands 0.802 0.675 Tourism has led to water quality deterioration 0.564 0.549 Tourism led to the generation of waste products 0.551 0.560 1.09 5.77 Life quality impacts α = 0.69 Tourism enhances the well-being of the local community 0.628 0.729 Meeting tourists is a valuable experience 0.607 0.705 The Standard of living is increasing due to tourism 0.597 0.625 Tourism improves the quality of public services 0.515 0.480 Tourism increases recreational facilities in the area 0.475 0.460 1.02 5.41 Overcrowding impacts α = 0.63 Tourism increases traffic problems during peak season 0.796 0.736 Tourism increases congestion 0.777 0.692 Total variance 60.48% The second factor, reckoned as "social-cultural impacts" of tourism, explains 8.60% of the total variance (Table 3), with a mean of (2.29; SD 1.31) loaded with four variables showing the perceived social effects of tourism. Given the socio-cultural point of view, indicators ‘Tourism interrupts the quiet life of the area', with a mean score of 1.91, factor loading 0.761, does not believe that tourism has any effect on their quiet life and has a friendly perception toward the growth of tourism in their region. Besides, from the perspective of evaluating social problems and tourism, residents’ perception signified the least impact, with (mean = 1.87; factor loading 0.587) perceiving tourism as a boon to their area. Furthermore, residents were in favorable agreement toward tourism development as tourism improves the ‘image of the area', having mean = 2.76; SD = 1.30. Concerning the preservation of traditional culture and tourism impacts, the perception of residents was mainly inclined toward the changing cultural signifiers and community values vis-à-vis tourism activities with a mean score of 2.64; Factor loading 0.617. The third factor, entitled "Environmental impacts," comprises three factors (destroying agricultural lands, water quality deterioration, and waste generation), registered 6.05 percent of the variation and had a (mean = 2.50; SD = 1.30). As per statistics, inhabitants in Doodhpathri witnessed significant adverse effects on the environment of tourism, particularly water quality degradation (mean 2.53; SD = 1.30) and garbage creation (mean 2.77; SD = 1.32). A portion of these negative impacts was due to the generation of solid waste, including water bottles, glass bottles, waste paper, and polyethylene bags throughout the peak season. The lack of dustbins at appropriate locations and waste management plans has led to the generation of waste products, impacting tourist satisfaction and water quality deterioration. The fourth factor, named "Life quality effects," consists of five variables and accounts for 5.77% of the variance with a (mean = 2.53; SD = 1.32). This is a constructive aspect since the residents of Doodhpathri have realized the benefits of tourism in their livelihoods. The sub-indicators viz., Tourism improves their quality of life (mean 2.50; factor loading 0.628), Meeting tourists is a valuable experience (mean 2.88; factor loading 0.607), Standard of living is increased by tourism (mean 2.89; factor loading 0.598), and Public facilities improvement (mean 2.57; factor loading 0.515) indicate that the locals believe that the growth of tourism has led to improved infrastructure and services like roads, shopping facilities, telecommunications, and public services that have been significantly enhanced by tourism. The fifth factor, labeled as "overcrowding impacts" of tourism with a 5.41% variance, having a mean score (mean = 1.82; SD = 1.16) concurs that there is no concern regarding the impacts of "traffic congestion’ during peak season (mean 2.03; Factor loading 0.796) and crowding (mean 1.61; Factor loading 0.777) on the local community. The residents were constructive regarding this factor and believed that tourism in Doodhpathri does not promote overcrowding issues. Tourism facilities should be increased to minimize the effects of the seasonality of visitation and explore the potential of tourism at Doodhpathri. Residents' support for potential tourism development The evaluation of the findings in Table 4 revealed that overall residents (mean 3.39, SD = 0.739) favor future tourism development in Doodhpathri. Most residents believed tourism should be actively developed (mean of 3.47) and were in favor of additional tourism facilities (mean of 3.42) so that more tourists would come to this destination. Moreover, residents perceive tourism as an essential part of their community (mean 3.40) and are in support of its long-term prosperity (mean 3.48).Table 4 Support for future tourism development (N = 344). Source: Primary survey, 2021 Items Descriptive statistics Mean (3.39) SD (0.739) Tourism in Doodhpathri should be actively developed 3.47 0.652 I' I will encourage new attractions for tourism that will draw more visitors to my region 3.42 0.729 I support tourism as an important part of my community 3.40 0.626 Tourism is vital for the long-term prosperity of my community 3.48 0.728 I believe that tourism will be crucial to boost our local economy 3.34 0.747 At Doodhpathri, the residents may have a crucial part in the creation of plans and projects 3.33 0.732 The neighborhood helps to address issues related to tourism growth 3.37 0.845 The positive benefits out weight the negative impacts 3.39 0.728 The future of Doodhpathri as a tourist destination is sustainable 3.33 0.824 Discussions The study results reveal that the indigenous community is well aware of the impact of tourism on their environment and significantly appreciates the positive ones. The study results illustrate that the income generated from tourism is made by local authorities and trickles down to the residents. The results of the economic impact of tourism were the same as in other studies concluding that the economic effects of tourism development are predominantly seen positively (Gössling 2001, pp. 436–437; Gursoy et al. 2010, p. 9; Samuelsson and Stage 2007, p. 51). Various studies, for instance, yielded comparable results (Hammad et al. 2017, section. discussion) in Abu Dhabi; Andereck et al. (2005, p. 1067) in the USA; Andereck and Nyaupane (2011, p. 257) in Arizona; Stylidis et al. (2014, p. 269) in Greece Latkov and Vogt (2012, p. 60) in Midwest State, USA; Rasoolimanesh et al. (2016, p. 11) in Malaysia, concluded that locals have a favorable view of the economic benefits of tourism. However, there were some negative impacts as perceived by residents, this may be attributed to the increasing land value, the cost of labor, and the exclusion of local control over the local resources, but overall, the locals at Doodhpathri agreed that tourism has a significant contribution by providing job opportunities, demand for labor, increasing household income, and the potential to propel the economy of the local community. This role of tourism development is perceived as the most significant benefit, as it offers the inhabitants job prospects and other economic benefits (Choi and Sirakaya 2005, p. 388). It is also argued that the positive argument in favor of the development of tourism in any region is related to the economic impact the local community is expected to achieve (Brankov et al. 2019, p. 136; Dyer et al. 2007, pp. 416–417). Regarding social and cultural impacts, the locals have a positive attitude toward tourism development. This may be due to the cultural fluxes of different consumption patterns, shifting occupational patterns, fashion, the commercialization of host community culture, diminishing community interconnection, and more (Boissevain 1979, p. 87; Choi and Sirakaya 2005, p. 388; Eraqi 2007, p. 195; Latip et al. 2018, section “discussion, Para. V”). This impact on changing traditional culture can be preserved by organizing cultural events and services and educating cultural consciousness, specifically among younger generations, which in turn will maintain the community’s unique traditional fabric. Overall, the analysis demonstrates that locals were highly apprehensive about tourism's impact on their culture. Similar results corroborate the findings of Brunt and Courtney (1999, p. 509), demonstrating that residents' ascribing tourism's social implications is significant. This might be due to the residents' sensitivity to undesirable socio-cultural costs compared to the predicted economic advantages of tourist flow. The residents show a negative perception of the environmental concerns, which appear to be more worrying given the contemporary emphasis on eco-tourism and sustainable tourism research methods.. These impacts were similar to the results of numerous studies (Aref et al. 2009, p. 133; Cohen 1978, p. 225; Bagri and Kala 2016, p. 31). Furthermore, as per KO and Stewart (2002), tourist growth has a detrimental influence on the natural environment and is intrinsically associated with changes in the quality of water and waste generation. The thrust of these works has been on the physical effects of tourism, like water quality deterioration, the production of waste, and other types of pollution. These problems associated with environmental impacts are also urged in the Doodhpathri Master Plan 2011–32. As a result, future tourism growth possibilities in Doodhpathri might face major obstacles if locals' attitudes are not handled appropriately. The locals at Doodhpathri believe that tourism has improved infrastructure and services like roads, shopping facilities, telecommunications, and public services. Residents also consider tourism an opportunity to meet people from diverse cultures. The results indicate that tourism offers the locals of Doodhpathri a feeling of community well-being and pleasure as their living standards have been significantly enriched due to tourism. Further, the results revealed how the growth of tourism at Doodhpathri has improved the living standards of the indigenous community as their standard of life has increased, public facilities such as roads, communication, and attractions, and meeting people of diverse cultures. According to the studies, the promotion of tourist products in any territory has a remarkable influence on the living standards of the local inhabitants (Andereck and Nyaupane 2011, p. 250). Because of their area’s booming tourism market, inhabitants could experience other festivals, hotels, and attractions. These results were consistent with similar works (Dyer et al. 2007, p. 418; Nunkoo and Ramkissoon 2010, p. 48). However, dissatisfaction with recreational facilities at Doodhpathri may be attributed to its initial stage of development, and addressing issues such as developing more recreational facilities needs a multifaceted, long-term approach so that the resident's quality of life will be enhanced and will attract more and more tourists, keeping in consideration the sustainability of Doodhpathri. Tourism at Doodhpathri is viewed as an economic driver, and its positive effects outweigh its negative impacts. As stated by Gursoy and Rutherford (2004, p. 508), the host community favors tourism growth if they see tourism as a way to produce revenue and generate jobs. Further, the residents perceive that Doodhpathri Development Authority has promoted the destination efficiently and is in support of providing knowledge about the destination while developing tourism strategies and plans, and accept that the future of Doodhpathri is sustainable. Generally speaking, the present study found that the local indigenous people perceived more positives and supported tourism growth in their area. According to the existing literature, residents' support for tourism development is determined by how they perceive the costs and benefits of it, which can result from a wide range of social, economic, and environmental factors (Rasooolimanesh et al. 2015, p. 338, Zuo et al. 2017, p. 52). The current study’s findings align with these findings and support SET. Social exchange theory makes assumptions about locals based on their experiences with the cost and benefits in the context of tourism. Positive impact perceptions were found to have a significant relationship with support for tourism due to the various benefits tourism brings to the community. Economic benefits are reported to be the most critical determinants of positive perceptions and support for future tourism development before the start of tourism growth; it is pivotal to ascertain the local inhabitants' attitudes and expectations regarding the expansion of tourism as well as the trade-off between the costs and advantages of such development. Butler’s (1980) “Tourism Area Life Cycle Model” explains the tourist destinations' life cycle in six stages namely, exploration, involvement, Development, consolidation, stagnation, and decline or rejuvenation. Butler's model predicts that the impacts of the tourism sector will alter over the various stages of tourism development. As a result, host perspectives will differ over the various development stages (Butler 1980, pp. 6–9). Residents' perceptions of the destination phase of the life cycle model confirm that Doodhpathri is in either the involvement stage of the destination life cycle since they indicated how excited visitors were to visit Doodhpathri. This research confirms that there were particular worries about tourism growth, which locals thought had both favorable and detrimental implications on their quality of life. Before beginning new tourism initiatives or modifying current destinations, developers and policymakers must know the dynamics of these effect elements and how the residents perceive them. Balancing the benefits and drawbacks of tourism development is critical, so it is necessary to gauge how locals perceive it regularly. Tourist planners must develop the optimum solutions for sustainable tourism growth to strengthen the aspects seen favorably. Additionally, they should work to minimize the effects of any issues or components that are viewed negatively. For policymakers, the present study has relevance as the indigenous community feels that tourism has led to negative environmental effects, and a specific environmental policy concerning the issue must be framed and implemented. Additionally, environmental education, waste management programs, resource conservation, etc. can improve locals' perceptions and trust in tourist growth. That will make it possible for tourism planners to comprehend the feelings of those who come into contact with tourists directly and give them essential information for developing strategic tourism development policies. The present study broadens the attention on the indigenous community's perceptions of the impact of tourism development by reviewing their insight on where there is the transition of the economy from being primarily agrarian to an emerging tourist industry. As an emerging tourist destination located in an ecologically fragile area, the study’s findings will form the basis for planners to formulate policies in consultation with the local bodies, as they are the stakeholders whose local knowledge will be of immense value. Doodhpathri has the potential to be the gateway to a plethora of potential tourist destinations like Diskhal, Dander, Ashtar, Pal maidan, Bargah, and Tosai maidan in the middle Himalayas. Due to its virginity, Doodhpathri has a huge potential that, if properly exploited, can enhance the economic development of the region as well as the poverty reduction strategy in the region. It is imperative that its biotic potential is properly analyzed and the infrastructure development to suit the region’s long-term sustainability. Besides, the destination provides visitors with a unique experience of exotic climate, beautiful landscape with bountiful high altitude meadows surrounded by dense, lush green forests, gushing milky waters of Shaliganga and Sukhnag, opportunities for adventure tourism, angling, pony ride, local indigenous food, and many more things. To maintain the socio-ecological fabric of the destination, the development of this ecologically sensitive area should be along the lines of sustainable development. This study has several limitations that must be addressed to provide further research opportunities. Because the study's data was limited to villages near/around the study area, the results could not be generalized to other villages. Further, it is possible if research could be conducted on individual demographic factors like age, gender, the status of occupation, education status, and duration of residence to establish whether there are any disparities or commonalities that may exist, resulting in a practical framework for a comprehensive and sustainable tourism approach. Conclusions Despite Doodhpathri being on the tourist map of the Kashmir Valley over the last decade and experiencing remarkable growth in tourism during recent years, no study has examined its potential in the regional tourism sector. This study focused on examining residents' opinions and experiences toward tourist development, which is still underexplored to its full potential. This study was conducted in response to concerns about potential tourism effects research in developing tourist destinations. The present study aimed to learn more about locals’ perceptions regarding tourism development implications and their support for potential sustainable tourism development at Doodhpathri, providing valuable inputs and recommendations for establishing preliminary data on visitor impacts on the local inhabitants in the comprehensive development plans. From a broader viewpoint, the study’s outcomes reveal that the indigenous community perceives that tourism development has brought many more positive impacts than negative impacts to the destination. The research output indicates that tourism development at Doodhpathri boosted economic avenues in terms of employment generation and household income and strengthened socio-cultural benefits and living standards indicators. Regarding local opinions and attitudes regarding the growth of tourism, the findings of this study will act as a road map for tourism regulators in locations that are still in the early stages of the process. The study findings have revealed significant evidence of local support for future tourist promotion. Planners should also be conscious that how locals perceive these effects are determined by a variety of factors, including the destination's economic situation, cultural values, and level of development, so to attain residents' acceptance, policymakers and entrepreneurs must strike a careful balance between residents' priorities and commercial priorities judiciously. Furthermore, since the nature of the destination may fluctuate over time, people's evaluations of tourist externalities and acceptance of future tourism growth must be monitored regularly. That will allow the relevant authorities to work with the native community to develop comprehensive plans and policy decisions for the overall sustainability of the destination. Authors’ contribution The research was conceived and supervised by MSB. MSW conducted the field survey, carried out the data analysis, and wrote the first draft. AA edited and revised the manuscript. SAM helped with the field survey and proofreading. All authors agreed to the present form of manuscript. Funding The author is highly indebted to the University Grants Commission (UGC-MANF) in New Delhi, India, for funding this research. Declarations Conflict of interest No potential competing interest was reported by the authors. 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==== Front Anaesthesiologie Anaesthesiologie Die Anaesthesiologie 2731-6858 2731-6866 Springer Medizin Heidelberg 36454255 1221 10.1007/s00101-022-01221-9 Leitthema Thrombotische Mikroangiopathien in der operativen Intensivmedizin unter besonderer Berücksichtigung des atypischen hämolytisch-urämischen Syndroms Thrombotic microangiopathies in surgical intensive care medicine with special consideration of atypical hemolytic uremic syndromeAlbrecht Sebastian [email protected] 1 Kamla Christine E. 2 Schönermarck Ulf 3 Wassilowsky Dietmar 1 1 grid.411095.8 0000 0004 0477 2585 Klinik für Anaesthesiologie, LMU Klinikum, Marchioninistr. 15, 81377 München, Deutschland 2 grid.411095.8 0000 0004 0477 2585 Herzchirurgische Klinik und Poliklinik, LMU Klinikum, München, Deutschland 3 grid.411095.8 0000 0004 0477 2585 Medizinische Klinik und Poliklinik IV/Nephrologisches Zentrum, LMU Klinikum, München, Deutschland 1 12 2022 19 17 10 2022 © The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Thrombotische Mikroangiopathien (TMA) sind seltene Krankheitsbilder, die durch die Trias mikroangiopathische hämolytische Anämie, Thrombozytopenie und eine Organschädigung definiert sowie mit einer hohen Morbidität und Letalität verbunden sind. Insbesondere im perioperativen und im intensivmedizinischen klinischen Alltag sind das Erkennen einer TMA („daran denken“) und die Abgrenzung wichtiger Differenzialdiagnosen die entscheidenden Faktoren. Die weitere Eingrenzung der TMA-Syndrome (thrombotisch-thrombozytopenische Purpura [TTP], hämolytisch-urämisches Syndrom durch shigatoxinbildende Escherichia coli [STEC-HUS], atypisches hämolytisch-urämisches Syndrom [aHUS]) anhand der zugrunde liegenden pathophysiologischen Prozesse und die Entwicklung zielgerichteter Therapien haben die Prognose in den letzten Jahren deutlich verbessert. Die rasche Diagnosestellung und Therapieeinleitung gelingen nur im Rahmen einer engen interdisziplinären Zusammenarbeit zwischen Intensivmedizinern und in der Therapie der TMA erfahrenen Fachdisziplinen. Thrombotic microangiopathies (TMA) are rare diseases, which are defined by the triad microangiopathic hemolytic anemia, thrombocytopenia and organ damage, and are associated with high morbidity and mortality. The recognition of a TMA and the distinction of important differential diagnoses are key factors, particularly in a perioperative context and in routine clinical intensive care. The further differentiation of the different TMA subtypes, such as thrombotic thrombocytopenic purpura (TTP), Shiga toxin-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) and atypical hemolytic uremic syndrome (aHUS), based on the underlying pathophysiology and the development of new targeted treatment options in recent years have significantly improved the prognosis. A close interdisciplinary cooperation between critical care specialists and specialist disciplines experienced in the treatment of TMA, is essential for a prompt diagnosis and the initiation of the appropriate treatment. Schlüsselwörter Thrombozytopenie Anämie Niereninsuffizienz Eculizumab Intensivmedizin Keywords Thrombocytopenia Anemia Eculizumab Renal insufficiency Critical care ==== Body pmcDas Auftreten einer Thrombozytopenie, einer Anämie oder eines akuten Nierenversagens in der operativen Intensivmedizin, insbesondere infolge großer Eingriffe oder nach Operationen bei Patienten mit hoher Komorbidität, ist selbst in Kombination keine Seltenheit und erscheint nicht außergewöhnlich. Jedes dieser Krankheitsbilder kann jedoch, für sich genommen, verschiedene, mannigfaltige Ursachen haben. Das wiederholte gleichzeitige Auftreten dieser Symptome im klinischen Alltag einer herzchirurgischen Intensivstation, häufig in Kombination mit qualitativen Bewusstseinsveränderungen, lenkte die Aufmerksamkeit der Autoren des vorliegenden Beitrags zu den seltenen Erkrankungen aus dem Formenkreis der thrombotischen Mikroangiopathien (TMA) und im Speziellen zum atypischen hämolytisch-urämischen Syndrom (aHUS) [10]. Diese Publikation soll den aktuellen Wissensstand hinsichtlich Ätiologie, Diagnostik und therapeutischen Optionen, ergänzt durch eigene klinische Erfahrungen, wiedergeben, den Leser für die zu den TMA gehörenden Syndrome, insbesondere das aHUS, sensibilisieren sowie eine Hilfestellung bei der Diagnostik und Therapie im klinischen Alltag geben. Einführung Das aHUS ist eine seltene, aber mit hoher Morbidität (insbesondere der terminalen Niereninsuffizienz) und Letalität einhergehende Erkrankung der kleinsten Blutgefäße, die dem Formenkreis der TMA zuzurechnen ist. Diese sind durch die Trias aus Thrombozytopenie, mikroangiopathischer Hämolyse und Organschädigung charakterisiert. Der Begriff thrombotische Mikroangiopathie beschreibt die gemeinsame pathophysiologische Endstrecke – die Thrombosierung kleinster Gefäße mit konsekutiver Ischämie im Endstromgebiet des jeweils betroffenen Organs. Merke. Eine TMA ist durch das Vorliegen einer Coombs-negativen hämolytischen Anämie und Thrombozytopenie in Kombination mit der Dysfunktion mindestens eines Organs definiert. Wenngleich sich die verschiedenen TMA-Formen klinisch mit ähnlichen Laborkonstellationen und überlappenden klinischen Symptomen präsentieren, zeigen sich klare Unterschiede hinsichtlich der Pathogenese. Das verbesserte pathophysiologische Verständnis hat in den letzten Jahren zunehmend Einfluss auf die Nomenklatur und spezifische therapeutische Strategien gefunden. Trotz der stetigen Erkenntnisgewinne bestehen jedoch teilweise uneinheitliche Terminologien [14]. Im klinischen Alltag sind das Erkennen einer TMA und die Abgrenzung wichtiger Differenzialdiagnosen essenziell. Diese eigentliche Herausforderung ist jedoch nicht einfach zu bewältigen, da nicht für jedes TMA-Syndrom spezifische Tests existieren oder in der Klinik schnell verfügbar sind. Entsprechend den unterschiedlichen pathophysiologischen Vorgängen können folgende 3 Syndrome unterschieden werden:die thrombotisch-thrombozytopenische Purpura (TTP), das („typische“) hämolytisch-urämische Syndrom (HUS) durch shigatoxinbildende Escherichia coli (STEC-HUS) und das atypische hämolytisch-urämische Syndrom (aHUS). Im Folgenden wird der Begriff atypisches hämolytisch-urämisches Syndrom nach Ausschluss von TTP und STEC-HUS für alle anderen Formen der TMA verwendet [2, 9], um eine im klinischen Alltag praktikable Einteilung zu präsentieren. Eine Einteilung in primäre und sekundäre Formen entsprechend der zugrunde liegenden Ursachen und Triggerfaktoren ergibt sich häufig erst im weiteren Krankheitsverlauf. Der Terminus atypisch soll keinesfalls dazu verleiten, eine besonders seltene Entität einer TMA zu vermuten, vielmehr werden hierunter „historisch bedingt“ verschiedenste Krankheitsbilder subsumiert, die weder STEC-HUS noch TTP sind. Diese sehr heterogene Gruppe des aHUS stellt jedoch mit einer relativen Inzidenz über 60 % den größten Anteil aller diagnostizierten TMA-Syndrome beim Erwachsenen dar [25]. Entsprechend den zugrunde liegenden Ursachen bzw. auslösenden Triggerfaktoren kann diese Krankheitsgruppe weiter unterteilt werden. Mit einer geschätzten absoluten Inzidenz von 0,2–2 Patienten/1 Mio. Einwohner zählt das aHUS zu den seltenen Erkrankungen („orphan disease“; [6, 29]). Pathophysiologie Allen TMA-Syndromen gemeinsam sind Endothelschäden und Thrombosen kleinster Gefäße. Hierdurch kommt es zu einer mechanischen („mikroangiopathischen“) Coombs-negativen Hämolyse sowie zum Verbrauch von Thrombozyten. Abhängig von dem oder den betroffenen Organsystemen können verschiedenste Symptome bzw. Organinsuffizienzen resultieren, am häufigsten sind Nieren und Zentralnervensystem (ZNS) betroffen [8]. Thrombotisch-thrombozytopenische Purpura Der TTP liegt ein Mangel der Von-Willebrand-Faktor-spaltenden Protease „a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13“ (ADAMTS-13) zugrunde. Dieser geht mit einer Anhäufung von ungewöhnlich großen Von-Willebrand-Faktor-Molekülen („ultralarge von Willebrand factor [UL-vWF] multimers“) einher, was zur gesteigerten Thrombozytenaggregation und Thrombosierung kleinster Gefäße führt. Der ADAMTS-13-Mangel kann im Sinne genetischer Mutationen angeboren oder – häufiger – erworben sein, Letzteres durch Vorliegen von Autoantikörpern gegen ADAMTS-13. Verglichen mit anderen TMA-Syndromen, stehen bei der TTP neurologische und kardiale Symptome im Vordergrund; schwere Nierenfunktionseinschränkungen sind selten [2]. Mithilfe der Bestimmung der ADAMTS-13-Aktivität kann die TTP sicher und schnell diagnostiziert werden. Dieser Test ist inzwischen in vielen Laboren auf Anforderung erhältlich. Merke. Für die Diagnose einer TTP ist eine fehlende oder stark verminderte ADAMTS-13-Aktivität (< 5–10 %) pathognomonisch. Hämolytisch-urämisches Syndrom durch shigatoxinbildende Escherichia coli Beim STEC-HUS wird der Endothelschaden durch die Bindung von in der Blutbahn zirkulierendem Shigatoxin – meist produziert von enterohämorrhagischen Escherichia coli (EHEC) – an Endothelzellen der Nieren eingeleitet. Neben der meist blutigen Diarrhö ist eine Nierenbeteiligung bis hin zur Dialysepflichtigkeit eine typische Manifestation. Das STEC-HUS tritt häufiger im Kindes- als im Erwachsenenalter auf. Merke. Die Diagnose eines STEC-HUS erfolgt aufgrund des Nachweises von Shigatoxin im Stuhl. Atypisches hämolytisch-urämisches Syndrom Beim aHUS können primäre und sekundäre Formen unterschieden werden. Insbesondere zu Beginn ist eine eindeutige pathophysiologische Zuordnung jedoch häufig nicht möglich. Verschiedene endo- und exogene Faktoren führen zu einer überschießenden Aktivierung des alternativen Komplementwegs und einer komplementvermittelten Endothelschädigung. Das Komplementsystem ist Teil des angeborenen Immunsystems, und die feine Balance dieses kaskadierten Systems durch aktivierende und inhibierende Faktoren ist für seine korrekte Funktion unerlässlich [22]. Auf dem Boden einer endogenen Prädisposition im Komplementsystem können komplementverstärkende Trigger eine TMA-Manifestation auslösen. Je nach Stärke der genetischen Prädisposition ist ein geringer oder starker exogener Trigger notwendig (Abb. 1). Bei Patienten mit primärem aHUS liegt eine klare genetische Prädisposition für eine komplementvermittelte TMA vor. Diese kann zum „loss of function“ inhibierender Faktoren (v. a. Faktor H, Faktor I oder „membrane cofactor protein“) bzw. zum „gain of function“ aktivierender Faktoren (Faktor B oder C3) führen. Selten lösen erworbene Autoantikörper gegen Faktor H eine ungebremste Aktivierung des Komplementsystems aus. Allerdings kann eine entsprechende Genmutation nur bei etwa 50–70 % der Patienten nachgewiesen werden [6, 23]. Als potenzielle Triggerfaktoren („komplementaktivierende Faktoren“) konnten verschiedene Medikamente (insbesondere Immunsuppressiva und Chemotherapeutika) und Infektionen (v. a. virale, z. B. mit „human immunodeficiency virus“ [HIV], Hepatitisviren, Zytomegalievirus [CMV], Influenzaviren oder „severe acute respiratory syndrome coronavirus 2“ [SARS-CoV-2] [15]) identifiziert werden. Auch Tumorleiden, Schwangerschaft oder große Operationen können als Trigger wirken [13]. Im Gegensatz zum STEC-HUS kann ein aHUS – einmal in Gang gesetzt – selbst nach Kontrolle und anschließender Abwesenheit des auslösenden Triggers (unbehandelt) fortschreiten. Aus dem eigenen klinischen Schwerpunkt, der intensivmedizinischen Behandlung herzchirurgischer Patienten, konnte im postoperativen Verlauf mit bis zu 5 % ein gehäuftes Auftreten von aHUS an einem Kollektiv von 245 Patienten nach Eingriffen an der herznahen Aorta beobachtet werden. Als unabhängige Risikofaktoren wurden das weibliche Geschlecht und der (Mit‑)Ersatz der Aortenklappe identifiziert. Außerdem scheint ein bereits erfolgter Einsatz der Herz-Lungen-Maschine im Rahmen einer kardialen Voroperation eine Schlüsselrolle in der Entwicklung eines postoperativen aHUS zu spielen, weitere Untersuchungen in dieser Hinsicht sind jedoch noch notwendig. Über 80 % dieser Patienten erlitten ein z. T. ersatzpflichtiges Nierenversagen. Mithilfe adäquater, aufwendiger Therapie kann jedoch ein Outcome erreicht werden, das dem der Patienten ohne aHUS vergleichbar ist [13]. Es muss hervorgehoben werden, dass der Nachweis einer genetischen Mutation nicht für die Diagnosestellung erforderlich ist. Auch steht diese Information erst später im Verlauf zur Verfügung. Nach exogenen Triggerfaktoren muss aktiv gesucht werden, da diese oft therapeutisch beeinflusst werden können. Merke. Für die Diagnose eines aHUS gibt es bis jetzt keinen in der Routine verfügbaren Labortest. Endogene Risikofaktoren und externe Trigger sind von Bedeutung. Klinische Präsentation und Diagnostik Abgrenzung wichtiger Differenzialdiagnosen Die Diagnose einer TMA im perioperativ-intensivmedizinischen Setting stellt nicht nur durch ihre Seltenheit und das Fehlen eines spezifischen Testverfahrens eine besondere Herausforderung dar, sondern auch, weil sie sich klinisch-phänomenologisch und dank ihrer Variabilität hinter weit gängigeren Symptomen verbergen kann. Anämie, Thrombozytopenie und eine Nierenfunktionsverschlechterung treten, auch in Kombination, nicht selten auf. Die Schwierigkeit besteht darin, überhaupt an diese seltene Differenzialdiagnose zu denken und unter dem Häufigen das Besondere zu erkennen. Schon die Bestimmung von Hämolyseparametern (Laktatdehydrogenase [LDH], Haptoglobin und Bilirubin) ist nicht immer Bestandteil der Routinediagnostik. Wesentliche Differenzialdiagnosen, die im jeweiligen klinischen Kontext betrachtet werden müssen, sind z. B.:Sepsis und disseminierte intravasale Koagulopathie, autoimmune Hämolyse und autoimmune Thrombozytopenie, hämorrhagische Fiebersyndrome und virale Infektionen, Malaria und Babesiose, Endokarditis und herzklappeninduzierte Hämolyse, katastrophales Antiphospholipid-Antikörper-Syndrom, schwere Vitamin‑B12-/Folsäuremangelzustände oder Thrombozytopenie und Hämolyse durch extrakorporale Verfahren. Vor der Applikation von Heparin muss das Vorliegen einer Heparin-induzierten Thrombozytopenie (HIT) ausgeschlossen werden. Merke. Im klinischen Alltag sind das Erkennen einer TMA („daran denken“) und die Abgrenzung wichtiger Differenzialdiagnosen besonders wichtig. Diagnostischer Algorithmus Im Folgenden wird es v. a. um die Diagnose und Therapie eines aHUS gehen, da STEC-HUS und TTP im perioperativen Setting deutlich seltener auftreten. An entsprechender Stelle wird aber auf die notwendige Diagnostik zur Abgrenzung der Syndrome eingegangen. Derzeit existieren keine spezifischen diagnostischen Tests, um die Diagnose eines aHUS zu stellen. Die Diagnosestellung ist komplex und erfolgt in mehreren Schritten. Sie beginnt mit dem Erkennen der Symptomentrias aus hämolytischer Anämie, neu aufgetretener Thrombozytopenie und einer Organschädigung, in der Mehrzahl der Fälle einer akuten Nierenfunktionsverschlechterung. Weitere Organinsuffizienzen sind möglich; oft tritt eine Bewusstseinsstörung hinzu, insbesondere in Form eines Delirs. In seltenen Fällen ersetzt Letzteres das Nierenversagen. Die potenzielle Mitbeteiligung weiterer Organe ergibt sich aus der systemischen Ätiologie, was auch die Variabilität der Erkrankung ausmacht. Merke. Der Verdacht auf das Vorliegen eines aHUS erhärtet sich insbesondere dann, wenn eine Anämie mit ausgeprägten Hämolysezeichen (meist erhöhte LDH-Konzentration als Routineparameter), ein für den klinischen Verlauf überraschend abruptes, anurisches Nierenversagen und/oder eine transfusionsrefraktäre, ausgeprägte Thrombozytopenie bestehen. Die folgenden Laboruntersuchungen dienen der Untermauerung der Diagnose einer TMA, der Abgrenzung von Differenzialdiagnosen und der Unterscheidung der einzelnen TMA-Formen: Laborchemisch sticht zuerst meist die stark erhöhte LDH-Konzentration als Zeichen der Hämolyse ins Auge, da dieser Parameter zum Portfolio des postoperativen Labors vieler Intensivstationen gehört. Nicht pathognomonisch für die mikroangiopathische Hämolyse, doch richtungweisend ist das Vorkommen von Fragmentozyten (Synonym Schistozyten) im Blutausstrich. Weitere Hämolyseparameter – erniedrigter Haptoglobinwert, erhöhte Konzentration des (indirekten) Bilirubins – unterstützen den Verdacht auf eine Hämolyse. Eine immunologische Genese der Anämie (z. B. autoimmunhämolytische Anämie) sollte mithilfe des direkten Antiglobulintests (Synonym Coombs-Test) ausgeschlossen werden; andere mögliche Auslöser einer Hämolyse wie eine medikamentös-toxische oder mechanische Ursache sollten beachtet werden. Die Thrombozytopenie ist oft ausgeprägt (Thrombozytenzahl < 50 G/l), kann jedoch auch nur gering ausfallen. Im Gegensatz zum aHUS beträgt die Thrombozytenzahl bei der TTP regelhaft < 30 G/l. Geradezu typisch ist ein nur geringer oder fehlender Anstieg der Thrombozytenzahl nach einer Transfusion von Thrombozytenkonzentraten. Differenzialdiagnostisch sind im intensivmedizinischen Setting eine HIT, der Verlust von Thrombozyten im Rahmen einer Blutung oder der Verbrauch bei disseminierter intravasaler Koagulopathie (DIC) sowie seltener das Vorliegen von antithrombozytären Allo- oder Autoantikörpern zu bedenken und auszuschließen. Wenn der Verdacht eines aHUS besteht, sollten weitere Thrombozytentransfusionen äußerst restriktiv erfolgen. Dies gilt zum einen, um den Pathomechanismus nicht weiter zu unterhalten. Zum anderen kommt es gemäß der Erfahrung der Autoren beim aHUS mit einer Thrombozytenzahl oberhalb 20 G/l auch im unmittelbar postoperativen Kontext nur selten zu Blutungen, sofern sonst regelrechte hämostaseologische und chirurgische Bedingungen gegeben sind. Das Nierenversagen beim aHUS sticht klinisch nicht selten gerade dadurch hervor, dass es, gemessen am klinischen perioperativen Verlauf, überraschend abrupt und häufig anurisch verläuft. Die empirische Volumengabe oder diuretische Stimulation – wie so häufig alltägliche Praxis – erzielt dann nicht den erwarteten Effekt. Selbstredend müssen dennoch die gängigen Ursachen eines akuten Nierenversagens (prä-, post- bzw. intrarenal) in Erwägung gezogen und mit geeigneten diagnostischen Maßnahmen ausgeschlossen werden. Die Einleitung einer Nierenersatztherapie ist trotz rascher Diagnostik häufig notwendig. Der Zeitpunkt ihrer Initiierung unterliegt den sonst üblichen Kriterien und hat keinen prognostischen Einfluss auf den Verlauf, weder den des Nierenversagens noch des aHUS insgesamt. Circa 20 % der Patienten weisen extrarenale Symptome auf, wovon zentralneurologische die häufigsten sind [27]. Diese äußern sich entweder als Delir oder Vigilanzminderung bis hin zum Koma. Hier zeigt sich das aHUS klinisch unspezifisch. Zur Diagnostik des Delirs kann die Confusion Assessment Method for the Intensive Care Unit (CAM-ICU) dienlich sein; Blutungen wären im CT sichtbar. Bei ausgeprägter Vigilanzminderung kann die Elektroenzephalographie (EEG) zur differenzialdiagnostischen Abgrenzung eines (nonkonvulsiven) Status epilepticus erforderlich werden. Fokal-neurologische Defizite auf dem Boden petechialer Blutungen sind bei der TTP häufiger zu finden, da die Thrombozytopenie ausgeprägter ist. Einen Überblick über die zur Diagnosestellung benötigten Parameter gibt Tab. 1.Thrombozytopenie Hämolytische Anämie Organdysfunktion Thrombozytenzahl < 150 G/l oder Abfall > 25 % + Hämoglobin ↓ ±LDH ↑ ±Haptoglobin ↓ ±Bilirubin (indirekt) ↑ ±Fragmentozyten ↑ + Akute Niereninsuffizienz ± Neurologisches Defizit ± Gastrointestinale Symptome LDH Laktatdehydrogenase Anhand der bisher erhobenen klinischen und laborchemischen Kriterien kann die Diagnose einer TMA bereits gestellt werden. Zur weiteren Differenzierung dieses für Intensivmediziner eher seltenen und zudem sehr variablen Krankheitsbildes empfiehlt sich, jetzt rasch die Expertise anderer Fachdisziplinen (z. B. Nephrologie, Hämatologie) einzubinden; diese besitzen Erfahrung in der Betreuung dieser Patienten. Mit Diagnosestellung sollte zeitnah das weitere diagnostische und therapeutische Vorgehen festgelegt werden. Merke. Eine Abgrenzung der TMA-Syndrome TTP, STEC-HUS und aHUS voneinander ist aufgrund der unterschiedlichen – pathophysiologisch orientierten – Therapieoptionen essenziell. Für die Abgrenzung einer TTP ist die Bestimmung der ADAMTS-13-Aktivität erforderlich. Eine stark vermindert ADAMTS-13-Aktivität (< 5–10 %) ist für die Diagnose einer TTP beweisend. Allerdings handelt es sich um keine Routinediagnostik, sodass von der Abnahme der Proben bis zur Befundübermittlung oft mehrere Tage vergehen können. Im klinischen Alltag kann die Bestimmung des PLASMIC-Scores (Tab. 2) oder die Beurteilung des Ausmaßes von Nierenfunktionseinschränkung bzw. Thrombozytopenie hilfreich sein und Hilfestellung für die Einleitung spezifischer Therapiemaßnahmen geben. Ein PLASMIC-Score von 6–7, eine Thrombozytopenie (Thrombozytenzahl < 30 G/l) und/oder eine Serum-Kreatinin-Konzentration < 1,8 mg/dl machen die Diagnose einer TTP gegenüber einem (a)HUS wahrscheinlicher; im umgekehrten Fall ist eine TTP unwahrscheinlich [7, 19]. Zu berücksichtigen ist allerdings, dass perioperative Komplikationen wie Nachblutung oder Schock diese Grenzen aufweichen. Bereits der hochgradige Verdacht auf eine TTP sollte eine unverzügliche Therapieentscheidung nach sich ziehen. Die Blutentnahme zur Bestimmung von ADAMTS-13-Aktivität, -Antigen und -Inhibitor vor Therapiebeginn bleibt als Goldstandard obligat.Parameter Punkte Thrombozyten < 30 G/l 1 Hämolyse (indirektes Bilirubin > 2 mg/dl, unkorrigierte Retikulozyten > 2,5 % oder Haptoglobin unterhalb der Nachweisgrenze) 1 Keine aktive Tumorerkrankung im letzten Jahr 1 Keine solide Organ- oder Stammzelltransplantation in der Vorgeschichte 1 „Mean cell volume“ (MCV) < 90 fl 1 International Normalized Ratio (INR) < 1,5 1 Serumkreatinin < 2,0 mg/dl 1 0 bis 4 Punkte: niedriges Risiko, 5 Punkte: intermediäres Risiko, 6 bis 7 Punkte hohes Risiko für ADAMTS13-Aktivität < 10 % Wenngleich das STEC-HUS aufgrund des überwiegenden Vorkommens im Kindesalter bei Erwachsenen die seltenste Form einer TMA darstellt, muss beim Vorliegen einer (blutigen) Diarrhö zum Ausschluss des STEC-HUS der Nachweis von Shigatoxinen in einer Stuhlkultur oder der Toxingennachweis (stx1 und/oder stx2) mithilfe der Polymerase-Kettenreaktion (PCR) erfolgen, da auch die TTP oder das aHUS mit einer Diarrhö einhergehen kann [16]. Einen eindeutigen und beweisenden Test für das aHUS – vergleichbar mit der ADAMTS-13-Aktivität bei der TTP bzw. dem Nachweis von Shigatoxin beim STEC-HUS – gibt es im klinischen Alltag nicht. Vielmehr kann die Diagnose aHUS grundsätzlich nach dem Ausschluss der anderen beiden TMA-Syndrome gestellt werden. Trotz allem sollte eine Komplementdiagnostik erfolgen. Die Bestimmungen von Komplement C3 und C4 sind zwar im Routinelabor rasch verfügbar, die Verminderung der Komplementfaktoren ist aber nur wenig sensitiv und spezifisch. Beim aHUS liegen meist eine normale Konzentration des Komplementfaktors C4, sowie – abhängig von der zugrunde liegenden Genmutation – eine normale oder verminderte Konzentration von C3 vor [28]. Die weiterführende Diagnostik im Speziallabor umfasst die Bestimmungen der allgemeinen Komplementaktivierung, des löslichen Komplementkomplexes sC5–9 und des Anti-Faktor H‑Autoantikörpers. Auch diese Diagnostik sollte vor Beginn einer Therapie durchgeführt werden. Eine genetische Diagnostik ist dem weiteren Verlauf vorbehalten und für die akute Therapieentscheidung nicht erforderlich. Eine Nierenbiopsie zur Diagnosesicherung ist möglich, wird beim kritisch kranken Patienten mit einer Thrombozytopenie in Abwägung vom therapeutischem Nutzen gegen das Blutungsrisiko jedoch nicht empfohlen. Im Verlauf kann die Nierenbiopsie aber bei fehlender Erholung der Organfunktion oder nichteindeutiger Differenzialdiagnose hilfreich sein. Bei der Suche nach potenziellen Triggern ist eine ausführliche Anamnese bzw. eine detaillierte Untersuchung hinsichtlich Malignomen, Medikamenten (insbesondere Immunsuppressiva und Chemotherapeutika), Infektionserkrankungen sowie Transplantationen unerlässlich. Zusätzlich wird empfohlen, einen HIV-Test und bei Frauen im gebärfähigen Alter einen Schwangerschaftstest durchzuführen. In Abb. 2 ist der auf der Intensivstation der Autoren angewendete diagnostische Algorithmus dargestellt. In Tab. 3 sind wichtige Unterscheidungsmerkmale der einzelnen TMA-Syndrome hinsichtlich Pathogenese, Diagnostik und Therapie zusammengefasst. TTP STEC-HUS aHUS Pathogenese ADAMTS-13 < 5–10 % Meist Antikörper nachweisbar Selten genetisch Shigatoxin-assoziiert Dysregulation des Komplementsystems, oft genetisch, seltener Anti-Faktor-H-Antikörper Typische Screeningparameter PLASMIC-Score 6–7 Thrombozytenzahl < 30 G/l Kreatinin < 1,8 mg/dl PLASMIC-Score < 6 Thrombozytenzahl > 30 G/l Kreatinin > 1,8 mg/dl Klinische Zeichen Petechien, neurologische Symptomatik, kardiale Beteiligung, selten Nierenbeteiligung Blutige Diarrhö, Nierenversagen Nierenbeteiligung im Vordergrund, andere Organmanifestationen möglich, oft Rezidive Therapie Immunsuppression, Plasmapherese, Caplacizumab Supportiv Triggerfaktoren stoppen, ggf. Plasmapherese, Komplementinhibition ADAMTS-13 „a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13“, aHUS atypisches hämolytisch-urämisches Syndrom, STEC-HUS hämolytisch-urämisches Syndrom durch shigatoxinbildende Escherichia coli, TTP thrombotisch-thrombozytopenische Purpura Intensivmedizinische Therapie Die Symptomatik bzw. (Verdachts‑)Diagnose einer TMA kann einerseits bereits die Aufnahme der Patienten auf die Intensivstation begründen oder sich vielmehr erst im weiteren intensivstationären Verlauf entwickeln bzw. aggravieren. In beiden Fällen ist es gleichermaßen wichtig, die medizinische Dringlichkeit des Krankheitsbildes zu erkennen und die Patienten zügig einer adäquaten Therapie zuzuführen. Allgemeine Leitlinien zur Diagnostik und zur Therapie der TMA liegen von verschiedenen Fachgesellschaften vor; für kritisch kranke Intensivpatienten gibt es bisher nur davon abgeleitete Expertenmeinungen [1, 20]. Die Therapie richtet sich nach dem zugrunde liegenden TMA-Syndrom und dem damit verbundenen Letalitätsrisiko. Aufgrund der hohen Akutletalität muss eine Therapie innerhalb weniger Stunden bereits bei hochgradigem Verdacht auf eine TTP eingeleitet und bis zu ihrem sicheren Ausschluss fortgeführt werden. Diese besteht aus einer Immunsuppression (initial mit hochdosierten Steroiden) sowie einer Plasmapherese mit Austausch des ein- bis 2‑fachen Plasmavolumens des Patienten gegen Fresh Frozen Plasma (FFP, [26]). Seit September 2018 ist der spezifische humanisierte Nanoantikörper Caplacizumab europaweit additiv zum Plasmaaustausch für die Therapie der erworbenen TTP zugelassen. Im Gegensatz dazu steht beim STEC-HUS die supportive Therapie ganz im Vordergrund. Merke. Im Fall der TTP muss die Therapie unverzüglich begonnen werden (bestehend aus Plasmapherese, Immunsuppression und Caplacizumab). Im Gegensatz dazu ist die Therapie beim STEC-HUS supportiv. Das optimale therapeutische Vorgehen beim aHUS setzt das Wissen um den individuellen Pathomechanismus des einzelnen Patienten voraus; dies ist zu Behandlungsbeginn oft nicht gegeben und muss individuell diskutiert werden. Wichtig ist die Beeinflussung der auslösenden Triggerfaktoren (z. B. Beendigung der auslösenden Medikation) bzw. der zugrunde liegenden Erkrankung. Dies kann im Einzelfall bereits ausreichen, um eine TMA-Episode zu beenden. Die Plasmapherese ist bei unklarer Differenzialdiagnose eine adäquate Therapieoption, da z. B. Autoantikörper entfernt und regulierende Komplementfaktoren substituiert werden. In diesem Sinn kann sie helfen, eine akute Dysregulation des Komplementsystems zu beheben. Außerdem ist sie insbesondere bei noch fehlendem Ausschluss einer TTP als Therapie der Wahl anzusehen. Allerdings führte dies in der Vergangenheit nur bei einem Teil der Patienten mit komplementvermitteltem (primären) aHUS zur Erholung der Nierenfunktion. Auch für andere (sekundäre) aHUS-Formen ist das therapeutische Ansprechen auf eine Plasmapherese nicht sicher. Mit der Zulassung des Komplementantikörpers Eculizumab für die Therapie des aHUS begann 2011 eine neue therapeutische Ära mit signifikanter Reduktion der Morbidität und Verbesserung der Lebensqualität der Patienten [18]. Eculizumab ist ein humanisierter monoklonaler IgG-Antikörper gegen den Komplementfaktor C5, wodurch dessen Spaltung in C5a und C5b unterbunden wird. Die gemeinsame terminale Endstrecke des Komplementsystems wird unterbrochen und die Bildung des Membranangriffskomplex („membrane attack complex“, MAC) verhindert [24, 30]. Mit Diagnose eines aHUS kann die Plasmapherese beendet und eine Therapie mit Eculizumab begonnen werden. Nach Therapiebeginn kommt es innerhalb von Stunden zu einer signifikanten Reduktion der überschießenden Komplementaktivität, was sich im weiteren Verlauf in der raschen Beendigung der Hämolyse, einer Normalisierung der Thrombozytenzahl sowie einer signifikanten und anhaltenden Verbesserung der Nierenfunktion innerhalb von Tagen bis Wochen zeigt [18]. Es muss jedoch betont werden, dass die Diagnose eines aHUS nicht gleichbedeutend mit der zwingenden Notwendigkeit einer Eculizumabtherapie ist, solange sich diese nicht allein auf das klar komplementvermittelte (primäre) aHUS bezieht. Nicht für jede sekundäre aHUS-Form ist die Wirksamkeit der Komplementblockade gut belegt. Bei der Therapie mit Eculizumab gilt es, Folgendes zu beachten:Initial werden 900 mg Eculizumab als Kurzinfusion einmal wöchentlich und für insgesamt 4 Wochen verabreicht, anschließend folgt alle 2 Wochen eine Erhaltungsdosis von 1200 mg. Es handelt sich um ein extrem teures Medikament mit Jahrestherapiekosten von mehreren Hunderttausend Euro. Generika werden in absehbarer Zeit verfügbar sein. Eculizumab ist für die Therapie des aHUS zugelassen. Allerdings sollte vorab geklärt werden, ob ein krankenhausindividuelles Zusatzendgeld (ZE) vereinbart wurde. Das Medikament ist grundsätzlich gut verträglich, birgt aber aufgrund seines Wirkmechanismus – der effektiven Inhibition des Komplementsystems – ein erhöhtes Risiko für Infektionen mit Meningokokken (Neisseria meningitidis). Darüber muss der Patient informiert und für die Krankheitssymptome sensibilisiert werden. Auch ist eine Meningokokkenimpfung – möglichst vor Therapiebeginn – erforderlich. Liegen zwischen Impfung und Therapiebeginn weniger als 2 Wochen, ist für diesen Zeitraum eine geeignete Antibiotikaprophylaxe (z. B. Penicillin G) notwendig, ggf. muss bei unzureichender Impfantwort (z. B. unter Immunsuppression) eine dauerhafte Fortführung der Antibiotikaprophylaxe erwogen werden. Empfehlenswert ist die Impfung mit beiden verfügbaren Meningokokkenimpfstoffen (ACWY-Konjugat- und MenB-Impfstoff; [12]). Bei einer floriden Meningokokkeninfektion verbietet sich eine Therapie. Die Therapiedauer sollte individuell angepasst werden. Aufgrund der hohen Therapiekosten und des erhöhten Risikos für Meningokokkeninfektionen wäre eine möglichst kurze Therapiedauer wünschenswert. Dem steht eine nichtunerhebliche Rezidivrate nach Absetzen des Medikaments – abhängig vom auslösenden Ereignis, möglichen Triggerfaktoren und dem individuellen genetischen Risiko – gegenüber. Insbesondere für Patienten ohne Nachweis einer Genmutation im Komplementsystem und nach Ausschaltung bzw. Kontrolle des vermuteten auslösenden Triggerfaktors erscheint ein therapeutisch-diagnostisches Regime mit kurzfristiger Therapie bis zur kompletten klinischen Remission (also Normalisierung der Nierenfunktion und Thrombozytenzahl sowie Ausbleiben der Hämolyse) und anschließender engmaschiger klinischer und laborchemischer Kontrolle in vielerlei Hinsicht als sinnvoller und sicherer Kompromiss [4, 21]. Für Patienten mit ursächlichen Genmutationen im Komplementbereich ist eine lebenslange Therapie sinnvoll. Seit 2019 ist in der EU mit Ravulizumab ein weiterer C5-Inhibitor zugelassen, der aufgrund seiner deutlich verlängerten Halbwertszeit – nach einer Induktionsphase – lediglich alle 8 Wochen verabreicht werden muss und mit langfristig geringeren Therapiekosten und mehr Patientenkomfort verbunden ist [17]. Weitere das Komplementsystem beeinflussende Medikamente sind in Entwicklung. Neben der spezifischen Therapie des aHUS bleibt die Therapie symptomatisch. Je nach Ausprägung der Organinsuffizienz(en) kann z. B. eine Dialyse notwendig werden. Hinsichtlich der Anämie können Transfusionen von Erythrozytenkonzentraten nach den aktuell geltenden Empfehlungen der Querschnittsleitlinie der Bundesärztekammer notwendig und sicher durchgeführt werden. Transfusionen von Thrombozytenkonzentraten sind lediglich bei schwerer Thrombozytopenie (< 20 G/l), bei symptomatischer Blutung oder vor Eingriffen mit erwartet hohem Blutverlust empfohlen [3]. Im Rahmen einer vermehrten Hämolyse ist eine Folsäuresubstitution sinnvoll. Prognose und Outcome Durch eine verbesserte Diagnostik, den Einsatz spezifischer Therapien und eine optimierte supportive Therapie hat sich die Prognose aller TMA-Formen in den letzten Jahren substanziell verbessert. Mithilfe des kombinierten Einsatzes von Plasmapherese, Immunsuppression und Caplacizumab konnte die Letalität der TTP von 90 % auf < 5 % gesenkt werden. Auch die Prognose des aHUS hat sich mit dem Einsatz des Komplement-C5-Inhibitors Eculizumab wesentlich geändert. Während es zuvor innerhalb eines Jahres bei über 50 % der Patienten zum Fortschreiten der Erkrankung bis zur Dialysepflichtigkeit kam [6], konnte diese Entwicklung deutlich < 10–15 % der Fälle reduziert werden [5]. Aktuell ist der entscheidendste Faktor für die Beeinflussung der Prognose die rasche Diagnosestellung, um die Therapie frühzeitig und zielgerichtet einzusetzen. Merke. Die Prognose konnte in den letzten Jahren durch eine schnellere Diagnosestellung und zielgerichtete Therapien deutlich verbessert werden. Die Beendigung der Komplementinhibition ist eine individuelle Therapieentscheidung. Sie ist mit dem Auftreten eines Rezidivs und dem damit verbundenen Risiko für einen irreversiblen Verlust an Nierenfunktion oder andere Organschäden verbunden. Diese Therapieentscheidung sollte in Abhängigkeit vom genetischen Hintergrund, einer positiven Familienanamnese, des Vorhandenseins auslösender exogener Triggerfaktoren, der Schwere der Erkrankung und der Erkrankungsmanifestation sowie der verbleibenden Nierenfunktionseinschränkung individuell mit dem Patienten getroffen werden. Insbesondere in den ersten Monaten nach dem Therapieende ist eine engmaschige Verlaufskontrolle erforderlich. Für die Zukunft sind diagnostische Tests zur besseren pathophysiologischen Unterteilung der TMA-Formen und rasch verfügbare Tests der Komplementaktivierung wünschenswert. Auch sind mit der Entwicklung und Zulassung weiterer Komplementinhibitoren eine individuelle Therapie und eine Kostenreduktion zu erhoffen. Fazit für die Praxis Thrombotische Mikroangiopathien (TMA) im perioperativ-intensivmedizinischen Setting sind zwar selten, doch aufgrund ihrer unspezifischen und variablen Symptomatik auch leicht zu übersehen. Daher sind das Erkennen einer TMA („daran denken“) und die Abgrenzung wichtiger Differenzialdiagnosen besonders essenziell. Die Diagnose einer TMA ergibt sich aus der Trias mikroangiopathische hämolytische Anämie, Thrombozytopenie und mindestens einer Organdysfunktion, hier meist der Niereninsuffizienz. Pathophysiologisch können die thrombotisch-thrombozytopenische Purpura (TTP), das hämolytisch-urämische Syndrom durch shigatoxinbildende Escherichia coli (STEC-HUS) und das atypische hämolytisch-urämische Syndrom (aHUS) unterschieden werden. Die TTP ist durch eine fehlende bzw. stark verminderte Aktivität der Protease „a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13“ (ADAMTS13; < 5–10 %) gekennzeichnet. Therapeutisch kommen Plasmapherese, Immunsuppression und Caplacizumab zum Einsatz. Die Diagnose eines STEC-HUS erfolgt durch den Nachweis von Shigatoxin im Stuhl. Die Therapie ist supportiv. Zur Diagnose eines aHUS gibt es keinen in der Routine verfügbaren, spezifischen Labortest. Im klinischen Alltag kann eine außergewöhnlich hohe Konzentration der Laktatdehydrogenase (LDH) den Verdacht in Richtung TMA lenken. Die Therapie des aHUS ist multifaktoriell ausgerichtet: supportive Maßnahmen, Beeinflussung der exogenen Triggerfaktoren, initial Plasmapherese und Komplementinhibition (Eculizumab). Eine schnelle Diagnosestellung und individualisierte, an der Pathophysiologie orientierte Therapien haben das Outcome bis heute deutlich verbessert. Einhaltung ethischer Richtlinien Interessenkonflikt S. Albrecht, C.E. Kamla, U. Schönermarck und D. Wassilowsky geben an, dass kein Interessenkonflikt besteht. Für diesen Beitrag wurden von den Autor/-innen keine Studien an Menschen oder Tieren durchgeführt. Für die aufgeführten Studien gelten die jeweils dort angegebenen ethischen Richtlinien. QR-Code scannen & Beitrag online lesen ==== Refs Literatur 1. 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==== Front Educ Inf Technol (Dordr) Educ Inf Technol (Dordr) Education and Information Technologies 1360-2357 1573-7608 Springer US New York 11478 10.1007/s10639-022-11478-7 Article Investigating the effect of multimodality and sentiments on speaking assessments: a facial emotional analysis http://orcid.org/0000-0002-5118-9742 Chong Joey Jia Qi [email protected] http://orcid.org/0000-0001-6960-2489 Aryadoust Vahid [email protected] grid.59025.3b 0000 0001 2224 0361 National Institute of Education, Nanyang Technological University, Singapore, Singapore 1 12 2022 124 22 8 2022 16 11 2022 © This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. This quasi-experimental study aimed to determine the relationship between (i) oral language ability and emotions represented by facial emotions, and (ii) modality of assessment (audios versus videos) and sentiments embedded in each modality. Sixty university students watched and/or listened to four selected audio-visual stimuli and orally answered follow-up comprehension questions. One stimulus was designed to evoke happiness while the other, sadness. Participants’ facial emotions during the answering were measured using the FaceReader technology. In addition, four trained raters assessed the responses of the participants. An analysis of the FaceReader data showed that there were significant main and interaction effects of sentiment and modality on participants’ facial emotional expression. Notably, there was a significant difference in the amount of facial emotions evoked by (i) the happy vs. sad sentiment videos and (ii) video vs. audio modalities. In contrast, sentiments embedded in the stimuli and modalities had no significant effect on the measured speaking performance of the participants. Nevertheless, we found a number of significant correlations between the participants’ test scores and some of their facial emotions evoked by the stimuli. Implications of these findings for the assessment of oral communication are discussed. Keywords Basic emotions Facial emotional analysis FaceReader Integrated speaking Listening Multimodality Sentiment analysis http://dx.doi.org/10.13039/501100001475 Nanyang Technological University Undergraduate Research on CAmpus (URECA) Programme ==== Body pmcThere is a wide array of factors that influence the speaking performance of individuals in assessments, such as task types (e.g., type of response format such as whether tasks are independent or integrated), types of stimuli used (e.g., sentiment and modality of stimuli) and emotions of participants (Butler et al., 2000; Ockey & Li, 2015). Among these, sentiments and modality of stimuli are under-researched in language assessment research, although there is an extensive body of the literature that investigates these factors in other contexts (Broadbent et al., 2017; Schreuder et al., 2016; Taffou et al., 2013; Takagi et al., 2015). In this study, we investigated the effect of sentiment and modality of stimuli on speaking performance in an integrated speaking (listen-to-speak) assessment. Research shows that sentiments embedded in stimuli (input) during speaking or other language activities have an impact on how efficiently language users communicate (e.g., Barabadi & Khajavy 2020; Karami et al., 2019; Quintelier et al., 2019). On the other hand, the effect of stimuli’s modality on speaking and language performance of students remains an open question. Some studies have indicated that visual stimuli can enhance the language performance of students under assessment conditions (e.g., Wagner, 2010). However, it has been suggested that video-mediated stimuli can have an adverse effect on language performance, likely because language users are distracted by the input they receive from several modalities (Pardo-Ballester, 2016). Following previous research (Arramreddy & Krishnan, 2016; Jung et al., 2014; Pardo-Ballester, 2016), we measured sentiments as emotions embedded in auditory-visual stimuli and examined their effects on participants’ emotions and oral language performance. In addition, the effect of the modality of stimuli (auditory vs. visual) on oral skills performance was investigated. To achieve the goals of the study, we leverage advanced face-reading technologies and sentiment analysis along with psychometric methods in a quasi-experimental study. Emotions and feelings Results from neurocognitive research show that there is a close affinity between affect and cognition (Klinger, 1996). Affect is a general term that consists of emotions (unconscious physio-neurological responses to external and internal stimuli) and feelings (subjective and conscious perceptions of emotions) (Tran, 2007). Based on Tran’s (2007) study, emotion is episodic and dynamic, of a relatively brief duration, event- or object-specific, and arises based on the cognitive and emotional content of the stimuli. Parkinson et al., (1996) found that both feelings (moods) and emotions can affect cognitive processes like memory and perception, and by extension other behaviours such as verbal interactions. Thus, emotions usually have specific implications for behaviours (Tran, 2007). Previous studies have shown that emotions can profoundly affect students’ academic engagement, learning and hence performance (Hascher, 2010; Linnenbrink-Garcia & Pekrun, 2011; Pekrun, 1992). The role and importance of emotions in academic settings has also been recognized in educational theories like control-value theory of achievement emotions (Pekrun, 2006). However, a study by Pekrun and Stephens (2010) showed that apart from test anxiety research and attributional studies, these emotions are often under-appreciated in psychological research. Previous research has employed various methods to measure feelings. Subjective measurements consist of self-reports questionnaires like the achievement emotions questionnaire (AEQ; Pekrun et al., 2011; Peixoto et al., 2015), positive and negative affect schedule (PANAS; Ketonen et al., 2019), and self-assessment manikin (SAM; Geethanjali et al., 2017). However, it is well-known that self-reports may not be accurate in measuring emotions, as people are not conscious about the physiological changes in their bodily chemistry and can overstate or understate their experience of their mood, thus consciously altering measured outcomes (Ciuk et al., 2015). Accordingly, many researchers have applied facial electromyography (EMG; Kulke et al., 2020), face-reading emotion recognition software such as FaceReader (e.g., Hirt et al., 2019), or Affectiva iMotions (e.g., Fasel & Luettin, 2003) to measure emotions expressed through facial emotions. These studies have attested to the utility of these techniques in educational and psychological research, although the techniques are not without limitation. For example, a study by Boxtel (2010) evaluating the strengths and limitations of facial EMG in measuring emotions discovered that it can be an obtrusive technique which requires the fitting of electrodes on face. In addition, the effectiveness of facial EMG can be influenced by nonaffective, behavioural factors such as mental fatigue, which may elicit affective responses in the face. FaceReader, on the other hand, has been shown to be a relatively reliable automated system for the recognition of a number of specific properties in facial images amongst the major software tools for emotion classification currently available (e.g., Uyl & Kuilenburg 2005). Emotions can be induced through different modalities, such as imagination, film (audio narration with visuals), sound (audio narration), music, images (see Fakhrhosseini & Jeon 2017), and their effect can be partially captured through the measurement of facial emotional expressions. FaceReader utilizes this fundamental principle and, through the use of machine learning, determines emotions of people with a high degree of accuracy. Sentiment analysis Different emotions can be induced by sentiments embedded in the stimuli; for example, happy stimulus would induce happy emotions in participants, regardless of the modality of the stimuli (Fakhrhosseini & Jeon, 2017). Previous research has shown the effect of sentiment on different aspects of language and educational assessment performance or test scores (Barabadi & Khajavy, 2020; Karami et al., 2019; Quintelier et al., 2019). The sentiment embedded in a stimulus can be determined through sentiment analysis of content which can identify emotions such as frustration, joy, anger, sadness, excitement, and so on (Mohammad, 2015). A stimulus targeted at inducing a particular emotion may also induce other emotions. In a study conducted by Hewig et al. (2005), sad stimuli evoked other negative emotions such as disgust and rage in addition to the primary sad emotions and an amusement stimulus caused higher intensity of positive emotions than any of the negative emotions. Some studies have shown that emotions do affect a participant’s performance, where better performance (i.e., higher test scores) is observed when the stimuli contained happy rather than sad sentiments (Arramreddy & Krishnan, 2016). In Jung et al.’s study (2014), participants who experienced positive emotions performed better than those experiencing negative emotions, and both groups of participants outperformed participants experiencing neutral emotions. In another study by Lochner (2015), however, the experimentally manipulated emotions did not affect test performance in an online reasoning test. There is comparatively little research on how induced emotions of people affect performance in integrated speaking tests, as much research only discussed how emotions affect test performance in other areas such as in listening tests (Stientjes, 2012; Wagner, 2010), general academic tests (Gumora & Arsenio, 2002) and logical reasoning tests (Jung et al., 2014). As performance on speaking tests (indicated by scores) is affected by many other factors such as task type and the participant’s ability (Tuan & Mai, 2015), it is difficult to generalise whether emotions do affect test performance and whether these effects are significant. In this study, we hypothesized that only a small share of variance in test scores is attributed to the emotions of participants. This assumption is based on the analysis of the first operationalisation of communicative competence (Canale & Swain, 1980) which included no mention of affects and emotions as a dimension that is related to spoken language. Even based on later formulations of communicative competence, it can be inferred that affective schema is not expected to have a significant impact on participants’ performance in standardized or formal situations. Modality of Stimuli and oral Language performance Previous research has examined the effect of modality of stimuli on oral language performance, but there is a dearth of research on the effect of mode on integrated listening test performance. Wagner (2010) discovered that participants who were exposed to video-audio stimuli performed better than (achieved higher scores) when pure audio stimuli was used in an auditory comprehension test and this difference was statistically significant. This suggests that the non-verbal visual information of spoken texts helped participants better comprehend aural information and contributed to the video group’s superior performance. Contrastingly, Suvorov (2008) found that participants scored significantly lower for video-mediated passages than for audio-only passages and photo-mediated passages in an auditory comprehension test upon comparing the mean scores. Further, Pardo-Ballester (2016) reported that learners of different proficiencies performed differently (achieved different scores) when different modalities are used. Learners with lower proficiency levels performed better with only audio, while learners with higher proficiency levels performed better with video-audio stimuli. A plausible reason provided was that the presence of distracting visual elements in video-audio stimuli could have obstructed learning (Pardo-Ballester, 2016). The Present Study While many research studies compare the effects of visual-auditory or auditory stimulus on either emotions (Riviello & Esposito, 2016) or performance (Pardo-Ballester, 2016; Wagner, 2010), there is little empirical research investigating and comparing the use of both auditory and visual stimulus, and their impact on emotions and integrated speaking test performance. Extending the existing body of research, this study investigates whether there is a relationship between two groups of variables: (i) the participants’ oral ability and emotions proxied by their facial emotions, and (ii) the modality of assessment (audios vs. videos) and sentiments embedded in each modality. The research questions of this paper are: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ integrated speaking abilities? Is there any significant effect of the sentiments and modalities of stimuli on the participants’ facial emotions? What is the relationship between integrated speaking performance and emotions evoked by various types of stimuli? Method Participants Sixty (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{a}\text{l}\text{e}=33, \text{f}\text{e}\text{m}\text{a}\text{l}\text{e}=27$$\end{document}) bilingual undergraduate students from a public university in Asia, aged between 18 and 29 years old, participated in the study. English was the first language of the participants. Participants who were under 21 years of age were asked to obtain parental consent before their allocated test session. Each participant was assessed on their English oral proficiency in answering 12 follow-up questions (three questions per stimulus) after watching or listening to four 2-minutes long stimuli. As discussed later, the stimuli and questions were all in English. Raters Four (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{a}\text{l}\text{e}=1, \text{f}\text{e}\text{m}\text{a}\text{l}\text{e}=3$$\end{document}) post-graduate university students from an Asian university between 29 and 38 years of age who were enrolled in a graduate course in applied linguistics participated in the study as raters. The raters had high proficiency levels in English for them to specialise in applied linguistic. Each rater assessed the performance of 18 assigned participants on 4 items for 4 stimuli (approximately 288 data points per rater) using the Internet-Based Test of English as a Foreign (TOEFL iBT) Integrated Speaking Rubrics. Measurement Instruments Integrated tests Participants were exposed to four 2-minutes long stimuli on two main topics (Education-Animals and Education-Earth), where two were designed in video forms and two were in audio forms. They were required to answer follow-up comprehension questions orally and their responses were recorded for coding. Special care was taken to select only 2-minutes portion of the videos that are formal, informational, and educational as studies conducted by Wistia (Fisherman, 2016; Guo et al., 2014) found that the optimal video length for maximum engagement is 2 min. Transcripts were then generated for selected videos and analysed using the Sentiment Analysis and Social Cognition Engine (SÉANCE) by Crossley et al. (2017) which provides data for further analysis. Accordingly, multiple indices that measure happiness were chosen, as they were the only relevant indices that directly measure happiness and sadness. They are: Happiness_GALC (Geneva affect label coder): Words with a positive valence which imply or indicate happiness. They were extracted and coded based on the GALC list (see Scherer, 2005). “Happiness_GALC_neg_3” is another index used which indicates vocabulary associated with the feeling of happiness. This index was computed based on the GALC list and includes words such as cheers and delight (“neg” stands for the negative filter). Joy_GALC: Positive emotion words describing joy based on the GALC list. Similarly, “Joy_EmoLex” (emotion lexicon) refers to positive emotion words describing joy such as tantalizing, loveable, etc. EmoLex is a list of English words annotated for basic emotions (anger, anticipation, surprise, fear, trust, sadness, joy, and disgust) and negative and positive sentiments (Mohammad & Turney, 2013, p. 451). Another related index used is “Joy_EmoLex_neg_3”, which also refers to positive emotion words describing joy based on the EmoLex list (“neg” stands for the negative word filter.). Likewise, “Joy_GALC_neg_3” represents the degree of positive emotions describing joy based on the GALC list. Finally, “Joy_component” describes positive emotions describing joy, and is derived from the principal component analysis. Anticipation_EmoLex: Vocabulary extracted from the EmoLex wordlist, indicating anticipation such as tantalizing or unbeaten. Relatedly, “Anticipation_EmoLex_neg_3” is an anticipation index computed with the negative filter on. The other two indices related to anticipation are “Surprise_EmoLex” and “Surprise_EmoLex_neg_3”. Four indices that measure sadness: “Sadness_GALC”, “Sadness_EmoLex”, “Sadness_GALC_neg_3”, and “Sadness_EmoLex_neg_3”. These indices were used to choose eight videos which were downloaded, and audio versions were generated for each of them. After each stimulus (video or audio), three follow-up questions would appear on the screen. For each question, the participants were given 30 seconds to construct their answers and 60 seconds to orally-present their answers to the camera. Each participant answered a total of 12 questions during the test. The sequence of videos was counterbalanced. FaceReader We used FaceReader 8.0, a software developed by Noldus Information Technology, to detect and classify the facial emotions of participants into seven categories: happy, sad, angry, surprised, scared, disgusted and neutral (see Fig. 1). These emotional categories were described by Ekman (1970) as basic or universal emotions (Loijens & Krips, 2018), which is sufficient for this research that works with basic emotions. Fig. 1 FaceReader Interface. (Note:FaceReader can detect and classify the facial emotions of participants into seven categories: happy, sad, angry, surprised, scared, disgusted, and neutral.) In this research, video files were processed through FaceReader to obtain frame-by-frame analysis. FaceReader works by first using the popular Viola-Jones algorithm (Viola & Jones, 2001) to detect the face followed by using the Active Appearance Method (AAM; Cootes & Taylor 2000) to analyse over 500 key points in the face, as well as facial texture to create an accurate 3D model of the face. FaceReader then uses an artificial neural network built by trained experts (Bishop, 1995) to classify facial emotion. The Deep Face classification method used in addition to the AAM allows for a better analysis of a face even if part of is hidden (Loijens & Krips, 2018). Additionally, the accuracy of FaceReader in measuring emotions has been validated in a recent validation study by Zumhasch (2018) where FaceReader showed 100% precision in measuring happy emotions, 97% in measuring neutral emotions and 91% precision in measuring scared and sad emotions, thus providing additional validation for the sentiment analysis carried out in this study. Assessment Rubrics We used the TOEFL iBT Test Integrated Speaking rubrics to rate participants’ performance. The Integrated Speaking test of the TOEFL iBT requires participants to utilise their reading and listening skills in addition to their speaking skills. As the designed test in this research requires participants to answer follow-up questions based on visual and/or auditory stimuli, the Integrated Speaking rubrics was used. There are 4 items for scoring in the Integrated Speaking rubrics (namely General Description, Delivery, Language Use and Topic Development), with scores ranging from 0 to 4. In this research, no score 0 was given to any participants as all participants made attempts to respond and all responses were related to the topic to various degrees. Procedures for participants Participants were seated comfortably in front of a computer monitor that was connected to researchers’ laptop which projected relevant content onto the computer monitor. They were first given a 5-minutes briefing. Next, they were asked to complete the consent form and a background questionnaire. Special care was taken to ensure that the participants were in good illumination, which is important for FaceReader to yield reliable results (Loijens & Krips, 2018). The participants were given a pen and some paper to take down notes during the test. Next, the participants were exposed to the four 2-minutes long stimuli on two main topics (Education-Animals and Education-Earth) and answered 12 follow-up questions (3 questions per stimulus). At the end of the session, participants were given $10 in cash. The sessions lasted 25 to 50 min. Procedures for raters The four raters were trained through a synchronous video chat with the researchers via WebEx due to the CoVID-19 pandemic. Prior to the training session, the TOEFEL Integrated Speaking rubrics, a marking guide and samples from level 1, 2, 3, and 4 were prepared and uploaded onto a folder on Google Drive which is shared with the four raters. The transcripts of the 4 stimuli, their YouTube links and relevant duration to watch, as well as suggested answers for each of the 12 questions were further included in the marking guide. During the training session, the 4 components of the TOEFEL Integrated Speaking rubrics (namely General Description, Delivery, Language Use and Topic Development) were explained and important points were highlighted. Ratings were given out for each stimulus (4 ratings per stimulus per participant) instead of each question (12 ratings per stimulus per participant) to normalize any variance in performance for the three questions of the same stimulus. Four samples from level 1, 2, 3 and 4 were presented to the raters where researchers explained the reasons for the gradings. It was followed by hands-on rating practices where all raters were asked to view some video snippets of participants uploaded on Google Drive and subsequently rate their performances based on the TOEFEL Integrated Speaking rubrics. Discrepancies in rating were addressed by directing the raters’ attention back to the rubrics as well as the four samples from level 1, 2, 3, and 4. After the training session, each rater was assigned 18 participants to assess where 4 sets of ratings were given per participant, amounting to a total of 72 sets of ratings per rater. As each set of rating includes 4 items, there was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$4 \text{X} 72= 288$$\end{document} data points per rater, thus yielding sufficient information for measuring their ability (Linacre, 2008). A cascading design was employed to assign the participants to raters where each rater had to assess 9 participants that were common to the rest of the raters in addition to the 9 participants that were only assigned to them. One of the researchers also rated a total of 24 participants (9 common participants, 9 exclusive participants and 6 participants) which were used as samples or for practice during the training session. Data Analysis Before answering the research questions of the study, we examined the reliability and psychometric validity of the data. This consisted of examining raters’ performance, functionality of the stimuli or assessment instruments, and participants’ scores. We chose the multi-faceted Rasch measurement (MFRM; Linacre 1994) for this purpose, as it provides very useful evidence concerning the functionality of the instrument and the performance of raters and participants. After establishing the reliability and psychometric validity of the data, we answered the research questions by using repeated-measures MANOVA and bivariate correlation analysis. We discuss the analytical procedures next. Reliability and psychometric Validity: multi-faceted Rasch Measurement (MFRM) We used the Facets computer package (version 3.83.2) (Linacre, 2008) to investigate the psychometric validity of the measurements and the reliability of rater performance. The MFRM model applied is, as follows:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{log}\frac{{p}_{nijk}}{{p}_{nijk-1}}={B}_{n}-{D}_{i}-{C}_{j}-{E}_{h}$$\end{document} where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${P}_{nijk}$$\end{document} is the probability that participant \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$n$$\end{document} will be awarded a rating of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} on item \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i$$\end{document} by rater \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j$$\end{document}; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${B}_{n}$$\end{document} is the ability of participant n; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${D}_{i}$$\end{document} is the difficulty level of item \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i$$\end{document}; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${C}_{j}$$\end{document} is the severity of rater j; and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${E}_{h}$$\end{document} is the difficulty of the threshold from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k-1$$\end{document} to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k$$\end{document} on the scale unique to item \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i$$\end{document}. We computed (i) infit mean square (MnSq) which is an index sensitive to disturbances near the ability of the participant, such as when a mid-ability student receives extremely high or low score by one or more raters, and (ii) outfit MnSq which is outlier sensitive where it highlights distortions in the data distant from the actual speaking ability of the participant, such as when a low-ability student is given an unexpectedly high score or vice versa (Linacre, 1994). MnSq values of the participants, raters, and stimuli (both audio and visual ones) fell between 0.5 and 1.5 indicating tolerable randomness in the data for these facets. This further lends evidence to the psychometric validity of participants’ scores, raters’ performance (i.e., their scoring practice), and the stimuli. In addition, the variance explained by Rasch measures in this analysis was 71.04%, which is a significantly large share of variance. This provides additional supporting evidence for the psychometric validity of the measurements conducted by the raters in the study (Linacre, 1994). The reliability of student scores was 0.79, with a strata coefficient equal to 2.89, indicating high reliability and the detection of roughly 3 (2.89) levels of ability or test performance among the students. Finally, the last piece of evidence backing up the reliability and psychometric validity of the measurement stimuli and raters’ performance was derived from the psychometric functionality of the scoring categories. The TOEFL rubrics adopted in the study comprised four scoring categories (1 to 4). This yields three “thresholds”, that is, one threshold between every two adjacent categories, i.e., a threshold between 1 and 2, a threshold between 2 and 3, and a threshold between 3 and 4. In MFRM, a threshold is a point on the ability continuum where the participant starts to have a higher probability of achieving a higher score. Thresholds should be adequately distant from each other, meaning that they should ascend monotonically (Linacre, 1994). We estimated the Rasch-Andrich thresholds in this study, which were − 3.38, -1.05, and 4.42, suggesting a monotonic increment in the difficulty level of the thresholds. In sum, we found the ratings provided by the raters, the instrument, and the stimuli to be psychometrically valid and reliable. Thus, we utilized the scores in follow-up analyses to answer the research questions of the study. Research Question 1: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ integrated speaking abilities? To address the first research question, we first performed a descriptive statistical test to investigate the normality of the data by examining skewness and kurtosis values. We applied a within-subject design consisting of sentiment in stimuli (sad and happy) and modalities (video vs. audio) as the independent variables, and measures of integrated speaking performance (general description, delivery, language use, & topic development) as the dependent variables. Next, we performed a 2 × 2 repeated-measures MANOVA on the measured integrated speaking performance (dependent variable), which was the average ratings for each of the 4 individual scoring categories provided by the raters. Research Question 2: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ facial emotions? To address the second question, we performed a 2 × 2 repeated-measures MANOVA with participants’ facial emotions (neutral, happy, sad, angry, surprised, scared & disgusted measured by FaceReader) as the dependent variable and sentiment and modality as independent variables. Research Question 3: What is the relationship between integrated speaking performance and emotions evoked by various types of stimuli? To address this question, the data from raters measuring participants’ speaking ability and data from facial emotions measured by FaceReader were arranged based on the modalities and sentiments of stimuli. The combined data was subjected to bivariate correlation analysis to investigate whether there is a relationship between the participants’ oral ability and emotions proxied by their facial emotions. The effect sizes of data that were significant at either 0.01 or 0.05 alpha levels were calculated by squaring the correlation coefficients. Results Descriptive statistics Table 1 presents the mean, standard deviation, skewness and kurtosis values of participants’ measured general performance for the 4 combinations of sentiment and modality of stimuli. As demonstrated, the skewness and kurtosis values fell between the range of -1.26 and 1.98, thus providing evidence for normality. The largest and smallest mean value occurred when the stimulus was in video form and embedded sad emotions (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}= 3.32$$\end{document}) and when the stimulus was in audio form and embedded sad emotions (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}= 3.19$$\end{document}), respectively. In addition, the mean value of participants’ measured general performance was higher when the stimuli embedded happy sentiments (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}=3.26$$\end{document}) rather than sad sentiments (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}=3.25$$\end{document}). The mean value of participants’ general performance was higher when the stimulus was in the video modality (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}=3.28$$\end{document}) rather than audio forms (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{m}\text{e}\text{a}\text{n}=3.24)$$\end{document}. Table 1 Descriptive Statistics of General Performance in the Integrated Listening Test Sentiment, mode of stimuli M SD Skewness Kurtosis Happy, Video 3.24 0.66 -0.93 0.81 Happy, Audio 3.29 0.68 -1.26 1.98 Sad, Video 3.32 0.64 -0.82 -0.10 Sad, Audio 3.19 0.67 -0.79 0.62 Note: M = Mean; SD = standard deviation. Range of the scale is from 0 to 4. Research Question 1: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ integrated speaking abilities? The results of the repeated-measures MANOVA test conducted for the 4 scoring categories (general description, delivery, language use, & topic development) showed no significant main and interactions effects of sentiments of stimuli (happy vs. sad) and modalities of stimuli (video vs. audio) on participants’ measured performance for each of the 4 individual scoring categories (p > .05). Research Question 2: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ facial emotions? The descriptive statistics of the seven emotions of participants proxied by facial emotion showed the data were normally distributed (skewness and kurtosis between − 2 and + 2). The Pillai’s Trace of the repeated-measures MANOVA test showed that the sentiment of stimuli had a significant main effect on the amount of facial emotional expression (F (7,33) = 59.35, p < .001), with a partial eta squared = 0.926, which indicates a very large effect. Similarly, there was a significant main effect for the modality of stimuli, F (7, 33) = 72.53, p < .001, partial eta squared = 0.94) as well as a significant interaction effect, F (7, 33) = 43.91, p < .001, partial eta squared = 0.90). Using a Bonferroni correction, we conducted multiple comparisons on the estimated marginal means. As Fig. 2 presents, there was a significant difference in the amount of facial emotion evoked by the happy vs. sad sentiment videos in four pairwise comparisons: neutral (p < .001), happy (p < .001), scared (p < .001), and disgusted (p = .025). Fig. 2 Pairwise comparisons of participant emotions based on the sentiment of the stimuli (sad vs. happy). (Note: 1 = stimulus with happy sentiment; 2 = stimulus with sad sentiment.) We also performed multiple comparisons on the estimated marginal means of the modality of stimuli and found significance differences in the amount of facial emotion evoked by video vs. audio modalities (Fig. 3). These consisted of neutral (p < .001), happy (p < .001), scared (p = .002), and disgusted (p = .002). Finally, the interaction analysis revealed 8 significant interactions out of 14 comparisons (Table 2). Fig. 3 Pairwise comparisons of participant emotions based on the mode of the stimuli (video vs. audio). (Note: 1 = video mode; 2 = audio mode.) Table 2 SentimentMode Interaction Effects on Facial Emotions Participants’ facial emotions measured by FaceReader Stimuli’s sentiment Mode 1 Mode 2 Mean Difference p value Neutral Happy Video Audio 0.411 0.00 Sad Video Audio -0.016* 0.016 Happy Happy Video Audio -0.042* 0.003 Sad Video Audio 0.360* 0.00 Sad Happy Video Audio 0.030* 0.01 Sad Video Audio 0.028 0.347 Angry Happy Video Audio 0.008 0.133 Sad Video Audio 0.002 0.924 Surprised Happy Video Audio -0.004 0.597 Sad Video Audio -0.005 0.81 Scared Happy Video Audio -0.003 0.431 Sad Video Audio 0.058* 0.001 Disgusted Happy Video Audio -0.005* 0.039 Sad Video Audio -0.033* 0.008 Research Question 3: What is the relationship between integrated speaking performance and emotions evoked by various types of stimuli? To answer research question 3, emotions of participants proxied by facial emotions were correlated with their scores to explore the relationship between the two variables. Table 3 presents statistically significant bivariate correlations (rounded up to two decimal values) and their respective effect sizes in brackets. In this table, the components of participants’ integrated speaking scores are presented in the left-hand column. For example, the “Happy Video_General description” represents the participants’ general score in the happy video, while the “Sad Audio_Language use” represents their language use score in the sad audio. In addition, the participants’ facial emotions captured by FaceReader are presented in the top row. For example, the “Happy Video_Neutral Emotions” indicates the amount of neutral facial emotions of the participants as they were watching the happy video, whereas the “Happy Video_Sad Emotion” represents their sad facial emotions (if any) while watching the happy video. Non-significant correlations are not presented. Table 3 Correlation Analysis of Average Ratings and Emotions Happy Video_Neutral Emotions Happy Video_Happy Emotions Happy Video_Sad Emotions Happy Video_Surprised Emotions Happy Audio_Neutral Emotions Happy Audio_Happy Emotions Happy Audio_Scared Emotions Sad Video_Happy Emotions Sad Video_Scared Emotions Sad Audio_Neutral Emotions Sad Audio_Happy Emotions Sad Audio_Surprised Emotions Sad Audio_Scared Emotions Happy Video_General description − 0.36* (0.13) − 0.38* (0.14) − 0.38** (0.14) Happy Video_Delivery 0.29* (0.08) − 0.42** (0.17) 0.33* (0.11) − 0.50 (0.25) − 0.52 (0.27) 0.28* (0.08) − 0.53** (0.28) Happy Video_Topic development − 0.30* (0.09) − 0.44** (0.19) − 0.37* (0.14) − 0.33* (0.11) Happy Video_General description 0.33* (0.11) − 0.41 (0.17) − 0.38 (0.15) 0.32* (0.10) − 0.39** (0.15) 0.34* (0.11) − 0.48** (0.23) Happy Audio_Delivery − 0.40* (0.16) 0.33* (0.11) − 0.48** (0.23) 0.30* (0.09) − 0.45** (0.20) 0.35* (0.12) − 0.56 (0.32) Happy Audio_Language use − 0.42 (0.17) − 0.41** (0.17) 0.28* (0.08) − 0.45** (0.20) 0.34* (0.11) − 0.54** (0.29) Happy Audio_Topic development 0.33* (0.11) − 0.43 (0.19) − 0.40 (0.16) 0.39 (0.15) − 0.44 (0.19) 0.27* (0.08) − 0.45** (0.21) 0.38* (0.15) Sad Video_General description 0.29* (0.08) Sad Video_Delivery − 0.50** (0.25) 0.33* (0.11) − 0.48 (0.23) 0.36 (0.13) − 0.49 (0.24) − 0.58 (0.34) Sad Video_Language use − 0.41** (0.16) − 0.30* (0.09) − 0.33* (0.11) − 0.38* (0.15) Sad Video_Topic development − 0.34* (0.11) − 0.28* (0.08) − 0.32* (0.10) − 0.36* (0.13) Sad Audio_General description 0.30* (0.09) − 0.33* (0.11) − 0.326* (0.11) − 0.34* (0.12) 0.30* (0.09) Sad Audio_Delivery − 0.33* (0.11) − 0.50 (0.25) − 0.48 (0.23) − 0.43** (0.18) Sad Audio_Language use − 0.34* (0.12) − 0.39* (0.09) Note. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${}_{ }{}^{}p<.05. {}_{ }{}^{*}p<.001.$$\end{document} The correlation coefficients have been rounded up to two decimal values Overall, the magnitude of effect sizes is low to medium, as indicated by the parenthesized coefficients of determination ranging from 0.08 to 0.34. For example, there was a medium correlation of 0.50 (effect size = 0.25) between the delivery scores in the video with embedded happy emotions and the “Happy Audio_Happy Emotions”. The findings indicate that a low-to-medium share of the variance in the participants’ test scores is associated with their facial emotions. Discussion This study set out to investigate the relationship between (i) the participants’ oral ability and emotions proxied by their facial emotions, and (ii) the modality of assessment (audios vs. videos) and sentiments embedded in each modality. The three research questions are discussed next. Research Question 1: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ integrated speaking abilities? Using repeated-measures MANOVA, we found that modality (video vs. audio) and sentiments of stimuli (happy vs. sad) had no significant effects on participants’ measured performance. Regarding the effects of modality, our observations exhibited a similar (though non-significant) trend with Wagner’s (2010) study wherein participants who were exposed to video-audio stimuli did not perform differently from when pure audio stimuli were used in a listening test. This suggests that the non-verbal visual information of spoken texts did not seem to help participants to better respond to the questions concerning the audio-visual modality. While this lends support to the validity of the test (as there was no test method effect), there are several observations that would be worth further investigation—with a larger sample size, the non-significant differences may tend to move towards conventionally accepted statistical significance (Faber & Fonseca, 2014; Mayo & Spanos, 2011). Based on Wagner’s (2010) argument, we speculate that some factors such as the nature of stimuli used (dialogue vs. lecturette texts) could have reduced the effectiveness of using video stimuli. An important advantage of videos in the assessment of spoken proficiency is their authenticity or their similarity with real-life situations (Douglas, 2000). In real academic environments, lectures constitute an important component of every subject. Accordingly, the content of spoken proficiency tests, which aim to predict participants’ spoken performance in these environments, should exhibit significant similarities with the content of the language use domain the tests aim to emulate. Regarding the effects of sentiments, the measured performances of participants were better when the stimuli embedded happy rather than sad sentiments, which shows a similar trend to Arramreddy & Krishnan (2016). This could be explained by the findings of Fredrickson (2001) which suggested that positive emotions led to better performance because they encourage exploring and integrating diverse materials, as well as better problem-solving skills. Nevertheless, test developers should note that that, as Valiente et al. (2012) showed, the intensity of positive emotions determines their impact on test performance or scores where high-arousal positive emotions (such as exuberance, excitedness, and elatedness) may instead lead to worse performance. This may not be applicable in this study as it involved happiness (rather than excitedness) which is a low-arousal positive emotion, but it is a point worth investigation in future research. Research Question 2: Is there any significant effect of the sentiments and modalities of stimuli on the participants’ facial emotions? There were significant main and interaction effects of sentiment and modality of stimuli on the amount of facial emotional expression. In particular, multiple comparisons on the estimated marginal means showed that there was a significant difference in the amount of facial emotion evoked by the happy vs. sad sentiment videos in four pairwise comparisons: neutral, happy, scared, and disgusted. As expected, the pairwise comparison of the mean values of neutral, happy, scared, and disgusted emotions for video stimuli evoking sad and happy emotions showed that the happy stimuli evoked higher intensities of happy emotions than the sad stimuli, while the sad stimuli evoked higher intensities of disgust emotions. This perhaps provides criterion-based validity evidence for the sentiment analysis that the four chosen stimuli evoked their targeted emotions where the happy stimuli evoked more happy emotions than sad stimuli, while the sad stimuli evoked higher intensity of some negative emotions (e.g., disgust) than the happy stimuli. Interestingly, the sad stimuli evoked lower intensity of some negative emotions like scared emotions compared to the happy stimuli. This has a theoretical and an empirical implication. Our findings provide evidence that sentiments embedded in auditory texts would have measurable effects on speakers’ sentiments expressed as facial emotions. In other words, measurable stimulus-response effects of emotions can be revealed via text-mining and biometric technologies. Based on the study by Ekman (1992) who investigated basic emotions, there is consistence evidence showing that facial emotions provide a good way to distinguish the different emotions, thus supporting the use of biometric technologies such as facial emotional analysis to measure stimulus-response effects of emotions. Just like the study by Hewig et al. (2005) where a sad stimulus evoked other negative emotions such as disgust and rage in addition to the primary sad emotions, our study showed a similar trend where the stimuli targeted at inducing sadness also induced higher intensity of other negative emotions such as disgust emotions as compared to the stimuli that embedded happy sentiments. This is consistent with another study by Schwartz and Weinberger (1980) that investigated the relations between happiness, sadness, anger, fear, depression, and anxiety where they discovered that emotions have similarities and typically interrelate during various affective situations with fear being a particular type of anxiety. We further found that there was a significance differences in the amount of facial emotions evoked by video vs. audio modalities. These consisted of neutral, happy, scared, and disgusted emotions. It was found that the video stimuli elicited higher intensities of a larger number of emotions (neutral, happy, and scared) compared to audio stimuli which only evoked higher intensities of disgusted emotions. This follows the findings of the meta-analysis of the elicitation techniques by Westermann et al. (1996) which found that videos are the most effective stimuli at eliciting emotions, whether positive or negative, among participants. The study by Murugappan et al., (2009) also confirmed that audio-visual stimulus performs superior in evoking emotions than visual stimulus. An explanation for videos being most effective could be due to additional non-verbal cues in them besides audio, which may help in inducing higher intensity of emotions (Yazdani et al., 2013). The deviation in this study where participants experienced higher intensity of disgust for audio than video stimuli could perhaps be due to the presence of visuals in videos which did not help in eliciting disgust and maybe elicited other emotions in addition to disgust. Further research could perhaps consider analysing tone in addition to facial emotions to gain an even more accurate measurement of emotions. Research Question 3: What is the relationship between integrated speaking performance and emotions evoked by various types of stimuli? The amount of variance in test scores attributed to the emotions of participants was low to medium, with an effect size ranging from 0.08 to 0.34. This aligns with our hypothesis that only some part of the observed variance in test scores can or should be associated with the emotions of participants when they are using academic language, while most of the variation should arise from the differences in participants’ speaking abilities. Thus, it might be said that similar to the study by Lochner (2015), the experimentally manipulated emotions did not have much significant impact on participants’ speaking performance. Lochner (2015) suggested several explanations to account for the deviation from the expected hypothesis such as reasoning tests being less susceptible to the influence of emotions than other types of tests. One reason could be the difficulty to detect the effect of affective state on test performance due to the comparatively unstandardized situations under non-laboratory conditions. Another reason might be the diversity of the sample or the possibility of participants entering a state of flow wherein thoughts or feelings do not affect their ability to perform the task. As the test situation was rather standardized (controlled) in our study, one possible explanation for the small correlation between participants’ measured performance and emotions could be the possibility of participants entering a state of flow where feelings do not affect their ability to perform the tasks. Nonetheless, this remains a speculation and further research is needed to examine its plausibility. Messick’s (1996) publication raised construct under-representation and construct-irrelevant variance as causes of invalidly high or low scores but as this study made conscious efforts to consider all the important aspects of what we intended to measure, construct under-representation does not seem to be the cause of low-to-medium correlations between emotions and test scores. A small amount of construct-irrelevant variance, on the other hand, seems to be a plausible explanation for the correlations observed. We suggest that future research on assessing speaking should take into consideration the effect of variables such as affect and modality of presentation of the stimuli on participants performance. It should be noted that research questions 1 and 3 serve different purposes in this study. While research question 1 examined the overall differences between test scores across sentiments and modalities, research question 3 examined the fine-grained association between test scores and the different types of facial emotions that were captured by FaceReader in different modalities. In addition, only the correlation between the facial emotions and test scores on unique stimuli should be considered pertinent to this research question. Implications for Assessment and Practice An implication of this study is that the effect of test takers’ affect (emotions and feelings) on the validity of oral proficiency tests is inconclusive. Particularly, as indicated in the MFRM analysis, we found that the test scores were psychometrically valid and reliable. However, there was evidence from research question 3 that some share of variance in some of the scores was associated with the participants’ facial emotions. The corollary of these two seemingly contrastive findings is that, as discussed by Low and Aryadoust (2021), while psychometric analysis is useful and necessary in assessment development, it is not sufficient. Thus, we suggest that assessment designers should exercise caution in developing multimodal assessments with emotionally bound content. Specifically, one should bear in mind that test takers’ affective and cognitive process cannot be merely represented by test scores. Test scores only represent the end product of the assessment and are completely “imperceptive” to the processes by which test takers answered the test items (Aryadoust, 2023). It is possible to access (some of) the affective and cognitive processes of test takers through adopting modern technologies such as facial emotional analysis in study design. The integration of psychometric analysis (e.g., MFRM) and technology would present a more reliable profile of the validity of assessment instruments. Limitations of the study This study is not without its limitations. There are three limitations of the study that should be addressed in future research. First, based on Wagner’s (2007) study, we suggest that the auditory stimuli should be diversified beyond the lecture style used in the study. As Wagner (2007) suggested, using lecturette texts could lead to participants having less interest and engagement with the stimuli than if dialogue texts were to be used, hence perhaps leading to reduced effectiveness of video stimuli in conveying additional contextual and non-verbal cues. Second, unlike the study by Jung et al. (2014) wherein participants experiencing positive and negative emotions both outperformed participants experiencing neutral emotions, in our study there were no stimuli exclusively embedding neutral emotions which could serve as a point of reference to determine if positive and negative emotions will lead to better measured performance than neutral emotions. Having a point of reference allows the determination of how emotions affect performance from the norm, regardless of whether it is positive or negative emotions. Finally, it is important to note that different individuals express emotions differently. Some individuals may show little emotions through facial expressions but their emotions may show clearly in the tone of their voice or in autonomic nervous system reactions, which may be measured using facial electromyography (Barrett, 2006), galvanic skin response, and/or pupil dilation analysis in eye-tracking. Analysing emotions using facial expressions could thus be improved by using biometric measures in speaking assessment research. Conclusion We investigated integrated speaking performance and found that there were no significant main and interaction effects of modality of stimuli (video vs. audio) and the sentiments of stimuli (happy vs. sad) on participants’ measured performance. Another finding was that the sentiment (happy vs. sad sentiment videos) and modality (video vs. audio) of stimuli had a statistically significant effect on the amount of facial emotional expression. It was also found that the video stimuli elicited a larger number of emotions (neutral, happy, and scared) with a higher intensity compared to the audio stimuli which only evoked higher intensities of disgusted emotions. This resonates with the finding of several studies that show video stimuli are more effective than audio stimuli in eliciting emotions. In addition, there were small to medium correlations among some of the measured facial emotions evoked by the audio and video stimuli and test scores of the participants. Hopefully, these findings will inform and inspire future research on the nature and functionality of multimodal tests of speaking. Acknowledgements We would like to acknowledge the funding support from Nanyang Technological University – URECA (Undergraduate Research Experience on Campus) for this research project. The IRB number for the study is IRB-2019-10-029. Data availability The datasets generated during and/or analyzed during the current study are not publicly available but can be made available upon request. Declarations Conflict of Interest None. 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[Blog post]. :text=A.,additionally%20the%20neutral%20facial%20expressions. https://www.noldus.com/blog/validation-study-facereader#:~.	36471775	PMC9713747	NO-CC CODE	2022-12-02 23:23:09	no	Educ Inf Technol (Dordr). 2022 Dec 1;:1-24	utf-8	Educ Inf Technol (Dordr)	2,022	10.1007/s10639-022-11478-7	oa_other
==== Front J Chem Sci (Bangalore) J Chem Sci (Bangalore) Journal of Chemical Sciences (Bangalore, India) 0974-3626 0973-7103 Springer India New Delhi 2109 10.1007/s12039-022-02109-2 Regular Article Synthesis, Spectroscopic Characterization, Biocidal evaluation Molecular Docking & DFT Investigation of 16-18 membered Macrocyclic Complexes of Cobalt (II) Subhash 1 http://orcid.org/0000-0003-3648-998X Chaudhary Ashu [email protected] 1 Jyoti 2 Kumar Manish 3 Kumar Naveen 4 Agarwal Neeraj K 4 1 grid.411194.8 0000 0001 0707 3796 Department of Chemistry, Kurukshetra University, Kurukshetra, 136119 Haryana India 2 grid.411194.8 0000 0001 0707 3796 Department of Applied Science, UIET, Kurukshetra University, Kurukshetra, 136119 Haryana India 3 Department of Chemistry, Maharishi Dayanand University, Rohtak, 124001 Haryana India 4 grid.411194.8 0000 0001 0707 3796 Department of Microbiology, Kurukshetra University, Kurukshetra, 136119 Haryana India 22 11 2022 2022 134 4 11316 8 2022 11 10 2022 13 10 2022 © Indian Academy of Sciences 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Macrocyclic ligands (MacL1-MacL3) and Co(II) complexes were synthesized via template condensation of o-phenylenediamine with various aromatic dicarboxylic acids. The elemental analysis, FT-IR, mass spectrometry, 1H NMR, 13C NMR, UV-vis, SEM analysis, powder X-ray diffraction, thermogravimetric (TG) analysis, electrochemical studies, and DFT analysis were used to characterize these synthesized ligands and their cobalt (II) complexes. TGA analysis to determine the stability and decomposition kinetic parameters. In element analysis, the percentage of different elements present and also the stoichiometry of compounds were confirmed. The proposed framework for tetraaza macrocyclic cobalt (II) complexes was supported by spectral analysis, which also revealed distorted octahedral geometry surrounding the central metal atom. The molecular structure of cobalt (II) complexes was also optimized theoretically, and their electronic or thermodynamic parameters were obtained from density functional theory (DFT). The synthesized ligands and their cobalt (II) complexes were tested against bacteria: Escherichia coli, Bacillus subtitles. Candida albicans were tested for antifungal properties. It was found that ligands and complexes show good antimicrobial results. Finally, using the Auto Dock VinaPyRx programme, molecular docking studies were used to evaluate the biological significance of the synthesized ligands to identify the probable and efficient binding mechanisms between the various ligands and the active site of the receptor protein. Graphical abstract Antimicrobial, DFT, and Docking representation of Cobalt (II) macrocyclic complexes Supplementary Information The online version contains supplementary material available at 10.1007/s12039-022-02109-2. Keywords Cobalt(II) Complexes Macrocycles Antimicrobial Docking DFT University Grants Commission (UGC) New Delhi IndiaRef. No.- 92(CSIR-UGC NET DEC. 2018 Subhash issue-copyright-statement© Indian Academy of Sciences 2022 ==== Body pmcIntroduction The current scenario of macrocyclic ligands with amide group and their transition metal complexes have been developing an active area of research interest from the last few decades, attracting the attention of the researchers due to the broad biological actions of transition metal complexes.1–4 An important class of tetraamide macrocyclic (MacL1-3) ligands are derived from the condensation of o-phenylenediamine with various aromatic dicarboxylic acids. Such type of ligands and their transition metal complexes have a wide-ranging field of pharmacological activities such as antitumor;5–7 antibacterial;8–10 antifungal;11,12 antioxidant;13,14 anti-inflammatory15 and analgesics16 activities. The formation of the macrocyclic ligands implicates the metal ion in the cavity of macrocyclic ligands and forms stable complexes, which makes the reactant groups in the desired conformation for the ring closer.17 The cyclization reaction is supported by the formation of metal-ligand bonds and the arrangement of multidentate ligands around the central metal atom, both favored by the formation's enthalpy, which also overcomes the unfavourable entropy.18 In general, the formation of stable macrocyclic complexes depends upon the size, ring structure, and rigidity of the cavity of the macrocycle. The strength and magnitude of crystal field effects are especially crucial in the case of transition metal complexes.19 The transition metal macrocyclic complexes formed by the macrocyclic ligands having the heterocyclic moieties are of prodigious importance due to their biocidal activities and good binding affinity with proteins.20 Hence, the biological activities of the macrocyclic complexes containing heterocyclic moieties in the macrocyclic ring can increase the selectivity of ligands for a given ion and enhance the metal complex biological properties.21 The substituted pyridine is a fortunate framework and the most important class of heterocyclic compounds. It shows the many interesting biological activities and is widely tolerated by people owing to their enormous biological activities. People widely tolerate it and show extensive applications in remedial chemistry.22 The macrocyclic complexes and their derivatives derived from the o-phenylenediamine have been studied, and due to their pharmacological and biological properties, the o-phenylenediamine containing macrocyclic complexes gives a broad class in synthetic inorganic chemistry.23 Following a thorough review of the literature, the DFT was used in the current study to calculate a number of macrocyclic molecules properties. DFT is unquestionably the best option for theoretical calculations. Cobalt(II) macrocyclic complexes possess significant antibacterial and antifungal as compared to other transition metal complexes.24 Here in the presented work, the macrocyclic ligands were synthesized by condensation of o-phenylenediamine with substituted pyridine and phthalic acid, and their coordination behaviour with cobalt(II) compounds was studied (Scheme 1). Numerous spectral analyses, including elemental analysis, FT-IR, 1H NMR, 13C NMR, mass spectrometry, UV-vis, SEM analysis, X-ray diffraction, DFT, and TG analysis, were used to explain the structure of these ligands and their complexes. The MacL1-3 ligands and their complexes were optimized theoretically. Molecular docking and antibacterial activity have also been investigated in the current work.Scheme 1 Synthetic route of the synthesis of tetraamide ligand (MacL1) and their cobalt(II) complex. Experimental Material and methods The solvents and other metals salt used were of high purity, purchased from Sigma-Aldrich, and utilized just as it arrived. The phthalic acid, pyridine-2,3-dicarboxylic acid, and pyridine-2,6-dicarboxylic acid from (fluka). All the solvents are of AR grade. Synthesis of ligand (MacL1-MacL3) The 2:2 molar ratios are used to carry out the reaction. Ethanolic solution of o-phenylene diamine (1.08 g, 0.01 M) was placed in a magnetically stirred round-bottomed flask, and ethanolic solution of Phthalic acid (1.66 g, 0.01 M), pyridine-2,3-dicarboxylic acid (1.67g, 0.01 M) and pyridine-2,6-dicarboxylic acid (1.67g, 0.01 M were refluxed at room temperature for about 8-10 h and to make the solution acidic add the few drops of conc. HCl (to facilitate the condensation reaction). The colored ppt are formed with % yield of 76%, 78%, and 75%, respectively. The formed precipitate was separated by filtration and repeatedly rinsed with ethanol and dried in vacuo. The synthetic compounds were once more recrystallized in benzene and dried in vacuo. MacL1: Off white solid; Yield 76%; M.p.; 107 °C; Analytical calculated for C28H20N4O4; C, 70.58; H, 4.23; N, 11.76; Found; C, 69.80; H, 4.04; N, 11.60; MS(ESI), m/z: 476.15 [M+H]+. MacL2: Off white solid; Yield 78%; M.p.; 142 °C; Analytical calculated for C26H18N6O4; C, 65.27; H, 3.79; N, 17.56; Found; C, 64.92; H, 3.45; N, 17.10; MS(ESI), m/z: 478.14 [M+H]+. MacL2: Off white solid; Yield 75%; M.p.; 169 °C; Analytical calculated for C26H18N6O4; C, 65.27; H, 3.79; N, 17.56; Found; C, 65.12; H, 3.15; N, 17.08; MS(ESI), m/z: 478.14 [M+H]+. Synthesis of complexes [Co(N4 MacL1)Cl2]– [Co(N6 MacL3)Cl2] The 1:1 molar ratio are used to carry out the reaction. The complexes were synthesized by template method. A weighed amount of CoCl2.6H2O (0.023 g, 0.0001 mol) dissolved in ethanol and added to the ethanolic solution of ligand MacL1 (0.047 g, 0.0001 mol) and mixed slowly with constant stirring. The resulting solution was refluxed for about 10-13 h at 45 °C. The solid Brown colored precipitate was obtained, separated by filtration, washed thoroughly with ethanol, and dried under vacuo (% yield=78%). Recrystallized from benzene. The same procedure has been used for the synthesis of Co(N6MacL2)Cl2 and Co(N6 MacL3)Cl2 by replacing MacL1 with MacL2 and MacL3, respectively, with % yield of 76% and 75%, respectively (Scheme 1). [Co(N4 MacL1)Cl2]: Reddish pink solid; Yield 78%; M.p. 147 °C; Analytical calculated for C28H20Cl2CoN4O4; C, 55.47; H, 3.32; N, 9.24; Found; C, 54.91; H, 3.08; N, 8.82; MS(ESI), m/z: 606.02 [M+H]+. [Co(N6 MacL2)Cl2]: Brown solid; Yield 76%; M.p. 134 °C; Analytical calculated for C26H18Cl2CoN6O4; C, 51.34; H, 2.98; N, 13.82; Found; C, 51.04; H, 2.51; N, 12.97; MS(ESI), m/z: 608.30 [M+H]+. [Co(N6 MacL2)Cl2]: Brown solid; Yield 76%; M.p. 134 °C; Analytical calculated for C26H18Cl2CoN6O4; C, 51.34; H, 2.98; N, 13.82; Found; C, 51.09; H, 2.62; N, 13.60; MS(ESI), m/z: 608.30 [M+H]+. The analytical data and physical properties The Rast Camphor method was used to determine the molecular weights. Conductivity Bridge 305 was used to evaluate conductivity in dry DMF (dimethylformamide). The CHNS Analyzer was used to determine the percentage of the various components (C, H, and N) present: ELEMENTAR Vario EL III at STIC Kerala, the FT-IR was recorded on FT-IR; Perkin Elmer Spectrum IR Version 10.6.2 using as KBr disc India. NMR spectra were recorded in DMSO-d6 using a JEOL400 ECZS NMR Spectrometer, and A SCIEX Triple TOF 5600 mass spectrometer was used to certify the mass spectra. The complexes in DMSO had their electronic spectra (10-3 M, 200–800 nm) measured at room temperature using a Perkin Elmer -25 spectrophotometer. Co(II) complexes ESR spectra were captured at liquid nitrogen temperature (LNT) using a JEOL JES - FA200 ESR Spectrometer with the X and Q bands. The powder X-ray diffraction was recorded on Bruker D8 Advance Twin- Twin at STIC Kerala, India. The morphological study was examined using a JEOL JSM6510 scanning electron microscope (SEM). TGA study has determined the stability and decomposition kinetic characteristics. Thermogravimetric analyzer (Hitachi STA 7200) TGA/DTA study was carried out with N2 flow (20 mL min-1) and heating rate of 10 °C/min. Results and Discussion The biological active tetraamide macrocyclic ligands (MacL1-MacL3) and their corresponding complexes [Co(II)(N4 MacL1)Cl2], were derived from o-phenylenediamine and aromatic dicarboxylic acid by template condensation reactions (Scheme 1). All the complexes are colored solids. All the synthesized macrocyclic ligands and cobalt (II) complexes were soluble in most of the organic solvents like benzene, methanol, DMF, and DMSO and sparingly soluble in water. They are non-electrolytic in nature since the observed molar conductance values for 10−3, mol L-1 solutions in dry DMF are in the range of 13-32 ohm-1 cm2, also suggesting that the anions are coordinating with the central metal atom in these complexes. The physical properties and analytical data for all the macrocyclic ligands and cobalt (II) complexes are given in Supplementary Information (Table S1, SI). The analytical data confirmed the proposed stereochemistry of the ligands and complexes. Numerous spectroscopic, physicochemical, and biological characterizations were done on the synthesized macrocyclic ligands and the related macrocyclic complexes. FT-IR The absence of -NH2 stretching vibrations of amino acids and -OH groups of dicarboxylic acids in the FT-IR spectra of macrocyclic ligands and their complexes is the first factor that conforms the synthesis of macrocyclic ligand. In the spectra of all the ligands band present in the region of 3244-3279 cm-1 assigned to ν(NH) of the amide group. The bands in the areas of 1648-1707, 1544-1587, 1252-1275, and 628-684 cm-1 in the IR spectra of the ligands and complexes can be attributed to the amide I. [ν(C=O)], amide II [ν(C-N) + δ(N-H)], amide III [δ(N-H), and amide IV wagging [Θ (C=O)] vibrations respectively which confirmed the closed cyclic products are present.26 The complexes [Co(N4MacL1)Cl2]-[Co(N6MacL3)Cl2] showed a minor negative value in the (NH) band in the area 3215-3235 cm−1 when compared to their ligands, which is well in keeping with the coordinated N-H stretching vibration.27 This is further supported by the possibility of assigning the (M-Cl) and (M-N) vibrations to all complexes' bands in the range of 330-336 cm-1 and 480-490 cm-1, respectively. The complexes absorption bands are attributed to the C-H stretching and bending vibrational modes, which exist in the areas 2815-2975 and 1420-1448 cm-1, respectively (Table 1, Figure 1).Table 1 Characteristic IR Spectral data of ligands (MacL1-MacL3) and Co(II) complexes (in cm-1). Compound ν(NH) Amide ν(Co-N) ν(Co-Cl) I II III IV [MacL1] 3244 1648 1558 1252 661 - - [MacL2] 3268 1669 1544 1265 665 - - [MacL3] 3279 1683 1567 1271 628 - - [Co(N4 MacL1)Cl2] 3215 1675 1571 1267 659 490 303 [Co(N6 MacL2)Cl2] 3226 1707 1580 1275 678 486 336 [Co(N6 MacL3)Cl2] 3235 1692 1587 1254 684 480 332 Figure 1 IR spectrum (a) ligand MacL1 (b) Complex [Co(N4MacL1)Cl2]. 1H NMR of the tetraamide macrocyclic ligands (MacL1-3) In the 1H NMR spectra of the macrocyclic ligands (MacL1-MacL3 were recorded in (400 MHz, DMSO-d6), the peaks indicated the mode of linkage in the compounds. The 1H NMR spectra of the precursors showed the signals of the -COOH proton and -NH2, but were absent in the synthesized ligands, indicating the removal of the -COOH and -NH2 protons for the formation of a macrocyclic structure. The information well accords with what the infrared data have revealed. The 1H NMR spectra of the ligands (MacL1-MacL3) presented in Table 2 exposed the signals predictable for the given Scheme. From the 1H NMR spectra of the (MacL1-MacL3) it is also concluded that no peak could be assigned for -OH or -NH2 groups, confirming the formation of proposed macrocyclic ligands after the condensation.28 A signal was detected in the region δ 9.10-9.60 ppm for amide protons in macrocyclic ligands.29 In the spectra of the macrocyclic ligands, the multiplets appearing in the region δ 8.11-8.21 ppm and δ 6.32-6.47 ppm correspond to the 1,4-and 1,2-phenyl ring protons, respectively, while multiplets appeared in the region δ 6.40-7.76 ppm was assigned to protons of a pyridine-2,3-dicarboxylic acid moiety which have different chemical environment (Figure 2).30Table 2 1H NMR data (in ppm) of ligands (MacL1- MacL3). Compound (CO-NH) (m) 1,4-C6H4 (m) 1,2-C6 H4 (m) C6H3N– MacL1 9.32 8.11, 8.21 6.47, 6.32 - MacL2 9.60 - - 6.40-7.76 MacL3 9.10 - - 6.53-7.66 Figure 2 1H NMR spectrum of ligand (MacL1). 13C NMR The results drawn from the FT-IR and 1H NMR spectra were also supported by the 13C NMR spectral data regarding the validity of the suggested Skelton. The carbons linked to the nitrogen atoms showed a chemical shift indicative of their suggested coordination in the ligands (Table 3) (Figure 3).Table 3 13C and NMR spectral data of macrocyclic ligands (MACL1-3). Compound (CO-NH) 1,4-C6H4 C6H3N- 1,2-C6H4 MacL1 169.08 115.4, 118.1 - 129.5, 133.3, 134.5 MacL2 169 - 150.8, 132.6, 128.2 - MacL3 164 - 151.3, 131.5, 127.7 - Figure 3 13C NMR spectrum of ligand MacL1. Mass spectrum of the ligands (MacL1-3) and cobalt(II) complexes The mass spectra of ligands (MacL1-MacL3) and their corresponding cobalt (II) complexes were recorded. The proposed molecular formula of the ligand [(C28H24N4O4)] was confirmed by the mass spectrum of the ligand (MacL1), which displayed a molecular ion peak at m/z 477.21 amu [M+] that corresponded to its molecular weight. The line (at m/z=325 amu (C18H14N2O2C2+)+1) in Figure 4 is called the base peak, and it is due to the commonest fragment ion. The intensity of all other peaks was measured with respect to this base peak. The other fragmentation peaks were also observed at 163(C6H6N2O2C2+)+1, 189(C8H8N2O2C2+)+1, 201(C10H8N2O2C2+)+1, 214(C10H8N2O2C+C)+1, 241(C13NO2HN+H10C2+)+2, 278(C15H13N2O2C2+)+1, 289(C16H16N2O2C2+)+1, 303(C17H13N2O2C2+)+1, 340(C20H14N2O2C2+)+2 and 371(C18H14N2O4C2+)+1 amu. The intensity of these peaks confirmed the stability of various fragments. In the mass spectra of all other ligands and their corresponding complexes MacL2, MacL3, [Co(N4MacL1)Cl2], [Co(N6MacL3)Cl2], and [Co(N6MacL3)Cl2] show the molecular ion peak at 481, 481, 607,609 and 609, respectively. This is comparable to the molecular mass of compounds.Figure 4 Mass spectrum of (a) ligand (MacL1) (b) Complex [Co(N4 MacL1)Cl2]. Electronic spectral data The observed magnetic moment of Co(II) complex at room temperature in the range of 4.80–4.87 B.M. corresponds to three unpaired electrons, which is substantially higher than the spin-only value, 3.87 B.M. The spin-orbit coupling may cause this deviation from the spin-only value. The electronic spectra of six coordinated cobalt (II) complexes were recorded using DMSO as solvent. For high spin d7 the bands are observed in the at 9663-12,620 (ν1), 13,242-13,979 (ν2) 19,498-24,701 cm-1 (ν3) and 26,298-32,359 cm-1 (ν4). The electronic spectrum of the Co(II) complex exhibits these bands, and the position of the bands is comparable to the characteristic features of distorted octahedral geometry and having the D4h Symmetry. These bands are corresponding to the following transition: 4T1g(F) → 4T2g(F) (ν1), 4T1g(F) → 4A2g(F) (ν2), 4T1g(F) → 4T2g(P) (ν3), respectively.31 The fourth band may be due to charge transfer (Table 4, Figure 5).Table 4 Electronic spectral data and magnetic moments of the Cobalt (II) macrocyclic complexes. Compound μeff. (BM) λmax (cm-1) [Co(N4 MacL1)Cl2] 4.82 9663, 13242, 19498, 26298 [Co(N6 MacL2)Cl2] 4.83 10948, 13575, 21575, 28705 [Co(N6 MacL2)Cl2] 4.87 12642, 13979, 24701, 32359 Table 5 (a) Thermal Parameters of tetraamide macrocyclic ligands and their Cobalt (II) complexes by applying Coats and Redfern method at a heating rate 10 °C/ min. Compound R (From Graph) TMAX. (From DTG) (Ea) (J/mol) (A) (min.-1) (∆G) (KJ/mol) (∆H) (KJ/mol (∆S) (J/mol) MacL1 0.70858 438.08 12.99 0.002312 130.780 -3.628 -290.31 MacL2 0.99354 510.00 36.48 0.004827 148.059 -4.203 -282.10 MacL3 0.99618 523.00 54.90 0.006879 148.102 -4.185 -282.19 [Co(N4MacL1)Cl2 0.76722 453.00 7.69 0.001271 137.638 -3.758 -295.54 [Co(N6MacL2)Cl2 0.67599 463.00 5.49 0.000872 142.197 -3.836 -298.55 [Co(N6MacL3)Cl2 0.54652 478.93 10.00 0.001480 145.107 -3.971 -294.68 (b) Thermal Parameters of tetraamide macrocyclic ligands and their Cobalt (II) complexes by applying the Ozawa-Flynn-wall method at a heating rate 10 °C/ min. Compound R (From Graph) TMAX. (From DTG) (Ea) (J/mol) (A) (min.-1) (∆G) (KJ/mol) (∆H) (KJ/mol (∆S) (J/mol) MacL1 0.88182 438.08 21.86 0.0039 128.891 -3.619 -286.00 MacL2 0.99521 510.00 44.46 0.00596 148.943 -4.195 -300.27 MacL3 0.99723 523.00 63.60 0.0080 151.588 -4.284 -281.65 [Co(N4MacL1)Cl2 0.95704 453.00 17.87 0.00310 125.619 -3.748 -269.03 [Co(N6MacL2)Cl2 0.90124 463.00 11.55 0.001839 139.562 -3.837 -293.14 [Co(N6MacL3)Cl2 0.84213 478.93 20.43 0.003047 160.606 -3.961 -327.03 Figure 5 UV-vis spectrum of Co(II) macrocyclic complex (1b). The EPR spectra of [Co(N4MacL1)Cl2]- [Co(N6MacL3)Cl2] complexes EPR spectroscopy can be used to investigate the paramagnetic properties of cobalt (II). In cobalt (II), due to the very fast spin-lattice relaxation time, the EPR measurements are possible only at liquid nitrogen temperature to avoid broadening the line at higher temperatures.32 The EPR spectra of the tetraaza macrocyclic cobalt(II) complex were recorded in dry DMF solution. The g\|\| and g⊥ values are found to be 2.12 and 1.78, respectively Figure 6. The spin-only value for g is 2.0023, and the difference in g values from the spin-only value may be due to the high angular momentum contribution.Figure 6 EPR spectrum of Co(II) macrocyclic complex. Thermogravimetric analysis The thermogravimetric (TG) analysis of macrocyclic ligands (MacL1-MacL3) and their corresponding cobalt (II) complexes was carried out with N2 flow (20 mL/min) and heating rate of 10 °C/min in the temperature range 45-650 °C. Thermogravimetric curve is given in Figure 7. The thermogravimetric curve for ligand (MacL3) shows the complete decomposition of 100% mass loss in one step. The [Co(N6 MacL3)Cl2] TG curve is a three-step breakdown with a mass loss of 70%. The fact that there is no weight loss before 180 °C implies that the complex is stable up to 180 °C in the absence of coordinated water molecules. First mass loss of 11.51% occurred between 180 and 220 °C, indicating the breakdown of the Cl2 group. The 2nd mass loss of 26.09% was discovered in the temperature range of 250-320 °C, showing the breakdown of the C10H6N2 group. In the range of 510-600 °C, C16H6N4O4 loses 52.82 % of its weight throughout the decomposition.33Figure 7 Thermogravimetric analysis of ligand (MacL3) and Complex [Co(N6 MacL3)Cl2]. The kinetic decomposition parameters in TGA have been calculated using two kinetic approaches: the COATS AND REDFERN method and the Ozawa-Flynn Wall (OFW) method. Furthermore, the thermodynamics parameter such as Arrhenius factor, enthalpy change, Gibbs free energy, and entropy change has been calculated.34 Kinetic Parameters The kinetics of macrocyclic compounds was studied by data obtained from TGA. The rate constant (k) and the degree of conversion (α), which both relate to thermal degradation, are two functions that describe the rate of reaction:1 dαdt=k(T)f(α) In the above equation, absolute temperature (T) in Kelvin, the time duration of a reaction (t) in minutes, the degree of conversion is (α), the differential form of the reaction model is f(α) is, and the reaction rate constant is k(T) is, α can be written as:2 α=mo-mtmo-m∞ where m0 represents the initial mass, mt represents the mass with respect to time, and m∞ represents the sample's final mass. The Arrhenius equation can be used to express the rate constant k(T):3 k(T)=Ae-EaRT where R= gas constant, Ea = activation energy, and A = pre-exponential factor. The equation for the rate of reaction with linear heating rate can be found by combining equations (1) and (2)4 dαdt=Aβe-EaRTf(α) Different iso-conversional kinetic approaches are developed based on Equation (4). These kinetic methods used different temperature integral approximations to calculate the kinetic parameters. The following can be stated as the generic equation that applies to all kinetic methods:5 lnβTB=constant-CEaRT In this study, the most popular kinetic methods, Ozawa-Flynn-Wall (OFW), Coats and Redfern, are utilized to compute the activation energy of the tetra azamacrocyclic ligands and their cobalt(II) complexes.35 The Doyle approximation is the foundation of the OFW technique, which is based on the idea that the variables (A, g(α), and Ea) are independent of the absolute temperature (T), and it is written as follows:6 lnβ=lnAEagαR-5.331-1.052EaRT where g(α) is an ideal reaction model's integral form. The Coats and Redfern approximation is the foundation of the Coats and Redfern method, which is defined by the equation given below:7 lnβT2=lnAREa-EaRT Analysis The overall activation energy was found to be around 107.953 kJ/mol. The activation energy obtained through Kissinger method is close to the activation energies obtained from OFW, KAS, and for conversion of 0.1 and 0.2. After the kinetic study, the following equation calculated the thermodynamic parameters such as Arrhenius factor, Enthalpy change, Gibbs free energy, and Entropy change.8 A=β×Ea×eEaRTmaxRTmax2 9 ΔG=Ea+R×TmaxlnKBTmaxhA 10 ΔH=Ea+RTmax 11 ΔS=ΔG-ΔHTmax where kB is Boltzmann’s constant (1.38×10-23 Js-1), and h is Planck constant (6.626×10-34Js-1). The result for Arrhenius the Factor uses different kinetic methods for different heating rates. Further, the result of ∆H, ∆G, and ∆S using the different kinetic methods for a 10 °C/min heating rate. The positive values of ∆H and ∆G show that the thermal decomposition of MacL2 is an endergonic, non-spontaneous, and unfavourable process (Figures 8, 9).Figure 8 The linear fitted curve obtained through the Coats-Redfern method at 10°/min heating rate of (MacL1-MacL3). Figure 9 The linear fitted curve was obtained through the Ozawa -Flynn-wall method at 10°/min. The heating rate of (MacL1-MacL3) (Table 5). X-ray powder diffraction The X-ray diffraction analysis of the tetraamide macrocyclic ligands (MacL1-3) and their complexes [Co(N4MacL1-3)(Cl)2] was obtained in the range of 2θ =10-80° Figure 10. Among all the compounds, the ligands in the diffractogram of ligands show sharp peaks, indicating their crystalline nature. Whereas in the diffractogram of all the cobalt (II) complexes, peaks expansion and less intensity suggest the semi-crystalline or amorphous nature of the complexes. The average crystalline size of the ligands and their corresponding cobalt (II) complexes were calculated by using Scherrer’s formula. The highest intensity peaks were at 2 θ = 22.8167° and 21.7313° for ligand and complex. The presence of metal in the ligand environment is confirmed by the powder X-ray diffraction.35D=kλβcosθ Where the λ=wave length of the X-rays used, θ = diffraction angle, β= (FWHM) full width at half maximum of the peak position, and K is Scherrer's constant. The ligand and complexes computed average crystalline size were discovered to be 51.34 and 45.68 nm, respectively. The contraction of Cobalt(II) macrocyclic complexes is a sign of macrocyclic ligand coordination with cobalt metal, which results in a contraction of the ligand in complexes. However, many attempts have been tried to form a single crystal but have not been successful in growing the single crystal. The powder X-ray diffraction study shows that the ligands, which have four coordination sites, function as tetradentate chelating agents. Additionally, a hexacoordinated environment for cobalt has been proposed since the Cl- anions coordinated to the cobalt atom.Figure 10 (a) The PXRD diffractogram of ligand (McL1) (b) The PXRD diffractogram of Complex [Co(N4MacL1)Cl2. SEM Analysis of Tetraamide Macrocyclic ligands and Cobalt (II) Complexes: The surface morphology of the tetraamide macrocyclic ligands (MacL1-3) and their complexes [Co(N4MacL1-3)(Cl)2] have been examined using scanning electron microscope (SEM) analysis, and it is observed that the SEM image of the ligand MacL1 revealed highly pure and small roads like structure revealing its crystalline nature in Figure 11 (a). While the irregular rough surface clouds indicated the SEM image of complexes the semi-crystalline character of the complexes in the SEM image. From the scanning electron microscope analysis, the change in surface morphology of the compounds also confirms the coordination of metal ions with the donor atom of ligands. The average crystalline size was found to be 50 and 45 nm for the ligand and complex, respectively the contraction in the size of complexes compared to ligand was is in good agreement with the results from the powder X-ray diffraction analysis.36 Although attempts were made to grow single crystals many times but failed.Figure 11 SEM image of (a) ligand MacL1; (b) complex [Co(N4 MacL1)Cl2]. Electrochemical study The oxidation levels of the metal centers in the cobalt(II) complexes were verified using cyclic voltammetry. In order to take into account the spectral and structural changes that come along with electron transfer, the electrochemical characteristics of metal complexes, particularly those with nitrogen donor atoms were explored. Using an electrochemical analyzer, the redox behaviour of cobalt(II) complexes in DMSO was investigated. Glassy carbon served as the working electrode. Only in relation to the metal core are any of these compounds electroactive. Cobalt(II) is oxidized to cobalt(III) on the cyclic voltametric time scale, and the detection of a single, strong peak that is not displayed by the matching ligand shows that the metal center is symmetrically situated in one complex. When compared to the Ag/AgCl, CI electrode, the cobalt (II) complexes exhibit both metal- and ligand-centered electrochemistry in the potential range of 1.7 V. Peak-to-peak separation of all the complexes is greater than 100 mV, indicating a single, nearly reversible oxidation process. They all display redox potentials between 1.0 and 1.25 V. The cathodic and anodic peak heights (Ipc and Ipa) are identical in all cobalt (II) complexes. The Co(III)/Co(II) pair is responsible for the cobalt (II) complexes' electrochemical behaviour. The positive potential shows that the ligand is tightly linked to a metal in a lower oxidation state (Figure 12).45Figure 12 Cyclic voltammogram of complex [Co(N4 MacL1)Cl2]. Molecular modeling The software Avogadro 4.0 was used to perform the geometry optimization. Figure 13 depicts the fully optimized molecular structure of the ligands and Cobalt (II) macrocyclic complexes. The bond lengths of the macrocyclic ligands and the cobalt (II) complexes are compared, with the bond angles form the Cobalt (II) complexes listed in Table 6 and Table 7, respectively. The proposed structure is verified by the bond angle values, which are approximately equal to the octahedral geometry of the complexes. The C=O and C-N bond lengths of the ligand are 1.2051 and 1.39048, respectively. In complexes, these C=O (1.2274 Å) and C-N (1.42685 Å) bond lengths become longer, indicating coordination of the ammine group via the N atom. Selected bond lengths of complexes are Co-N and Co-Cl, confirming the distorted octahedral geometry of the Cobalt(II) complexes.37Figure 13 Optimized structure of ligand (MacL1) and the Co(II) macrocyclic complex. Color code: blue-N; red-O; grey-C; white-H; green-Cl; peach-Co. Table 6 The calculated bond lengths (Å) of the ligand and the complexes Compound C=O C-N N-H Co-N Co-Cl MacL1 1.2051 1.39048 1.01095 - - MacL2 1.21695 1.37088 1.02246 - - MacL3 1.21309 1.38103 1.01201 - - [Co(N4 MacL1)Cl2] 1.22746 1.42685 1.02993 1.98771 2.24491 [Co(N6 MacL2)Cl2] 1.22669 1.43265 1.03269 2.18037 2.23325 [Co(N6 MacL3)Cl2] 1.22627 1.42447 1.03063 2.01901 2.25196 Table 7 Selected bond angles (°) for the Cobalt(II) complexes. Atom connectivity Bond angles (°) Atom connectivity Bond angles (°) N-Co-N 86.7523 N-Co-Cl 89.2919 N-Co-Cl 179.6951 N-Co-Cl 90.6023 N-Co-Cl 89.6435 N-Co-Cl 90.4243 N-Co-N 179.7675 N-Co-Cl 89.5951 N-Co-Cl 90.7574 Cl-Co-Cl 179.8913 N-Co-Cl 89.3485 N-Co-N 86.8228 Density Functional Theory (DFT) Study The DFT calculations have been carried out by using Avogadro 4.0 with the ORCA program. The frontier molecular orbitals of a molecule are its highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), and they play a significant role in regulating molecular chemical reactions. It is also confirmed from the previous investigations that the gap in energy (ΔE) between the HOMO-LUMO is explained the stability of the molecule. The energy gap (ΔE) of these frontier molecular orbitals for macrocyclic ligands and complexes are used to predict the stability and strength of these compounds are obtained from DFT calculations. The Koopman’s Theorem specifies that the energy of HOMO-LUMO corresponds to ionization energy (IE) and electron affinity (EA), respectively. According to Koopman's Theorem, calculations of HOMO-LUMO energies values presented in Table 8 can be used to determine quantum chemical parameters38–41 for example, electrophilicity index (ω), chemical potential (Pi), electronegativity (χ), softness (S) and hardness (η).Table 8 Theoretically calculated quantum parameters of the macrocyclic ligands and complexes. Parameters (MacL1) (MacL2) (MacL3) 1(a) 1(b) 1(c) EHOMO (eV) -8.687 -8.438 -8.617 -15.25 -11.44 -17.15 ELUMO (eV) -1.135 -1.223 -1.017 -9.535 -9.238 -15.28 ∆E (eV) 7.552 7.215 7.6 5.715 2.208 1.87 IE (eV) 8.687 8.438 8.617 15.25 11.446 17.15 χ (eV) 4.911 4.830 4.817 12.3925 10.342 16.2195 η (eV) 3.776 3.607 3.8 2.8575 1.104 0.935 S (eV)-1 0.132 0.138 0.131 0.1749 0.4528 0.5347 ω (eV) 3.19 3.23 3.053 26.8721 48.4402 140.68 Pi -4.911 -4.830 -4.817 -12.392 -10.342 -16.219 The HOMO and LUMO energy gap for ligands is found to be 7.552 eV, 7.215 eV, 7.6 eV. The energy gap is reasonably reduced in complexes, respectively (Figure 14). HOMO and LUMO are distributed throughout the entire π -moiety of the ligand. HOMO is distributed across the entire π-moiety, and LUMO is distributed over cobalt metal in complexes.Figure 14 (a) The HOMO-LUMO energy gap of the tetraamide macrocyclic ligands (MacL1-3). (b) The HOMO-LUMO energy gap of the Cobalt(II) complexes. Antimicrobial assays Antibacterial activity The Inhibition Zone Technique was used to assess the bacterium-fighting ability of the bacteria.25 In this approach, 5 mm diameter Whatman No. 1 paper plates with the supplement agar medium (composed of 5 g peptone, 5 g meat extract, 5 g NaCl, 20 g agar, and 1000 mL distilled water) were used. The Petri dish was filled with pipetted agar media. 5 mL of heated seeded agar was added after it had set. The molten agar was cooled to 40 °C to create the seeded agar, and the bacterial solution was then added. In dimethylformamide, the compounds were dissolved at 80 ppm concentrations. We soaked 5 mm diameter paper discs of Whatman No. 1 filter paper in these solutions at 80 ppm concentrations. The petri plates were dried and positioned at the proper distance on the medium previously seeded with organisms in petri plates. For 24 h, the petri plates were kept in an incubator set at 30±2 °C. The precise measurement in mm of the inhibitory zone that subsequently formed around each disc containing the test chemicals. The synthesized compounds are tested against Bacillus subtitles (+) and Escherichia coli (-) by taking Ampicillin as standard at 80 ppm concentration. The results are shown in Table 10. The results of antibacterial screening show the maximum inhibition by Cobalt (II) complexes compared to ligands because complexes formed as a result of chelation become more lipophilic. When chelating, the polarity of the metal ion disappears, which causes the orbitals of the ligands to overlap and the metal's positive charge to be shared with donor groups. The invasion of complexes into the lipid membranes is efficiently induced by the delocalization of electrons on the chelate ring, which inhibits microbial growth. Antifungal activity The Radial Growth Method25 was used to assess the antifungal activity of the ligands and their complexes with divalent cobalt using Czapeks agar medium, which is (composed of 20 g of starch, 20 g of agar-agar, 20 g of glucose, and 1000 mL of distilled water). The Solutions of the test ligands and Cobalt(II) complexes at 80 ppm concentration in dimethylformamide were prepared. The agar medium was then emptied into the Petridis’s, and by using an inoculum needle, the fungi were placed on the medium. Petri plates wrapped in polythene and placed in an incubator set at 28±2 °C with a few drops of alcohol. By measuring the fungal region's diameter, the fungus's linear growth was determined. The Candida albicans organisms used in these investigations for all synthesized compounds the results are in Table 9 (Figure 15)Table 9 Antibacterial and antifungal studies of ligands and their Cobalt(II) complexes. Antibacterial and antifungal studies of ligands and their Cobalt(II) complexes Compound Antibacterial activity (Zone inhibition diameter in mm) Antifungal activity (Zone inhibition diameter in mm) E Coli Bacillus subtilis Candida albicans [MacL1] 08 10 09 [MacL2] 10 12 12 [MacL3] 09 08 08 [Co(N4 MacL1)Cl2] 13 12 15 [Co(N6 MacL2)Cl2] 15 17 20 [Co(N6 MacL3)Cl2] 10 12 11 Ampicillin 20 20 Amphotericin-B - - 22 Table 10 The binding affinity (kcal/mol) of ligands (MacL1-3) with different proteins. Active Protein (PDB ID) Binding Affinity (kcal/mol) MacL1 MacL2 MacL3 Sars-Cov-2 (2ajf) -10.4 -9.8 -10.0 C. Albicans (3dra) -10.7 -10.1 -10.4 X. Campestris (3row) -10.8 -10.7 -10.4 E. Coli (3t88) -11.2 -14.0 -9.9 B. subtilis (5h67) -11.7 -10.5 -10.6 Figure 15 Zone inhibition diameter(mm) data of macrocyclic ligands and complexes. (a) Antibacterial activity; (b) Antifungal activity. Antifungal activity results show that the ligand Macl2 is more active than macL1 and MacL3, and all the complexes [Co(N6 MacL2)Cl2] are more active against C. Albicans. The coordination of metal to the ligands influences the magnetic property, conductance, and solubility. The high solubility of the complexes, which makes it easy to aggregate in bacterial and fungal cells and activate enzymes, is another explanation for their enhanced activity. The antimicrobial potential of Schiff base macrocyclic complexes of Transition metal has been reported by Chandra et. al.19 However, In the present investigation, N4 tetraaza macrocyclic complexes of Co(II) show superiority over these complexes. Molecular docking study The Molecular Docking study is an effective Computer-aided drug designing method for predicting the binding association and binding site between the drug and active site of bio-molecules (Proteins and CT-DNA). Docking also played a major role in advancing therapeutically significant molecules for over four decades.42 In this study, molecular docking was used to examine the particular groove binding characteristics and provide useful details about the way drugs bind in the active site. Molecular docking is an important computational technique in structural biochemistry and computer-aided drug design. Molecular modeling studies were carried in order to validate the results of our studies.43 Discovery studio with Auto Dock VinaPyRx software was used to assess the biological importance of synthesized ligands.44 Tetra amide macrocyclic ligands were docked with various receptor protein targets, including E. coli (3t88), C. albicans (3dra), X. campestris (3row), SARS-COV-2 (2ajf), and B. subtilis (5h67). Higher negative binding energy means more affinity of the ligand for receptors, which is the binding affinity utilized to select the best-docked structure from the output. The binding modalities of proteins amino acids are explained using 2D plots. Within the docking compounds, typical hydrogen bonds were discovered, and several hydrophobic non-polar interactions were found between the aromatic carbons of the macrocyclic ligands and the amino acids of the active protein. The docking site on the protein targets was defined and then run with pyrx AutoDockVina was performed with all ligand structure. The best pose for each run was saved. The interactions of ligands (MacL1-3) with protein sars-cov-2 (PDB: 2AJF). The interactions between the protein and ligand, including hydrogen bonding and hydrophobic interactions, were analyzed in Figure 16, and out of these ligands, MacL1 shows a good binding affinity. The MacL1 ligand was docked with the active site of the protein sars-cov-2 and showed various interactions. A conventional hydrogen bonding interaction (green color) is observed in phe390, asn390, and tyr394 with a bond length of 3.08 Å. Some pi-pi stacked interactions were also observed in phe40 and his401. The best-docked ligand MacL1 selected has a binding score of -10.4 kcal/mol with binding site of SARS-Cov 2 (2ajf) protein, as shown in Figure 16.Figure 16 (a) 2D interaction of ligand MacL1 with protein 2ajf. (b) 3D image of ligand MacL1 in the active site of protein 2ajf. The interactions of ligands with protein C. Albicans (PDB: 3DRA). The interactions between the protein and ligand, including hydrogen bonding and hydrophobic interactions, were analyzed in Figure 17, and out of these ligands, MacL1 shows a good binding affinity. The MacL1 ligand was docked with the active site of the protein C. Albicans and showed various interactions. The conventional hydrogen bonding interaction in asn435 with a bond length of 2.93 Å is observed (Light green color in 2D plot). Some pi-pi T-shaped interactions were observed in trp150, pi-sigma with thr154, pi-alkyl ILE434, pi-anion asp153, and van der Waals interactions with arg491 were observed. The best-docked ligand MacL1 selected has a binding score of -10.4 kcal/mol with a binding site of 3dra protein as shown in Figure 17.Figure 17 (a) 2D interaction of ligand MacL1 with protein 3dra; (b) 3D image of ligand MacL1 in the active site of protein 3dra. The interactions between the protein X. Campestris (PDB: 3ROW) and ligand, including hydrogen bonding and hydrophobic interactions, were analyzed, and out of these ligands, MacL1 shows a good binding affinity, as in Figure 18. The MacL1 ligand was docked with the active site of the protein X. Campestris and showed various interactions. The van der Waals interactions is observed in asn627, pro630, glu495, and ieu102. The interactions were also observed in pi-alkyl ILE434 and pi-anion asp153. The best binding mode of docked MacL1 ligands with the binding site of 3ROW protein is shown in Figure 18, with a binding affinity of -10.8 kcal/mol.Figure 18 (a) 2D interaction of ligand MacL2 with protein 3row; (b) 3D image of ligand MacL2 in the active site of protein 3row. The interactions between the protein E. Coli (PDB: 3T88) and ligand, including hydrogen bonding and hydrophobic interactions, were analyzed, and out of these ligands, MacL2 shows a good binding affinity, as in Figure 19. The MacL2 ligand was docked with the active site of the protein E. Coli and showed various interactions. A conventional hydrogen bonding interaction (green color) is observed in thr1518 and thr394 with a bond length of 2.32 Å. pi-cation and pi-anion interactions were observed in arg341, asp1511, and asp387, arg1465, respectively. Some pi-alkyl interactions were also observed in ala1462, arg390, arg1514, and ala338. The best-docked ligand MacL2 selected, has a binding score of -14 kcal/mol with binding site of 3t88 protein, as shown in Figure 19.Figure 19 (a) 2D interaction of ligand MacL2 with protein 3t88; (b) 3D image of ligand MacL2 in the active site of protein 3t88. The interactions between the protein B. subtilis (PDB: 5H67) and ligand, including hydrogen bonding and hydrophobic interactions, were analyzed, and out of these ligands, MacL2 shows a good binding affinity, as shown in Figure 20. The MacL2 ligand was docked with the active site of the protein B. subtilis and showed various interactions. A pi-donor hydrogen bonding interaction is observed in gly144 with a bond length of 3.08 Å. pi-cation, and pi-anion interactions were observed in glu148, lys145, and glu163, respectively. Some pi-alkyl interactions were also observed in lys135 and leu168. The best-docked ligand MacL2 selected has a binding score of -11.7 kcal/mol with a binding site of 3dra protein, as shown in Figure 20.Figure 20 (a) 2D interaction of ligand MacL1 with protein 5h67; (b) 3D image of ligand MacL1 in the active site of protein 5h67. Conclusions Novel biologically active tetraamide macrocyclic ligands (MacL1-3) and their Cobalt(II) complexes were synthesized by templet condensation reactions. Analytical and spectroscopic methods characterized the prepared ligands (MacL1-3) and their complexes. FT-IR and 1H NMR spectroscopic results clearly indicate that the nitrogen atoms of amide group are coordinated to the Co(II) metal atom. The oxidation state was explained by an electrochemical study. The proposed structure of the synthesized Cobalt (II) macrocyclic complexes were characterise with has various spectroscopic analysis that confirmed octahedral geometry. DFT calculations of tetraaza macrocyclic ligands and their cobalt (II) complexes have been calculated and agreed with experimental results. The experimental results and molecular docking investigations are relevant because ligands can be effective antibacterial agents against known microbes. It has been determined how well (MacL1- MacL3) ligands and their Cobalt(II) macrocyclic complexes inhibit the growth of bacterial and fungal strains. According to the study, Cobalt (II) complexes are more active than macrocyclic ligands. All Cobalt(II) macrocyclic complexes have good activity against the B. subtilis. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (PDF 1873 KB) Acknowledgements The authors are grateful to K. U. Kurukshetra, for providing the necessary facilities to carry out IR, 1H NMR,13C NMR, UV-Vis, and thermal analysis studies. The author Subhash is thankful to University Grants Commission (UGC), New Delhi, for financial assistance in the form of SRF (Ref. No.- 92(CSIR-UGC NET DEC. 2018). We are also thankful to IIT Bombay for ESR, SAIF Kerala for CHN & PXRD analysis, and G. J. University Hisar for Mass Analysis. Declarations Conflict of interest The authors declare no conflict of interest. Supplementary Information (SI) Supplementary information associated with this article is available at www.ias.ac.in/chemsci. ==== Refs References 1. Indira S Vinoth G Bharathi M Bharathi KS Synthesis, spectral, electrochemical, in-vitro antimicrobial and antioxidant activities of bisphenolic mannich base and 8-hydroxyquinoline based mixed ligands and their transition metal complexes J. Mol. Struct. 2019 1198 126886 10.1016/j.molstruc.2019.126886 2. 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==== Front Environ Sci Pollut Res Int Environ Sci Pollut Res Int Environmental Science and Pollution Research International 0944-1344 1614-7499 Springer Berlin Heidelberg Berlin/Heidelberg 36454526 24032 10.1007/s11356-022-24032-2 Review Article Focusing COVID-19-associated mucormycosis: a major threat to immunocompromised COVID-19 Sharma Neelam 1 Wani Shahid Nazir 2 Behl Tapan 3 Singh Sukhbir [email protected] 1 Zahoor Ishrat 2 Sehgal Aayush 4 Bhatia Saurabh 5 Al-Harrasi Ahmed 5 Aleya Lotfi 6 Bungau Simona 7 1 Department of Pharmaceutics, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, Haryana 133207 India 2 grid.428245.d 0000 0004 1765 3753 Chitkara College of Pharmacy, Chitkara University, Punjab, India 3 grid.444415.4 0000 0004 1759 0860 School of Health Sciences and Technology, University of Petroleum and Energy Studies, Dehradun, Uttarakhand India 4 GHG Khalsa College of Pharmacy, Gurusar Sadhar, Ludhiana, Punjab India 5 grid.444752.4 0000 0004 0377 8002 Natural & Medical Sciences Research Center, University of Nizwa, Nizwa, Oman 6 grid.7459.f 0000 0001 2188 3779 Chrono-Environment Laboratory, UMR CNRS 6249, Bourgogne Franche-Comté University, Besançon, France 7 grid.19723.3e 0000 0001 1087 4092 Department of Pharmacy, Faculty of Medicine and Pharmacy, University of Oradea, Oradea, Romania Responsible Editor: Philippe Garrigues 1 12 2022 120 22 2 2022 2 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. COVID-19 disease has been identified to cause remarkable increase of mucormycosis infection cases in India, with the majority of cases being observed in individuals recovering from COVID-19. Mucormycosis has emanated as an outcome of the recent COVID-19 pandemic outbreak as rapidly developing fatal illness which was acquired by Mucorales fungus which is a subcategory of molds known as mucormycetes. Mucormycosis is one of the serious, sporadic mycotic illnesses which is a great threat to immunocompromised COVID-19 patients and affects people of all ages, including children with COVID-19 infections. This is associated with tissue damaging property and, therefore, causes serious clinical complications and elevated death rate. The COVID-19-associated mucormycosis or “black fungus” are the terms used interchangeably. The rapid growth of tissue necrosis presenting as “rhino-orbital-cerebral, pulmonary, cutaneous, gastrointestinal, and disseminated disease” are various clinical forms of mucormycosis. The patient’s prognosis and survival can be improved with proper surgeries using an endoscopic approach for local tissue protection in conjunction with course of appropriate conventional antifungal drug like Amphotericin-B and novel drugs like Rezafungin, encochleated Amphotericin B, Orolofim, and SCY-078 which have been explored in last few years. This review provides an overview of mucormycosis including its epidemiology, pathophysiology, risk factors, its clinical forms, and therapeutic approaches for disease management like antifungal therapy, surgical debridement, and iron chelators. The published patents and ongoing clinical trials related to mucormycosis have also been mentioned in this review. Graphical abstract Keywords Antifungal therapy Black fungus COVID-19-associated mucormycosis Pulmonary mucormycosis Rhino-orbital-cerebral mucormycosis ==== Body pmcIntroduction Mucormycosis which is known by the term black fungus is a mycotic illness caused by a rising number of zygomatic fungi. It is a fungal disease which gets spread by fungus of Mucorales order and zygomycotic families (Kwon-Chung 2012). The main cause of this infection is exposure to air contaminated with spores which affects the lower and upper part of the respiratory tract leads to sinusitis, pulmonary infection, and rhino-cerebral mucormycosis. This infection rarely spread to cutaneous tissues, central nervous system, and other body organs, but infection can spread to various adjacent organs and is quite prevalent if infected patients will not undergo immediate surgeries and medical treatment. Rhino-orbitocerebral, cutaneous, disseminated, gastrointestinal, and pulmonary mucormycosis are the major clinical forms observed in infected individuals. Immediate treatment is required due to quick progression and tissue destruction characteristics of this infection. The delayed commencement of mucormycosis treatment is related with high death rate (Chamilos et al. 2008a, b). Early diagnosis and adopting a therapeutic approach, as well as prompt participation of an integrative radiological, surgical, medical, and lab-based team is required to maximize survival rates. The mucormycosis is commonly observed in patients having uncontrolled Diabeties mellitus (DM) with ketoacidosis and metabolic acidosis, corticosteroid therapy, bone marrow or organ transplantation, neutropenia, trauma, wounds, cancerous hematological abnormalities, and in hemodialysis patients on deferoxamine therapy (Ibrahim et al. 2012). Immunocompromised individuals having weaker immune systems, such as those who abuse drugs, take insulin for diabetes, undergo cancer treatment, or have AIDS/HIV are more likely to get this infection. Disfiguring surgical debridements and other antifungal therapies cannot save the lives of people infected with mucormycosis; the overall mortality rate is approximately 50% and can arrive at 100% in people with underlying diseases or prolonged neutropenia (Gleissner et al. 2004; Spellberg et al. 2005). In Wuhan (China), an unusual zoonotic epidemic first emerged in the month of December 2019 and the Coronavirus disease 2019 (COVID-19) started to spread, which is acquired by novel severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), and have tendency to induce variety of symptoms ranging from moderate to high-grade pneumonia. Coronavirus is classified as RNA virus of the coronaviridae subfamily (Khan et al. 2021; Umakanthan et al. 2020). The incubation time of COVID-19 is 1–2 weeks and is transmitted by both symptomatic and asymptomatic infected patients (Hojyo et al. 2020). The clinical signs of COVID-19 infection are Pyrexia, dry cough, chest congestion, muscle weakness, difficulty in breathing, muscle pain, along with other clinical complications. Over 200 million people have been infected and over 4 million died as a result of this virus. The COVID-19 is associated with hypoxia, suppression of immune system, iron loss in the host, hyperglycemia related to diabetes mellitus, and longer hospitalizations. These clinical complications create an ideal environment for opportunistic fungal infections in individuals suffering from COVID-19. The immune system gets activated in response to a fungal infection, which can be the primary cause of illness. The mechanism of infection and the risk of complications vary depending on the type of fungus responsible for the infection. The multi-infected condition may act to enhance the inflammation of systemic components that leads to delayed recovery, resulting in requirement of higher treatment approaches, the need for intensive care, and the danger of mortality. When a virus binds to a target host cell, it produces various cytokines mainly interleukin-6 (IL-6) and activates the nuclear factor kappa B which leads to pro-inflammatory condition marked by a rise in macrophages and cytokines levels. The prevailing cytokines storm and immunological imbalance in COVID-19 may result in failure of the respiratory system, coagulation, failure of body organs, and other clinical complications (Hariharan et al. 2021). The increased level of cytokine reaction can result in T-cell depletion which is common in persistent viral conditions. COVID-19 individuals have less numbers of CD4 + helper T cells (200 cells/L), which enhances vulnerability to fungal infection development. Because CD4 + helper T-cells play an important role in successful immune reaction against invading microbes, and gives signal of immunogenic condition of patients (Diao et al. 2020; Gangneux et al. 2020; Luckheeram et al. 2012; Song et al. 2020). According to International Diabetes Federation, India has an extremely high incidence rate of type 2 diabetes mellitus and it is a well-known risk factor for mucomycosis (Mehta and Pandey 2020a, b). The patients suffering from COVID-19 are identified with additional serious opportunistic infections like gram-negative bacteria, Pneumocystis jiroveci pneumonia (PCP), oropharyngeal candidiasis, Staphylococcus aureus, pulmonary aspergillosis, and bloodstream candidiasis (Szarpak et al. 2021). Due to availability of more adequate diagnostic approaches at the turn of the twenty-first century, the mucormycosis has been classified as an emerging invasive fungal infection with high fatality rate of approximately 75–87% (Pagano et al. 2020). The objectives of this comprehensive review are focused on overview of mucormycosis including its pathophysiology and epidemiology, various predisposing factors, common clinical forms of mucormycosis and various therapeutic approaches. For this purpose, an extensive search of the literature was conducted using the Google Scholar, PubMed, and ScienceDirect databases. Literature review was executed from papers published in peer reviewed journals from the year 2000 to year 2022. The recently published patents and clinical status related to mucormycosis has also been focused in this review. Pathogenesis and pathophysiology of mucormycosis The mucormycosis infection is caused by inhalation and entry of mucorales spores into the respiratory tract. These spores can reach inside the nasal cones or in the pulmonary alveolar region. The sequence of events that leads to tissue hyphal growth is mostly unknown. Mucormycosis is characterized by hyphal invasion of blood vessels which is the most prominent characteristic of this infection. Hemorrhage, thrombosis, infarction, and tissue necrosis are all symptoms of this invasion. Mucormycosis and zygomycosis are two terms that are frequently used interchangeably. The latter term refers to diseases caused by fungi belonging to the Zygomycota phylum (which included Entomophthorales and Mucorales). Entomophthorales fungi are uncommon pathogens that cause persistent cutaneous as well as sub-cutaneous infections which are usually restricted to tropical environments. The organisms Rhizopus, Rhizomucor, and Absidia are most typically isolated from mucormycosis patients. Mucor, Cunninghamella, Mortierella, Saksenaea, and Apophysomyces were also found in mucormycosis patients, although these were diagnosed in considerably lower numbers (Toumi et al. 2012). The order Mucorales contains 14 families out of which various species of few families has been depicted in Table 1 (Onyango et al. 2002). The Rhizopus oryzae is the prevalent causative pathogen recovered from infected mucormycosis individuals, accounting for 70% of all mucormycosis cases (Ibrahim et al. 2012). Mucorales are widespread causative agents that play an important role in starting and promoting the decomposition of organic matter. Because exposure to these fungi spores is unavoidable, the rarity of infection caused by Mucorales is an indicator of their relative avirulence. Infection caused by one of the Mucorales, on the other hand, indicates a serious underlying susceptibility. Mucorales are non-fastidious organisms that can survive at a broad range of climatic conditions (25 to 55 °C) while the ideal temperature for significant Mucorales species is 28–30 °C (Pagano et al. 2020). At a temperature of 37 °C, isolates collected from clinical specimens will also proliferate. These organisms are aerobic which can proliferate after 2–5 days of incubation in the microbiology laboratory. In a single hospital in Spain, Lichtheimia spp. was found as the leading cause of mucormycosis, showing geographic diversity and the need to understand local epidemiology (Cornelyet al. 2019; Guinea et al. 2017). Apophysomyces spp. was the second most usually isolated agent in Indian investigations (Chakrabarti et al. 2006; Chakrabarti and Singh 2014). Apophysomyces and Saksenaea spp., which are most prevalent in Asia, are virtually invariably accountable for cutaneous mucormycosis infection in immunocompromised patients (Prakash et al. 2016). Rhizopus homothallicus, Saksenaea erythrospora, Thamnostylum lucknowense, and Mucor irregularis are among the new species that have emerged in recent years (Chakrabarti et al. 2010; Lu et al. 2013; Xess et al. 2012).Table 1 Representation of various families of pathogenic mucorales and their species Family Species Mucoraceae Rhizopus oryzae, R. rhizopodiformis, R. rhizopodiformis, R. microspores, Absidia corymbifera, Mucor crircinelloides, M. pusillus, M. miehei, Rhizopus homothallicus, Mucor irregularis Mortierellaceae Mortierella wolfii Cunninghamellaceae Cunninghamella elegans, C. bertholletiae Saksenaeaceae Saksenaea vasiformis, Saksenaea erythrospora Syncephalastraceae Syncephalastrum sp. Patients who have less number of phagocytes or have decreased phagocytic activity are obviously more prone to mucormycosis, according to clinical and experimental findings. Patients who are severely neutropenic are more susceptible to get mucormycosis (Binder et al. 2014). The neutrophils are playing an important role in suppressing fungal spore growth. Furthermore, typical host mononuclear and polymorphonucleated phagocytes destroy Mucorales by production of oxidative metabolites, defensins, and cationic peptides. Ketoacidosis, diabetes, and corticosteroids all affect phagocytes, but the exact mechanisms are unknown (Binder et al. 2014; Spellberg et al. 2005). The mucorales must have distinct virulence features that allow the organism to take advantage of particular condition of immunosuppressive and physiologic impairment that incline individuals to mucormycosis. One such attribute is capacity to get iron from infectious host. Iron is a necessary element since this participates in many key cellular activities for growth and development of cell. The pathogenic viruses adopt numerous ways to extract iron from their hosts. The recent research has shown that persons with ketoacidosis who are susceptible to mucormycosis depend critically on amount of available free iron in their plasma. The iron is bounded to carrier proteins of host like ferritin, lactoferrin, and transferrin in mammals. This sequestration process prevents the harmful effects of free iron (Ibrahim et al. 2012; Ibrahim 2014; Tabassum et al. 2021). Mucormycosis infections are distinguished by widespread angioinvasion, which results in thrombosis of vascular compartment and subsequently leads to tissue necrosis (Ibrahim et al. 2012; Mehta and Pandey 2020a, b; Tabassum et al. 2021). The ischemic necrosis of damaged tissue can hinder white blood cells and antifungal drugs from reaching infection sites. This angioinvasion most likely adds the ability of organisms to hematogenously spread to additional target organs of body. Consequently, dissemination through endothelium lining of the blood arteries is thought to be significant phase in R. oryzae’s pathogenetic approach. Determining the mechanisms behind these pathogenic processes may result to novel techniques for preventing and/or treating mucormycosis (Ben-Ami et al. 2009; Chayakulkeeree et al. 2006; Spellberg et al. 2005). Figure 1 describes various steps involved in pathogenesis and pathophysiology involved during progression of Mucormycosis. Mucormycosis gets spread by inhaling, ingesting, or directly inoculating Mucorales spores into punctured skin. Because of epithelial cell injury in a susceptible host, these spores infiltrate tissues by attaching to exposed extracellular matrix (ECM) proteins. Mucorales infiltrate epithelial cells and secreted proteases to enter into ECM protein. Mucorales avoid tissue macrophage mediated death and infiltrate blood arteries by attaching to ECM proteins and invades endothelial cells. Mucorales proliferate in an infiltrated blood vessel in affected host in presence of free iron (e.g., iron release from transferrin (T) through a proton-mediated process in DKA individuals) and due to inefficient polymorphonuclear leukocytes. Mucorales penetrate endothelial cells by adhering to GRP78, which is highly expressed and relocalized to cell surface endothelium in DKA individuals with elevated glucose and iron levels and finally, fungal hyphae enter the blood vessels.Fig. 1 Schematic depiction of pathogenesis and pathophysiology involved during Mucormycosis progression. PMNL, polymorphonuclear leukocytes; DKA, diabetic ketoacidosis; Fe, iron; T, transferrin; GRP-78, Glucose Regulated Protein-78 Epidemiology of mucormycosis The inhalation of fungal spores which are present in atmospheric air or direct injection of any pathogenic agents in mucus membrane or damaged skin can cause the majority of human illnesses. Mucorales are thermo-tolerant and prefer to live in decomposing organic materials (Petrikkos et al. 2012). Cases of mucormycosis have been reported from all over the globe. There appear to be differences in mucormycosis epidemiology between industrialized and poor countries. Transplantation of hematopoietic stem cells and hematologic malignancies are some of the predominant underlying conditions of mucormycosis infection in well-developed countries. The uncontrollable diabetes mellitus and injuries are well known risk factors to get this infection in underdeveloped countries, particularly in India (Challa 2019; Petrikkos et al. 2012; Prabhu and Patel 2004; Skiada et al. 2011). Due to challenges in clinical diagnosis, exact information on incidence of mucormycosis is limited. On the other hand, as per recent global autopsy data, the most prevalent cause of invasive mycotic illness is mucormycosis, after Candidiasis and Aspergillosis, with the increased number of cases observed in adults and children who are less than one year in age (Dignani 2014). In accord to published research, mucormycosis is 5–10 times less prevalent than the other mycotic infections such as aspergillosis (Chamilos et al. 2006a, b; Kontoyiannis et al. 2005). Nonetheless, mucormycosis is a rare illness, even in individuals who are more prone to get an infection, accounting for 8.3–13% of maximum mycotic infections discovered at autopsy in these individuals (Kume et al. 2003; Suzuki et al. 2013). According to postmortem prevalence analysis, mucormycosis is 10–50 times rarer than candidiasis or aspergillosis with 1–5 instances per 10,000 autopsies (Petrikkos et al. 2012; Suzuki et al. 2013). In addition, diabetes mellitus is a significant underlying condition for mucormycosis in India, but there is a difference in the number of cases in different regions of India i.e., 67% in the north and 22% in the south (Prakash et al. 2019; Sharma et al. 2021). In COVID-19 individuals, the factual occurrence of rhino-orbital mucormycosis is unknown. However, there are many reports of mucormycosis infection in COVID-19, most of which are from India, particularly in diabetic patients who have undergone COVID-19 treatment and recovered, where corticosteroids are administered inadvertently to control severity, resulting in a higher fatality rate and complicating the pandemic (Gupta and Singh 2021; Singh et al. 2021a, b). Mucormycosis has been found in 1–5 instances per 10 thousand autopsies in autopsy studies, leading the illness 10–50 times less prevalent than persistent Candidiasis or Aspergillosis infections (Suzuki et al. 2013; Tietz et al. 1998; Yamazaki et al. 1999). The occurrence of mucormycosis infection is quite high as 2–3% in individuals who are at higher risk, especially those who have undergone transplantation of bone marrow (Maertens et al. 1999). Risk factors causing mucormycosis Mucormycosis, often called as black fungus infection, is a new opportunistic infection having an estimated prevalence of 0.005–1.7 per million people (Siddiqui et al. 2006). Nevertheless, the occurrence of mucormycosis infection in India is quite close to 0.14 cases for every 1000 individuals that are about 80 times more than the incidence in well-established nations (Chander et al. 2017). In India, the rise in COVID-19 incidence is associated to rise in reports of extensive mucormycosis infection post-COVID-19, and this trend is expected to continue. While numerous therapy options for COVID-19 have been investigated, glucocorticoids have been proven to enhance survival but can also result in secondary fungal infections. The SARS-CoV-2, corticosteroid usage, and uncontrollable diabetes, these predisposing factors are responsible to rise in the number of invasive mucormycosis infection (Amin et al. 2021). The triggering factors of mucormycosis are mentioned below in Fig. 2.Fig. 2 Classification of risk factors causing mucormycosis Diabetes mellitus DM has been proved to be a main underlying condition among 36–88% of total number of mucormycosis incidences (Roden et al. 2005). Individuals with high sugar levels, especially patients with ketoacidosis, are more prone to get the mucormycosis infection. The mucormycosis infection is mostly observed in patients having uncontrolled DM, but this is uncommon in patients with controlled diabetes. Mucormycosis infection has been linked to type-1 DM, type-2 DM, and also with the secondary DM (Bhansali et al. 2004; Chamilos et al. 2006a, b; Kontoyiannis 2007). DM is a major predisposing factor for intense COVID-19 infection and is also linked with high death rate due to COVID-19 (Wu and Mc Googan 2020). DM alters the innate immunity by altering the function of phagocytes that improves considerably after controlling sugar level (Shodja et al. 2017). Furthermore, affected dendritic cells respond and postpone the stimulation of adaptive immunity. On the other hand, COVID-19 can cause arrival of DM and also DKA which is observed in the recently diagnosed diabetic patients subsequent to COVID-19 infection (Heaney et al. 2020; Hodgson et al. 2015). The ACE-2 is mostly present in lungs, as well as in pancreas which act as COVID-19 doorway receptor. The ACE-2 proteins promote the SARS-CoV-2 entry into the pancreatic islets and may harm beta cells. Furthermore, intense COVID-19 causes resistance to insulin by increasing the release of most of stress hormone like hydrocortisone and various cytokines. Furthermore, uncontrolled DM is a common predisposing factor for mucormycosis, equally in COVID-19 and non-COVID-19 individuals. Mucormycosis is more common in DKA patients. On the human endothelial cells, expression of glucose-regulated protein 78 kDa (GRP78) increases in hyperglycemia which is a critical binding receptor for many mucorales vascular invasion via the spores coat protein (CotH) (Gebremariam et al. 2014; Liu et al. 2010). Also, Rhizopus is involved with the GRP78 on epithelial cells of nasal mucosa, mostly through CotH3 protein in order to attack and disrupt the cells. High levels of glucose, iron, and ketones (the hallmarks of DKA) drastically increase the proteins expression namely GRP78 and CotH3 that potentially causing ROCM (Alqarihi et al. 2020; Muthu et al. 2021). Use of corticosteroids The use of corticosteroids is also one major underlying condition for COVID-19-associated mucormycosis (CAM). On innate and adaptive immune systems, they are acting as powerful immunosuppressant and are producing an extensive range of effects. The corticosteroid-induced hyperglycemia increases the susceptibility to mucormycosis infection since elevated hyperglycemia affects neutrophil and phagocyte activities i.e. impair the ability of phagocytes to eliminate fungal spores (Fuji et al. 2017; Hoang et al. 2020). Dexamethasone had completely inhibited the Aspergillus and Rhizopus phagocytosis in a Drosophila melanogaster model (Chamilos et al. 2008a, b). Dexamethasone and various other steroidal drugs are mostly considered to treat COVID-19 infection; however, their role in progress of COVID-19-associated mucormycosis (CAM) appears undisputed (Patel et al. 2021). The corticosteroids are familiar to increase threat of invasive infections like mucormycosis and aspergillosis. Persistent usage of steroids with a dose of 0.3 mg/kg of steroids for about 21 days in the earlier two months and that has been identified as a determinant of invasive fungal diseases (Donnelly et al. 2020). Recently, a research study found that the doses and durations higher than the current COVID-19 recommendation (Dexamethasone 6 mg for up to 10 days) were linked with an increased incidence of CAM (Patel et al. 2021). More research is needed to determine whether even lower doses are harmful or whether other unknown factors contribute to CAM. Iron overload Hyperferritinemia is a defining feature of COVID-19 and it is linked to a higher risk of mortality. Remarkably, ferritin-associated iron causes imperfection in both the adaptive (T-lymphocytes) and innate (neutrophils) immunity in mouse models (Deng et al. 2021; Edeas et al. 2020; Kuvibidila and Warrier 2004). This has been found in an in vitro model that deficiency of iron can cause apoptosis in R. arrhizus. Therefore, it is necessary for mucorales to obtain free iron from patients in order to acquire their development and maturation. (Ibrahim 2014; Shirazi et al. 2015). Deferasirox is an iron chelator which prevents the mice from mucormycosis infection by deprivation of iron but it is not useful in patients. Individuals with excess iron are highly susceptible to develop mucormycosis infection, particularly patients taking iron chelator namely deferoxamine (Ibrahim et al. 2007; Soman et al. 2012; Spellberg et al. 2012). In patients with DKA, acidosis causes a partial dissociation of iron that is bonded to transferrin. The ability of transferrin to chelate iron is eventually impacted by the ketoacid b-hydroxybutyrate. The high level of iron may allow R. arrhizus to grow. Deferoxamine therapy chelates the iron and is used to treat iron and/or aluminum overloads in individuals undergoing dialysis, and has been linked to angioinvasive mucormycosis infection. Iron overload, whether from dyserythropoiesis or transfusion, is also a main underlying condition for mucormycosis infection (Vigouroux et al. 2005). Disseminated mucormycosis is the common clinical type of mucormycosis in those patients who are taking Deferoxamine (44%), and this clinical form is linked with elevated mortality rate reaching upto 80% (Torres-Narbona et al. 2007). Nowadays, the Deferoxamine is not used due to emergence of novel iron chelators such as deferasirox and deferiprone, due to which individuals are not getting more susceptible to mucormycosis infection. Hematological malignancies and transplantation of hematopoietic stem cell In the USA and Europe, one of the most common diseases for mucormycosis infection was hematological malignancy which ranged from 38 to 62%. Mucormycosis is more common in individuals who suffer from acute myeloid leukemia, undergone transplantation of hematopoietic stem cells, having myelodysplastic syndrome and acute lymphoblastic leukemia during the stage of neutropenia (Pagano et al. 2004; Roden et al. 2005). According to the findings of a multicenter cohort research that was conducted in France, 0.4% of patients of allogeneic hematopoietic stem cell transplantation (HSCT) were found to have mucormycosis (Xhaard et al. 2012). In Italy, one cohort study of patients undergone HSCT found 0.1% cases of mucormycosis between 1999 and 2003 (Pagano et al. 2007). The TRANSNET research on HSCT patients in the USA found that mucormycosis attributed for 8% of invasive mycotic infections with a yearly collective occurrence of 0.29% (Kontoyiannis et al. 2010; Park et al. 2011). The occurrence of mucormycosis in individuals undergoing allogeneic stem cell transplantation or autologous is less than that observed in individuals with myeloid leukemia, that ranges from 0.9 to 2.0%, with the maximum proportion experienced by individuals with the graft-versus-host disease (Baddley et al. 2001; Neofytos et al. 2009; Petrikkos et al. 2012). Solid organ malignancies and transplantation The malignancies of solid organ and recipients of solid organ transplants (SOT) are also key underlying conditions for mucormycosis infection, even though only in a small percentage of incidence. Mucormycosis accounts a less percentage of invasive mycotic infections in case of SOT individuals. Furthermore, it is related to a high mortality rate. Depending on SOT type, the prevalence rate ranges from 0.4 to 16.0%, 0.2 to 1.2% in kidney transplant patients, 0 to 1.6% in patients who have undergone a liver transplant, 0 to 0.6% in heart transplant patients, and 0 to 1.5% in lung transplant patients (Bitar et al. 2009; Petrikkos et al. 2012; Roden et al. 2005). Moreover, mucormycosis infection spread to other organs frequently after graft rejection and treatment. Individuals with kidney failure (58%), DM (38%), and earlier antifungal treatment of caspofungin or voriconazole (26%) are highly susceptible to get mucormycosis (Singh et al. 2009). Long-term use of voriconazole In many research centres, the development and broad use of anti-Aspergillus agents, particularly voriconazole, in people with hematological malignancies and recipients of hematopoietic stem cell transplants increase the mucormycosis cases (Kontoyiannis et al. 2005; Trifilio et al. 2007). The prospective randomized studies comparing voriconazole prophylaxes with itraconazole or fluconazole prophylaxes in allogeneic transplant patients did not prove this finding (Antinori et al. 2009). Because the patients in both studies had a low risk of invasive fungal infections and mucormycosis infection, the utility of voriconazole is still controversial. Despite the ambiguity around this risk, clinical professionals must be aware that mucormycosis infection can develop in high-risk patients receiving voriconazole. Mucormycosis developed after voriconazole treatment of persistent aspergillosis in individuals with blood cancer has a high mortality rate. The awareness of this entity for clinical professionals is critical in patient management. It is vital to have strong index of suspicion for mucormycosis for proper diagnosis and accurate laboratory diagnostic approaches using molecular and conventional techniques are the needful methods for managing this catastrophic infection (Sharifpour et al. 2018). Dysfunction of endothelial cells COVID-19 is linked with dysfunction of endothelium. In individuals with intense COVID-19, autopsy studies revealed rigorous injury of endothelial cells and is associated with the existence of intracellular virus as well as disruption of cell membranes (Meini et al. 2020). Another explanation for the rise in mucormycosis cases is the damage of endothelium and endothelialitis observed in severe COVID-19 patients. ACE-2 receptors are present in endothelial cells of the lungs and endothelialitis could be an immune reaction in response to direct viral infection. In COVID-19 individuals, autopsy reports revealed the existence of severe injury of endothelium with interrupted cellular membrane, microangiopathy, extensive thrombosis, and angiogenesis in the pulmonary vasculature (Meini et al. 2020). Adherence to endothelium, as well as penetration, is crucially important in initial stages of mucormycosis infection. The vascular endothelitis also provides an easy path for Mucorales to enter inside the bloodstream which leads to increase the risk of complications (Rudrabhatla et al. 2021). Human immunodeficiency virus or AIDS Mucormycosis is extremely uncommon in individuals having human immunodeficiency virus (HIV) or AIDS. Only two patients with mucormycosis were found in an extensive research examination of 1630 autopsies that died of AIDS (Antinori et al. 2009). This low occurrence shows the rarity of mucormycosis infection in HIV-infected individuals in comparison to other immunocompromised patients. The majority of mucormycosis incidences in HIV-infected individuals are linked to the use of intravenous drugs (Roden et al. 2005; Skiada et al. 2011). In 2016, approximately 67 cases of mucormycosis infection were reviewed among HIV patients and found that intravenous drug use was the predominant risk factor, followed by 29.7% neutropenia, 15% steroid usage, and 10% diabetes mellitus as the other underlying diseases (Moreira et al. 2016). Environmental factors The fungi that cause mucormycosis infection and its spores are found in decomposed matter and soil that belongs to order mucorales. The organism reproduces quickly in favorable conditions and the percentage of spores in the air increases. In spite of the fungal prevalence, mucormycosis infection is most commonly seen as an opportunistic illness in immunocompromised patients. In hospitals, outbreaks of mucorales cutaneous infection have been linked with contaminated sticky tape bandages, cloths, and tongue depressors made up of wood (Ribes et al. 2000; Spellberg 2017; Walther et al. 2020). Pulmonary and rhino-orbital mucormycosis infection has been seen less frequently upon contact contaminated air (from dirty air conditioners, ongoing building constructions, or ventilating systems) (Walther et al. 2020). In an Indian study, approximately 9% of the mucormycosis infection (largely cutaneous) incidents were nosocomial (Chakrabarti et al. 2009). Miscellaneous factors Repurposing of some biological drugs for the treatment of COVID-19 infection, like interleukin-1 and interleukin-6 inhibitors, Janus kinase inhibitors, tumor necrosis factor may raise the susceptibility for infections (Cavalli et al. 2020). Several unproven COVID-19 therapies were widely used around the world, especially in India (Beović et al. 2020). The impact of antibiotic overuse on various superadded infections is well documented. Staphylococcus epidermidis as well as Staphylococcus aureus both are frequent nasal flora constituents and were shown to prevent the development of common saprophytic fungi Rhizopus arrhizus (Singh and Kumari 2021). The improper use of antifungal and antibacterial drugs may disrupt the fine balance of the nasopharyngeal and respiratory epithelial mycobiome and microbiome (Muthu et al. 2021). Mucormycosis infection is not only observed in immunocompromised patients, significant incidences are observed in immunocompetent individuals as well with no known underlying predisposing conditions. Two meta-analyses from different time periods found that 19% cases of mucormycosis infection happen in immunocompetent patients also (Jeong et al. 2019; Wu and Mc Googan 2020). These patients frequently develop cutaneous mucormycosis as a result of burns, surgery, trauma, contaminated dressings, or injections (Cavalli et al. 2020; Chakrabarti et al. 2006). Clinical forms of mucormycosis Mucormycosis infection is an invasive mycotic illness associated with COVID-19 with an increased mortality rate acquired by widespread fungi having hyphae which belongs to the Mucorales order. Due to an increase in the population at risk and improving diagnostic techniques, the reported incidence of mucormycosis has risen over time (Bitar et al. 2009). This fungal disease is distinguished by widespread necrotizing vasculitis, which leads to thrombosis and subsequent tissue destruction. The primary site of mucormycosis infection differs depending on the order of Mucorales or the predisposing conditions of susceptible hosts (Jeong et al. 2019; Wu and Mc Googan 2020). The common clinical forms of mucormycosis are mentioned below in Fig. 3.Fig. 3 Various types of common clinical forms of mucormycosis Rhino-orbito-cerebral mucormycosis (ROCM) ROCM is inevitably a deadly mycotic disease which primarily influences the immunocompromised individuals. There is insufficient data from India on the outcomes of individuals with ROCM (Singh et al. 2021a, b). It is one of the prevalent clinical forms of mucormycosis. This is acquired by inhalation of spores which permits the mold to expand in adjoining nasal cavities. This disease can spread quickly to adjoining tissues, including the sphenoid sinuses, palate, orbits, and cavernous sinuses, subsequently spread to various cerebral regions. The destruction of these infected tissues was reported as black necrotic tissue and is a concerning indication of local extension. DM is the commonest predisposing risk factor in a meta-analysis of 175 ROCM incidences which was later published between 1994 and 2005 and contributed to 64% cases of ROCM, followed by hematological malignancy and kidney diseases which produced 15% and 13% cases of ROCM, respectively (Vaughan et al. 2018). ROCM remains the main popular type of infectious ailment, contributing for one-third to one-half of all incidences of mucormycosis. Approximately 70% of rhino-cerebral incidence which is also known as craniofacial has been observed in diabetic individuals having ketoacidosis (Spellberg et al. 2005). ROCM has also been reported in individuals who have undergone SOT or have chronic neutropenia (Gleissner et al. 2004; Petrikkos et al. 2003). The early symptoms of ROCM infection are similar to periorbital cellulitis or sinusitis including eye or face pain and lack of sensation in facial area and subsequent conjunctival suffusion, soft tissue swelling and blurred vision (Dhiwakar et al. 2003; Talmi et al. 2002). Fever is quite uneven which may be absent in up to 50% of the patients. Leukocyte count usually remains high as the host has functional bone marrow (Spellberg et al. 2005). Even though the nasal and paranasal sinuses are primary locations of mold inoculation, these combative fungi propagate and extend to orbital and various parts of central nervous system through haematogenous route or direct extension, resulting in development of life-threatening ROCM. Infection of SARS-CoV-2 causes immunosuppression by disturbing the T-lymphocytes, resulting in lymphopenia, which plays a critical role in cell-mediated immunogenicity and altering the ratio of neutrophils and lymphocytes (Gangneux et al. 2020; Song et al. 2020). The existence of comorbid illness such as DM with simultaneous corticosteroid and other immunomodulating agent therapy for treatment of normal to severe cases suppresses the immune system of patients and leads to aggressive mycotic infections throughout the duration of the disease (Garg et al. 2021; Mehta and Pandey 2020a, b). Pulmonary mucormycosis Another common site for mucormycosis infection is involvement of the pulmonary tract, which is frequently seen in organ transplant recipients and patients having hematological disorders (Jeong et al. 2019; Prakash et al. 2019). The foremost underlying condition for pulmonary mucormycosis has been hematological malignancy while other causal conditions includes transplant of hematopoietic stem cells, diabetes, transplant of solid organs, and renal diseases (Feng and Sun 2018; Prakash and Chakrabarti 2019). The high fever, chest pain, persistent cough, dyspnoea, and hemoptysis are the common symptoms of pulmonary mucormycosis. The diagnosis of pulmonary mucormycosis infection is still challenging. Pulmonary mucormycosis is generally unilateral, but it can be bilateral, hilar, or mediastinal. Upper lobe involvement is common in unilateral lung disease, followed by involvement of lower and middle lobes. Involvement of multi-lobes is observed in about half of all pulmonary mucormycosis scenarios (Feng and Sun 2018). Diagnostic imaging studies of pulmonary mucormycosis patients have indicated the presence of pulmonary infiltrates and consolidation, pleural effusion, thickly walled cavities, multiple nodules, pneumothorax, mediastinal lymphadenopathy, and air crescent. The therapies of pulmonary mucormycosis include combination of antifungal and surgical treatment. Pulmonary mucormycosis has a higher mortality rate than other localized types of mucormycosis infections (Prakash and Chakrabarti 2019; Spellberg et al. 2005). Cutaneous mucormycosis Cutaneous intervention in mucormycosis infection occurs as a result of either direct fungus inoculation by traumatic injuries known as primary mucormycosis or dissemination by blood-borne infectivity or spreaded contiguously called as secondary mucormycosis (Vinay et al. 2014). In general, there are two types of primary cutaneous mucormycosis which includes rapidly developing angioinvasive necrotising type or slowly chronic granulomatous type. The penetrating trauma is the most common cause of cutaneous mucormycosis while other predisposing factors includes intramuscular injection in substandard healthcare premises, car accidents, surgery, open wound trauma, contaminated dressings, natural disasters, burns, animal bites, and scratches (Wu and Mc Googan 2020). The most common symptoms of cutaneous mucormycosis are necrosis, purulent discharge, redness, swelling, and mouldy appearance. The localized infection is observed in 32–56% of hosts and is typically limited to subcutaneous and cutaneous tissue without intruding into adjoining sites. The deeper extension includes involvement of muscles, tendons, and bones that is generally observed in 24–52% of patients. In these patients, erythematous necrotic eschar with necrotizing fasciitis appears as common manifestation of infection. Cutaneous mucormycosis is a type of disseminated infection that involves other non-contiguous sites apart from the cutaneous sites and is observed in 16 to 20% of cutaneous infections (Feng and Sun 2018; Wu and Mc Googan 2020). The treatment for cutaneous mucormycosis includes the combination of systemic antifungal agents and surgical procedures (Lelievre et al. 2014). European conference on infections in leukemia-6 recommends surgery in conjunction with Amphotericin-B for all grades of soft tissue disease (Tissot et al. 2017). Gastrointestinal mucormycosis The least common type of mucormycosis is primary gastrointestinal disease. It can spread by eating unhygienic food, such as dried bread products and fermented milk, but it can also be acquired through healthcare-associated contact with dirty surgical devices. The most prevalent site of infection has been presented as the stomach, and thereafter the colon, small intestine, and also esophagus (Serris et al. 2019). In a research study of 31 cases, the main prevalent type was intestinal disease followed by gastric disease (Dioverti et al. 2015). Hematologic malignancy and SOT are the two the most prevalent causes. The premature neonates have also been reported to have gastrointestinal mucormycosis (Rammaert et al. 2012). Peritoneal dialysis and DM are the most common underlying conditions in adults; however, malnutrition and prolonged use of antibiotics are associated extensively with children. The most complicated form of the disease to diagnose ante-mortem is gastrointestinal mucormycosis, which is frequently observed in underweight babies, malnourished individuals, and peritoneal dialysis patients (Kaur et al. 2018; Wu and Mc Googan 2020). Individuals with hematological malignancy, neutropenia, and solid organ transplants are majority of immunocompromised individuals with gastrointestinal mucormycosis infection (Kaur et al. 2018). Emergency surgery along with intravenous Amphotericin B is the most recommended treatment strategy for gastrointestinal mucormycosis. Since GI symptoms are quite non-specific, therefore diagnosis is frequently deferred or missed due to which the mortality rate remains high at 57% (Dioverti et al. 2015). Disseminated mucormycosis The mucormycosis infection can spread from one to other body organs hematogenously (Liu et al. 2000; Tomita et al. 2005). The lungs are usually associated with dissemination process; however, this can also occur through extensive cutaneous lesions, gastrointestinal tract, and burns. Although the cenral nervous system is a common location of spread, lesions in the liver, heart, spleen, and other organs can also be causative reasons identified in patients (Petrikkos et al. 2012). Individuals with high iron intake, particularly those on Deferoxamine therapy, significant suppression of immune system, e.g., patients who have undergone transplants of allogeneic stem cell with graft-versus-host disease, the patients on steroid therapy, intense neutropenia, and active leukemia, are typical risk individuals for the disseminated type of mucormycosis (Gonzalez et al. 2002; Petrikkos et al. 2012; Prabhu and Patel 2004). The systemic staging with cerebral MRI and a sinus thoracoabdominal CT scan must be executed to diagnose disseminated mucormycosis. The patients infected with disseminated mucormycosis had the maximum death rate (58–79%) in comparison to other clinical presentations of mucormycosis (Lanternier et al. 2012; Skiada et al. 2011). Renal mucormycosis Several Indian studies found that the patients of isolated renal mucormycosis infection increased from 5.4 to 14% of entire incidences of mucormycosis (Chakrabarti et al. 2006; Prakash et al. 2019). There was no underlying disease in approximately 33–100% of renal mucormycosis infection cases in China and India (Ambrosioni et al. 2010; Prakash et al. 2019). Early detection of renal mucormycosis can be aided by computer tomography and ultrasound scans. A CT scan of abdominal part reveals bilaterally inflamed kidneys, bulging of the renal pelvis, and infarction of parenchyma (Bhadauria et al. 2018). In India, renal mucormycosis infection in patients having the normal immune system is another clinical entity. The analysis of several mucormycosis case series from India illustrated that 33–100% of renal mucormycosis infection were observed in immunocompetent patients (Gebremariam et al. 2015). Hemodialysis and CKD were considered critical risk determinants in patients with renal mucormycosis (Prakash et al. 2019). Renal mucormycosis can affect the kidneys unilaterally or bilaterally. The fever, haematuria, flank pain, severe kidney injury, flaky white crystals in the urine, and acute kidney injury are common symptoms reported in patients (Bhadauria et al. 2018). Approaches for treatment of mucormycosis Mucormycosis can be effectively managed if various sectors work together in collaborative and interdisciplinary manner. Because of the high mortality rate, even the smallest clinical suspicion would prompt the start of antifungal treatment. Surgical debridements, as well as antifungal medicines, are used to treat this condition. Roden et al. discovered that antifungal therapy and combined surgery was highly related to higher survival rates (69%) in a multivariate investigation of 929 recorded cases of mucormycosis, whereas fatality was virtually definite (97%) for individuals who received no treatments (Sipsas et al. 2018). Antifungal therapy Amphotericin B (AMB) is the primary treatment for this disease, and it has a significant impact on patient outcomes. This was demonstrated in a research study of 70 individuals having mucormycosis who did not receive AMB therapy on time (starting therapy 6 days following diagnosis), which resulted in an almost doubling in mortality 12 weeks after diagnosis (Chamilos et al. 2008a, b). AMB therapy is needed until clinical recovery is shown, which generally involves a couple of weeks. Compared to AMB, deoxycholate, a less expensive and more toxic alternative, intravenous AMB lipid formulation is commonly used (Honavar 2021). Metabolite repletion should also be monitored after Amphotericin therapy. Regular intravenous saline hydration and electrolyte replenishment were found to reduce metabolic irregularities and renal problems related with AMB poisoning in a clinical cohort trial of 368 individuals (Bicanic et al. 2015). AMB has been found ineffective against Apophysomyces and Cunninghamella isolates (Alastruey-Izquierdo et al. 2009). Because of its nephrotoxic properties, patients with compromised kidney function must be put on triazoles, such as Isavuconazole and Posaconazole (PSZ), which suppress ergosterol production in the cell membrane of fungal cells. In individuals who could tolerate AMB, these can also be administered as rescue treatment and as a step-down therapy (Hof 2006). Despite the fact that antifungal combination treatment is not currently recommended in any of the major clinical guidelines, more trials are required to determine its efficacy (Sipsas et al. 2018). Isavuconazole, a drug for mucormycosis that was newly approved in the Europe and U.S., has the possibility to become the standard treatment for fungal infections. The absence of cyclodextrins group which cause nephrotoxicity is a considerable advantage of Isavuconazole as compared to voriconazole. A once-daily regimen is also possible due to its long half-life (Spellberg and Ibrahim 2010). According to multicentric clinical trials, individuals receiving Isavuconazole or Amphotericin B have same mortality rate. In DKA mice infected by Rhizopus spp., combined therapy of Amphotericin B Lipid Complex and caspofungin was found to have a considerably higher survival rate than placebo or monotherapy. Liposomal Amphotericin-B along with anidulafungin or micafungin has shown improved results in disseminated mucormycosis. In the present circumstances, lipid formulations of Amphotericin B (LFAB)-echinocandin therapy for mucormycosis must be given at levels permitted by USFDA (Falci and Pasqualotto 2013). Rezafungin, encochleated Amphotericin B, orolofim, and SCY-078 are some of the new antifungal drugs currently being tested (Brunet and Rammaert 2020; Van Daele et al. 2019). Olorofim belongs to the orotomides, a novel antifungal agent that block the activity of dihydroorotate dehydrogenase (DHODH), an important enzyme for pyrimidine synthesis. A new glucan synthase inhibitor SCY-078 is ineffective against Mucorales (Lamoth and Alexander 2015). It also has low antimicrobial activity against Mucorales (Jørgensen et al. 2018). Antifungal drugs that are effective against saprophytic fungi are currently developed. VT-1161 is a new blocker of CYP-51 which is a fungal enzyme with mucorales activity in vitro. In R. arrhizus models, VT-1161 used as a curative or prophylactic therapy increased the survival of mice with neutropenic (Gebremariam et al. 2015, 2017). In a murine model, SCH 42427, a broadly acting triazole, was proven to be effective (Sugar and Liu 2000). APX001A (previously E1210) is an antifungal drug which blocks the Gwt1 protein. Gwt1 is a “glycosylphosphotidyl inositol post-translational modification pathway” surface protein. Despite the fact minimal inhibitory concentrations for mucorales are quite high, several researchers have demonstrated that APX001A is as efficacious as AMB in protecting mice in a R. delamar model (Miyazak et al. 2011; Rivero-Menendez et al. 2019; Sipsas et al. 2018). Finally, PC1244, a novel prolong acting antifungal azole, has been proven to have antifungal action against Mucorales with MICs ranging from 0.25 to 2 mg/L (Colley et al. 2018), and yet it has not been evaluated in vivo. Colistin has shown moderate in vivo and in vitro action against Mucorales among antibiotics (Ben-Ami et al. 2010). Adjunctive therapies In individuals having blood-related problems, any attempt to reverse neutropenia must be attempted, either employing hematopoietic growth agents or, in some cases, infusions of white blood cells. Persons suffering from corticosteroid-induced immunosuppression, like those suffering from immunological diseases must be weaned or shifted to non-steroidal medication if possible. Individuals having HIV/AIDS must start taking antiretroviral therapy to strengthen their immune system. Sugar control is crucial for people who have uncontrolled diabetes and/or ketoacidosis. Iron chelator therapies are still a possible treatment option for individuals with DKA. Individuals with diabetes particularly those having ketoacidosis could benefit from chelating with unbound iron. Physicians should also emphasize the treatment of any additional comorbidities that may exist (Alekseyev et al. 2021). Hyperbaric oxygen treatment has been demonstrated to be an adjuvant therapy when used in combination with other therapies. Increased oxygen pressure is suggested to stimulate the neutrophil function and enhance AMB activity by lowering acidosis. Finally, raising oxygen pressure promotes wound healing and reduces fungal development through inhibition of spore germination. Consequently, hyperbaric oxygen therapy (HBOT) for mucormycosis is generally suggested as an adjunct to surgical and antifungal treatment (Sipsas et al. 2018). In such situations, healthcare practitioners can play an important role to enhance the clinical result. Because of the difficulty in diagnosing these diseases, the late initiation of antifungal therapy can be linked with a significant incidence of deaths. At present, the most prevalent form of diagnosis is blood culture. Due to their poor sensitivity, cultures need a considerable time period to give the result. To effectively identify the species and evaluate resistance development, rapid, and quite exact technique like enzyme-linked immunosorbent assays must be employed in combination with cultures. Any clinical indication of fungus should be evaluated, and antifungal treatment should be given as early as possible (Yasmin et al. 2021). Surgical debridement Low drug bioavailability to site of infection can be caused by thrombosis of blood vessels and angioinvasion. Even if there is a slight possibility of mucormycosis, individuals should be prepared and prioritized for surgery. Surgical debridement of infected site has been found to reduce death rates by a significant amount (Spellberg and Ibrahim 2010). The MRI/CT-guided endoscopic sinus technique must be used to remove the afflicted tissue. Orbital exenteration and aggressive paranasal sinus debridement should be used to handle a rapid invasion of orbits (less than 72 h). Individuals should continue to receive intravenous AMB before beginning step-down therapy. Triazoles should be used to treat refractory infections. In the highly immunocompromised patients, it is preferable to take preventative measures to avoid immunosuppression before beginning antifungal treatment (Honavar 2021). Iron chelators The key function of metabolism of iron towards aetiology of mucormycosis suggests that potent iron chelators could be used as supplement to antifungal therapy. In fact, R. oryzae has been tested in vitro with two experimental iron chelators (Brunet and Rammaert 2020). The iron chelators like deferiprone and deferasirox did not permit the pathogen to absorb iron and presents an obstacle in development of micomycosis. Novel therapies In the recent years, the development of novel perspectives on the relationship that exists between the host, the fungus, and antifungal medication has been observed (lamoth and Kontoyiannis 2019). Some authors have emphasized PSZ’s ability to accumulate inside the leukocyte membranes due to its lipophilic characteristics. PSZ was loaded into an HL-60 leukemia cell line that had been differentiated to a neutrophil-like phenotype and is also used in aspergillosis mouse model to distribute PSZ effectively into infectious area (Baistrocchi et al. 2017). This innovative approach, however, has not been evaluated against Mucorales. There has been significant advancement in bioengineering, particularly in the field of genetically engineered cytotoxic T-cells. The beta-glucan in the fungus cell wall can be specifically targeted by these modified T-cells (Baistrocchi et al. 2017). However, only an aspergillosis model has been used to test this approach. CotH3, a Mucorales peptide that binds the human endothelial cell receptor GRP78, has recently been referred to mucormycosis endothelial invasion. Anti-CotH3 antibodies have prevented mucormycosis in neutropenic and diabetic mice and worked effectively in conjunction with antifungal agents (Gebremariam et al. 2019). Furthermore, other researchers have discovered that blocking the GRP78 cell receptor with GRP78-specific immune serum might help to protect diabetic mice from mucormycosis infection (Brunet and Rammaert 2020; Rocamora-Reverte et al. 2022). The interaction of this peptide-receptor could be a promising novel therapeutic avenue to investigate. Nutraceuticals may play a vital role in the treatment of COVID-19. Natural compounds such as theaflavin, gallic acid, berberine, nimbin, curcumin, withaferin A, andrographolide, naringenin, mangiferin, luteolin, quercetin, piperine, resveratrol, and zingiberene bind to ACE-2 receptors and prevent the SARS-CoV-2 virus from attaching to host cell (Kunnumakkara et al. 2021; Maurya et al. 2020). The plant species with anti-inflammatory properties can be effectively used to decrease cytokine storm in COVID-19 individuals (Agnihotri et al. 2021; Kunnumakkara et al. 2021). Diet may be able to reduce inflammation, and also, nutraceutical may be proficient in stopping viruses from entering inside the body. Dietary nutraceuticals could be assessed as a complementary dimension in the management of CAM. Patents and clinical status of in-progress treatment strategies for mucormycosis From 2019 to 2022, patent and related data were searched on the World Intellectual Property Organization’s official website using analytics to assess and organize current work related to mucormycosis (Table 2). Clinical trials have been conducted to evaluate the novel treatment for mucormycosis. In order to address the current treatment challenges, clinical trials on novel antifungal drugs and combinations of conventional agents are important. For better therapeutic recommendations, definitive clinical data from randomized prospective and observational research will be useful. Various clinical trials related to treatment approaches of mucormycosis are under different stages and few of them are enlisted in Table 3.Table 2 Summary of patents literature related to mucormycosis Patent name Patent number Applicant Publication date Reference An innovative portable handheld IOT enabled device for the identification of mucormycosis infected images considering the symptom severity for the focused treatment of patients IN202241041139 Senthil Velan Suganantham C. Sugunadevi Sam Gilvine Samuvel CMR Institute of Technology 29.07.2022 (Suganantham et al. 2022) A systematic model to detect black fungus appeared during COVID -19 using 3D convolutional neural networks IN202211042624 Career Point University Kota 29.07.2022 (Hussain et al. 2022) Kit and method for detecting mucormycosis pathogens CN114574609 Guangdong Runpeng Biotechnology Co., Ltd 03.06.2022 (Chaojie et al. 2022) Inhibitory potential of resveratrol and its natural analogues against RNA dependant RNA polymerase (RDRP) of rhizopus oryzae in mucormycosis through in silico investigations IN202221008681 Mithun Rudrapal Ismail Celik Sampath Chinnam 11.03.2022 (Rudrapal et al. 2022) Mucormycosis treatment agent JP2021134176 Univ Chiba Nat Univ Corp Drug Genomics Co Ltd 13.09.2021 (Hiroharu 2021) Novel fungal toxins and methods related to the same US20210179695 Los Angeles Biomedical Research Institute at Harbor-Ucla Medical Center 17.06.2021 (Ibrahim et al. 2021) Antifungal macrocyclic polyene novel compound CN112175029 Liu Li 05.01.2021 (Liu 2021) Methods of treating or preventing mucormycosis WO2020006438 University of Maryland, Baltimore Los Angeles Biomedical Research Institute at Harborat [US]/[US]; UCLA Medical Center 1124 Carson Torrance, CA 90502, US; Los Angeles Biomedical Research Institute at Harborat [US]/[US] 02.01.2020 Bruno et al. 2020 Immunotherapy and diagnosis of mucormycosis using coth US20190194301 Los Angeles Biomedical Research Institute at Harbor-Ucla Medical Center 27.06.2019 (Ibrahim et al. 2019a) Fungal toxins and methods related to the same US20190265238 Los Angeles Biomedical Research Institute at Harbor-Ucla Medical Center 29.08.2019 (Ibrahim et al. 2019b) Method for inoculating fermented bean curd blank CN110089577 Zhejiang University 06.08.2019 (Yuanming et al. 2019) Volatile metabolite profiles for the diagnosis and treatment of mucorales fungi US20190183887 The Brigham and Women’s Hospital, Inc 20.06.2019 (Koo and Marty 2019) Table 3 An outline of ongoing clinical trials invesigations of mucormycosis Study title Sponsor Study type/allocation/intervention model NCT no Phase Combined inhalational with intravenous Amphotericin B versus intravenous Amphotericin B alone for pulmonary mucormycosis Postgraduate Institute of Medical Education and Research Interventional/randomized/parallel assignment NCT04502381 Phase 2 The Deferasirox-AmBisome therapy for mucormycosis (DEFEAT Mucor) study Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center Interventional/Randomized/Parallel assignment NCT00419770 Phase 2 A study to evaluate isavuconazonium sulfate for the treatment of invasive aspergillosis (IA) or invasive mucormycosis (IM) in pediatric participants Astellas Pharma Global Development, Inc Interventional/non-randomized/single group assignment NCT03816176 Phase 2 Pilot study of high dose liposomal Amphotericin B efficacy in initial zygomycosis treatment (AMBIZYGO) Assistance Publique—Hôpitaux de Paris Interventional/NA/single group assignment NCT00467883 Phase 2 PK/PD study of Posaconazole for empiric treatment of invasive fungal infections in neutropenic patients or treatment of refractory invasive fungal infections (Study P01893) Merck Sharp & Dohme Corp Interventional/randomized/parallel assignment NCT00034671 Phase 2 Clinical study of AK1820 (isavuconazonium sulfate) for the treatment of deep mycosis Asahi Kasei Pharma Corporation Interventional/randomized/parallel assignment NCT03471988 Phase 3 Conclusions and future prospectives Mucormycosis (black fungus) is a deadly opportunistic illness that mostly manifests as a rhino-orbito-cerebral infection. According to studies, the life-threatening CAM is becoming more common in both COVID-19 infected as well as recovered patients all around the world. It was discovered that, due to a severe shortage of sterile oxygen in few countries, a quick production of industrial oxygen is provided to save the patient, allowing pathogens of mucormycosis to enter immunocompromised patients, causing them to contract black fungus in addition to COVID-19. As a direct consequence of this, therapy and diagnosis of COVID-19 patients become challenging. People who take Deferoxamine, an iron overload treatment are quite susceptible to get black fungus. The diabetic individual’s who have undergone treatment for COVID-19; a significant raise in CAM cases is linked with use of systemic corticosteroids which leads to immunosuppression. SARS-CoV-2 insulin resistance via the cytokine flow must be investigated as a separate risk factor for CAM because it causes immunosuppression. Due to quick development and angioinvasive nature of mucormycosis, prompt diagnosis and treatment should be started whenever it is suspected. Therefore, a high index of suspicion, initial diagnosis, stringent glycemic management, and avoidance from corticosteroids are all recommended. The physicians and healthcare professionals must be aware of implications of invasive supplementary mycotic infections in patients having COVID-19, particularly in those with already existing underlying conditions and comorbidities, and be able to detect and treat them immediately in order to reduce mortality and morbidity. Since then, Amphotericin B has been widely used for treatment, but Posaconazole and Caspofungin in combination have been proved to be efficacious due to possible synergistic action. As of now, the standard of care is immediate radical surgical debridement, lipid-based AMB, and Posaconazole. The promising future prospects for diagnosis and treatment of mucormycosis might emphasize upon rapid progression in chip-based sensors or implanted biosensors for active tracking of fungal analyte throughout the course of therapeutic intervention. In addition, to address the quick detection challenge of mucormycosis, the emerging biosensor devices could be developed by integrating particular molecular biological markers or species-specific identification elements. The current difficulties associated with infection necessitate clinical investigations of novel antifungal medications and combinations of existing antifungal treatments. The investigation of immune-boosting dietary nutraceutical that modify metabolic irregularities could be promising approach for management of COVID-19-associated mucormycosis. Acknowledgements The authors would like to thank Department of Pharmaceutics, MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, Haryana, India, 133207 and School of Health Science, University of Petroleum and Energy Studies, Dehradun, Uttarakhand, India, for providing facilities for the completion of this review. Author contribution NS, SNW, and TB: conceived the study and wrote the first draft of the paper; SS, IZ, and AS: data compilation; SB, AAH, and LA: editing; SBU and TB: proof read. Data availability Not applicable. Declarations Ethical approval Not applicable. Consent to participate Not applicable. Consent for publication All the authors have approved the manuscript for publication. Competing interests The authors declare no competing interests. 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==== Front Asian Bioeth Rev Asian Bioeth Rev Asian Bioethics Review 1793-8759 1793-9453 Springer Nature Singapore Singapore 235 10.1007/s41649-022-00235-0 Editorial Notes Promoting Dialogue through Diversity in Bioethics Laurie Graeme T. [email protected] grid.4305.2 0000 0004 1936 7988 School of Law, University of Edinburgh, Edinburgh, Scotland 1 12 2022 13 © National University of Singapore and Springer Nature Singapore Pte Ltd. 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcThe articles that open and close this January 2023 issue of the Asian Bioethics Review each advocate for greater dialogue in bioethics, both in the realm of academia and more broadly in practice. As such, they frame the other contributions in this issue that amply demonstrate how diverse understandings of the role of bioethics across and within human cultures can inform and drive any such commitment to dialogue. The objective of promoting dialogue through diversity is central to the mission of the Asian Bioethics Review. We are very pleased to publish such a representative range of contributions that support this objective. The first article of this new year issue is published on the first anniversary of the coming into force of the piece of legislation that it critiques. In their piece, Kashyap and Tripathi offer insightful commentary on India’s Surrogacy (Regulation) Act 2021, which came into effect on 25 January 2022 [https://link.springer.com/article/10.1007/s41649-022-00222-5]. Against the social and commercial backdrop of the growing Indian reproductive sector, this piece of legislation was introduced ostensibly as a response to concerns about exploitation of women, abuses of human rights, and the so-called ‘commodification’ of motherhood. While undoubtedly stronger protections have been introduced in some respects, the authors highlight multiple ways in which this new law—driven by a ‘moral conservatism’—continues to fail groups and communities within India, particularly members of the LGBTQ+ population. In doing so, the authors argue for a wider, more realistic, and more inclusive dialogue about what kind of laws of this nature are required for India, now and in the future. A failure to engage robustly risks a failure to protect the rich diversity of human and cultural identities that make up modern India. Risks of a different kind are the focus of the paper by Silva and Smith that examines the imposition of risk on some groups in an effort to promote public health for all [https://link.springer.com/article/10.1007/s41649-022-00218-1]. The authors point out that a robust account of the circumstances in which such risk imposition is legitimate remains elusive, and they argue for an approach that combines relational autonomy and relational justice to begin to remedy this concern. The explanatory power of such an approach lies partly in the ways in which it can be deployed to reveal imbalances of power and to inform dialogue and decision-making dynamics accordingly. The theoretical and practical unpacking of this approach is done through two examples drawn from (i) the ongoing COVID-19 pandemic and (ii) the regulation of sugar-sweetened beverages. Informed consent has long been understood as having risk communication at its centre. And while this remains true today, ethical and legal developments now emphasise the equal importance of seeing consent as a dialogic process rather than a ‘moment’ in the provision of healthcare or recruitment to a research protocol. But understandings of the nature of informed consent, what is involved, the role of risk communication, and the respective rights and responsibilities of the parties involved invariably change across countries and cultures. This is well illustrated by the paper from Aluko-Arowolo et al. [https://link.springer.com/article/10.1007/s41649-022-00223-4] in the context of clinical trials in Nigeria. Using well-established empirical research methodology, the authors engaged with members of a local community involved in running clinical trials (or in supporting participants) to reveal rich data about understandings of informed consent. Then, through a lens of ethical relativism, they argue for practical ways in which researchers can be more sensitised to local ethical issues and better supported to design research accordingly, all the while under the appropriate scrutiny of a legitimate ethics review body. The next two papers in this issue continue a theme that necessarily cuts across the work of a journal like the Asian Bioethics Review: capturing and understanding diversity of understandings and approaches to bioethics, particularly as these are informed by religious faith. The paper by Farid and Tasnim is an excellent illustration of this for its engagement with Judaism, Christianity, and Islam on various aspects of artificial reproductive technologies (ARTs) [https://link.springer.com/article/10.1007/s41649-022-00224-3]. The paper is valuable in two key respects; first, it reveals nuance and difference even within each of these Abrahamic faiths concerning ethico-religious responses to increasingly novel ARTs and practices, and secondly, it exposes many commonalities between these three leading global religions as well. This last point is particularly important because it suggests that productive dialogue about how to move forward cross-culturally might be more achievable than perhaps first thought. Invariably, religious perspectives are most acutely called into question with the advent of new technological developments. The paper by Sabri et al. illustrates this well with respect to its discussion of Islamic law in the face of three-dimensional (3D) printing of organs for transplantation purposes [https://link.springer.com/article/10.1007/s41649-022-00210-9]. This technological possibility returns Islamic scholars and bioethicists to the enduring question under Islam: when, if at all, is it permissible to change the creation of Allah? Deploying various methods used under Islamic law, the authors make the case that necessary uses of this technology are permissible to save a life. They acknowledge, however, that this itself raises questions about what counts as ‘necessary’ in the context of elective use of a new technology, and it also leaves open some doubt as to whether ‘desirable’ uses then raise problems from Islamic law and its followers. The authors of the previous article adopt a first principles approach to their interpretation of Islamic law. In much the same way, we see a similar approach being followed by Park in their analysis of the justification of a policy of mandatory vaccination for COVID-19 in South Korea [https://link.springer.com/article/10.1007/s41649-022-00221-6]. Here, however, the analysis is not of law but of the foundational principles concerning utilitarianism, as expounded in some of the classic texts that define this area of philosophical and bioethical scholarship. Park characterises the policy approach of South Korea as determinedly utilitarian, and then proceeds to dissect it by reference to the two levels of utilitarianism presented by R.M. Hare. This policy is found wanting, and this is doubly so when subjected to critique from the principles of fairness laid down in the works by Rawls and Hart. The author invites all of us, but law and policy-makers in particular, to think harder and more deeply about how to justify intrusive policies such as this one. Our final contribution is a Perspectives Article that engages directly with recent work by Crump et al. (2022) that has sought to improve on models and guidelines relating to the evaluation of whether animals can feel pain. In the present piece, Veit applauds these efforts but seeks to go further [https://link.springer.com/article/10.1007/s41649-022-00230-5]. In particular, the author argues that more must be done to bridge the divide between scientific understandings of sentience and the downstream legal and ethical protections that might—eventually—result. To overcome any such divide, Veit argues for a multi-method and multidisciplinary approach that acknowledges that animal sentience itself is a multidimensional phenomenon. Ethical discourse, debate, and dialogue are central to the approach. Moreover, and to return to the theme of dialogue that frames this editorial, this last paper is an excellent example of the kind of dialogue we wish to support and promote in this journal. It is through direct engagement with the ideas and perspectives of others that the best hope emerges of identifying productive ways forward in bioethics. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Crump A Browning H Schnell A Burn C Birch J Sentience in decapod crustaceans: a general framework and review of the evidence Animal Sentience 2022 32 1 10.51291/2377-7478.1691	36471826	PMC9713751	NO-CC CODE	2022-12-02 23:23:09	no	Asian Bioeth Rev. 2022 Dec 1;:1-3	utf-8	Asian Bioeth Rev	2,022	10.1007/s41649-022-00235-0	oa_other
==== Front Small Bus Econ Small Business Economics 0921-898X 1573-0913 Springer US New York 711 10.1007/s11187-022-00711-9 Article Financing decisions in private family firms: a family firm pecking order http://orcid.org/0000-0002-8806-0707 Jansen Katrien [email protected] 12 http://orcid.org/0000-0002-2417-0106 Michiels Anneleen [email protected] 1 http://orcid.org/0000-0001-7041-3933 Voordeckers Wim [email protected] 1 http://orcid.org/0000-0001-6947-4421 Steijvers Tensie [email protected] 1 1 grid.12155.32 0000 0001 0604 5662 RCEF - Research Center for Entrepreneurship and Family Firms, Hasselt University, Agoralaan Building D, B-3590 Diepenbeek, Belgium 2 grid.5284.b 0000 0001 0790 3681 Department of Accounting and Finance, University of Antwerp, Prinsstraat 13, 2000, Antwerpen, Belgium 1 12 2022 121 18 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Abstract Family firms are one of the most ubiquitous forms of business organizations worldwide. Their survival and growth are thus not only crucial for the firms themselves but also for the overall economy. One of the factors that influence their survival and development are their financing decisions. These decisions are generally described through the pecking order theory. However, not much is known about the applicability of this theory in private family firms. Given the shortcomings (both theoretically and empirically) of the current literature, we analyze 1087 incremental financing decisions from 277 family firms to develop and test a specific family firm pecking order. We integrate the elements of the socioemotional wealth perspective to theoretically explain the preferred order and introduce family capital into the pecking order model. Our findings indicate that family firms first prefer internal financing, next debt financing, followed by family capital, and last external capital. We also find that SEW considerations play a role in this financing decision. Especially the retention of control over the firm and the aim to pass the firm to the next generation appear to play an important role in determining this order. These dimensions ensure that family firms try to avoid extra capital. However, when it is needed, they will opt for family capital over external capital. This paper thus provides more insight into the reasoning behind financing decisions in private family firms. Plain English Summary How do family firms finance their investments? When looking for ways to finance their investments, firms have several options. According to traditional finance theories, they generally follow a so-called pecking order: they prefer to first use their internal funds, before turning to external financing. For family firms, the most ubiquitous form of business organization worldwide, two important aspects have been ignored in this research until now. First, socioemotional aspects influence decision-making in family firms and thus probably also financing decisions. Next, the business family itself can act as an external source of finance, which is not yet accounted for in the current pecking order model. In this research, we take these issues into account in order to develop—theoretically and empirically—a family firm pecking order. We investigate over a thousand financing decisions of 277 privately held family firms. Our results show that they prefer internal financing, followed by bank debt, family capital, and external capital. Especially the retention of control over the firm and the aim to pass the firm to the next generation appear to play an important role in determining this order. Our research thus indicates that future research should pay attention to the peculiarities of family firms when investigating their financing decision. Keywords Family firms Small- and medium-sized firms Pecking order Financing decisions Socioemotional wealth ==== Body pmcIntroduction Firms use multiple sources of funds to finance their overall operations, investments, and growth (Martinez et al., 2019). The financing decisions they make are essential for a firm’s survival (Koropp et al., 2014) and result in a particular capital structure (Martinez et al., 2019). Over the years, several theories have tried to explain these financing decisions. One of the most prominent theories is the pecking order theory (Myers, 1984), which describes a preferred order in the various financing sources that firms use to finance their investments. According to this theory, firms will first use internally generated funds, such as retained earnings. When the internal funds are insufficient, the firm will resort to external funds, with a preference for debt, and, lastly, equity. The pecking order theory is based on the problem of information asymmetry (Myers, 1984; Myers & Majluf, 1984) and assumes that due to incomplete information for investors, borrowing costs will increase (Degryse et al., 2012). The highest costs arise with the issue of equity, as the risks for the investors will be higher with this financing type because they are not sure whether the firm is overvalued or not (Myers & Majluf, 1984). In that case, the added value of the new investors will flow towards the current investors. The costs arising from this asymmetric information problem can thus explain the hierarchical order. However, empirical research is inconclusive about the pecking order: some studies support a pecking order in financing decisions (Lin et al., 2008; López-Gracia & Sogorb-Mira, 2008; McNamara et al., 2017), while others find little evidence (Fama & French, 2005; Frank & Goyal, 2003). Additionally, some research has shown that information asymmetry may not fully explain the pecking order theoretically (Fama & French, 2005; Frank & Goyal, 2003; Lin et al., 2008). Especially in private firms, of which the majority is family owned, separation of ownership and control is less prevalent, making information asymmetries related to equity financing less of a problem (Burgstaller & Wagner, 2015; Fama & French, 2005; Jensen & Meckling, 1976). In this paper, we revisit the pecking order—from both a theoretical and empirical point of view—with a focus on private family firms. We define a family firm as a firm in which a family has at least half of the shares and/or a firm that is perceived to be a family firm (Chua et al., 1999; Miller & Le Breton-Miller, 2006). Previous research about whether financing decisions of private family firms follow a pecking order is relatively scant, despite their abundant presence and influence on the overall economy and the essential nature of financing decisions in family firms (Michiels & Molly, 2017). Additionally, the problem of asymmetric information cannot fully explain the reasoning behind the preferred order in family firms (Gottardo & Maria Moisello, 2014; Romano et al., 2001) which calls for more in depth theorizing. Indeed, family firms have a “peculiar financial logic” that characterizes them (Gallo et al., 2004) and complicates the financing decision-making process. The choice for financing sources is often determined by control considerations in family firms (Schmid, 2013). They are reluctant to use financing sources, such as external equity, that dilute their perceived control over the firm (Koropp et al., 2014). The socioemotional wealth (SEW) perspective (Gómez-Mejía et al., 2007) takes these elements of family control and loss aversion into account and may thus help to explain a family firm pecking order. The SEW perspective suggests that the motives of individuals in a company go beyond purely economic goals, such as maximization of shareholder value. Family owners may use gains or losses in their SEW as the main frame of reference when taking strategic (financing) decisions. It may thus be essential to take these family firm-specific elements into account when researching their financing decisions. Previous empirical research has revealed findings that are in line with a pecking order in financing decisions in family firms. Some studies focused on the attitude toward different financing sources (Lappalainen & Niskanen, 2014; Romano et al., 2001), while others studied the relation between profitability and debt (Burgstaller & Wagner, 2015; López-Gracia & Sánchez-Andújar, 2007). These previous tests on capital structures often use the ratio of debt over assets, thereby taking into account all accumulated liabilities since the establishment of the firm and thus ignoring the timing of the acquired debt or equity (de Haan & Hinloopen, 2003). Additionally, these debt ratios do not distinguish between internal equity and external equity, which is necessary to empirically test a pecking order. Thus, none of these studies used methods that enabled them to focus on the actual financing decision made, which is surprising, as this is the core of the pecking order theory. Additionally, in order to obtain a family firm-specific pecking order, the integration of family firm-specific financing types, such as family equity, is required. However, this integration is missing in current empirical research. We argue that especially a more fine-grained distinction in the equity category is needed. As the theoretical argument for the use of family capital is totally different than for external equity, the integration of this specific financing type in the pecking order is important. In this paper, we develop and test a specific pecking order for family firms based on incremental financing decisions made for investments, using a unique dataset consisting of 1087 financing decisions from 277 small- and medium-sized family firms in Belgium. By applying the methodological strategy proposed by de Haan and Hinloopen (2003), we determine the hierarchy of the financing sources used. We distinguish between four different financing types: internal financing, bank debt, family capital, and external capital. A multinomial logit model first distinguishes the different financing types used by family firms. Afterwards, an ordered probit analysis is conducted to determine the hierarchy of the financing types. For each possible financing hierarchy, a separate ordered probit model is estimated, which shows the hierarchy that suits the data best. Finally, a continuation-ratio logit model is used to test whether there is a sequential mechanism that determines the response outcome. This paper contributes to the existing literature in four ways. First, we respond to the call of Reay and Whetten (2011) to modify existing theories to the specific context of family businesses to improve their explanatory power. We do this by expanding the pecking order theory in order to integrate family firm-specific elements to explain the preferred order. After all, traditional asymmetric information arguments (Myers, 1984) cannot fully explain the reasoning behind the preferred order in family firms (Gottardo & Maria Moisello, 2014; Romano et al., 2001), as several family-specific elements complicate the financing decision-making process in family firms (Michiels & Molly, 2017). These elements can be captured by the socioemotional wealth theory (Gómez-Mejía et al., 2007). The integration of this perspective, next to the problem of asymmetric information, will enable us to explain the theoretical reasoning behind the preferred financing order. Second, we do not only expand the pecking order theory by integrating family firm-specific elements to explain the preferred order but also by adding a family-specific financing type. Michiels and Molly (2017) argued that traditional frameworks, such as the pecking order theory, need a more extended view beyond the use of the classic financing types. Lappalainen and Niskanen (2014) and Romano et al. (2001) found that family firms have different attitudes towards family capital and external capital when increasing equity financing. That is why we make a distinction between these two types of capital. We will thus test a pecking order with four financing types: internal financing, bank debt, family capital, and external capital. Third, we take the heterogeneity of family firms into account when researching their financing decisions. As a majority of previous studies about financing decisions in family firms only make the oversimplified comparison between family and non-family firms, there is a need for research that takes the differences between family firms into account (Daspit et al., 2021; Michiels & Molly, 2017). We answer these calls and acknowledge goal-based heterogeneity by integrating the SEW perspective. After all, family firms can differ on their various family-related goals. For example, for some family firms, control considerations are more important than for others. In addition, not all family firms have the same dynastic succession intentions. We aim to measure these different SEW dimensions in a direct way, in contrast to prior studies building on the SEW perspective in the finance field which use indirect measures (e.g., Molly et al., 2019) or a composite direct measure (Baixauli-Soler et al., 2021). Additionally, we take governance-based heterogeneity into account and examine the role of governance mechanisms in explaining the pecking order, being the presence of a non-family CEO and a family charter. Finally, previous research mostly relied on methods based on debt ratios or attitudes to test whether family firms follow pecking order behavior (Burgstaller & Wagner, 2015; Lappalainen & Niskanen, 2014; López-Gracia & Sánchez-Andújar, 2007; Romano et al., 2001). We contribute to the literature by empirically testing the financing hierarchy of family firms based on incremental financing decisions. For every year, we know which financing type (e.g., internal financing or bank debt) is used for investments of every family firm in the sample. By looking into these incremental financing decisions, we are able to establish the relevant determinants for the choice of a specific financing source. Hereby, we are thus able to not only theoretically describe a family firm pecking order, but also to empirically test this order. The remainder of the paper is organized as follows: The next section gives an overview of the literature. After that, the data are described, followed by a discussion of the method and the results. The final section concludes and provides opportunities for future research. Literature Pecking order theory Financing decisions and capital structures have been important topics of research in business economics. Over the years, several theories have tried to explain the financing decisions that are taken. One of the traditional finance theories is the pecking order theory (Myers, 1984). This model focuses on a hierarchical order in which financing sources are chosen to finance investments. According to this theory, firms prefer internal over external financing. When internal funds are inadequate, bank debt will be used first, and equity funding will be considered as a last resort. As a consequence, the theory assumes there is no optimal capital structure or target debt level (Degryse et al., 2012). The pecking order theory is based on the problems related to the presence of asymmetric information (Myers, 1984; Myers & Majluf, 1984). Typically, managers have more information about the firms’ value than outsiders. When there is a high level of asymmetric information, investors will not have complete borrower information, which results in increased borrowing costs (Degryse et al., 2012). Investors will mostly be more suspicious in providing equity due to the risks associated with this transaction (Myers & Majluf, 1984). It is only interesting for them if it reveals a growth opportunity for the firm. The costs associated with the issue of debt or equity, such as transaction costs, can thus explain the preferred order of the financing options. Because the use of internal funds has the lowest costs, this funding will be preferred first. When an external party is needed, debt will be chosen above equity due to the lower transaction cost associated with the former. Empirical research shows mixed results about the presence of a pecking order. Some studies support a traditional pecking order in financing decisions (e.g., Lin et al., 2008; López-Gracia & Sogorb-Mira, 2008; McNamara et al., 2017), while others find little or no evidence of the pecking order (e.g. Fama & French, 2005; Frank & Goyal, 2003; Fulghieri et al., 2020). Additionally, several researchers investigated a modified version of the order (Bartholdy et al., 2012; de Haan & Hinloopen, 2003) by dividing the traditional financing types into different categories. For example, de Haan and Hinloopen (2003) split up the external equity into bonds and shares. These inconsistent empirical results may be caused by the variety of methodologies used to test the pecking order in these papers. Some studies focus for example on the ratio of debt over assets in order to explain a pecking order. However, by using this method the timing of the acquired debt and equity is ignored because all accumulated liabilities since the establishment of the firm are taken into account (de Haan & Hinloopen, 2003). Moreover, there is no distinction between internal equity and external equity, which is necessary to empirically test a pecking order. By relying on incremental financing decisions—as we are doing in this study—these concerns are substantially mitigated. Additionally, there is also an indication that information asymmetry may not fully explain the pecking order in most private firms (Fama & French, 2005; Frank & Goyal, 2003; Lin et al., 2008). For example, Frank and Goyal (2003) and Fulghieri et al. (2020) found that small high-growth firms do not follow the predetermined order. Debt and equity are shifting places in this “pecking disorder” (Fulghieri et al., 2020). The traditional pecking order works best for large, well-established, and publicly traded firms (Frank & Goyal, 2003). However, because large firms are often well known, with long uninterrupted trading records, investors have enough information about the firms when they need financing. Accordingly, information asymmetry problems are expected to be less severe in these firms and can therefore not explain the presence of the pecking order, which calls for more in-depth theoretical exploration of pecking order behavior in private firms. Some alternative explanations can be found in agency theory (Jensen & Meckling, 1976; Myers, 2003; Xiang & Worthington, 2015). Indeed, when a firm is only funded with internal funds, agency costs are minimal. However, when external funding is needed, agency costs will occur, especially when external funding is provided by an outside investor. Lin et al. (2008) found an explanation in the behavior of the manager, being the manager’s optimistic earnings forecasts. This managerial optimism may lead to the manager’s pecking order preference. Their results show that managers who are more optimistic will issue more debt. Additionally, as the traditional pecking order generally focuses on listed firms, the question arises whether the theory also applies in the context of private SMEs. After all it is inherent to all types of capital structure research that outcomes can be influenced by both supply (e.g., availability, access) and demand considerations (e.g., control motives) which may be different in private SMEs. In addition, although traditional external financing sources such as the public capital market may not be easily available to them, SMEs do have access to alternative sources of external finance such as capital from friends, family, angel investors, crowdfunding, or venture capital (Schickinger et al., 2018). Also the theoretical reasoning behind financing decisions in private firms can be substantially different in comparison to listed firms (Ampenberger et al., 2013; Gottardo & Maria Moisello, 2014) More specific, the problem of asymmetric information may differ in private firms because there is usually no separation between ownership and control. Costs associated with equity financing, especially from existing owners, may thus be less present (Burgstaller & Wagner, 2015; Fama & French, 2005; Jensen & Meckling, 1976). In the next section, we will revisit the pecking order in private family firms and its theoretical drivers. A family firm pecking order When further looking into privately held family firms, some studies also point towards a pecking order. Lappalainen and Niskanen (2014) indicated that due to differences in the attitude towards different financing types between family firms and non-family firms, it could be expected that the pecking order may differ between these types of firms. Burgstaller and Wagner (2015) and López-Gracia and Sánchez-Andújar (2007) used panel data to study the debt levels of family firms. Based on a negative relationship between profitability and debt, they favored the pecking order as an explanation of the financing decisions in family firms. However, Gottardo and Maria Moisello (2014) and Romano et al. (2001) indicated that the traditional pecking order theory building on informational asymmetry cannot fully explain family firms’ financial choices. Romano et al. (2001) indicated that a complex array of factors influence family firm owner’s financing decisions and Koropp et al. (2014) showed empirically that financing decisions are indeed influenced by the manager’s preferred choice and even non-rational elements. Therefore, family firms’ financing behavior is likely to be driven by non-economic considerations such as risk-taking propensity, emotions, and family goals (Berrone et al., 2012; Romano et al., 2001). Although family firms are often aware of the economic consequences (e.g., a lower growth rate) of their financing decisions, family business owners may consider non-economic goals more critical than these economic goals (Motylska-Kuzma, 2017). Especially family control and loss aversion considerations are crucial in understanding financing decisions in family firms (Burgstaller & Wagner, 2015; González et al., 2013; Schmid, 2013). López-Gracia and Sogorb-Mira (2008) indicated that in small- and medium-sized family firms, owner-managers will be more hesitant to seek financing that limits their ability to act. For example, additional external equity can reduce the owners’ shareholding in the company. Indeed, family firms have to make a trade-off between retention of control, which favors the use of debt financing, and risk aversion, which stimulates the company to adopt more cautious attitudes toward debt (Burgstaller & Wagner, 2015; González et al., 2013; Schmid, 2013). On the one hand, family owners are reluctant to use financing sources, which dilute their perceived control over the family firm. On the other hand, using more debt increases the probability of default and is thus risk enhancing. This illustrates the complexity of the financing decisions in family firms. These two key elements, family control and loss aversion, are core concepts of the socioemotional wealth (SEW) perspective (Gómez-Mejía et al., 2007). This perspective suggests that the motives of individuals in a company go beyond purely economic goals. The firm’s non-financial aspects have to meet the family’s affective needs. Some recent finance studies started to point to this perspective as an explanation for financing decisions in private family firms (Baixauli-Soler et al., 2021; Molly et al., 2019). However, these studies only focused on the use of debt. We argue that the SEW perspective has also high relevance as a theoretical explanation for pecking order behavior in private family firms. The SEW concept is multidimensional including the dimensions family control and influence (F), family members’ identification with the firm (I), binding social ties (B), emotional attachment (E), and renewal of family bonds to the firm through dynastic succession (R) (Berrone et al., 2012). Two of these dimensions are especially relevant for this study, as they might influence the willingness to attract specific financing types: the F- and the R-dimensions. Indeed, prior family firm finance papers pointed to the importance of control considerations (e.g., Ampenberger et al., 2013; Croci et al., 2011; Schmid, 2013) and dynastic (managerial) succession intentions (e.g., Amore et al., 2011; Koropp et al., 2013; Molly et al., 2010) as drivers of capital structure decisions. The key of the F-dimension is that the family exerts control over the strategic decisions (Berrone et al., 2012). To preserve SEW, family members require continued control of the firm, regardless of financial considerations (Gómez-Mejía et al., 2007). This control can be carried out directly, for example, by providing the CEO, or more indirectly, for example, by having family members in the top management team. By having control over the firm, the family also has an influence over the financing decisions made, making them capable of avoiding financing sources that dilute their control over the family firm. The R-dimension focuses on the intention of handing the business down to future generations (Berrone et al., 2012). This implies, among other things, that the family has a long time horizon in the decision-making process (Berrone et al., 2010). It measures to which degree the family sees the firm as a long-term family investment, which influences the financing decisions made. Both dimensions indicate that family firms may not strive toward the most optimal debt level; instead, their desire to attract debt determines their debt level (Molly et al., 2012). These desires determine the willingness to attract some financing types and the different attitudes towards the various financing types. Lappalainen and Niskanen (2014) found, for example, that the attitude towards additional equity from current owners is more positive in family firms than in non-family firms. Additionally, smaller family businesses have a substantial amount of their funding provided by internally generated funds such as owner capital (Lappalainen & Niskanen, 2014; Romano et al., 2001), while public markets are not used that often (Romano et al., 2001). Thus, there is a clear distinction in attitude towards additional capital injections from the current shareholders versus those from external parties (Neubauer & Lank, 1998). This distinction in preference suggests that these two different financing types should be separately integrated in a family firm-specific pecking order. The question is then where this family capital is positioned in the pecking order. As capital from family members can be issued with modest information asymmetry problems and thus low transaction costs (Fama & French, 2005), it will not be the last resort in the pecking order. In contrast, for debt financing and external equity, costs arise due to the fact that the interests between the family and creditor or family and external shareholders do not align (Xiang & Worthington, 2015). However, Croci et al. (2011) argued that the cost of debt in family firms is lower than in non-family firms. Due to their long-term orientation and good connections with their stakeholders (Carney, 2005) in combination with their preference for low-risk investment (Croci et al., 2011), credit markets are less reluctant to offer them debt financing. Based on these arguments, family capital could be placed between bank debt and external capital. By adding family capital as financing source and using the SEW perspective as additional theoretical justification, we are able to develop and test a family firm-specific pecking order. We expect that this pecking order will have the following sequence of preferred financing types: first internal financing, next bank debt, followed by family capital, and last external capital. We argue that, based on the arguments of the SEW perspective, internal financing will be chosen first, especially when keeping control in the hands of the family is considered important. These control considerations also explain the preference for family capital over external capital. Additionally, the position of these two financing sources can also be explained by the desire to hand over the firm to the next generations. When dynastic succession intentions are considered important, the family views the business as a long-term investment and will therefore be more open to provide additional capital. Bank debt will still be preferred over family capital due to minor risks of losing control of the firm and the reasonable costs associated with the issue of debt. Therefore, we argue that firms will rely on the following pecking order: (1) internal financing, (2) bank debt, (3) family capital, and (4) external capital. So far, we revisited the pecking order from a general family firm perspective, considering family firms as a uniform group. However, a growing body of research revealed a high degree of heterogeneity among family firms (Neubaum et al., 2019). In the next section, we focus on two important sources of family firm heterogeneity, namely goal-based and governance-based heterogeneity (Chua et al., 2012) as drivers of pecking order behavior in private family firms. Family firm heterogeneity as a driver of a family firm pecking order Goal-based heterogeneity Prior research proposed that socioemotional wealth is a main driver of distinct family business behavior vis-à-vis non-family firms. However, this assumption has been questioned recently (Hasenzagl et al., 2018). Indeed, family firms are a very heterogeneous population and show a wide variation regarding the different dimensions of SEW (Gerken et al., 2022). For example, although several family firms consider absolute control of the firm as the main reference point in their financial decision making (Berrone et al., 2012), the existence of many listed and venture capital backed family firms (Chemmanur et al., 2021) are exemplary of a more flexible attitude towards outsiders. In a similar vein, family firms also differ in the degree to which they aim to renew the family bonds through dynastic succession (Gerken et al., 2022). Such variations in the importance of SEW dimensions may lead to heterogeneous strategic (financing) behavior among family firms (Debicki et al., 2016). Therefore, we expect that internal finance and family capital will be preferred more and external capital less when family firms attach a higher value to control and dynastic succession considerations. For bank debt, family firms have to make a trade-off between retention of control, which favors the use of debt financing, and risk aversion, which stimulates the company to adopt more cautious attitudes towards debt (Burgstaller & Wagner, 2015; González et al., 2013; Schmid, 2013). Governance-based heterogeneity Apart from their goals, family businesses also differ in terms of their governance structure. In this regard, we focus on two important sources of governance-based heterogeneity, namely a family charter and a non-family CEO. First, a family charter is a mechanism to establish an effective family governance system (Suess, 2014). The family charter (also called family constitution or family protocol) can be defined as a formal agreement in which fundamental principles and guidelines on how the family organizes its relationship with the business are formulated (Berent-Braun & Uhlaner, 2012; Suess, 2014) and finds its theoretical roots in the relational governance perspective (Mustakallio et al., 2002; Poppo & Zenger, 2002; Uhlaner et al., 2007). This perspective proposes that “governance emerges from the values and agreed-upon processes found in social relationships” (Poppo & Zenger, 2002, p. 709). Relationally governed exchanges happen through social processes that promote norms of flexibility, solidarity, and information exchange which will lead to trust, solve potential high costs of exchange hazards, and ultimately lead to expectations of continuity and longevity (Poppo & Zenger, 2002). In family firms, a family charter can play a pivotal role in establishing effective relational governance. Indeed, a family charter should ideally be the result of a lengthy developmental process in which multiple family members articulate in advance the expectations concerning the firm and try to reach a shared vision. This process view of the family charter proposes that the development process is much more important from a relational perspective (e.g., relational dynamics characterized by open communication develop feelings of fairness, trust, and family unity) than the document itself (Botero et al., 2015). The relational process and the final document will ultimately lead to strong family owner commitment and responsible ownership (Uhlaner et al., 2007). Thus, developing a family charter helps to formally describe the social capital in the family firm, which results in a more structured organization for family and business, fewer conflicts, and a better view of the long run of the business (Suess, 2014). Leana and Van Buren (1999) indicated that good managed social capital leads to increased access to (external financial) resources, improved group communication, and efficient collective actions. Further, a shared vision of the firm and its future is created, which also results in a shared vision about financing decisions to be taken in the future. Due to the formal agreements about the future of the firm, it is likely that the family firm will be more open towards external parties (Suess, 2014). This is because the position of the family and external parties in the firm is well thought and clearly described in advance (Mustakallio et al., 2002). Moreover, from an external investor (supply side) perspective, responsible family ownership and commitment (as a result of the family charter development process) will mitigate potential agency conflicts (Arteaga & Menéndez-Requejo, 2017) which may increase their willingness to invest. Accordingly, we expect a positive relationship between having a family charter and external capital. Second, non-family CEOs represent an important stakeholder group across listed and private family firms (Waldkirch, 2020) and are an important source of heterogeneity in family firm governance. Due to a lack of sufficient human resources inside the family, non-family managers can be included to guarantee the firm’s survival and growth (Block, 2011; Klein, 2000; Sonfield & Lussier, 2009). This may be an indication that the family is open for external partners in the firm. By having a non-family CEO, the family can prevent emotions severely influencing the decision-making process (Goel et al., 2013). From a cognitive perspective, family CEOs might have had limited exposure to the external environment and make decisions “by intuition” and with emotions, as opposed to non-family CEOs who make decisions “based on logic and rational analysis” (Block, 2011, p. 11). Therefore, we argue that a non-family CEO will rather make decisions, including financing decisions that are best for the organization. This will limit the influence of family goals on the decisions made and thus limit the higher preference for internal financing and family capital. Data and variable definition Data We analyze a unique dataset based on survey data combined with financial data from the Bel-first database (Bureau Van Dijk). The survey was sent out to CEOs of 5005 Belgian companies with 10 to 500 employees in the Flemish region. The firms were all (private) limited companies, not active in the financial or governmental sector, and no holdings were included. The emails were sent out in December 2019, followed by two reminders (8 days and 29 days after the first email). We received 546 responses, which is a response rate of 10.91%. This response rate is in line with previous studies of privately held firms that target CEOs (Berent-Braun & Uhlaner, 2012; Cruz et al., 2010; Michiels et al., 2015). As it was not possible to select only family firms ex-ante, we coded these firms afterwards. For the purpose of this study, we define a family firm as a firm in which a family has at least half of the shares and/or a firm that is perceived to be a family firm (Chua et al., 1999; Miller & Le Breton-Miller, 2006; Vandekerkhof et al., 2014; Voordeckers et al., 2007). Based on this definition, our database contains information on 427 family firms. The survey data is supplemented with data from a secondary source: the Bel-First database by Bureau Van Dijk, which contains accounting statements of all Belgian firms. Using two different data sources, the risk of common method bias is mitigated since several control variables result from a database external to the survey. Due to missing variables in the survey or the Bel-first database, our research is based on 277 family firms and 1087 incremental financing choices. Variables Dependent variable The dependent variable of our models is the incremental financing decision made for an investment. To capture these incremental financing decisions, the respondents were asked to indicate which financing types were used for investments every year in the period from 2014 until 2018. Based on the literature, we selected the following financing types in order to determine a family firm pecking order: internal financing, bank debt, family loans and equity (family capital), and outside equity (external capital). We make a clear distinction between capital from family members and external equity. When more than one financing type was indicated for 1 year, we code it under one primary financing type. If only retained earnings are used, the firm is coded under “internal financing.” If a firm uses bank debt or a combination between bank debt and internal funding, we code the financing decision under “bank debt.” It is common practice in the Belgian context for banks to ask for firms to partly finance an investment with internal resources before the firm receives a bank loan. When a firm uses a family loan or family equity, even in combination with internal financing and/or bank debt, we classify it under “family capital.” The choice to involve the family in a particular decision is the most important. Lastly, every financing decision where an external partner is involved will be classified under “external capital.” The dependent variable is thus an ordinal variable with four categories: 0 = internal financing, 1 = bank debt, 2 = family capital, and 3 = external capital. Our final sample consists of 1087 financing choices from 277 family SMEs. Explanatory variables The models are supplemented with explanatory variables based on financial variables linked to the capital structure and family firm-specific variables. Financial variables The financial variables are based on previous capital structure research and are collected from the Bel-first database for the years 2013 until 2017. To avoid constructed correlations between explanatory variables and recorded financing types, all financial explanatory variables are lagged 1 year (de Haan & Hinloopen, 2003). To control for outliers, the variables are winsorized at 1% and 99% (Bacci et al., 2017). The first financial variables explain the finance decisions from the pecking order theory (Myers, 1984) and are also used by de Haan and Hinloopen (2003). Liquidity (liquid assets/total assets) and profitability (earnings/total assets) capture the availability of internal funds (Burgstaller & Wagner, 2015; de Haan & Hinloopen, 2003; Myers & Majluf, 1984). We can expect that they will be positively related to internal financing and negatively related with the other financing types. Next, the firm’s size (log(total assets)) will be positively related with debt and external financing and negatively related with internal financing. Large firms are more diversified, have less risk for bankruptcy, and have more bargaining power (Burgstaller & Wagner, 2015). This results in fewer information problems and, thus, higher levels of debt and external equity. This results in a lower cost to acquire external finance. Lastly, the age (ln(age)) of the firm is added. When a firm is older, information asymmetry should be less present due to the known history of the firm (Burgstaller & Wagner, 2015; Frank & Goyal, 2003). This reduces borrowing costs and thus results in higher debt levels. However, older firms do have more internal funds and thus less likely need external financing (Burgstaller & Wagner, 2015). We thus expect a positive relationship with internal financing and debt financing. Additionally, we will include two other capital structure determinants. The first variable is firm risk (proxied by the absolute value of the difference between the annual percentage change in net income and the average of this change over 5 years). Riskier firms are expected to have lower leverage because they have a higher chance of entering into financial distress (Burgstaller & Wagner, 2015). We thus expect a negative relationship with debt and with external capital. The second variable is the effective tax rate. When this tax rate is high, there will be higher benefits of having debt (Bigelli et al., 2014). We thus expect a positive relationship with bank debt. Family firm-specific variables To capture the particular context of family firms, we will also add family firm-specific variables to our model. As discussed in Sect. 2, the socioemotional wealth (SEW) perspective (Gómez-Mejía et al., 2007) proposes family control and dynastic succession considerations as important reference points for financial decision-making. The seminal paper of Berrone et al. (2012) discussed five different dimensions of SEW which form the basis of several scale development efforts. Two dimensions are highly relevant in our analyses: the F-dimension “Family Control and Influence” and the R-dimension “Renewal of Family Bonds Through Dynastic Succession.” We measure the R-dimension by the 3 items of the REI scale of Hauck et al. (2016) (α = 0.78). Although the F-dimension did not show up as a strong factor in scale development studies (Gerken et al., 2022), prior finance studies (e.g., Martínez Romero & Rojo Ramírez, 2017) tested the direct effect of this dimension based on the items proposed by Berrone et al. (2012) and found some interesting results. Therefore, we went back to the original 6 items for the F-dimension as proposed by Berrone et al. (2012) (α = 0.77). Finally, we control for the influence of governance-related factors by adding two family firm-specific variables that might have an influence on the financing decisions. First, we take the presence of formalized family governance practices into account. The family charter (dummy variable with a value of 1 when the firm has a family charter; 0 otherwise) is a mechanism to establish an effective family governance system (Suess, 2014). Our sample was gathered in Belgium, which was one of the first countries worldwide with a corporate governance code for private firm (Code Buysse), which includes a specific section on family firms. This code contains several guidelines concerning the role of a family charter, the development process and its content, which substantially increases the likelihood that the charter is the result of a dynamic development process and contains a shared vision concerning the financing strategy of the firm. Second, we take family involvement in the top management of the firm into account (non-family CEO versus family CEO). Therefore, we include a dummy variable with a value of 1 for having a non-family CEO and 0 otherwise. Method and results Method Our analyses are based on the methodology used by de Haan and Hinloopen (2003). Our dependent variable is considered as ordinal and consists out of four categories: internal financing, bank debt, family capital, and external capital. After discussing the descriptives, we estimate a multinomial logit model which explains the drivers of the financial choices. Based on these results, we can conclude if the drivers behind the different financing types differ and we can thus make a distinction between the different financing types. Second, we use ordered probit analyses to test every possible hierarchy of the different financing types. Based on these results, we can see which hierarchy suits the data best and is thus the preferred order. Descriptive analyses Table 1 gives an overview of the sample. Most firms are between 20 and 50 years old (86.64%) and have between 10 and 50 employees (79.78%). The firms are mostly situated in the manufacturing (33.21%) and wholesale and retail (30.69%) industry. Table 2 gives an overview of the financial variables and family firm variables. The mean liquidity and profitability are 11.8% and 4.4%, respectively. On average, firms pay 25.6% taxes. The average score on the F-dimension is 5.15 out of 7 and on the R-dimension 5.46 out of 7. The governance variables show that less than 24% of the family firms have a non-family CEO and that 18% have a family charter. Table 3 shows the correlation table. The highest (0.448) correlation can be found between the F- and R-dimension of SEW. Based on the correlations, we can conclude that there is no problem with multicollinearity.Table 1 Overview of the sample Variable Number of firms Percentage of total sample Firm age 0–20 years 6 2.16% 21–50 years 240 86.64% More than 50 years 31 11.19% Employees 10–50 221 79.78% 51–100 32 11.55% 100–250 24 8.66% Industry Manufacturing 92 33.21% Construction 32 11.55% Wholesale and retail 85 30.69% Services 68 24.55% Table 2 Descriptives Variable Mean Std. Dev Min Max Liquidity 0.118 0.124 0.000 0.646 Profitability 0.044 0.074 − 0.250 0.317 Sizea 8,047,336 1.08e+07 320,213.9 6.97e+07 Agea 33.897 13.682 16 89 Risk 4.531 11.480 0.007 139.523 Effective tax rate 0.256 0.284 − 0.766 2.006 SEW F 5.146 1.309 1 7 SEW R 5.460 1.437 1 7 Family charter 0.181 0.385 0 1 Non-family CEO 0.238 0.426 0 1 aFor these variables, we report the absolute value (whereas in the regression analyses, natural logarithm of these variables is included) Table 3 Correlation table , , and significant at the 10%, 5%, and 1% level, respectively Table 4 gives an overview of the firm characteristics by financing choice. For every variable, the mean value per financing choice is given. An ANOVA test is executed to test whether there are differences, with respect to these firm characteristics, among the different groups with another choice of financing, based on the between and within variances of the groups. The results show that firms that finance more internally are relatively more profitable and have higher liquidity. External capital is used more by larger firms. This is in line with what we would expect from the problem of asymmetric information: larger firms should use more external capital due to the lower costs. Regarding firm age, the results show that older firms have a higher average value on internal financing and external capital. Thus, we see both effects that could be expected from the traditional pecking order literature: older firms have more internal funds and have more access to external funding. The highest level of risk can be found with family capital. We expected that riskier firms do not have access to bank debt or external financing. We thus see that, if there is a need for financing, these firms will use the only option left, namely family capital. When looking at the family firm-specific variables, we see that the scores on the F-dimension of SEW are higher when the firm chooses for internal financing or family capital. For the R-dimension, the highest score is seen with firms financing with family capital. In other words, firms that attach importance to retention of control or who aim to pass the firm to the next generation will limit the use of external parties when seeking for financing and mostly use internal generated funds or extra capital from family members. Having a family charter occurs more in the case with firms that use external capital. Family firms that have recorded agreements in official documents thus seem to be more open to external capital. Family firms with a non-family CEO are less likely to opt for family capital.Table 4 Firm characteristics by financing type Internal financing Bank debt Family capital External capital F Financial variables Liquidity 0.187 0.098 0.091 0.103 52.07* Profitability 0.064 0.044 0.025 0.039 15.71* Sizea 7,443,721.6 7,932,043 8,937,831 10,017,527 4.40* Agea 36.433 33.091 32.879 36.276 8.18*** Risk 3.060 4.479 5.424 4.657 2.42* Effective tax rate 0.255 0.280 0.205 0.230 5.77* Family firm variables SEW F 5.243 5.111 5.247 4.688 4.33 SEW R 5.581 5.300 5.707 5.667 6.34* Family charter 0.174 0.142 0.203 0.432 14.33* Non-family CEO 0.278 0.249 0.176 0.238 2.74** aFor these variables, we report the absolute value (whereas in the regression analyses, natural logarithm of these variables is included). This table provides the mean values of the explanatory variables for every financing type. The F-score of the analysis of variance is given. , , and significant at the 10%, 5%, and 1% level, respectively Drivers of the incremental financing choices A multinomial logit model is used to explain the drivers behind the financing choices. The marginal effects, the partial derivatives of the probabilities concerning the explanatory variables evaluated at their respective means, are used because they are directly interpretable (de Haan & Hinloopen, 2003). The results are shown in Table 5.Table 5 Multinomial logit model Marginal effects Internal financing Bank debt Family capital External capital Financial variables Liquidity 0.900 (7.69)* − 0.830 (− 4.95)* 0.147 (1.11) − 0.216 (− 3.11)* Profitability 0.348 (1.72)* 0.535 (2.06) − 0.779 (− 3.59)* − 0.103 (− 1.13) Size 0.004 (0.31) − 0.011 (− 0.64) 0.015 (1.07) − 0.008 (− 1.50) Age 0.171 (4.59)* − 0.076 (− 1.62) − 0.093 (− 2.41) − 0.002 (− 0.11) Risk − 0.003 (− 1.48) 0.000 (0.20) 0.002 (1.71)* 0.000 (0.96) Effective tax rate − 0.046 (− 0.84) 0.168 (2.73)* − 0.127 (− 2.55) 0.005 (0.31) Family firm variables SEW F-dimension 0.002 (0.14) 0.020 (1.43) − 0.007 (− 0.61) − 0.015 (− 3.47)* SEW R-dimension 0.015 (1.31) − 0.047 (− 3.53)* 0.029 (2.63)* 0.003 (0.82) Family charter 0.054 (1.40) − 0.151 (− 3.49)* − 0.002 (− 0.06) 0.099 (3.43)* Non-family CEO 0.084 (2.36) 0.008 (0.19) − 0.090 (− 3.23)*** − 0.001 (− 0.12) Controlled for year Yes Controlled for industry Yes Log likelihood − 1133.237 Pseudo-R2 0.1079 Number of observ 1,087 Multinomial logit model with categories defined as 0 = internal financing, 1 = bank loans, 2 = family capital, and 3 = external capital. Absolute value of z-statistics between parentheses. , , and significant at the 10%, 5%, and 1% level, respectively The results show that liquidity is highly positively related with the probability of using internal financing but negatively related with the probability of attracting bank debt and external capital. These findings are in line with the traditional pecking order. The availability of internal funds causes a higher probability of using these funds and a lower probability of using external funds. Profitability is positively related with the probability of using internal funds and bank debt and negatively related with the probability of using family capital. This indicates that firms prefer to use the profits, maybe in combination with bank debt, to fund new investments. There are no significant effects for size. For age, we find that older firms are more likely to use internal financing and less likely to use family capital. Because of their age, they seem to have more internal financing available and have no need for other financing types. Additionally, the effects of the risk variable are small and only significant and positively related with family capital. This finding can be an indication that riskier firms can only use family capital as financing source. We see a positive relation between the effective tax rate and the use of debt but a negative relation with family capital. Firms which have to pay higher taxes will profit from the benefits of bank debt. Next, we also see negative and positive effects from the family firm variables. When looking at the specific dimensions of SEW, the R-dimension is negatively related with the probability of using bank debt and positively related with the probability of using family capital. The F-dimension is negatively related with the probability of using external capital. Thus, when families find it important that the firm is transferred to the next generation, they are more prepared to choose for family capital. They will also be reluctant to use bank debt. When retention of control is important for the family, there is a higher chance on a negative attitude toward external parties in the firm. Having a family charter is negatively related with bank debt and positively with external financing, which means that there is a higher chance that the family has formalized agreements in a document before introducing external capital. Having a non-family CEO is positively related with the probability of using internal financing and negatively related with the probability of using family capital. Thus, the non-family CEO seems to be more reluctant to use family capital and primarily focuses on the traditional financing types whereby internal financing is preferred. This is in contrast with what we expected. Another explanation might be found in the behavior of the family: it is possible that the family does not want to invest more in the firm because a non-family CEO is appointed. The presence of a non-family CEO does not limit the preference for debt financing and does not increase the openness to external capital. To summarize, we find indications for a pecking order in financing in the financial variables. Based on the effects found with liquidity, profitability, and age, we see a preference of internal funding and a limited use of debt and especially external capital. Additionally, the family firm-related variables have clearly an influence on the preferred financing types. Therefore, the integration of these variables is a valuable addition to the literature. Also, the drivers behind the four financing types are different, which indicates that the financing types differ from each other. As different marginal effects occur for family capital and external capital, we can split up external funding from the traditional pecking order into “family capital” and “external capital” in our family firm pecking order. Therefore, we can base our pecking order on the four proposed financing types. Financing hierarchy As a next step, ordered probit models are estimated to determine the most preferred hierarchy of the financing types (de Haan & Hinloopen, 2003). The distinct financing types are coded with ordinal variables, which impose the pecking order hierarchy when estimating the model, for example, internal financing, bank debt, family capital, and external capital = [0, 1, 2, 3]. There is one reference financing type and three alternative choices with an a priori imposed hierarchy. The next choice in the hierarchy is chosen when a threshold parameter’s value has trespassed. For every possible hierarchy, an ordered probit model is tested. There are 24 different orders, but every order has a twin with an opposite sign and thus a perfect inverse correlation. Because of this, we consider 12 ordered probit estimates, which results in 12 log likelihoods, one for every model. These numbers will then be compared by likelihood ratio tests to see if the hierarchies differ significantly from each other and to reveal which hierarchy fits the data best. Table 6 reports all 66 pairwise likelihood ratio tests. The results of these tests show if the hierarchies differ from each other and make it possible to determine a ranking of the hierarchies. The hierarchies in the columns and rows are sorted by their likelihood values, from lowest to the highest (de Haan & Hinloopen, 2003). The likelihood ratio tests are computed as − 2 [ln(likelihoodcol)-ln(likelihoodrow)]. Significance values at 5% and 1% level are 3.84 and 6.63, respectively. Out of these analyses, we can draw two conclusions. First, as seen in Table 6, most of the hierarchies differ significantly from each other. For every significant result, we can thus conclude that the two hierarchies are not equally preferred, but one is preferred over the other. Only four pairs are not significantly different at the 5% level: [hj,hh], [hd,hf], [hb,hd], and [hc,hb]. Four additional pairs ([hh,hi], [hg,hh], [hg,hj], and [hd,hc]) are not significant different at the 1% level.Table 6 Likelihood ratio test results and * significant at the 5% and 1% level, respectively Second, Table 7 shows the resulting ranking of the 12 hierarchies. Both on the 5% and 1% significance level, one order is preferred the most in our sample: ha. The family firms in our sample first opt for internal finance, followed by bank debt. Then, family capital will be preferred over external capital. So, when including family capital into the traditional pecking order, it will be preferred over external financing. This is what we could expect from the peculiar financial logic of family firms. For these firms, it is important to lower risk and to retain control over the family firm (Gallo et al., 2004), which explains why family capital will be preferred over external finance.Table 7 Hierarchies and their ranking according to their likelihood Hierarchy Internal finance Bank loans Family External Ln(likelihood) Pseudo-R2 Rank at 1% Rank at 5% ha 0 1 2 3 − 1194.945 0.0593 1 1 hb 0 1 3 2 − 1202.606 0.0533 2 2 hc 0 2 1 3 − 1201.916 0.0538 2 2 hd 0 2 3 1 − 1204.378 0.0519 2 2 he 0 3 1 2 − 1214.874 0.0436 3 3 hf 0 3 2 1 − 1206.212 0.0504 2 2 hg 1 0 2 3 − 1232.909 0.0294 4 5 hh 1 0 3 2 − 1235.615 0.0273 4 6 hi 1 2 0 3 − 1224.751 0.0358 4 4 hj 1 3 0 2 − 1235.269 0.0276 4 6 hk 2 0 1 3 − 1241.973 0.0223 5 8 hl 2 1 0 3 − 1238.649 0.0249 6 7 The estimation results of the ordered probit regression for the preferred order further are analyzed.1 However, only two cut-off points are significant: there is a clear distinction between internal financing and debt financing and between debt financing and family capital. However, between family capital and external capital, there is no significant difference. Additionally, the pseudo-R2 we obtain from the ordered probit regression is quite low (0.0593). In order to dig deeper into these results, additional analyses are needed, making use of a continuation-ratio logit estimation (Agresti, 2003). This estimation technique is useful when a sequential mechanism determines the response outcome. In the traditional pecking order theory (Myers, 1984), firms first make their choice between internal financing and external financing. If external financing is needed, they will first choose for debt and then equity. This reasoning can be an explanation of our results: in the last step, when equity is chosen, there can be an additional decision between family and external capital. The existence of a sequential mechanism can be tested by a continuation-ratio logit estimation. This technique allows us to check whether there is indeed a first choice between internal and external financing. Next, in case they opt for external financing, we can analyze whether they choose between debt and capital. Lastly, if family firms prefer to finance with an increase in capital, we can check if they choose between family capital and external capital. We will thus get a better view on how the financing decision is made. Following an order in financing decisions Table 8 gives the results of the continuation-ratio logit estimation. The results show that every model is significant. This means that family firms first choose between internal and external financing. When external financing is selected, the firm will compare debt against extra capital. Last, in case an increase in capital is chosen, the firm will make a choice between family capital and external capital.Table 8 Continuation-ratio logit estimations Model 1 Model 2 Model 3 Financial variables Liquidity − 5.515 (− 7.62)* 1.453 (1.74)* − 3.522 (− 1.64) Profitability − 2.209 (− 1.76)* − 4.827 (− 3.60)* − 0.383 (− 0.13) Size − 0.031 (− 0.37) 0.058 (0.72) − 0.257 (− 1.44) Age − 1.060 (− 4.54)* − 0.251 (− 1.09) 0.523 (1.02) Risk 0.019 (1.59) 0.011 (1.50) − 0.007 (− 0.54) Effective tax rate 0.269 (0.81) − 0.733 (− 2.68)* 0.716 (1.44) Family firm variables SEW F − 0.016 (− 0.22) − 0.141 (− 2.14) − 0.468 (− 3.13)* SEW R − 0.089 (− 1.29) 0.198 (3.15)* − 0.014 (− 0.10) Family Charter − 0.321 (− 1.49) 0.728 (3.68)* 1.425 (3.78)* Non-family CEO − 0.486 (− 2.51) − 0.443 (− 2.19) 0.259 (0.57) Constant included Yes Yes Yes Controlled for year Yes Yes Yes Controlled for industry Yes Yes Yes X2 161.56* 72.69* 36.43*** Pseudo-R2 0.1381 0.0680 0.1208 Number of observ 1,087 838 281 In model 1, the choice between internal and external financing is investigated; in model 2, the choice between bank debt and extra capital is investigated, and in model 3, the choice between family capital and external capital is investigated. Absolute value of z-statistics between parentheses. , , and ** significant at the 10%, 5%, and 1% level, respectively As indicated in Table 8, financial variables clearly have an influence on the decision between internal and external financing (model 1). The coefficient estimates of liquidity, profitability, and age are significant and negative. Profitable, older firms and firms with more liquidity will choose for internal financing over external financing to finance investments. Only the non-family CEO variable is significant when looking at the family-specific variables. Family firms managed by a non-family CEO more often use internal finance instead of external finance. When looking at the decisions between bank debt and extra capital (model 2), some other effects are shown. Profitability has a negative significant effect which may indicate that profitable firms have more access to bank debt due to their better repayment capacity and will thus choose for this financing type over extra capital. This is in contrast with the positive effect we find concerning liquidity. Family firms with more liquidity have a higher probability to choose external capital. Investors are more open to invest in firms with high liquidity, which makes access to extra capital easier for these firms. Retention of control (F-dimension of SEW) will cause family firms to prefer bank debt over extra capital. However, the opposite effect is seen with the R-dimension. The presence of a long-term view will make family firms prefer extra capital over bank debt. Thus, the control aspect and the long-term view give different results. This finding shows that it is important to look at the different dimensions of SEW separately. Debt may be seen as a way to decrease risk to lose control over the family firm; however, it may not be good from a long-term point of view. This can also be seen in the result of the coefficient estimate of the charter variable, which is positive. When the family firm has made agreements about the future of the family firm, external capital will be considered more. A negative coefficient estimate is found for the non-family CEO. The influence of the non-family CEO seems to ensure that debt will be chosen over extra capital. Lastly, the choice between family capital and external capital is tested (model 3). Concerning the financial variables, none of our variable has an influence on this decision. However, concerning the family firm-specific variables, we find that the decision is influenced by the F-dimension of SEW. The retention of control will cause that family firms choose for family capital over external capital if a capital increase is needed. The variable family charter shows a significant positive coefficient: external capital is chosen over family capital. This openness towards external parties might be explained by the availability of clear agreements about external parties made by the family. Based on these agreements, introducing an external party will contain less risk of losing family control over the firm. Discussion and conclusions In this paper, we analyze 1087 incremental financing choices of 277 Flemish small- and medium-sized family firms, made between 2014 and 2018. We distinguish four different financing types: internal financing, bank debt, family capital, and external capital. This is the first paper that uses incremental financing decisions to empirically test a family firm pecking order. Using these incremental financing decisions enables us to truly test if there is a hierarchy in between the different financing types and to specifically look into the drivers behind every financing decision. Both these elements lack in previous research about a family firm-specific pecking order. Following de Haan and Hinloopen (2003), a multinomial logit regression is performed, which shows that there are indeed different drivers for family capital than for the other financing types. Based on these results, we use these financing types to create a family-specific pecking order. We modify the traditional pecking order by adding family-specific theoretical drivers and a family-specific financing type being “family capital,” which includes family equity and family loans. Based on an ordered probit model, we determine the preferred hierarchy between the financing types. The results show that first internal financing is preferred, followed by bank debt, family capital, and external capital. This is in line with the traditional pecking order of Myers (1984). However, family capital is added in the pecking order as a family-specific financing type between bank debt and external capital. Last, we use a continuation-ratio logit estimation to show that family firms first make a choice between internal financing and external financing. When external financing is needed, a choice will be made between debt financing and extra capital. And lastly, when extra capital is needed, the family firm chooses between family capital and external capital. Our tests did not only focus on the addition of a family firm-specific financing type. As it is clear that problems related to asymmetric information are not fully capable of explaining the pecking order theory (Fama & French, 2005; Frank & Goyal, 2003; Lin et al., 2008), we look into different family firm variables linked towards the socioemotional wealth theory (Gómez-Mejía et al., 2007) as possible explanations of the financing choices. We thus used the socioemotional wealth theory (Gómez-Mejía et al., 2007) as additional theoretical base in our family firm-specific pecking order. We selected two dimensions of the FIBER scale (Berrone et al., 2012): the F-dimension family control and influence and the R-dimension renewal of family bonds through dynastic succession. Through our analyses, we see that both the R-dimension and the F-dimension have an influence on the financing decisions. Especially a higher score on the F-dimension will limit the chances that family firms will use any kind of external funding. When it is thus important that the control over the family firms stays within the family, the family will be less willing to use extra capital. The R-dimension will cause family firms to choose for family capital when external funding is needed. The long-term view of the family over the firm makes family members more open to invest in the firm. Further, we test two other governance variables with a link to financing decisions, specific to family firms: having a family charter and a non-family CEO. Two important conclusions can be made. First, when family firms prefer to use family capital or external capital, it is more likely that they will have written down agreements in a family charter. These agreements will help to preserve the family character of the firm and make the long-term view on the firm clear. This can make the firm more open towards external financing parties because the agreements enable the integration of these parties in the firm without losing the family character of the firm. Second, even if a family firm has a non-family CEO, external funds will not be preferred more. The traditional pecking order will still be applicable: internal funds will be preferred over external funds and debt over extra capital (family capital or external capital) in case external funding is needed. Our results contribute to the literature in at least four different ways. First, we contribute to the pecking order theory research by integrating family firm-specific elements in order to explain the preferred order. We use the socioemotional wealth theory as an additional theoretical basis for our pecking order. We conclude that not only information asymmetry has an influence on the preferred order, but also family firm-specific variables like the F- and R-dimensions of SEW. This indicates that family firm-specific elements, such as control considerations and long-term view on the family firm, will influence the financing decisions made and the order in which different financing types are preferred. Second, we tested a pecking order specifically for family firms by adding a specific family firm financing type: family capital. It is necessary to distinguish between these two types of extra capital, as the reasoning behind the choice for family capital and for external capital is not the same. For example, the family remains in control over the family firm if family capital is used, while this is not the case when external capital is integrated. Third, we empirically contribute to the pecking order literature by using a method based on incremental financing decisions. Previous research used methods based on debt ratios or attitudes to test if family firms follow a pecking order behavior. However, based on these methods, it is not possible to formally test a hierarchy. This is only possible if incremental financing decisions are used, which we do in this paper. Finally, we contribute to family firm research by taking the heterogeneity of family firms into account when researching financing decisions. The results indicate that both goal-based differences as governance-based differences have an influence on the financing decisions made. In addition to these contributions to extant research, our study also holds various practical implications. Our findings indicate that family firms who find retention of control important, or whose aim is to pass the firm to the next generation, will limit the use of any form of external financing. Yet, this is limiting the growth of family firms (Molly et al., 2012). Previous studies have highlighted the crucial role of external finance for SMEs (Beck & Demirguc-Kunt, 2006; Wright et al., 2015). Business advisors and policymakers should understand that it is SEW preservation that appears to hinder family businesses to open up to external financing through bank loans or external equity. Additionally, previous studies found that family business owners’ intention to use external equity is influenced by their knowledge of it (Graves et al., 2022). Policymakers and family business advisors might therefore consider ways to reduce family business owners’ fear of losing control—and thereby fostering firm growth—by enhancing their financial knowledge. For example, illustrating that incremental increases in the debt level not necessarily mean higher risks of bankruptcy might engender a more positive attitude towards external financing in optimizing their capital structure. From another point of view, the lower indebtedness of family businesses can have advantages as well. In a crisis situation, such as the COVID-19 pandemic, these lower debt levels may have led to higher survival rates for family firms. Business advisors and policymakers should thus be aware that it gives family firms financial flexibility (Andrieu et al., 2017; Canton et al., 2013) in difficult situations. This research has some limitations, which can provide fruitful avenues for future research. First, the pecking order theory is only one theoretical angle to look into financing decisions. There are several other theories, such as the trade-off theory, target adjustment behavior, or agency theory, that can be used to explain the financing decisions in family firms (Colli, 2012; Kayhan & Titman, 2007). It is certainly useful to test if firms also strive towards a target debt level, using incremental financing decisions. Second, our results show different effects from the two dimensions of SEW we use. This finding indicates that the concept SEW can thus not be seen as unidimensional but should be analyzed taking into account its different dimensions. Further research on financing decisions in family firms should focus on the potential opposite effects of different SEW dimensions. Third, we included a family firm-specific financing type: family capital. We show that this kind of capital is different from capital from external parties and therefore, these two types of capital should be distinguished from each other. However, in the current literature, still little is known about family capital. There may be elements, such as family conflicts and disagreement about risk-return characteristics, that may influence the use of this financing type. Additional research that focuses on this specific type of financing is thus necessary. Fourth, the sample used in this research consists of Belgian family firms. Belgium is a bank-based economy with a less developed bond market. A different institutional context may influence the outcomes of the tests. In addition, contextual differences may also apply to some key variables in our analysis like the family charter. We assumed that the family charter is actually the result of a dynamic development process to which all family members could contribute, leading to a shared vision concerning the financing strategy of the firm. We argued that this assumption is valid in the Belgian context, given its long-standing corporate governance code for private firms and years of efforts from employers organizations to raise the awareness on the importance of the process in developing a family charter to reach a shared vision. However, this assumption might not always be valid in other institutional contexts. Therefore, future research may investigate the relationship between how a charter is developed and potential firm outcomes such as the financing strategy. Fifth, this paper focuses on small- and medium-sized family firms. However, not all family firms are small- or medium-sized. Therefore, it may be interesting in future research to look for a confirmation of our findings on a sample of large family firms. 1 Results available on request. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Agresti, A. (2003). 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==== Front Int J Sociol Leis International Journal of the Sociology of Leisure 2520-8683 2520-8691 Springer International Publishing Cham 123 10.1007/s41978-022-00123-9 Original Paper Retiring from ‘University Life’: Critical Reflections on a Retirement Lifestyle Planning Program http://orcid.org/0000-0001-6406-5127 Woodford Kimberley [email protected] Hutchinson Susan [email protected] Ausman Christine [email protected] grid.55602.34 0000 0004 1936 8200 School of Health and Human Performance, Dalhousie University, Halifax, NS Canada 1 12 2022 123 24 1 2022 4 11 2022 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. While assisting individual workers to prepare or plan for a successful transition to retirement is a key responsibility of human resource (HR) departments, within many large organizations (including universities) preparations related to financial planning are prioritized, with limited evidence of consideration for the lifestyle preparations needed. The purpose of this study was to evaluate a series of leisure education-based webinars focused on supporting university employees to engage in lifestyle planning associated with the transition to retirement. In addition to live sessions, a learning management system provided access to discussion boards and resource materials with senior students available to provide individualized assistance. Participants (n = 44 across two implementations) indicated wanting assistance to make retirement fulfilling or rewarding. Participants were very-to-highly satisfied with the sessions, with the most highly valued focused on self-exploration (e.g., considering values, beliefs and strengths to bring into retirement). Participants also valued opportunities to reflect on what aspects of their work life they want to bring with them into retirement, and what they want to leave behind. Although a ‘readiness’ for self-exploration seemed important, opportunities for leisure-related self-reflection and assessment seemed particularly beneficial. Findings are discussed in relation to considering HR departments’ responsibilities to assist university workers to prepare for the retirement transition. Leisure education as a tool for facilitating retirement planning in the university context is warranted. Possibilities for incorporating peer-to-peer education and support—as well as tailored educational sessions—are discussed. Keywords Leisure education Lifestyle planning Retirement Transition University ==== Body pmcIntroduction Retirement is a paradoxical life transition. On one hand, it can be an exciting time in which one looks forward to connecting with family, having more time to do valued activities free from obligations, and to leaving behind stresses of work (Freedman, 2013; Miron et al., 2021). However, it also a major life event that can create upheaval in social connections and people’s sense of self (or identity) (Haslam et al., 2018). Similarly, work life in a university is also paradoxical. University staff and faculty are part of an institution that is often widely recognized and valued within their community. A distributed model of governance within many universities is foundational to fostering a sense of collegiality. Further, working in partnership with diverse unit-level, university- and community ‘partners’ further reinforces the social world (Unruh, 1979) that comprises university life. In fact, it is this social world that Stebbins (2019) and others (e.g., Dorfman, 2009; Rowson & Phillipson, 2020) argue is the most difficult feature to leave behind in retirement from a university position. Alongside this commitment to service, university life is also often associated with freedom and autonomy (e.g., for self-directed tasks like research that reflect personal passions) and social identity that transcends mundane work responsibilities. Thus, working within a university is viewed by sociologist and leisure scholar Robert Stebbins (2019) as “occupational heaven” that is contrasted with an “incongruous lifestyle…[and] lonely, unsettling existence” that follows retirement (p. 447). Although this may not be true for all university employees, retirement from university life is an adjustment with many factors that impact well-being in the transition, such as connections to community, social connections, and identity (Cook, 2015). For organizations, employees delaying retirement can result in negative impacts. Diminishing motivation and increasing ‘disengagement’ can occur amongst older workers (Rau & Adams, 2013), therefore employers have a large stake in facilitating retirement transition processes. Henkens and van Dalen (2013) suggested that “employers are key players in defining the opportunities for retirement” (p. 215) and often maintain an “implicit contract” (p. 216) with their employees regarding the relationship between productivity and earnings. They noted that this is “unsustainable if workers work beyond the age at which the net present value of wages exceeds that of the productivity profile” and further noted that additional factors, such as social security, health care, and pension premiums “make an aging population an even more serious liability” (p. 216). Academics who continue to work beyond their eligible years of retirement affect a university’s ability to attract (and retain) highly productive and skilled younger workers (Henkens & van Dalen, 2013). Assisting individual workers to prepare or plan for a successful transition to retirement is a key responsibility of human resource departments (Rau & Adams, 2013). Many large organizations, including universities, offer informational or educational sessions designed to facilitate planning for the retirement transition, such as offering workshops on government or university pension plans. We could find little evidence of universities caring for the future lifestyles of their workers. The purpose of this study was to evaluate a series of leisure education-based webinars focused on supporting university employees (academics and staff) to engage in lifestyle planning associated with the transitional challenges of retirement. Before presenting the study context we will briefly describe retirement, and review literature about organizations’ interest in retirement, what is known about retiring from an academic position, and propositions regarding the role of leisure education in preparing for the retirement transition shifting focus to what is lost in retirement to what can be gained. Literature Review The Retirement Transition Retirement is viewed as an “expected life course transition in contemporary, industrialized societies” (Wheaton & Crimmins, 2013, p. 22) largely defined and structured by national social security retirement benefits programs (e.g., Canada Pension Plan). At the individual level, retirement is also viewed as a “major life changing event” (Wang, 2013, p. 3), which can have direct impacts on one’s subjective well-being. Retirement typically signifies a discrete ‘ending’ or termination from paid employment, although many people may elect to continue or return to work for the same or a different employer (e.g., bridge employment or contract work). Given mandatory retirement has been abolished in many jurisdictions, this could mean some workers choose to work long after what is considered transitional retirement age of 65. Across the Canadian workforce in general there was a 178% rise from 2012 to 2022 in the number of Canadians aged 65 years or older who are still employed (Statistics Canada, 2022). There are noted similarities for individuals working in a university setting (e.g., Dorfman, 2009). While retirement is influenced in large part by (real or perceived) economic conditions (Wang & Shultz, 2010), many university employees have a relatively secure pension (although staff and faculty unions are typically separate); nonetheless many choose to continue to work long after age 65. According to a report from the Higher Education Quality Council of Ontario, the proportion of professors in Ontario over the age of 65 has grown almost nine percent, with 1,239 working professors older than 65 years old in 2016 (Chiose, 2018). At our own institution approximately 4% of employees (n = 182 staff and faculty) continued to work past the age of 65 in 2021, compared with 2.5% in 2017. With our focus on retirement from a university context, we explore reasons why university-affiliated workers seem reluctant to retire in the following section. Retiring from University Life Efforts were made to identify literature that addressed not only academics’ transition to retirement but other university employees as well. There is a surprising lack of research in the peer-reviewed literature that addresses university staff (non-academic) roles. We were able to find only one study, out of Australia, that examined university employees’ (including academic and non-academic staff) preferences regarding retirement and found most preferred to reduce the number of days worked per week or to continue working. There were significant gender differences however; women were more likely to desire a reduction in workdays per week whereas men were more likely to prefer to continue working as currently or to reduce responsibility. “Economic reasons are more likely to be the barrier for women, while organisational reasons are more likely to be the barrier for men” (Maloney, 2022, p. 93). Most of the literature we found focused on academics’ transition from university life, where one’s professional identity as a recognized ‘expert’ appears to be make the transition to retirement more challenging. We briefly review them below but, taken together, these studies highlight the centrality of professional or social identity associated with the professoriate. Stebbins (2019), a long-time expert in sociological approaches to leisure, lamented the loss of a valued social world associated with university life when one retires from an academic position. Stebbins argued that, associated with this loss of a social world, comes further losses in one’s personal and social identity including the loss of “locally recognized organizational entity, source of livelihood, set of like-minded friends and colleagues” (p. 446) and other “prized features of everyday living” (p. 447). He particularly focused on the features of a university that contribute to creating a vibrant social world and suggested ways that serious leisure pursuits can substitute for this. Ellis and colleagues (Ellis et al., 2017) shared that it was the change in the culture of the university, especially with the shift to computer technology (resulting in increasingly working alone in one’s office), that led to the decision to retire. Most expressed delight at leaving behind never-ending forms of ‘administrivia’ but also sadness at leaving behind a connection to the university and not being sought out as an influential expert. Cahill et al. (2019) conducted a systematic literature search to identify qualitative studies (n = 20 included) focusing on academics’ experiences of retirement. The majority of studies noted how many academics continue to work in retirement, but leave behind aspects of their work they disliked (e.g., teaching and administration). The biggest challenge identified across the studies was the impact on the academics’ identity. As they noted “For many academics, retirement is associated with feelings of fear regarding the potential loss of identity in retirement” (p. e187) including threats to self-worth, self-esteem, feelings of belonging to a professional ‘community.’ The other significant ‘loss’ identified with retirement was the result of changing relationships, again highlighting valuing of the social world of university life. Dorfman (2009) noted that academics share many of the attributes of other highly educated professionals (e.g., engineers) who are employed in occupations characterized by job complexity, high levels of autonomy, and low physical demands. She conducted interviews with faculty (n = 13) at a research-intensive university who elected not to retire after the abolition of mandatory retirement (at 70). Those still working in their 80 s were doing so because they enjoyed it or felt it was important to continue the research they were doing. The reasons given for retiring were changes in students or the ‘culture’ of the unit, faculty or university. As one participant explained “The old collegial climate was changing. It wasn’t unpleasant, it was just different. It just didn’t provide the old excitement…” (in Dorfman, 2009, p. 1019). As it relates to retirement planning, Dorfman (2009) noted that: “Neither employed nor retired professors engaged in substantial planning for retirement; when such planning did occur, it tended to involve financial planning and checking on retirement benefits with the university” (p. 1043). Similar themes were identified by Rowson and Phillipson (2020) who interviewed retiring male professors in Brazil and the United Kingdom. They also found that for many it was difficult to imagine a life outside the university, with their work described as “a passion, or part of a legacy contributing to a better world – whether through teaching or research” (p. 5). While changes in the university culture or senior leadership led many to feeling less connected, a lifetime of developing international networks and recognition also made it difficult to leave. Loss of identity and a social world of universities was also noted in another study of Brazilian professors (n = 11, 8 females; Nascimento & Polia, 2019). The authors found that most they interviewed were struggling to imagine their lives outside of university and added: “Usually, these professors are those who have devoted most of their lives to the university and have developed few activities out of this environment” (p. 394). Within the Brazilian culture Nascimento and Polia noted that retirement is associated with marginalization and perceived uselessness, whereas being a university professor offers prestige. The authors suggested that universities invest in the creation of groups focused specifically on planning for retirement, including focusing specifically on the factors that prevent them from retiring, in order that the benefits of retiring can be perceived as more rewarding than what they are leaving behind. In the next section we review literature on the role of leisure and leisure education in supporting successful retirement transitions. Leisure and Leisure Education as Potential Solutions As identified in the literature reviewed above, there is considerable evidence the transition to retirement (especially for academics), has a significant impact on people’s sense of self or identity and perceptions of loss of belonging to a valued social world (e.g., Cahill et al., 2019; Ellis et al., 2017; Stebbins, 2019). Retirement in particular can pose one of the greatest threats to one’s sense of self, as identity is often closely related to what we do (Unruh, 2004). Although this is likely not the entirety of university employees’ experiences when they retire, we believed that leisure could be part of the ‘solution’ to adapting to changes experienced in this transition to retirement, as there is substantial evidence of the ways leisure serves as a context for meaning-making (Iwasaki et al., 2018), identity development and expression (e.g., Mai & Hao, 2020), and for experiencing a sense of belonging and purpose (e.g., Liechty et al., 2012) in adulthood. In fact, because of its very nature, leisure can be a ‘liberating’ context that, according to Deschenes (2011, as cited in Iwasaki et al., 2018), can free people from the push for productivity. Across both anticipated and unanticipated life transitions (e.g., in health, relationships, residential relocation and retirement) leisure researchers have examined the impacts of these changes on people’s leisure participation but also how people make sense of these changes and adapt or grow from them (e.g., Hutchinson & Kleiber, 2005; Kleiber et al., 2002; Liechty et al., 2012; Nimrod, 2007; Prentice et al., 2022). While transitions can be opportunities for personal growth and exploration, they also give rise to the need to maintain valued self-perceptions and social roles. In the face of changes in adulthood, Kleiber and Nimrod (2008) argued for attending to leisure innovation, which they argued “can be growth promoting and liberating … while at the same time generally protecting a sense of internal continuity” (p. 1), with the potential for fostering personal growth, interest renewal and identity reconstruction. In a study of retirement-aged women Liechty and colleagues (2012) found that leisure innovations contributed to increased feelings of joy, self-confidence, and independence along with improved social connections. Yet, there is other retirement literature that suggests that people, for the most part, continue patterns of activity from work into retirement (Rosenkoetter et al., 2001). Cook (2015) explored how volunteering played a role in supporting retirees in their transition in that they used their career skills and identity in a meaningful and purposeful way while also feeling connected to others and their community. Taken together, this suggests that there is “a need for early planning and programs on use of time activities and management designed to meet individualized needs in preparation for retirement and during postretirement adjustment” (Rosenkoetter et al., 2001, p. 1). To this end, we believed there is value in helping people experiencing the transition to retirement to develop leisure-related knowledge, skills and awareness so they may feel more confident and prepared for the transition to retirement. Leisure education is the ideal tool to do this (Kleiber & Linde, 2014; Kleiber et al., 2012). Leisure education applies theory and evidence about leisure and leisure behaviour to the design of experientially or learning-oriented activities designed to enhance participants’ knowledge, skills, awareness and confidence related to leisure/recreation participation (Dattilo, 2015). Although there have been several recent studies examining the role and value of leisure in retirement (e.g., Earl et al., 2015; Lee et al., 2020; Nimrod, 2007), from what we have found, Kleiber and colleagues (Kleiber & Linde, 2014; Kleiber et al., 2012) are the only leisure scholars to argue for leisure education focused on the retirement transition. Kleiber and Linde (2014) reviewed a wide range of retirement and leisure-related evidence to suggest that leisure education could address the following topics to support successful adjustment in retirement: (1) Examine what is lost in the transition to retirement (e.g., opportunities to express competence) and how leisure can serve as a context for meeting these enduring needs (i.e., to compensate for or replace what is most valued about work); (2) Focus on examining the various ways different forms of leisure can be a ‘restorative resource’ for health and wellbeing, including a focus on addressing social needs. As part of the latter Kleiber and Linde encouraged an examination of the purposeful roles people can assume in retirement, including volunteering. For both of the above goal areas, Kleiber and Linde (2014) suggested that retirement planning programs focus on helping people: (a) deepen self-awareness and clarify values, (b) identify resources or opportunities (e.g., within one’s neighbourhood or community), (c) have the chance to ‘experience’ leisure (e.g., leisure sampling), and (d) engage in problem solving that supports making decisions and commitments. These recommendations align with the needs and challenges identified in other research on leisure and adjustment in the transition to retirement, so were the basis for program development and evaluation. Further description of the leisure education areas of concentration (Dattilo, 2015) used to frame program development are described in the Program Development section that follows. Summary of Purpose As noted above, supporting employees to prepare for the retirement transition is a responsibility of human resources departments within organizations. Yet, to date, their primary response is in the form of pensions or other incentives, with education focusing mostly on financial planning. Therefore, although the retirement transition is a major life event for most individuals, there is limited support for planning for this life change (except for the financial aspects). From the literature reviewed on university professors’ attitudes towards retirement it does appear many struggle with this transition, especially associating it with a loss of social role identity and the social world of university life. Although there are several recommendations to develop holistic and comprehensive educational opportunities that address retirement planning, including planning for what is lost and gained in retirement, we could not find another study that has evaluated a developed retirement lifestyle planning program. Below is an overview of the program, and results from evaluating content/processes and outcomes associated with two iterations of its implementation. Methods Program Context/Overview It was through our university’s HR department that we received a small grant, called the Workplace Wellness Grant, to develop, implement and evaluate the retirement lifestyle planning program that is the subject of this paper. Program development and implementation was completed by the authors and undergraduate students whose responsibilities were to be student coaches. Two members of the team (second author and one student coach) had/were experiencing the transition to retirement which added to the richness of the support provided as they had that extra connection with participants. The aim of the program was to offer a series of interactive workshops focused on different aspects of planning for a fulfilling retirement life, along with access to individualized follow-up ‘coaching.’ Notably, we informed program participants that the program was not designed to address financial planning. Program Development Program development was informed by key journal articles discussing retirement and lifestyle planning (e.g., Froidevaux et al., 2016; Kleiber & Linde, 2014; Moffatt & Heaven, 2017; Osborne, 2012). Content areas were also developed according to leisure education strategies for retirement preparation programs (Kleiber et al., 2012) as well as existing leisure education models. Specifically, Dattilo’s (2015) areas of concentration for leisure education informed the program development. Dattilo suggests that leisure education should be designed in a way to assist participants to: “explore various perspectives, develop an ethic conducive to leisure, and cultivate self-awareness” (p. 65). These recommendations were reflected not only in the overall emphasis of the program on self-reflection and self-assessment, but also in relation to specific topic areas such as ‘re-imaging the self’ and identifying personal values, strengths, and interests (See Table 1). Dattilo also recommends focusing on building participants’ knowledge and/or skills related to goal-setting, decision-making and social connections. Again, these were key foci of the session’s topics. A final aspect of Dattilo’s areas of concentration for leisure education focuses on helping participants to manage challenges; an entire session was devoted to this in the webinar series (Session 5: What might get in the way).Table 1 Program outline Session # and name Objectives (W1Ob1 = Webinar 1, Objective 1) Content Homework Webinar 1: Retirement, Your Transition 1. Understand the importance of planning retirement (W1Ob1) 2. Identify personal goals for their transition into retirement (W1Ob2) 3. Understand the concept of transition and the role it plays in retirement (W1Ob3) Introduction Goal setting Webinar 2: Gains, Losses, and Re-Imagining1 1. Assess work-related gains and losses that will be brought forward into retirement (W2Ob1) 2. Reflect on personal values and beliefs in order to consider who they want to be in retirement (W2Ob2) 3. Identify personal strengths which may help them with their transition into retirement (W2Ob3) Exploring what changes in retirement (e.g., things lost and gained) and personal values, needs, and strengths Identifying personal values, needs, and writing down how they envision their future retirement Webinar 3: Leisure in Retirement 1. Understand the importance of engaging in meaningful leisure in retirement to support health and well-being (W3Ob1) 2. Identify leisure/recreation activities which align with personal values, belief, and strengths from Webinar 2 (W3Ob2) The importance of leisure and planning leisure Completing a leisure inventory and identifying personally meaningful leisure benefits Webinar 4: What Really Matters 1. Reflect on different personally meaningful ways they can give back or leave something behind in retirement (W4Ob1) 2. Develop a short-term plan for daily life in retirement, including meaningful activities related to leisure/recreation, and giving back/leaving something behind (W4Ob2) 3. Develop a long-term plan for retirement including bucket list items, meaningful milestones, and goals (W4Ob3) Time use, finding purpose, and planning Practice in making a long-term plan in relation to goals from homework 1 while incorporating leisure from homework 3 Webinar 5: What Might Stop You 1. Identify potential barriers to meaningful engagement in – or planning for – retirement (W5Ob1) 2. Practice problem solving and discussing strategies for overcoming these barriers and unexpected challenges to engagement or planning (W5Ob2) Barriers and unexpected challenges in retirement Problem solving personal barriers to planning and/or personally meaningful experiences/ Leisure Webinar 6: Resources, Mastery, and Connection2 1. Be aware of the importance of building resources for retirement (W6Ob1) 2. Build confidence and belief in one’s own ability to be in control of their retirement transition (W6Ob2) 3. Understand the importance of connectedness in retirement (W6Ob3) Concept of mastery and importance of connection, along with sharing resources No homework 1First program implementation: Re-Imagining was a separate session 2First program implementation: Connection and a second discussion regarding resources were in a separate session Therefore, the program was developed to include content across several different topics and concepts: goal setting; exploring changes in retirement (e.g., what is gained and lost); personal values, needs, strengths; anticipated experiences in retirement; the importance of leisure and making time for it; time use and the importance of finding purpose; planning both short- and long-term; barriers and unexpected challenges in retirement; mastery; the importance of staying connected; and resources. The program outline, including each session’s title, objectives, content, and homework can be found in Table 1. This table is based on the second implementation of the program, and notes have been made to highlight any major changes from the first implementation. Program Implementation Program delivery and implementation was the same across both implementations. The program was originally designed to be delivered in-person in a ‘lunch & learn’ style series, but the COVID-19 pandemic led to an online delivery format. Webinars took place on the same day of the week and time of the day (e.g., Mondays, 12:00 pm-12:45 pm) with an optional discussion time (15 min) for anyone who wanted to talk with facilitators after the webinar content was delivered. Each webinar started with informal discussion as participants arrived, and formally started with a land acknowledgement, a quick introduction to the day’s topic, and was then followed by a ‘Poll of the Day’ activity where participants were asked a fun question related to the topic (e.g., What is your comfort level with planning? Answers: 1) I’m a planning wizard, 2) I’m pretty good, 3) Average Joe/Joanne, 4) Not great but willing to learn, 5) What’s planning?). Then, each webinar contained educational content for the day’s topic along with one-two activities using the online platform Poll Everywhere (www.polleverywhere.com). For example, a Wordle activity was used in the first webinar asking participants to answer the following question using one-word answers: What does retirement mean to you? Top answers included: “freedom,” “time,” and “relaxation.” Finally, each webinar ended with some form of takeaway message, an explanation of that webinar’s homework, and reminders for the following week. Outside the webinars, student coaches provided program participants one-on-one support and feedback throughout the program through the participant homework and by email. Program Evaluation Unlike research studies, program evaluation does not require ethical approval from the institutional research ethics board. According to the Tri-Council Policy Statement (TCPS2), the agency that governs research ethics in Canada: “[…] program evaluation activities […] do not constitute research for the purposes of this Policy, and do not fall within the scope of REB review” (TCPS2 (2018) – Article 2.5, Chapter 2: Scope and Approach, 2018). Authors confirmed with the host university research ethics board that ethics was not required for this program evaluation. This also means the gathering of evaluation results could not be burdensome to participants and required targeted questions to improve the program rather than questions regarding personal experiences of the program. The program evaluation was developed based on the intended objectives for each session; evaluative questions were to assess the content and processes as well as participant satisfaction for continued feasibility of the program. Program evaluation was mainly comprised of pre-, mid-, and post-program surveys with varying completion rates among participants. The pre-program survey gathered data regarding age, gender, participants’ retirement timeline (e.g., when will you be starting your transition into retirement?), reasons for participating in the program, if participants have done any retirement workshops before and what they were and, finally, Likert scale questions regarding participants’ confidence and preparedness in regards to their transition into retirement. The mid- and post-program surveys asked participants to score their satisfaction with each webinar (webinars 1–3 in mid-program survey, webinars 4–6 in post-program survey). Participants were also asked to score the extent to which each webinar objective was met on a scale of 1 to 5 (1 = not at all, 5 = extremely). The mid- and post-program surveys also collected additional comments from participants. The post-program survey additionally collected information regarding how participants chose to attend the program (live session or watching recordings), their confidence and preparedness for their transition to retirement after having participated in the program, and participant feedback on program delivery (e.g., time/day of webinars, favourite topic, topic that can be improved, missing topics, missing resources). Finally, during each webinar, notes were taken regarding participant attendance, participation in learning activities, and any comments made by participants deemed pertinent by the program facilitators. Analysis Statistical analysis was completed using SPSS v.26. Because the number of participants who completed the evaluation surveys was too low for statistical relevance, descriptive analyses helped to frame or contextualize the qualitative data. Descriptive statistics (e.g., counts, means, percentages) were used for demographic data, reports of confidence and perceived preparedness, as well as achievement and ratings for the webinars and their objectives. As the evaluation is used for program evaluation, qualitative comments were simply summarized and organized by similarity in their contents rather than being analyzed through thematic of content analysis as this is not necessary in program evaluation. Results Participants and Outcomes Demographics of participants who completed program surveys, as well as webinar attendance data can be found in Table 2 below. Although information on gender and anticipated retirement timeline was asked, participants were not asked to disclose their role within the university (e.g., staff or academic, full or part-time) or the Department they were affiliated with. Live attendance was noted by facilitators half-way through each webinar to allow for late arrivals. Recording views were tabulated by the learning management system (Brightspace) through which the program was being delivered.Table 2 Summary demographics First Implementation Second Implementation Average Age 59.5 58.2 Gender • Women • Men • Did not answer 21 (77.8%) 5 (18.5%) 1 (3.7%) 15 (88.2%) 2 (11.8%) Retirement Timeline • Less than 1.5 years • Less than 5 years • More than 5 years • Not clear/Did not answer 7 (25.9%) 14 (51.8%) 5 (18.5%) 1 (3.7%) 6 (35.3%) 11 (64.7%) 0 0 Previously attended retirement workshop • Yes • No 20 (74.1%)a 7(25.9%) 10 (58.8%)a 7 (41.2%) aPrevious retirement planning/webinar/workshops attended by our participants included: Financial (e.g., with financial planners; about the Canadian Pension Plan, Old Age Security, University Pension Plan), and Human Resources-hosted retirement courses/workshops Webinar attendance varied, with a minimum of ten and maximum of 23 live participants, though recordings were available for anyone who could not attend. Participants provided several motivations or reasons for participating in this program. Many participants reported wanting to better prepare for retirement: “To prepare for a fulfilling retirement”; “To start [preparing] for retirement […] so that it is as seamless and easy as possible”; “I want to make sure I am prepared for retirement, not just financially but mentally as well.” Participants also described wanting more information to educate themselves and build their knowledge regarding what to expect in retirement. Finally, some participants had specific motivations related to their well-being, with one participant explaining “I want to see how we can balance still raising kids and retirement”, and another describing exactly what they hoped to learn in this program: “Health and wellness aspects of retirement.” Due to the small sample size and attrition of participants participating in the program surveys, scores of participants’ confidence and preparedness for their transition into retirement are presented in averages. In terms of confidence, the average reported score was a 2.86/5 prior to the start of the program, and a 4.11/5 after the completion of the program. For preparedness, the average reported score before starting the program was a 2.80/5, and a 3.67/5 after the completion of the program. Program Satisfaction In terms of program satisfaction, ratings of each webinar and perceived achievement of webinar learning objectives can be found in Tables 3 and 4, respectively. Webinar ratings were based only on the second implementation due to the changes in course delivery from the first to the second implementation, making it difficult to combine scores. However, learning objectives were reorganized from the first to the second implementation, meaning scores of learning objective achievement of these learning objectives could still be calculated across both implementations.Table 3 Webinar ratings Webinar Ratingsab Webinar 1: Retirement, Your Transition 4.16/5 Webinar 2: Gains, Losses, and Re-Imagining 4.33/5 Webinar 3: Leisure in Retirement 4.16/5 Webinar 4: What Really Matters 4.50/5 Webinar 5: What Might Stop You 3.75/5 Webinar 6: Resources, Mastery, and Connection 4.00/5 aRatings were based on second implementation only because first implementation outline was different and therefore scores cannot be combined bAnchors provided for webinar ratings were based on the question: “How would you rate webinar #?”: 5 = excellent, 1 = poor Table 4 Perceived achievement of webinar learning objectives Webinar Objectives Perceived Achievementab Webinar 1 1. W1Ob1 2. W1Ob2 3. W1Ob3 4.56/5 4.37/5 4.71/5 Webinar 2 1. W2Ob1 2. W2Ob2 3. W2Ob3 4.57/5 4.53/5 4.53/5 Webinar 3 1. W3Ob1 2. W3Ob2 4.45/5 4.45/5 Webinar 4 1. W4Ob1 2. W4Ob2 3. W4Ob3 4.42/5 4.14/5 4.42/5 Webinar 5 1. W5Ob1 2. W5Ob2 3.80/5 3.20/5 Webinar 6 1. W6Ob1 2. W6Ob2 3. W6Ob3 4.16/5 4.33/5 4.5/5 aRatings were based on second implementation only because first implementation outline was different and therefore scores cannot be combined bAnchors provided for learning objective achievement were based on the question: “To what extent do you feel the following learning objectives were reached through this webinar and its associated homework?”: 5 = Excellent, 4 = Good, 3 = Average/Neutral, 2 = Could be better, 1 = Not at all Participants who completed the program surveys also provided additional comments regarding the program as a whole. Complimentary feedback was provided regarding the approachable and thoughtful facilitation of the program. For example, one participant shared: “The retirement planning sessions were excellent—they really gave me some concrete areas that I need to consider in the lead-up to retirement.” Participants also reported enjoying the topics that seemed meaningful to them in terms of the reflective nature of many activities embedded in the program. One participant added:…. Last year, I was essentially afraid of retirement and had no idea how to get my head around it. I started planning, however, and have been dealing with the reality of the exit from the office, [host university] requirements and getting our finances in order. These Webinars were a welcome bonus and have really helped me think more deeply about what retirement may or will look like for me. Finally, many participants mentioned having enjoyed reflecting on, and planning for, the lifestyle component of their retirement transition as it was something they had not considered before. One participant noted:It really got me thinking very hard of goals that are important to me leading into retirement such as health, learning new things, keeping busy, more time with family and friends, leisure time (relaxation) and travel (amongst other things). You got me in the right frame of mind that I believe will get me on the right track leading into my retirement. One common request for any future facilitation of this program would be to have recent University retirees as guest speakers in order to hear from folks who have not only retired recently, but from the same institution. Participants felt this could add value to the information as it would be contextualized by organization-specific experiences. Feedback that could be used for continued program improvement mainly focused on the repetitiveness of topics and the need for more concrete examples and activities. For example, one participant described wanting to create a plan in relation to the resources they have received and how they will explore or use them. As was expected by the program facilitators, participants did occasionally mention wanting information and resources related to financial planning, though the program facilitators reiterated that the focus of the program was on lifestyle as opposed to financial planning. Discussion It is clear from the literature reviewed that many academics struggle to give up their professional role, social connections within their institution, and what it means to be a highly regarded ‘expert’ in their field. In considering the question of how universities can better support their workers in this transition from university life, we believe there is merit in helping employees to both re-imagine their lives and selves in retirement and to consider ways to maintain or rebuild valued social connections and roles. Leisure is clearly central to these processes of both innovation and continuity (self-preservation; Nimrod & Kleiber, 2007) and there is ample evidence of leisure’s role in supporting both adaptations to and personal growth following other life transitions (e.g., Kleiber et al., 2002). Program evaluation findings provide evidence to support Kleiber and Linde’s (2014) recommendations for leisure education as a tool for assisting people to prepare for their transition to retirement. Further, building on Dattilo’s (2015) recommended areas of concentration for leisure education (e.g., developing a leisure ethic, leisure awareness) within a retirement transition context, the retirement webinar program met its intended goals and objectives. Through the retirement leisure education webinars, participants reported increased confidence in feeling better prepared for retirement and enjoyed reflecting on, and planning for, the lifestyle component of their retirement transition that they had not considered before. In addition, the activities incorporated within the webinar sessions are well-aligned with recommendations to incorporate a broader focus on psychosocial and lifestyle factors within retirement planning education (i.e., incorporating social, generative and work-oriented information into retirement seminars, as well as taking inventory of one’s own personal and social resources; (e.g., Nascimento & Polia, 2019; Taylor & Schaffer, 2013; Kleiber & Nimrod, 2008). The content and activities were also aligned with participants’ reports in our retirement webinar series. As noted, participants reported that their motivation or reason for attending this program was to better prepare for life in retirement (e.g., considerations for health and well-being). Therefore, it appears there is value in universities investing in supporting older employees in preparing for the psychosocial and lifestyle factors associated with the retirement transition. University and Human Resources’ Role in Retirement Planning (More than Financial) Recognizing that organizations, including universities, need a plan to gain and retain competitive advantage, human resource (HR) management practices are key (Rau & Adams, 2013). Providing information about aspects of retirement is seen as a key strategy in managing workplace exits within organizations, with pre-retirement financial education a primary focus (e.g., Hershey et al., 2013). Retirement researchers Taylor and Schaffer (2013) argue that nudging or supporting employees in preparing for and deciding to retire necessarily involves education on not only financial planning, but also attending to personal and social needs:Incorporating comprehensive financial, social, generative, and work-oriented information into seminars may be more beneficial than focusing on only one aspect. Even group-oriented seminars can incorporate discussions of financial and social needs, since these are likely to be relevant to a number of individuals involved in planning. (pp. 262-263) And while they and others provide several compelling arguments for attending to the personal and social (in additional to financial) resources and needs (e.g., for social connections, generativity, etc.) individuals bring with them into retirement, we could find no examples of published research on educational programs that addressed these broader lifestyle planning factors. Brown and Jones (2018) indicated that few post-secondary institutions have substantial retirement strategies that help their faculty make the transition to retirement, nor do they provide opportunities to continue to meaningfully engage with the institution post-retirement. The concept of having ongoing education for the transition to retirement as well as opportunity to retain social connections are also common themes from data collected on retirees in higher education (Brown & Jones, 2018). Brown and Jones explored findings from surveys conducted from 2008, 2012, and 2016 by the Association of Retirement Organizations in Higher Education (AROHE) and found programs and activities that related to purposeful work, advocacy, service, teaching and retirement transition education were top priorities for retirees in addition to keeping connected, maintaining university and community relationships, and preserving university history. As a result, we are convinced of the return on investment that a leisure education program on retirement lifestyle planning can provide HR departments within universities; it enables the university to be seen as not only socially responsible but also help their employees more clearly envision a life after/outside the university. This supports Nascimento and Polia’s (2019) recommendations that, to mitigate against a sense of perceived marginalization and uselessness, universities invest in the creation of groups focused specifically on planning for retirement, including focusing specifically on the factors that prevent them from retiring. In the following section we provide reflections on the program evaluation and recommendations for future program implementation. Key Take-ways for Future Program Implementation In reflecting on our retirement webinars for university faculty and staff, we have identified three key learnings for future program implementations. The first learning is in relation to financial planning in retirement. As noted previously, in planning for the webinar sessions, we did not want to focus on the financial piece as many retirement planning sessions already do this. Upon reflection, however, we have come to appreciate that planning one’s lifestyle in retirement does necessarily require considering one’s financial resources. Our dilemma is that we are leisure scholars, not financial experts! In future we would invite a financial planning specialist from the university to discuss financial planning as well as incorporate a ‘leisure budgeting’ exercise where participants are asked to consider the real costs of their current and future leisure-related interests. We anticipate the latter will address fears people often have that they won’t have enough money to sustain their lifestyle in retirement. A second key learning is related to the idea of incorporating ‘peer support’ opportunities within the program. Beyond facilitating opportunities for informal connections through the discussion board on the virtual learning platform used to support the webinars, or within the webinars themselves, we believe peer-assisted learning could be a key factor in better supporting university workers in planning for the retirement transition. Many participants suggested having individuals who have recently retired from the university to come to the webinar to share their experiences of transitioning into retirement. This supports Brown and Jones’ (2018) ideas regarding the role of Retirement Organizations (RO) in universities. ROs, whether developed and organized by senior administrators or entirely by retirees, have shown to have benefits in helping with the retirement transition and provides an opportunity for retirees to maintain social connections with the university community (Brown & Jones, 2018). At our university, we have a ‘Retirees and Pensioners Association’ that primarily supports informal social gatherings and provides information updates related to the university pension. The most successful ROs tend to be ones that are valued and supported by senior administration and funded by the institution and, depending on their structure (e.g., association, emeriti colleges or retiree centres), can have shared benefits for both retirees and current employees (Brown & Jones, 2018). We believe it would be a highly relevant collaboration if our university’s HR department and the ‘Association of Retirees and Pensioners’ teamed up to offer peer-facilitated retirement planning, drawing on the wealth of ‘expertise’ within retirees from our university who have lived through the retirement transition. The final take-away for future research and retirement program implementation is that, in our retirement webinars, we did not consider the job roles and job-related identities that our participants held. Yet, it is clear from the literature of retirement from academia (e.g., Cahill et al., 2019; Dorfman, 2009), the professoriate tends to have a deep, embedded identity associated with their work role making the transition to retirement more challenging. In one of our first sessions of our second webinar implementation, a participant who identified as an academic and researcher inquired if there were specialized content or resources for the professoriate. At first, we were surprised by the inquiry as we had developed the content and resources based on the belief that everyone could benefit no matter of job title. Upon further reflection and deeper dive in the literature on retirement from university life (e.g., Cahill et al., 2019; Ellis et al., 2017; Stebbins, 2019) we have come to believe there may be value to tailor some of the session content to address specific job roles, as well as other barriers related to retirement (e.g., being a caregiver to family in retirement, individuals who live alone or empty nesters in retirement). Post-program implementation we discussed different ways or methods for retirement planning within the university, such as having key sessions that provide everyone with education and skills for retirement planning, and then offering ‘special topics’ in which they can apply the knowledge and skills learned to specific contexts. Limitations and Future Research The evaluation results presented here are limited by number of factors. First, because the program implementation occurred within a University-sponsored retirement lifestyle planning program it could be that the results are positively biased because all the people attended elected to attend on their own non-work time (e.g., lunch hour). Thus, while we anticipate there was some ‘readiness’ to participate in the learning and reflective activities associated with the program, we also think a measure of readiness is required for people to engage deeply with the self-assessment exercises within the program. It would be interesting, in future, to see if similar results would be available from an implementation with an entire administrative/management (e.g., the maintenance staff) or academic unit. Second, as previously noted, evidence of academics’ experiences of the retirement transition largely informed that program we developed (e.g., Cahill et al., 2019; Dorfman, 2009; Ellis et al., 2017; Nascimento & Polia, 2019; Rowson & Phillipson, 2020). Clearly this literature paints a relatively ‘positive’ view of university life and ignores the breadth of a diverse range of employees’ experiences. For example, the literature ties together people’s role (e.g., as an academic) with their place of employment; in reality someone may love the type of work they do as a researcher or educator but feel alienated from their institution (e.g., because of increasing push for ‘commercialization’ or the changes associated with increased focus on issues related to equity or diversity). Further, as noted, evidence within the peer-reviewed literature of non-academics' experiences of their work life was very limited and we anticipate other university staff (e.g., food services, facilities maintenance, or contract research staff) may have quite different experiences of work and retirement than the professoriate. Additional studies are needed that explicitly address the beliefs, expectations and perspectives of non-academic employees and seek diverse perspectives of work in and retirement from universities. Third, related to the above, although there is considerable literature of people’s experiences of the transition to retirement, and the role of leisure in this transition, there were no studies we could find that had previously investigated the use of leisure education to support people in this transition. Kleiber and Linde’s (2014) recommendations for leisure education for the retirement transition, along with Dattilo’s (2015) areas of concentration, served as the primary foundations for program development. Further studies are needed to investigate the use of leisure education to support employees and others in this transition, with a specific focus on identifying the key change factors that are most relevant to helping people prepare for this next life stage. As part of this there would be value to ask participants to identify their key concerns or worries about retiring. Fourth, because this was a program evaluation study, we felt it was important to reduce the burden on program participants related to data collection. In future there would be value in employing measures used by retirement researchers that more rigorously evaluate readiness for the retirement transition or other aspects of social role identity. Further, completion of the program evaluation surveys was not a requirement for program participation; as a result the low number of responses meant that more robust analyses could not be completed. Fifth, an additional limitation of the webinars (and, by extension, the evaluation) is that these implementations occurred at the height of the pandemic when all teaching and meeting shifted to virtual implementation. As a result, it could have been university workers—whether faculty or staff—were experiencing high levels of technology (i.e., virtual meetings and teaching) fatigue. Finally, this evaluation study was limited in providing a ‘snapshot’ of participants’ reactions to and perspectives on this program as the sessions were occurring. In future it would be interesting to conduct a study which followed-up participants post-program to see if the program resulted in any decisions to retire, perhaps sooner than originally planned. Conclusion Retirement in contemporary, industrialized societies is an expected life course transition, and planning has traditionally been focused on finances. However, financial security is not the only barrier that may prevent one from transitioning to retirement; for many there are other losses in departing from their working world such as identity and social connections. Many academics continue to work beyond their eligible years of retirement, and the literature has shown that much of this fear has been associated with potential loss of identity, and loss of ties to their professional community (Cahill et al., 2019; Ellis et al., 2017; Stebbins, 2019). Eisenberg (2006) argues that the first step in planning for retirement should be envisioning the life you want to live in retirement, and then determining what you need in order to live that life. Overall, there does seem to be merit in a University-sponsored retirement planning education program such as the one presented in this paper, which focuses on reflecting deeply on and planning for various aspects of one’s lifestyle in retirement. Clearly there is value for universities to invest in leisure-related retirement planning to not only enact its social responsibility but to assist university workers to imagine and plan for a life outside the university. Hopefully this will dispel some of the hopelessness reflected in Stebbin’s (2019) descriptions of what it means to retire from university life. Exploring ways for ROs to team up with a university’s HR department can provide social opportunities that can help with the transition to retirement and provide a valued role for retirees post-retirement. A university is a pillar of the community; having some type of peer-to-peer learning model in which retirees have opportunity to continue to be involved in this valued community will not only help facilitate the retirement transition of its workers as well as strengthen the sense of community associated with university life. We are very lucky to work for a university whose HR department is dedicated to innovative ways to support its workers’ well-being by offering funding initiatives such as the Workplace Wellness Grant that funded this project. Finally, while we focused on the retirement transition, in reality many people who struggle with work-life balance could benefit from stepping back to assess and reconsider their everyday lifestyle and life priorities. Within the productivity-driven culture of universities, leisure can be a potential antidote to this. Finding ways to help university workers achieve greater work-life balance is certainly warranted as academic and work demands increase (Torp et al., 2018) and leisure education seems to be a relevant tool to address this need. Funding Webinar development was supported by a Workplace Wellness grant from Dalhousie University, Halifax, Nova Scotia. Declarations Conflict of Interest The authors declare that they have no conflict of interest. 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==== Front Curr Radiol Rep Curr Radiol Rep Current Radiology Reports 2167-4825 Springer US New York 405 10.1007/s40134-022-00405-w Geriatrics (giuseppe Guglielmi, Section Editor) Aging-Related Findings of the Respiratory System in Chest Imaging: Pearls and Pitfalls http://orcid.org/0000-0002-1061-9507 Baratella Elisa [email protected] 1 Fiorese Ilaria 1 Minelli Pierluca 1 Veiluva Alberto 1 Marrocchio Cristina 1 Ruaro Barbara 2 Cova Maria Assunta 1 1 grid.5133.4 0000 0001 1941 4308 Department of Radiology, Cattinara Hospital, University of Trieste, 34127 Trieste, Italy 2 grid.5133.4 0000 0001 1941 4308 Department of Pulmonology, Cattinara Hospital, University of Trieste, 34127 Trieste, Italy 1 12 2022 111 19 10 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose of Review The purpose of this review is to describe the main features of the aging chest, studied through different imaging modalities. Recent Findings Aging-related changes of the respiratory system are inevitable. Therefore, it is mandatory to be familiar with the para-physiological changes that occurs, in order to avoid inappropriate interpretation of radiological findings that put patients at risk of over or undertreatment. Summary The role of the radiologist is fundamental in evaluating aging-related processes affecting the respiratory system and in distinguishing them from frank diseases. Keywords Aging chest Chest CT Chest X-ray Digital tomosynthesis Artificial intelligence ==== Body pmcIntroduction Population is aging, with a global life expectancy of around 77.2 years in 2050, as reported by the United Nation Department of Economic and Social Affairs. It is estimated that in 2030 the percentage of population aged 65 years will reach 22% in Europe and Northern America, 19% in Australia/New Zealand, and 16% in Eastern and Southern Asia [1]. Conventionally, an individual older than 65 years of age is defined an elderly [2]. Physiologically, aging involves functional processes and anatomical structures and it is well known that there is a wide spectrum of CT findings that do not represent relevant diseases in elderly asymptomatic patients [3]; however, differentiating between para-physiological changes and pathological features can be particularly challenging in the elderly. Therefore, knowing aging-related changes is mandatory for a correct interpretation of the images. In this review, the main para-physiological changes in the geriatric population will be discussed, with a focus on the possible application of Artificial Intelligence (AI) in this specific area of interest (Fig. 1).Fig. 1 Main alterations of thoracic structures in the geriatric population Main Text Morphologic Changes of Chest Wall and Thoracic Muscles In elderly patients, thorax morphological changes are frequent and need to be known, in order to avoid improper images interpretation. These changes involve the chest wall, thoracic spine, diaphragm, and muscles, with costal cartilage calcification and muscle mass loss representing the most common findings [4]. The chest wall changes in shape and dimension, with an increase of its diameter in the sagittal view and a reduction in the lateral view (Fig. 2A, B).Fig. 2 Chest X-ray in postero-anterior (A) and lateral projection (B) shows typical chest wall morphologic changes in shape and dimension: a reduction in the lateral diameter and an increase in the sagittal diameter. On the lateral view a shortening of the thoracic spine is evident, along with the deformation of vertebral bodies and increasing of dorsal kyphosis Asymmetry of the lungs may be subsequent to the elevation of the hemidiaphragm, frequently observed after cardiothoracic surgery related to a iatrogenic lesion of the phrenic nerve, or to the lowering of the left hemidiaphragm due to the increase in the heart volume [5]. Moreover, an elevation of the right diaphragm is commonly seen in elderly, an alteration that can be related to the anatomical relationship between the lung and the liver (Fig. 3A, B).Fig. 3 On chest X-ray of the elderly, a common finding is the elevation of the left hemidiaphragm related to cardiothoracic surgery (A); on the contrary a lowering of the left hemidiaphragm due to increasing heart volume can be frequently seen as well (B). On sagittal MPR reconstruction, typings of the diaphragm caused by chronic obstructive pulmonary disease should not be misinterpreted as pathological findings (C) Typing of the diaphragm, typically caused by chronic obstructive pulmonary disease, is a frequent imaging finding in the older adult (Fig. 3C). Large hiatal hernia occurs more frequently in older age, particularly in females, due to the weakened or torn phrenoesophageal membrane [6]. Chondro-sternal ossifications frequently occur in elderly, typically centrally distributed in female and peripheric in male. (Fig. 4).Fig. 4 Three-dimensional images of thoracic wall show chondro-sternal ossification, typically located centrally in females (A) and peripherally in males (B) The so-called “senile osteoporosis” is an age-related process not associated with other diseases, more commonly seen in women. Osteoporosis may lead to a height reduction and deformation of vertebral bodies, resulting in an increase of the kyphosis and in a shortening of the spine. A bridging osteophyte in spondylodiscoarthrosis, costal arthrosic hypertrophy, and bony island can simulate parenchymal opacities on Chest X-ray (CXR), representing the most common cause of doubtful nodular lesion. In these cases, the use of Digital Tomosynthesis (DTS) could be useful to solve the problem. Aging determines gradual and inevitable changes of functional and structural features of the musculoskeletal system that leads to a loss of volume, leading to a diffuse decrease in attenuation on the CXR of the elderly patient, consequent to the reduced attenuation of fat tissue against muscles, thus simulating emphysema [4]. In addition, along with the structural musculoskeletal changes, a decrease in contractility and elasticity of muscles occurs. This is physiologically due to the reduction of water content, as well as the increase of connective tissue and lipids. These processes, although their extreme individual variability, depending on genetic, environmental, and lifestyle factors, are significantly reduced in subjects who perform constant physical activity [7]. Several studies have proved the link between sarcopenia and chronic obstructive pulmonary disease (COPD): above all, a systematic review and meta-analysis by Benz et al. showed that the overall presence of sarcopenia in patients with COPD was 21.6%, with a peak of 63% of COPD patients residing in nursing homes [••8]. The combination of sarcopenia and frailty with factors such as impairment of respiratory functions and inactivity are associated with the poorest clinical outcomes [9, 10]. There is a proved vicious circle between sarcopenia and COVID-19: patients with sarcopenia are expected to have increased infection rates and worsen prognoses, as well as COVID-19 itself tends to aggravate sarcopenia because of systemic inflammation and lack of exercise. The assessment of thoracic muscles may be performed with different imaging methods: in case of sarcopenia, lung ultrasound shows thinning of pectoral muscles, with decreased echogenicity and upheaval of the typical fibrillar appearance [11]. Chest CT allows Hounsfield Unit (HU) average calculation for the evaluation of myosteatosis and measurement of area and perimeter of paraspinal and pectoral muscles [12] (Fig. 5). MRI is superior for the assessment of intramuscular fat because of its better contrast between muscles and fatty tissue [13].Fig. 5 Using commercial software, it is possible to quantify the volume and the density of paraspinal muscles, commonly atrophic in elderly patients Morphological Changes of the Airways There is a lack of studies in literature regarding the normal radiological appearance of airways in the elderly [14]. One of the most frequent changes affecting the airways is represented by tracheobronchial cartilage calcifications, which are found in about 40–65% of people aged 60–79 years (Fig. 6). The displacement of the trachea on the right side can be frequently observed, due to the enlargement of the aortic arch.Fig. 6 Chest X-ray of a 74-year-old female shows diffuse tracheobronchial calcifications (A), better visualized on coronal MPR reconstruction (B) Trachea is typically characterized by oval or round shape, non-calcified, with a diameter of 25–27 mm in men and 21–23 mm in women. A tracheal deformity, the so-called “saber-sheath” trachea, is a common finding in the elderlies and is considered a pathognomonic sing of COPD (chronic obstructive pulmonary disease), a feature occurring mostly in men (Fig. 7).Fig. 7 Axial high-resolution CT image in inspiratory phase of a 78-year-old male with a severe COPD shows a saber-sheath trachea characterized by a reduction in coronal diameter with an increase in sagittal diameter (tracheal index < 0.67). Moreover, ossification of tracheal ring can be noticed On CT scan it is possible to detect airway structure alterations, such as bronchial wall thickening and bronchial dilatation [15], whose degree correlates with lung function [16]. In asymptomatic elderlies, bronchial dilatation and bronchial wall thickening related to collagen deposition are frequently seen [3] (Fig. 8). Even if Matsuoka et al. reported a significant relationship between bronchoarterial ratio and aging [17], it is still not clear how to interpret these radiological findings; therefore, particular attention should be paid in diagnosing bronchiectasis in asymptomatic elderly patients. Moreover, Lee et al. demonstrated that air trapping is present in more than 50% of asymptomatic subjects older than 61 years [18].Fig. 8 Axial high-resolution CT shows bronchial wall thickening and mild dilatation in the lower lobes in an asymptomatic 75-year-old female with normal functional pulmonary tests (A). A normal bronchoarterial ratio requires the bronchus and the adjacent pulmonary artery to have the same diameter (D diameter, T thickness of the bronchial wall) (B) COPD is a clinically relevant disease, commonly occurring in the elderlies and affecting about 10% of adults over the age of 40. COPD is characterized by airway inflammation and subsequent remodeling, with emphysema and bronchial alterations being the main pulmonary manifestations. COPD is not limited to the lung and extrapulmonary complications are common, including vascular alterations [15]. Nowadays, spirometry tests alone are not adequate to categorize these heterogeneous group of diseases. Chest X-ray represents the first imaging modality of choice in patients with known or suspected pulmonary disease. On CXR, indirect signs of pulmonary emphysema may be recognized and are represented by an increase in the radiolucency of the lungs, flattened hemidiaphragms, an increase in the retrosternal airspace (normally < 25 mm), and widening of space ribs (Fig. 9). The only direct sign of emphysema detectable on CXR is the presence of bullae [19]. Extrapulmonary alterations, such as cardiac enlargement, vascular and cardiac valve calcifications, may also be recognized on CXR. Even though CXR is not sensible enough to recognize and properly assess the degree of interstitial involvement and airway alterations, it remains the first step to exclude other causes of lung symptoms.Fig. 9 Chest-X-ray in two projections (postero-anterior and latero-lateral) shows a typical example of lung emphysema with pulmonary hyperinflation, flattened hemidiaphragms, and increased lung (A) and retrosternal (B) radiolucency High-resolution CT scan is the gold standard to assess interstitial lung disease. Pulmonary emphysema is the typical smoking-related parenchymal alteration, and, from a pathological point of view, it is defined as the “abnormal permanent enlargement of the airspaces distal to the terminal bronchioles accompanied by destruction of the alveolar wall.” Depending on which part of the acinus is more affected, there are three forms of pulmonary emphysema (centrilobular, paraseptal, and panlobular) that can be easily identified on CT [20] (Fig. 10).Fig. 10 Different types of emphysema: centrilobular, in which the central part of the acinus is predominantly affected (A); paraseptal, in which the distal part of the acinus is predominantly involved (B), and panlobular, in which the whole acinus is affected (C) In elderly patients, para-physiological changes as alveolar dilatation in association with a decrease in vascular structures, the so-called “senile lung,” can simulate an early stage of COPD [5]. It has to be considered that patients with similar spirometry tests may suffer from different COPD phenotypes (more emphysematous type or more small airway disease involvement) and these different entities can be recognized and quantified by using CT [21] (Fig. 11).Fig. 11 Quantitative analysis of airways and pulmonary emphysema can be done with commercial software, in order to quantify the extent of the disease and to differentiate the contribution of small airways and emphysema in COPD (Courtesy of Lucio Calandriello) Moreover, patients suffering from emphysema may benefit from the quantification of the extent of the disease on CT in order to distinguish which ones can benefit from endoscopy treatment or from surgery [22, 23]. Morphologic Changes of Lung Parenchyma Interstitial Lung Diseases Signs of aging include fibrotic changes, such as linear and reticular opacities, which are common findings in the elderly [14, 24]. Copley et al. demonstrated that a basal subpleural reticular pattern not associated with traction bronchiectasis on CT is frequently seen in asymptomatic elderly patients as a normal aging-related finding, independently of the smoking history. In this context, performing a correct differential diagnosis with clinically relevant interstitial lung diseases or interstitial lung abnormalities (ILA) could be particularly challenging [3, 25]. A focal area of ground glass opacity close to dorsal column osteophytosis is a common imaging finding in elderly patients and it represents an area of dysventilation that should not to be confused with a pathological finding (Fig. 12).Fig. 12 Axial CT scan shows a focal ground glass opacity (arrow) close to an osteophyte of the thoracic spine, representing a focal area of atelectasis Idiopathic pulmonary fibrosis is a condition related to smoking habit and to pulmonary senescence, rare in individuals younger than 65 years [26]. In order to differentiate age-related interstitial changes from the onset of interstitial lung disease, it is mandatory to evaluate the extension of the interstitial alterations and the longitudinal behavior of the disease and correlate these findings with clinical features. The presence of honeycombing and extensive traction bronchiectasis are suggestive for fibrotic disease rather than age-related changes (Fig. 13) [••27].Fig. 13 HRCT demonstrates subpleural basal reticulations in a never-smoker 78-year-old man (arrows) (A). Axial image in a 67-year-old man with a diagnosis of idiopathic pulmonary fibrosis shows diffuse irregular septal thickening, traction bronchiectasis, and honeycombing (B) Lung Nodules One of the most challenging tasks in thoracic imaging is the detection and characterization of lung nodules, especially on CXR, because of the poor conspicuity of lesions and the overlapping anatomical structures. In the past decades, oblique X-ray projections were usually employed to solve doubtful or equivocal cases, while in recent years DTS has demonstrated to be a useful technique to improve chest lesion diagnosis [28–30] (Fig. 14).Fig. 14 Chest X-ray shows a doubtful nodule in the right lung (arrow). DTS demonstrates that this opacity is due to a bony island of sclerosis in the costal arch (enostosis) (B). Enostosis is a typical imaging finding which can mimic a pulmonary opacity in the elderly A small solid nodule can be detected on the chest CT of the majority of elderly patients and, due to the increasing numbers of imaging exams, its characterization has become a daily clinical practice problem [5]. There are some specific radiological findings which indicate the probable benign nature of a nodule, such as fat components or pop-corn calcifications. However, the majority of incidentally detected lung nodules on CT scan remain of uncertain nature and need a follow-up CT, according to the Fleischner recommendations for the managing of lung nodules [31]. It is interesting that these guidelines require the quantification of the nodule’s diameter, but also of its volume to classify the risk of malignancy. In addition, considering that a doubling time of more than 500 days for solid nodules has a 98% negative predictive value of malignancy, it is mandatory for radiologists to calculate, as precisely as possible, the volume of the lung nodule [32]. Nevertheless, the limits of manual measurements are well known. In this scenario, artificial intelligence systems, and in particular computer-aided diagnosis (CAD), could be helpful for detection (CADe) and characterization (CADx) of lung nodules on both CXR and CT scan [•33]. A large number of different commercial software can be used to calculate volume and doubling time of nodules to evaluate the probability of malignant growth (Fig. 15).Fig. 15 A baseline CT scan shows an incidental lung nodule in the right apex (A); 13 months later a follow-up CT scan was performed (B). By applying a CAD system, the diameter, the volume, and the doubling time can be calculated In specific cases, lung biopsy or surgery need to be performed to characterize the nature of a lung nodule; however, elderly patients are at higher risk to develop both minor and major complications when undergoing this kind of procedures. Thus, it emerges the need for non-invasive systems to estimate the pre-test probability of malignancy. A recent application of AI systems in characterizing lung nodules is represented by radiomics, a procedure that quantitatively analyzes imaging features to provide information regarding the nature of the nodule, but also to predict the mutational profile of the tumor and to assess the response to therapy or the tumor aggressiveness [34]. Pleural Diseases Pleural Effusion Pleural effusion is widely more frequent in elderly patients, since pathological conditions causing an imbalance between fluid production and absorption, like cardiopulmonary disorders, malignant tumors, cirrhosis, inflammatory diseases, or pulmonary fibrosis, strongly develop from older age. On CXR in standing position, pleural effusion manifests with blunting of the costophrenic and/or the cardiophrenic angle, sometimes with a meniscus and with contralateral mediastinal shift when large amount of fluid is present. However, on account of the fragility of elderly patients, CXR is frequently performed in supine position and pleural effusion may manifests with a reduction of parenchymal transparency only. Therefore, in the supine antero-posterior view, when increased opacity of a hemithorax without obscuration of vascular markings is seen, pleural effusion should be suspected. In addition, adhesions between visceral and parietal pleura in elderly patients can lead to loculated effusion that can simulate a mass [35, 36]. CT is more sensible than x-ray to identify pleural effusion, even with low amount of fluid. CT is useful in recognizing acute hemorrhage, based on its HU values and on the presence of fluid–fluid levels, in differentiating between pleural effusion and empyema and in identifying the underlying causes of pleural effusion (like malignant tumors). Malignant effusions are usually greater than 500 mL and they often represent the first evidence of malignancy. For lung, breast, and ovarian metastases, 92% of pleural effusions are ipsilateral to the primary lesion. These lesions spread via hematogenous dissemination, by pleural invasion (as in T3 bronchogenic cancers), or by direct pleural seeding [35, 37]. Pleural Plaques Pleural plaques are the main and most frequent benign pleural pathology consequent to asbestos exposure, definitely more frequent in elderly subjects, as long as that they manifest after 15–20 years at least from initial exposure. They are 1–10 mm thick (thicker when developing adjacent to ribs) and predominantly develop in the chest wall, usually in the anterior upper region, or in the diaphragmatic pleura (sparing the costophrenic recesses), while the mediastinal region is rarely affected [35] (Fig. 16).Fig. 16 Axial CT image shows bilateral pleural plaques in a 76-year-old male with previous asbestos exposure (A). Three-dimensional reconstructions show the typical distribution of the pleural plaques (B, C) On CXR, pleural plaques present as focal pleural thickening with a well-defined inner margin, usually bilateral and asymmetrical. Typically, they involve posterolateral regions, along the costal margins and the diaphragm. They are frequently calcified, with punctate, linear, or “cake-like” calcifications. The main differential diagnoses are rib fractures, extra-pleural fat, and pleural tumors, mainly mesothelioma. On CXR, mesothelioma presents with pleural plaques, volume loss of the hemithorax involved, ipsilateral shift of the mediastinum, lymphadenopathy, and pleural effusion [35]. CT allows a better detection and location of pleural plaques, whether they are calcified or not. CT permits to differentiate between pleural plaques and subpleural fat deposits (typically bilateral and symmetrical), between benign pleural plaques and mesothelioma, presenting on CT as a soft tissue attenuation pleural mass or nodulation that penetrates the interlobar fissures and completely encases the lung in advanced stages. CT identifies also mesothelioma spread within the pleura and the local invasion of the chest wall, of the diaphragm and of the lymph nodes [35, 37]. Late Sequelae of Tuberculosis Nowadays, tuberculosis (TB) is still a worrying cause of death worldwide and it represents a pathology of special interest in the geriatric population; indeed, the elderlies are at higher risk for pulmonary infections, for reactivation of a latent TB (tuberculosis) and for acquisition of a new TB infection [38]. The availability of antibiotics, after the Second World War, has dramatically changed the treatment and prognosis of patients suffering of TB. Therefore, late sequelae of an old TB infection or surgical TB treatment, the last no more in use, are typically seen in the elderlies and are represented by fibrothorax, extra-pleural artificial pneumothorax, and thoracoplasty. Fibrothorax is defined as fibrosis within the pleural space, occurring after the inflammatory response to a series of pathological conditions, as tuberculosis, thoracic empyema, asbestos-related pleural disease, rheumatoid arthritis, and hemothorax. Fibrothorax is characterized by pleural thickening, which may be calcified, and volume loss of the affected hemithorax (Fig. 17A). The mediastinal pleura is usually spared [39].Fig. 17 Chest X-ray shows diffuse calcified pleural plaques on the right hemithorax due to the presence of extensive and coarse unilateral calcified pleural thickening (fibrothorax) (A). Chest X-ray shows an extensive opacity of the right upper lung with smooth internal margins and an obtuse angle with the lateral chest wall due to the sequelae of an extra-pleural artificial pneumothorax (B). Chest X-ray of an 83-year-old female shows multiple ribs resection following surgical treatment of TB (thoracoplasty) (C) Extrapleural artificial pneumothorax was a procedure for the treatment of TB, consisting in the introduction of air into the pleural cavity or between the coverings of the lung, that lead to the collapse of the diseased area and to a complete recovery (Fig. 17B). This procedure was first performed by Tuffier in 1891. Before the use of air to fill the created space, many different substances were used, including paraffin packs; however, these substances could erode the pulmonary tissue, or the chest wall and skin, making this procedure, at least, not a very satisfactory form of treatment [40]. This method is not used anymore since 1960 in Italy. Thoracoplasty is a surgical procedure that was originally designed to permanently collapse the cavities of pulmonary tuberculosis. It consists in the surgical removal of rib bones from the chest wall (generally 2–3 ribs), resection of the parietal pleura, periosteum, and intercostal muscles (Fig. 17C). Until supplanted by effective chemotherapy, it was one of several methods used to put the lung to rest, in order to inactivate the disease (41). Conclusions Diagnostic imaging has an important role in recognizing para-physiological changes of the respiratory system and to differentiate them from pathological conditions. In an increasingly older population, Radiologists have to become familiar with chest imaging findings physiologically related to aging, in order to reduce misinterpretations. Declarations Conflict of interest For all authors none were declared. Ethical Approval This article does not contain any studies with human or animal subjects performed by any of the authors. 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==== Front Biol Psychiatry Glob Open Sci Biol Psychiatry Glob Open Sci Biological Psychiatry Global Open Science 2667-1743 Published by Elsevier Inc. on behalf of Society of Biological Psychiatry. S2667-1743(22)00142-2 10.1016/j.bpsgos.2022.11.002 Archival Report Effects of the COVID-19 Pandemic on Mental Health and Brain Maturation in Adolescents: Implications for Analyzing Longitudinal Data Gotlib Ian H. 1∗ Miller Jonas G. 1 Borchers Lauren R. 1 Coury Sache M. 1 Costello Lauren A. 1 Garcia Jordan M. 1 Ho Tiffany C. 2 1 Department of Psychology, Stanford University 2 Department of Psychiatry and Weill Institute for Neurosciences, University of California, San Francisco ∗ Corresponding author: Ian H. Gotlib 1 12 2022 1 12 2022 8 8 2022 5 11 2022 7 11 2022 © 2022 Published by Elsevier Inc. on behalf of Society of Biological Psychiatry. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Background The COVID-19 pandemic has caused significant stress and disruption for young people, likely leading to alterations in their mental health and neurodevelopment. In this context, it is not clear whether youth who lived through the pandemic and its shutdowns are comparable psychobiologically to their age- and sex-matched peers assessed before the pandemic. This question is particularly important for researchers who are analyzing longitudinal data that span the pandemic. Methods In this study we compared carefully matched youth assessed before the pandemic (n=81) and after the pandemic-related shutdowns ended (n=82). Results We found that youth assessed after the pandemic shutdowns had more severe internalizing mental health problems, reduced cortical thickness, larger hippocampal and amygdala volume, and more advanced brain age. Conclusions Thus, not only does the COVID-19 pandemic appear to have led to poorer mental health and accelerated brain aging in adolescents, but it also poses significant challenges to researchers analyzing data from longitudinal studies of normative development that were interrupted by the pandemic. Keywords COVID-19 adolescent neurodevelopment youth mental health brain age analyzing longitudinal data ==== Body pmcThe COVID-19 pandemic has been a generation-defining event and a major source of adversity. Given the shelter-in-place orders in the Spring of 2020 that led to school closures, academic disruptions, social restrictions, and reduced access to school-based mental health services (1), the pandemic appears to have been particularly difficult for children and adolescents (2, 3, 4). In fact, a recent meta-analysis found that the prevalence of internalizing symptoms in youth has doubled since the onset of the COVID-19 pandemic (5). Despite this alarming statistic, however, the potential implications of the pandemic for children’s neurodevelopment have not been delineated. Research conducted prior to the pandemic has found that exposure to early life adversity, including violence, neglect, and family dysfunction, is associated not only with poorer mental health, but also with maladaptive neurodevelopmental outcomes that indicate accelerated brain maturation or aging (6). For example, cortical thickness, which decreases with age (7), is further reduced in youth with a history of early adversity (6). Recently, researchers have used machine learning algorithms to predict individuals’ ages from their neuroanatomical features (8). In adolescents, exposure to adversity has been associated with a brain age gap estimate (BrainAGE) suggestive of accelerated aging (i.e., having a predicted brain age older than one’s chronological age) (9). As a result of social isolation and distancing during the shut-down, virtually all youth experienced adversity in the form of significant departures from their normal routines. In addition, financial strain, threats to physical health, and exposure to increased familial violence were alarmingly common during the pandemic (10, 11). If the pandemic has adversely affected adolescents’ mental health and neurodevelopment, such that adolescents who are assessed now differ in significant respects from their age-and sex-matched peers who were assessed prior to the pandemic, researchers must give serious consideration to how they accurately analyze and interpret longitudinal developmental data that span years on both sides of this extraordinary event. In this study we matched a group of adolescents who experienced the pandemic shut-down (the “peri-COVID” group) with a group of adolescents, matched on age, sex, puberty, exposure to early life stress, and socioeconomic status, who underwent the same assessment before the pandemic (the “pre-COVID” group). We expected that compared with the pre-COVID group, the peri-COVID group would report more severe mental health problems and have older, or more mature, brains. Methods Participants Participants in this study were 163 adolescents (103 females) living in the San Francisco Bay Area who were participating in a larger longitudinal study assessing the effects of early life stress on psychobiology across puberty (N=214) (12, 13, 14). Exclusion criteria were post-pubertal status, non-fluency in English, inability to undergo magnetic resonance imaging, and history of neurological disorder or major medical illness. Participants were invited to return for follow-up assessments approximately every two years; however, the approximately one-year-long COVID-19 pandemic shut-down beginning in March 2020 interrupted participants’ in-person assessments (see 15 for more details). All participants and their legal guardians gave informed assent and consent, respectively, and were compensated for their time. All study procedures were approved by the Stanford University Institutional Review Board. From this larger cohort, we constructed two matched subsamples using data collected either before the pandemic (from November 2016 to November 2019; “pre-COVID group,” n=81) or during the pandemic but following the end of the Bay Area shutdown (from October 2020 to March 2022; “peri-COVID group,” n=82). We constructed these subsamples to maximize group sizes and to match the two groups on sex, age, pubertal status, race/ethnicity, parental education, annual household income, and severity of early life stress based on panel ratings of participants’ responses to interview (12, 13). Specifically, we attempted to match the peri-COVID participants with pre-COVID participants with respect to age and sex as closely as possible at the group level. Not all peri-COVID participants could be matched to pre-COVID participants given their older age, and not all pre-COVID participants were needed to be matched to the smaller peri-COVID group (and the youngest pre-COVID participants were too young to be matched to the peri-COVID participants). For the “mental health symptoms” sample of 81 pre-COVID and 82 peri-COVID participants (see below), we excluded from analyses 50 pre-COVID and 12 peri-COVID participants who could not be appropriately matched. For the “neuroimaging” sample, we were able to age- and sex-match 64 of the 104 participants who were scanned peri-COVID with 64 pre-COVID participants. Mental Health Symptoms Participants self-reported their depressive symptoms using the 10-item version of the Children’s Depression Inventory (16). This widely used reliable measure (17) has been shown to have convergent validity with clinician ratings of depression symptoms and diagnosis (18). We assessed anxiety symptoms using total score of the Social Anxiety and Physical Symptom subscales of the Multidimensional Anxiety Scale for Children (MASC; 19). The full MASC assesses a wide range of anxiety symptoms, including those that are not as relevant for the age range of our participants (e.g., Separation Anxiety). For this reason and to reduce participant burden, we administered only the Social Anxiety and Physical Symptoms subscales of the MASC for this study; therefore, the MASC total score in this study reflects the sum of these two subscales. Finally, we assessed internalizing and externalizing symptoms using the validated subscales of the Youth Self Report version of the Child Behavior Checklist (20). Neuroimaging A subset of these participants (matched n=64 per group) completed a T1-weighted magnetic resonance imaging (MRI) scan at the Center for Cognitive and Neurobiological Imaging at Stanford University. All participants in the pre-COVID group completed their scans using a 3T Discovery MR750 (GE Medical Systems, Milwaukee, WI, USA). As of 03/16/2020, the Discovery MR750 was upgraded to an Ultra High Performance (UHP) system. Thus, all peri-COVID participants were scanned on the upgraded scanner. Participants in both groups were scanned using a 32-channel head coil (Nova Medical, Wilmington, MA, USA). Prior work suggests that FreeSurfer-based cortical thickness and subcortical measures are highly reliable across scanner upgrades (21, 22, 23). For example, Han et al. (22) did not find evidence that scanner upgrades introduce bias for cortical thickness measures, and Brown et al. (23) found that hippocampal measures are reliable across scanners. In addition, we conducted analyses with our own data to assess potential differences in T1-weighted image quality related to scanner upgrade. Specifically, in a subset of 31 participants with imaging data before and after the scanner upgrade, we tested within-participant changes in gray-white matter contrast-to-noise ratio (CNR) using FreeSurfer’s mri_cnr quality metric command. We did not find significant differences in CNR from pre- to post-upgrade in either the left (t(30)=0.81, p=.425) or the right hemisphere (t(30)=0.66, p=.513). Thus, the scanner upgrade does not appear to have introduced a systematic bias in image quality. Whole-brain T1-weighted images were collected for all participants using the following spoiled gradient echo (SPGR) pulse sequence: 186 sagittal slices; TR (repetition time)/TE (echo time)/TI (inversion time)=6.24/2.34/450ms; flip angle=12°; voxel size = 0.9 mm×0.9 mm×0.9 mm; scan duration=315s. The SPGR sequence was repeated up to two additional times if the first acquisition did not yield clear images. For each participant with multiple acquisitions, the single SPGR image with the clearest structural boundaries (i.e., that was free from motion or other artifacts) was used for further analysis. Segmentation of Cortical and Subcortical Regions We used FreeSurfer v. 6.0 (http://surfer.nmr.mgh.harvard.edu/) recon-all function to automatically skull strip and segment cortical and subcortical volumes from the T1-weighted structural images (24), which has been shown to have acceptable scan-rescan reliability (21) and comparable accuracy to manual labeling techniques (24, 25, 26). We implemented structural image processing protocols established by ENIGMA (Enhancing Neuro Imaging Genetics through Meta Analysis) (http://enigma.ini.usc.edu/protocols/imaging-protocols) to extract and perform quality assurance checks on the cortical thickness and subcortical volume estimates from the FreeSurfer outputs. Using FreeView image viewer, all cortical and subcortical outputs were visually inspected to quality check for processing and segmentation errors. As previously described (27, 28), we converted gray matter volumes from each hemisphere into z-scores; volumes with z-scores greater than 2.5 or less than -2.5 were visually examined again for accuracy and any segmentations that failed any of these steps were removed from final analyses. We focused on mean cortical thickness (average of cortical thickness values across individual regions as defined by the Desikan-Killany atlas) (29) and unstandardized residuals of subcortical volumes regressed on total intracranial volume. Brain Age Gap Estimates Based on cortical and subcortical features, we computed BrainAGE (brain age gap estimate) values for male and female participants using sex-specific machine learning-based models developed by the ENIGMA-Brain Age working group (30). These models use data from 14 subcortical gray matter volumes, 2 lateral ventricles, 68 cortical thickness measures, 68 surface area measures, and total intracranial volume to predict chronological age (i.e., predicted brain age). We computed brain age gap estimates by subtracting chronological age from predicted brain age. Given that BrainAGE values are often overestimated in younger individuals and underestimated in older individuals, Le et al. (31) proposed adjusting for chronological age in analyses of BrainAGE. Therefore, we regressed gap estimates onto chronological age and used the unstandardized residuals as the BrainAGE outcome variable in our statistical analyses. Statistical Approach All statistical analyses were conducted using R v. 4.0.2. To examine whether adolescents who experienced the pandemic differed from their pre-pandemic peers, we conducted between-group tests on measures of internalizing and externalizing symptoms, cortical thickness, subcortical volume (regions of interest: the bilateral amygdala, hippocampus, and nucleus accumbens). For analyses of mental health problems, we first conducted a one-way multiple analysis of variance (MANOVA) test to examine whether there were group differences in overall mental health scores across measures. We conducted follow-up independent sample t-tests to examine whether the pre-COVID and peri-COVID groups differed in specific aspects of mental health as assessed by different measures. We repeated these steps for analyses of brain metrics. Given our expectations based on recent work suggesting that mental health problems have increased during the pandemic (5), we used one-tailed hypothesis tests for follow-up analyses of mental health outcomes. We used two-tailed hypothesis tests for follow-up analyses of brain outcomes. All participants and their parent(s)/legal guardian(s) signed assent and consent forms, respectively, and were compensated for their participation in the study. This study was approved by the Stanford University Institutional Review Board. Results Participant Characteristics The demographic and clinical characteristics of the pre- and peri-COVID subgroups of participants are presented in Table 1 . Participants’ parents reported on their annual household income, from which we computed an income-to-needs ratio by dividing the midpoint of their reported income bin by the low-income value for Santa Clara County. Importantly, this calculation considers the number of people in the home and the time period in which the study occurred (https://www.huduser.gov/portal/datasets/il/il2017/2017summary.odn; (32). Attesting to the success of our careful matching procedure, there were no significant group differences in participant characteristics between the pre-COVID and peri-COVID subgroups for either the “mental health” or the “brain” samples (all individual ps>.06).Table 1 Participant characteristics. Variable Pre-COVID “Mental Health” (n=81) M (SD) or n Peri-COVID “Mental Health” (n=82) M (SD) or n Pre-COVID “Brain” (n=64) M (SD) or n Peri-COVID “Brain” (n=64) M (SD) or n Sex (female) 51 (63%) 52 (63%) 34 (53%) 34 (53%) Age 15.87 (1.14) 16.17 (0.93) 16.08 (0.90) 16.43 (1.19) Race White 43 (53%) 33 (40%) 35 (55%) 26 (41%) Asian / Asian American 13 (16%) 11 (13%) 7 (11%) 9 (14%) Hispanic / Latin-X 5 (6%) 7 (9%) 4 (6%) 6 (9%) Black / African American 6 (7%) 7 (9%) 3 (5%) 7 (11%) Biracial 9 (11%) 21 (26%) 11 (17%) 12 (19%) Other race 5 (6%) 3 (4%) 4 (6%) 4 (6%) Income-to-needs ratio 1.37 (0.53) 1.27 (0.54) 1.30 (0.59 1.31 (0.53) Early Life Stress 6.60 (4.89) 6.31 (4.97) 7.30 (5.88) 5.84 (4.68) Parental Education No GED/No High School Diploma 0 (0%) 1 (1%) 0 (0%) 1 (%) GED/High School Diploma 0 (0%) 4 (5%) 0 (0%) 0 (0%) Some College 10 (12%) 15 (18%) 4 (6%) 10 (%) 2-year College Degree 5 (6%) 7 (9%) 5 (8%) 5 (%) 4-year College Degree 29 (36%) 25 (30%) 24 (38%) 27 (%) Master’s Degree 30 (37%) 20 (24%) 20 (31%) 17 (%) Professional Degree 1 (1%) 5 (6%) 3 (5%) 3 (%) Doctorate 4 (5%) 2 (2%) 2 (3%) 0 (0%) Not reported 2 (2%) 3 (4%) 6 (9%) 1 (%) COVID-19 Impact Individual Diagnosis n/a 1 (1%) n/a 1 (1%) Household Diagnosis n/a 3 (4%) n/a 2 (3%) Financial Strain n/a 13 (16%) n/a 11 (17%) Job Loss n/a 7 (9%) n/a 8 (13%) Note. “Mental Health” refers to the subsample of participants who completed the measures of mental health; “Brain” refers to the subsample of these participants who also successfully completed the neuroimaging protocol. Mental Health Group differences on the mental health measures are presented in Fig. 1 . A one-way multivariate analysis of variance (MANOVA) indicated that the pre- (n=81) and peri-COVID (n=82) groups differed significantly in their self-reported mental health difficulties (F(4,158)=2.67, p=.034). Follow-up t-tests showed that the peri-COVID group reported more severe symptoms of anxiety (t(161)=3.15, p<.001; Cohen’s d=0.49), depression (t(161)=1.92, p=.029; d=0.30), and internalizing problems (t(161)=1.77, p=.039; d=0.28); the two groups did not differ in externalizing problems (t(161)=1.25, p=.108).Figure 1 Group differences on the Children’s Depression Inventory (CDI), Multidimensional Anxiety Scale for Children (MASC; sum of the Social Anxiety and Physical Symptom subscales), and Youth Self-Report (YSR) internalizing and externalizing. p<.05, p<.01, ∗∗∗p<.001. Brain Metrics Group differences in cortical thickness, subcortical volumes, and BrainAGE are shown in Fig. 2 . A MANOVA conducted on all of the brain metrics yielded a significant difference between the pre-COVID (n=61-64) and peri-COVID (n=63-64) groups (F(5,116)=7.13, p<.001). Follow-up tests indicated that the peri-COVID group had reduced bilateral cortical thickness (t(122)=3.67, p<.001; d=0.66) and, controlling for intracranial volume, larger bilateral hippocampal volume (t(125)=3.56, p<.001; d=0.63) and bilateral amygdala volume (t(125)=2.01, p=.047; d=0.36); the two groups did not differ in bilateral nucleus accumbens volume (t(125)=0.68, p=.248; d=0.12). Finally, despite the fact that the two groups were matched on age and other relevant demographic characteristics, adolescents in the peri-COVID group had an older BrainAGE than did their peers who were assessed before the pandemic (t(125)=2.31, p=.022; d=0.41).Figure 2 Raw data are plotted for visualization. Significance levels are based on group differences on subcortical volumes (in mm) adjusted for intracranial volume, cortical thickness, and on BrainAGE adjusted for chronological age. p<.05, p<.01., *p<.001. Interval Between the COVID-19 Shutdown and the peri-COVID Assessments Finally, given the possibility that participants’ mental health difficulties and their brain metrics increased with the duration of the pandemic, we examined our clinical functioning and brain metrics as a function of time since the Bay Area shelter-in-place orders were initiated (March 17, 2020). The peri-COVID participants completed measures of clinical functioning between 01/10/21 and 09/30/2021 and MRI scans between 10/13/2020 and 03/22/2022. Within the peri-COVID group, we examined associations between the number of days from the start of shelter-in-place orders to the dates that participants completed measures of psychopathology (M=346.49 days, SD=131.70 days, range=133-720 days). There were no significant associations between this interval and participants’ scores on the measures of depression (r(80)=0.01, p=.901), anxiety (r(80)=-0.06, p=.544), internalizing symptoms (r(80)=0.07, p=.506), or externalizing symptoms (r(80)=0.00, p=.980). We repeated these analyses for the brain metrics (mean interval = 379.00 days, SD=119.24 days, range=210-735 days). Again, there were no significant associations between the interval and residuals of amygdala volume (r(62)=0.01, p=.935), hippocampal volume (r(62)=0.15, p=.245), NAcc volume (r(62)=0.05, p=.681), mean cortical thickness (r(61)=0.04, p=.303), or residuals of brainAGE (r(62)=-0.09, p=.459). Discussion In addition to replicating prior findings that the pandemic has adversely affected the mental health of young people (5), we found that adolescents assessed during the pandemic have neuroanatomical features that are more typical of individuals who are older or who experienced significant adversity in childhood. Compared to carefully matched peers assessed before the pandemic, adolescents assessed during the pandemic showed signs of advanced cortical thinning and had larger bilateral hippocampal and amygdala volumes. Given that volume in these structures typically increases over adolescence (33), these neural alterations may reflect accelerated brain maturation in the context of the pandemic. Indeed, adolescents assessed during the pandemic also had larger positive brain age gap estimates, indicative of older-appearing brains. It appears, therefore, that the pandemic not only has adversely affected adolescents’ mental health, but also has accelerated their brain maturation. These findings have critical implications for researchers who are conducting longitudinal studies that were interrupted due to pandemic-related shutdowns. In our own longitudinal study, we had been assessing a sample of approximately 200 adolescents at each of four timepoints, at two-year intervals, to examine the effects of early adversity on trajectories of neurodevelopment and clinical symptoms. At the time of the shutdown, we were two-thirds of the way through the third assessment, when our participants were 13-17 years of age. We had originally planned to simply use participants’ age in analyzing trajectories from our four timepoints of data. Although some participants would have had a longer interval than others between assessments that bracketed the shut-down, we would control statistically for those differences. It is important to recognize that this analytic approach assumes that, for example, 16-year-olds who were assessed after the shutdown ended are equivalent in their clinical functioning and neurodevelopment to 16-year-olds who were assessed before the pandemic, and would simply be grouped together. Our results suggest that this assumption is not correct. Rather, the pandemic appears to have altered adolescent mental health and neurodevelopment, at least in the short term, which will present a challenge for researchers in analyzing longitudinal data from studies of normative development that were interrupted by the pandemic. In order to not confound age-related changes in brain maturation with experiences and consequences of the COVID-19 pandemic, some researchers, including our group, have used a dummy-coded variable to control statistically for whether participants were assessed pre- or during the pandemic (e.g., 34). Nevertheless, restrictions around COVID-19 are constantly changing; therefore, additional measures may need to be used as covariates, including the interval between shelter-in-place orders and time of assessment, as well as the nature and severity of the individual’s stress and experience during the pandemic (e.g., COVID-19 infection, upheaval in living situation, financial strain, etc.). We should note that our sample is of relatively high socioeconomic status and represents the racial/ethnic composition of the San Francisco Bay Area. Researchers have reported that sample composition influences age-related effects on brain structure (35) and, more specifically, that the psychosocial and health consequences of the pandemic have been more severe among individuals from socially marginalized groups (e.g., lower socioeconomic status; 36–38). Therefore, it is important that investigators examine the effects of the COVID-19 pandemic on psychopathology and brain metrics in more diverse samples of adolescents that are representative of the broader population. Another critical task for future research is to determine whether these alterations are temporary effects of the pandemic or stable changes that will characterize the current generation of youth. If these changes are found to be enduring, accounting for and interpreting data acquired during this period will require additional attention and consideration. For example, as more researchers publish data concerning normative developmental trajectories of MRI-derived anatomical features (e.g., 39), it will be possible to compare COVID-impacted neurodevelopmental trajectories with normative trajectories and, indeed, to compute COVID-adjusted metrics of brain maturation. Regardless, however, we emphasize that it is important that we continue to follow and assess individuals who were recruited and assessed prior to the pandemic; this type of research offers the strongest possibility for us to examine the effects of a major stressor experienced on a global scale. Uncited reference 15., 19., 34., 36., 37., 38., 39.. Financial Disclosures All authors report no biomedical financial interests or potential conflicts of interest. Supplementary Material Key Resources Table for Biological Acknowledgments The authors thank the participants and their families for participating in this research. This research was supported by the National Institutes of Health (R37MH101495 to IHG). Author Contributions: IHG designed the study and helped to write the manuscript; JGM, LRB, SMC, and TCH analyzed the data and helped to write the manuscript; SMC, LAC, and JMG helped to collect the data and write the manuscript. Competing Interest Statement: The authors report no biomedical financial interests or potential conflicts of interest. ==== Refs References 1 E. Golberstein, H. Wen, B. F. Miller, Coronavirus Disease 2019 (COVID-19) and Mental Health for Children and Adolescents. JAMA Pediatr (2020) https:/doi.org/10.1001/jamapediatrics.2020.1456 (April 26, 2020). 2 Jiao W.Y. Wang L.N. Liu J. Fang S.F. Jiao F.Y. Pettoello-Mantovani M. Somekh E. Behavioral and Emotional Disorders in Children during the COVID-19 Epidemic J Pediatr 221 2020 264 266.e1 32248989 3 X. Xie, Q. Xue, Y. Zhou, K. Zhu, Q. Liu, J. Zhang, & R.X. Song. Mental Health Status Among Children in Home Confinement During the Coronavirus Disease 2019 Outbreak in Hubei Province, China. JAMA Pediatr (2020) https:/doi.org/10.1001/jamapediatrics.2020.1619 (April 26, 2020). 4 S.J. Zhou, L.G. Zhang, L.L. Wang, Z.C. Guo, J.Q. Wang, J.C. Chen, et al. Prevalence and socio-demographic correlates of psychological health problems in Chinese adolescents during the outbreak of COVID-19. Eur Child Adolesc Psychiatry (2020) https:/doi.org/10.1007/s00787-020-01541-4. 5 Racine N. McArthur B.A. Cooke J.E. Eirich R. Zhu J. Madigan S.N. Global Prevalence of Depressive and Anxiety Symptoms in Children and Adolescents During COVID-19: A Meta-analysis JAMA Pediatrics 175 2021 1142 1150 34369987 6 Colich N.L. Rosen M.L. Williams E.S. McLaughlin K.A. Biological Aging in Childhood and Adolescence Following Experiences of Threat and Deprivation: A Systematic Review and Meta-Analysis Psychol Bull 146 2020 721 764 32744840 7 Wierenga L.M. Langen M. Oranje B. Durston S. Unique developmental trajectories of cortical thickness and surface area Neuroimage 87 2014 120 126 24246495 8 Cole J.H. Franke K. Predicting Age Using Neuroimaging: Innovative Brain Ageing Biomarkers Trends Neurosci 40 2017 681 690 29074032 9 Drobinin V. Van Gestel H. Helmick C.A. 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Measuring the thickness of the human cerebral cortex from magnetic resonance images Proceedings of the National Academy of Sciences of the United States of America 97 2000 11050 11055 10984517 26 Morey R.A. Petty C.M. Xu Y. Hayes J.P. Wagner H.R. II Lewis D.V. A comparison of automated segmentation and manual tracing for quantifying hippocampal and amygdala volumes NeuroImage 45 2009 855 866 19162198 27 Ho T.C. Teresi G.I. Ojha A. Walker J.C. Kirshenbaum J.S. Singh M.K. Gotlib I.H. Smaller caudate gray matter volume is associated with greater implicit suicidal ideation in depressed adolescents Journal of Affective Disorders 278 2021 650 657 33039875 28 Ho T.C. Cichocki A.C. Gifuni A.J. Camacho M.C. Ordaz S.J. Singh M.K. Gotlib I.H. Reduced dorsal striatal gray matter volume predicts implicit suicidal ideation in adolescents Soc Cogn Affect Neurosci 13 2018 1215 1224 30256980 29 Desikan R.S. Ségonne F. Fischl B. Quinn B.T. Dickerson B.C. Blacker D. An automated labeling system for subdividing the human cerebral cortex on MRI scans into gyral based regions of interest NeuroImage 31 2006 968 980 16530430 30 Han L.K. Dinga R. Hahn T. Ching C.R. Eyler L.T. Aftanas L. Brain aging in major depressive disorder: results from the ENIGMA major depressive disorder working group Mol Psychiatry 26 2021 5124 5139 32424236 31 T. T. Le, R.T. Kuplicki, B.A. McKinney, H.W. Yeh, W.K. Thompson, M.P. Paulus, and Tulsa 1000 Investigators. A Nonlinear Simulation Framework Supports Adjusting for Age When Analyzing BrainAGE. Front Aging Neurosci 10, 317 (2018). 32 King L.S. Dennis E.L. Humphreys K.L. Thompson P.M. Gotlib I.H. Cross-sectional and longitudinal associations of family income-to-needs ratio with cortical and subcortical brain volume in adolescent boys and girls Developmental Cognitive Neuroscience 44 2020 100796 33 Coupé P. Catheline G. Lanuza E. Manjón J.V. Alzheimer’s Disease Neuroimaging Initiative, Towards a unified analysis of brain maturation and aging across the entire lifespan: A MRI analysis Hum Brain Mapp 38 2017 5501 5518 28737295 34 Ho T.C. Kulla A. Teresi G.I. Sisk L.M. Rosenberg-Hasson Y. Maecker H.T. Gotlib I.H. Inflammatory cytokines and callosal white matter microstructure in adolescents Brain, Behavior, and Immunity 100 2022 321 331 34896593 35 LeWinn K.Z. Sheridan M.A. Keyes K.M. Hamilton A. McLaughlin K.A. Sample composition alters associations between age and brain structure Nat Commun 8 2017 874 29026076 36 Lee H. Singh G.K. Monthly trends in self-reported health status and depression by race/ethnicity and socioeconomic status during the COVID-19 Pandemic, United States, April 2020 - May 2021 Ann Epidemiol 63 2021 52 62 34358622 37 Lopez L. III Hart L.H. III Katz M.H. Racial and Ethnic Health Disparities Related to COVID-19 JAMA 325 2021 719 720 33480972 38 Mena G.E. Martinez P.P. Mahmud A.S. Marquet P.A. Buckee C.O. Santillana M. Socioeconomic status determines COVID-19 incidence and related mortality in Santiago, Chile Science 372 2021 eabg5298 39 Ball G. Beare R. Seal M.L. Charting shared developmental trajectories of cortical thickness and structural connectivity in childhood and adolescence Hum Brain Mapp 40 2019 4630 4644 31313446	36471743	PMC9713854	NO-CC CODE	2022-12-02 23:23:13	no	Biol Psychiatry Glob Open Sci. 2022 Dec 1; doi: 10.1016/j.bpsgos.2022.11.002	utf-8	Biol Psychiatry Glob Open Sci	2,022	10.1016/j.bpsgos.2022.11.002	oa_other
==== Front Actas Dermosifiliogr Actas Dermosifiliogr Actas Dermo-Sifiliograficas 0001-7310 1578-2190 AEDV. Published by Elsevier España, S.L.U. S0001-7310(22)01019-5 10.1016/j.ad.2022.05.032 Carta Científico-Clínica Dermatomiositis juvenil durante la pandemia por SARS-CoV-2: afectación acral y de la cavidad oral [[Translated article]]Juvenile Dermatomyositis During the SARS-CoV-2 Pandemic: Acral and Oral Mucosal InvolvementGiacaman A. Dra 1⁎ Mir Perelló MC. 2 Rodríguez Diez L. 2 Martín-Santiago A. 1 1 Departamento de Dermatología, Hospital Universitari Son Espases, Palma de Mallorca, España 2 Departamento de Reumatología Pediátrica, Hospital Universitari Son Espases, Palma de Mallorca, España ⁎ Correspondencia 1 12 2022 1 12 2022 26 1 2022 22 5 2022 © 2022 AEDV. Published by Elsevier España, S.L.U. 2022 AEDV Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmc	36464006	PMC9714077	NO-CC CODE	2022-12-02 23:24:30	no	Actas Dermosifiliogr. 2022 Dec 1; doi: 10.1016/j.ad.2022.05.032	utf-8	Actas Dermosifiliogr	2,022	10.1016/j.ad.2022.05.032	oa_other
==== Front Clin Microbiol Infect Clin Microbiol Infect Clinical Microbiology and Infection 1198-743X 1469-0691 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. S1198-743X(22)00599-7 10.1016/j.cmi.2022.11.026 Research Note Increased Reporting of Venous and Arterial Thromboembolic events reported with Tixagevimab–Cilgavimab for COVID-19 Montastruc François 12∗ Lafaurie Margaux 12 Flumian Clara 1 de Canecaude Claire 1 1 Department of Medical and Clinical Pharmacology, Centre of PharmacoVigilance and Pharmacoepidemiology, Toulouse University Hospital (CHU), Faculty of Medicine, Toulouse, France 2 Centre d’Investigation Clinique 1436, Team PEPSS « Pharmacologie En Population cohorteS et biobanqueS », Toulouse University Hospital, France ∗ Corresponding author: Department of Medical and Clinical Pharmacology, Centre of PharmacoVigilance and Pharmacoepidemiology, Toulouse University Hospital, Faculty of Medicine, Toulouse, France 37 Allées Jules Guesde, 31000 Toulouse, France Tel.: +335 61 14 59 60; fax: +335 61 25 51 16 1 12 2022 1 12 2022 18 7 2022 6 11 2022 27 11 2022 © 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objectives Since in two phase 3 clinical trials, there were a disproportion of number of thromboembolic events in the tixagevimab/cilgavimab group than in placebo group, there is a cardiovascular safety concerns with the use of this Anti-SARS-COV-2 Monoclonal Antibody. Whether tixagevimab/cilgavimab use in real life context increases the risk for of thromboembolic events is unclear. Methods We used VigiBase, the World Health Organization’s individual case safety reports database, to assess the risk of reporting arterial or venous thromboembolic events in COVID-19 patients (≥12 years) exposed to tixagevimab/cilgavimab compared with COVID-19 patients exposed to other anti-SARS-CoV-2 mAbs, including casirivimab/imdevimab, bamlanivimab/etesevimab and sotrovimab. Results Among the 8,952 reports of patients with an anti-SARS-CoV-2 mAb, 31 reports of thromboembolic events associated with tixagevimab/cilgavimab, mainly deep vein thrombosis (10), pulmonary embolism (8) and myocardial infarction (7). Compared with other anti-SARS-CoV-2 mAbs, the use of tixagevimab/cilgavimab was associated with an increased risk of reporting arterial thromboembolic events (Reporting Odds Ratio (ROR) 3.25; 95%CI 1.73, 6.10). Concerning venous thromboembolic events, a significant increase in the risk of reporting was observed with use of tixagevimab/cilgavimab (ROR 3.59; 95%CI 2.16, 5.96). Conclusions This observational study corroborate in a real-world setting, the cardiovascular safety signal already found with tixagevimab/cilgavimab in two clinical trials. Owing these thromboembolic safety concerns and considering the lack of clinical trials supporting a protection against the omicron variant, there is an urgent need to improve knowledge on the effectiveness of tixagevimab/cilgavimab with new COVID-19 variants. Keywords Arterial thromboembolic events COVID-19 Myocardial infarction Pharmacovigilance Pulmonary embolism Tixagevimab–cilgavimab Vigibase Editor: Luigia Scudeller ==== Body pmcIntroduction Tixagevimab/cilgavimab, casirivimab/imdevimab, bamlanivimab/etesevimab and sotrovimab are monoclonal-antibodies (mAbs) with neutralizing activity against the spike protein of SARS-CoV.[1] They have been recommended by the Food and Drug Agency and the European Medicines Agency to prevent COVID-19 (pre or post-exposure prophylaxis of COVID-19) or to treat early-stage COVID-19 in patients who are at high risk for progressing to severe COVID-19. While data suggest efficacy against alpha or delta variants of SARS-CoV-2, there are concerns about the risk of arterial or venous thromboembolic events with tixagevimab/cilgavimab.[2] In the phase 3 PROVENT trial, evaluating the effect of tixagevimab/cilgavimab for the prevention of symptomatic and severe Covid-19 in adults, there were a higher number of serious thromboembolic events in the tixagevimab/cilgavimab group than placebo group (5‰ versus 2‰).[2,3] Events reported with tixagevimab/cilgavimab were myocardial infarction (n=9, with one death), pulmonary embolism (n=2) and deep vein thrombosis. In another phase 3 clinical trial (TACKLE study), assessing whether tixagevimab/cilgavimab can treat outpatient adults with COVID-19 and prevent severe COVID-19, a higher thromboembolic events was reported in tixagevimab/cilgavimab group than in placebo group (1,3% versus 0,4%).[2,4] Considering this safety concern and the lack of real-word data, there is an urgent need for more information on the risk of thromboembolic events with tixagevimab/cilgavimab. We performed a large pharmacovigilance study to investigate the risk of reporting arterial or venous thromboembolic events in COVID-19 patients treated by tixagevimab/cilgavimab compared with COVID-19 patients treated by other anti-SARS-CoV-2 mAbs (Table 1 ).Table 1 Reporting Odds Ratios for the Association Between Thromboembolic Events and the Use of tixagevimab/cilgavimab for coronavirus disease 2019 in Vigibasea Table 1 Casesb Non-Casesc ROR (95% CI) Arterial Thromboembolic events Primary analysisrowhead anti-SARS-CoV-2 mAbsd 56 8,334 1 (reference) tixagevimab/cilgavimab 12 550 3.25 (1.73, 6.10) Restricting to reports of serious adverse effectse anti-SARS-CoV-2 mAbsd 49 5,118 1 (reference) tixagevimab/cilgavimab 12 344 3.64 (1.92, 6.91) Restricting to one anti-SARS-CoV-2 mAbs bamlanivimab/etesivimab 9 1,987 1 (reference) tixagevimab/cilgavimab 12 550 4.82 (2.02, 11.50) casirivimab/imdevimab 35 4,962 1 (reference) tixagevimab/cilgavimab 12 550 3.09 (1.59, 5.99) sotrovimab 12 1,386 1 (reference) tixagevimab/cilgavimab 12 550 2.52 (1.13, 5.64) Venous Thromboembolic events Primary analysisd anti-SARS-CoV-2 mAbsd 81 8,309 1 (reference) tixagevimab/cilgavimab 19 543 3.59 (2.16, 5.96) Restricting to reports of serious adverse effectse anti-SARS-CoV-2 mAbsd 77 5,090 1 (reference) tixagevimab/cilgavimab 17 339 3.31 (1.94, 5.66) Restricting to one anti-SARS-CoV-2 mAbsrowhead bamlanivimab/etesivimab 25 1,971 1 (reference) tixagevimab/cilgavimab 19 543 2.76 (1.51, 5.05) casirivimab/imdevimab 47 4,950 1 (reference) tixagevimab/cilgavimab 19 543 3.69 (2.15, 6.33) sotrovimab 9 1,389 1 (reference) tixagevimab/cilgavimab 19 543 5.25 (2.43, 12.01) a ROR is a ratio similar in concept to the odds ratio in case-control studies and corresponds to the exposure odds among reported cases of bradycardia over the exposure odds among reported non-case. b reports containing any terms including in the standard medDra Queries “Embolic and thrombotic events, arterial” or “Embolic and thrombotic events, venous” found in MedDRA dictionary https://www.meddra.org/. c all other adverse events. d casirivimab/imdevimab, bamlanivimab/etesevimab and sotrovimab. e occurrence of death, a life-threatening adverse event, inpatient hospitalisation or prolongation of hospitalisation, significant disability, congenital anomaly. Methods We used VigiBase, the World Health Organization’s individual case safety reports database, to assess the risk of reporting arterial or venous thromboembolic events in COVID-19 patients exposed to tixagevimab/cilgavimab compared with COVID-19 patients exposed to other anti-SARS-CoV-2 mAbs (including casirivimab/imdevimab, bamlanivimab/etesevimab and sotrovimab). VigiBase contains more than 30 million spontaneous generated adverse drug reactions, from 136 countries, covering more than 90% of the world’s population.[5] We considered only reports of patients (≥12 years) with COVID-19 registered up to the 12th of July 2022. The risk of arterial or venous thromboembolic events was calculated using the Reporting Odds Ratios (ROR) with their 95% confidence interval (CI), a ratio similar in concept to the Odds Ratio in case-control studies. The ROR corresponds to the exposure odds among reported cases of thromboembolic events over the exposure odds among reported non-cases.[6] As conducted in previous pharmacovigilance studies and to assess the robustness of the primary analysis, we made sensitivity analyses restricting to serious cases or limiting to one anti-SARS-CoV-2 mAb in the comparator group.[7,8] We also used the PRR method which compares the rate of reporting if one event (here, arterial or venous thromboembolic events) among all reports for a given drug (tixagevimab/cilgavimab) with the rate of the same event among the other drugs (other anti-SARS-CoV-2 mAbs). A signal of disproportionate reporting was defined as a drug-event pair for which the statistical analyses led to a PRR≥2 associated with a Chi square≥4. According to the clinical research French law, review from an ethics committee is not required for such observational studies. As all data from VigiBase® were deidentified, patient informed consent was not necessary. Results Among the 8,952 reports of patients with an anti-SARS-CoV-2 mAb registered up to the 12th of July 2022, we found 164 thromboembolic events (1.8%), with 68 arterial and 100 venous thromboembolic events. Thromboembolic events originated from US (118), Europe (42), Australia (2) and Singapore (1) involving mostly men (103, 63%), with a mean age of 64.5 years. We found 31 reports of thromboembolic events associated with tixagevimab/cilgavimab, mainly deep vein thrombosis (10), pulmonary embolism (8) and myocardial infarction (7). Most of reports with tixagevimab/cilgavimab (n=29, 94%) were serious according the WHO definition.[9]. Compared with other anti-SARS-CoV-2 mAbs, the use of tixagevimab/cilgavimab was associated with an increased risk of reporting arterial thromboembolic events (Reporting Odds Ratio (ROR) 3.25; 95%CI 1.73, 6.10) (Table). Concerning venous thromboembolic events, a significant increase in the risk of reporting was observed with use of tixagevimab/cilgavimab (ROR 3.59; 95%CI 2.16, 5.96). Results were consistent in sensitivity analyses, in particular, when we restricted to reports with serious effects. Using PRR method, we found disproportionate reporting of arterial thromboembolic events (PRR=3.20, chi square =15.1) and of venous thromboembolic events (PRR=3.5, chi square =27.8). Discussion Such pharmacovigilance analysis could be subject to limitations as confounding by indication. To mitigate the confounding, we used as comparator group, anti-SARS-CoV-2 mAbs used in post-exposure prophylaxis (casirivimab/imdevimab) or as a curative COVID-19 treatment (sotrovimab) where the risk of thromboembolic events is higher than pre-exposure prophylaxis (cilgavimab/tixagevimab). Despide these methods, confounders, including risk factors of thromboembolic events could affect our findings. Another limitation is the under-reporting that could be differential between groups of comparison. In addition, due to the nature of VigiBase (spontaneous reports), it could be that all the events recorded were not completely attributable to the drugs reported. We were unable to clinically validate the reports, in particular data concerning thromboembolic events. Given that arterial and venous thromboembolic events are previously identified safety concerns, it could be that findings are due to surveillance bias, with more proactive screening finding of thromboembolic events. However, we can also assume that this monitoring applies to the whole class of anti-SARS-CoV-2 mAbs and therefore does not explain the disproportionality found. The study corroborate in a real-world setting, the cardiovascular safety signal already found with tixagevimab/cilgavimab in two clinical trials.[[2], [3], [4]]. Owing these thromboembolic safety concerns and considering the lack of clinical trials supporting a protection against the omicron variant, there is an urgent need to improve knowledge on the effectiveness of tixagevimab/cilgavimab with new COVID-19 variants.[10]. Article information Contributions FM conceived and designed the study. FM acquired the data and did the statistical analyses. All authors analyzed and interpreted the data. FM wrote the manuscript, and all authors critically revised the manuscript. FM supervised the study and is the guarantor. All authors approved the final version of the manuscript and are accountable for its accuracy. Funding Doctor François Montastruc has received funding under the Vigi-Drugs COVID-19 project from the French National Research Agency (ANR Agence Nationale de la Recherche) for the evaluation of pharmacovigilance data of drugs and vaccines used in the management or prevention of COVID-19. The other authors certify that they have not received any funding from any institution, including personal relationships, interests, grants, employment, affiliations, patents, inventions, honoraria, consultancies, royalties, stock options/ownership, or expert testimony related to this topic. Role Of The Funding Source NA. Conflict Of Interest None disclosed. Acknowledgements The authors acknowledge the Uppsala Monitoring Centre (UMC) who provided and gave permission to use the data analyzed in the present study. Access to the World Health Organization global individual case safety report database, VigiBase®, is available without fees to Dr. Montastruc. The authors are indebted to the National Pharmacovigilance Centers that contributed data. The opinions and conclusions in this study are not necessarily those of the various centers or of the World Health Organization. Dr François Montastruc and Dr Claire de Canecaude have participated in the pharmacovigilance surveillance of monoclonal antibodies used against COVID-19 in France as external experts for the French Drug Agency (ANSM). The research is made outside the French drug agency investigation. Dr François Montastruc and Dr Claire de Canecaude have not received funding from the ANSM for this research. ==== Refs References 1 Sheward D.J. Kim C. Ehling R.A. Pankow A. Castro Dopico X. Dyrdak R. Neutralisation sensitivity of the SARS-CoV-2 omicron (B.1.1.529) variant: a cross-sectional study Lancet Infect Dis S1473-3099 22 2022 10.1016/S1473-3099(22)00129-3 00129-3 2 Summary of Product Characteristics for Evusheld. GOVUK n.d. https://www.gov.uk/government/publications/regulatory-approval-of-evusheld-tixagevimabcilgavimab/summary-of-product-characteristics-for-evusheld (accessed May 17, 2022). 3 Levin M.J. Ustianowski A. De Wit S. Launay O. Avila M. Templeton A. Intramuscular AZD7442 (Tixagevimab–Cilgavimab) for Prevention of Covid-19 N Engl J Med 386 2022 2188 2200 10.1056/NEJMoa2116620 35443106 4 Montgomery H. Hobbs F.D.R. Padilla F. Arbetter D. Templeton A. Seegobin S. Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early outpatient treatment of COVID-19 (TACKLE): a phase 3, randomised, double-blind, placebo-controlled trial Lancet Respir Med S2213-2600 22 2022 10.1016/S2213-2600(22)00180-1 00180-00181. 5 Lindquist M. VigiBase, the WHO Global ICSR Database System: Basic Facts Drug Information Journal 42 2008 409 419 10.1177/009286150804200501 6 Faillie J.-L. Case-non-case studies: Principle, methods, bias and interpretation Therapie 74 2019 225 232 10.1016/j.therap.2019.01.006 30773344 7 Touafchia A. Bagheri H. Carrié D. Durrieu G. Sommet A. Chouchana L. Serious bradycardia and remdesivir for coronavirus 2019 (COVID-19): a new safety concerns Clinical Microbiology and Infection 27 2021 791 10.1016/j.cmi.2021.02.013 e5-791.e8 8 Montastruc F. Thuriot S. Durrieu G. Hepatic Disorders With the Use of Remdesivir for Coronavirus 2019 Clin Gastroenterol Hepatol 18 2020 2835 2836 10.1016/j.cgh.2020.07.050 32721580 9 Edwards I.R. Aronson J.K. Adverse drug reactions: definitions, diagnosis, and management Lancet 356 2000 1255 1259 10.1016/S0140-6736(00)02799-9 11072960 10 Mahase E. Covid-19: Has the spread of omicron BA.2 made antibody treatments redundant? BMJ 377 2022 o1009 10.1136/bmj.o1009 35444011	36464214	PMC9714079	NO-CC CODE	2022-12-02 23:24:41	no	Clin Microbiol Infect. 2022 Dec 1; doi: 10.1016/j.cmi.2022.11.026	utf-8	Clin Microbiol Infect	2,022	10.1016/j.cmi.2022.11.026	oa_other
==== Front Am J Infect Control Am J Infect Control American Journal of Infection Control 0196-6553 1527-3296 Published by Elsevier Inc. on behalf of Association for Professionals in Infection Control and Epidemiology, Inc. S0196-6553(22)00842-2 10.1016/j.ajic.2022.11.023 Major Article Infection Preventionists’ Experiences During the Second Year of the COVID-19 Pandemic: Findings From Focus Groups Conducted with Association for Professionals in Infection Control & Epidemiology (APIC) Members Rebmann Terri PhD, RN, CIC, FAPIC 1⁎ Alvino Rebecca T. RN, MS, CNS, CIC 2 Lugo Kaeli A. BS 1 Holdsworth Jill E. MS, CIC, FAPIC, NREMT, CRCST 3 Gomel Ashley MPH 1 1 Institute for Biosecurity, College for Public Health and Social Justice, Saint Louis University. St Louis, MO, USA 2 UCSF Health San Francisco, CA, USA 3 Emory University Hospital Midtown, Atlanta, GA ⁎ Corresponding author: Terri Rebmann, PhD, RN, CIC, FAPIC, Special Assistant to the President, Director, Institute for Biosecurity, Professor, Department of Epidemiology & Biostatistics, Saint Louis University, College for Public Health and Social Justice, 1 North Grand DuBourg Room 101A, Saint Louis, Missouri 63108, Phone: (314) 977-8260 1 12 2022 1 12 2022 © 2022 Published by Elsevier Inc. on behalf of Association for Professionals in Infection Control and Epidemiology, Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction COVID-19 epidemiology changed dramatically in spring 2021 when vaccine became widely available and the Delta variant emerged. There was a need to identify current infection prevention challenges due to changing pandemic epidemiology. Methods Six focus groups were conducted via Zoom with APIC members in November and December, 2021 to elicit infection preventionists’ (IP) experiences with the COVID-19 pandemic after the Delta variant had emerged. Each focus group was audio recorded then transcribed verbatim. Content analysis was used to identify major themes. Results In total, 90 IPs participated (average of 15 IPs per focus group). Participating IPs described multiple issues they have faced during the second year of the COVID-19 pandemic after the Delta variant emerged, including continuing challenges with personal protective equipment, changes in pandemic restrictions that caused confusion and pushback, the hope when vaccine first became available and then despair when there was more vaccine breakthrough than anticipated, staffing and medical supply shortages, overwhelming workloads, and anger towards healthcare personnel and IPs. However, IPs felt more valued by leadership, and reported greater internal collaboration and external coordination of care. Conclusions The second year of the pandemic brought ongoing and new challenges for IPs, but also better coordination of care. Strategic initiatives are needed to address the identified challenges, such as how to prioritize tasks when IPs are overwhelmed. ==== Body pmcIntroduction The COVID-19 pandemic, caused by the SARS-CoV-2 virus, began in spring 2020 and has resulted in numerous challenges for healthcare and public health systems. In fall 2020, the Association for Professionals in Infection Control and Epidemiology (APIC) COVID-19 Task Force conducted focus groups with infection preventionist (IP) members to understand their experiences during the first nine months of the COVID-19 pandemic. That project identified multiple challenges faced by U.S.-based IPs, including unavailability of setting-specific guidelines for non-acute care facilities, a lack of personal protective equipment (PPE), high IP workloads, and healthcare associated infection (HAI) increases.1 Challenges specific to rural healthcare settings were also identified, including the need to address inaccurate social media messages and polarization of the pandemic among rural community members.2 In spring 2021, the COVID-19 vaccine became widely available throughout the U.S. There was hope that the pandemic was ending or that COVID-19 was shifting to become an endemic illness due to the widespread availability of vaccine and the rapidly dropping infection rates seen at that time. The Centers for Disease Control and Prevention (CDC) dropped the federal mask requirement for those who had received the primary COVID-19 vaccination series.3 However, late that spring and over the summer, the pandemic epidemiology changed dramatically when the Delta variant emerged. COVID-19 cases once again began to surge in the U.S., including cases of breakthrough infection.4 The APIC COVID-19 Task Force recognized the need to identify current infection prevention challenges due to the changing epidemiology of the pandemic. The purpose of this project was to conduct a formal assessment to understand the infection prevention challenges posed by emergence of the Delta variant and perceived benefits derived from widespread availability of the COVID-19 vaccine. Methods Six focus groups were conducted with Association for Professionals in Infection Control & Epidemiology (APIC) members in November and December, 2021. APIC headquarters staff distributed a recruitment statement through their member newsletter. All APIC members located in the U.S. were eligible to participate, regardless of employer size, type, or practice setting. The six focus groups consisted of two sessions with infection prevention and control (IPC) System Directors, and one session in the following areas: 1) long-term care (LTC), 2) acute care, 3) rural areas/settings, and 4) behavioral health, home health, hospice, and ambulatory care. APIC members self-identified into the focus group session they believed applied to them. All focus groups were conducted via virtual online platform Zoom®. Members of the APIC COVID-19 Task Force and APIC staff developed the focus group questionnaire and methodology. Focus group sessions consisted of a series of open-ended questions intended to elicit participants’ comments about their thoughts, opinions, and experiences during the second year of the COVID-19 pandemic after the Delta variant emerged. IPs were also asked to complete a short demographic survey. Focus group sessions were audio recorded and then transcribed verbatim. IP participants’ comments from the Zoom chat box were downloaded and included in analysis. Data analysis was conducted using content analysis to identify and categorize major ideas and themes. Themes were reported with relevant quotations used to provide context and nuance. Words in brackets within quotes are not the participants’ words, but are provided to give context or explain the IPs’ quotes. The Saint Louis University Institutional Review Board approved this study. Results In total, 90 IPs participated (average of 15 IPs per focus group; see Table 1 ). Almost all were female (94.4%, n=85) and white (87.8%, n=79), which reflects the current diversity of APIC membership as a whole5. Most had a Bachelor's or Master's degree (43.3% and 46.7% respectively; Table 1). About two-thirds (61.1%, n=55) held the CIC credential. IPs’ work experience ranged from those with less than one year (11.4%, n=11) to those with more than 11 years (38.1%, n=37). Approximately half (48.95, n=44) indicated they work in a hospital. Of those who work in a hospital (44), about a third (36.4%, n=16) work in the smallest sized facility of 50 or fewer beds (Table 1). Participating IPs were from facilities across the U.S., with approximately a quarter of IPs from each of the four U.S. regions (Table 1). A full list of participant demographics is outlined in Table 1.Table 1 Focus Group Participants’ Demographic Characteristics Table 1: N = 90% (n) Focus Group System Directors (2 sessions) 25.5 (23) Acute care 18.9 (17) Long term care 18.9 (17) Rural areas/settings 18.9 (17) Ambulatory care, behavioral health, home health, hospice 17.8 (16) Gender Female 94.4 (85) Male 5.6 (5) Age 21 – 30 years 1.1 (1) 31 – 40 years 24.4 (22) 41 – 50 years 25.6 (23) 51 – 60 years 37.8 (34) ≥ 61 years 11.1 (10) Race White 87.8 (79) Black 6.7 (6) Asian or Pacific Islander 3.3 (3) American Indian or Alaskan Native 2.2 (2) Highest Education Level Associate's Degree 6.7 (6) Bachelor's Degree 43.3 (39) Master's Degree 46.7 (42) MD or PhD 3.3 (13) Certification Status CIC 61.1 (55) Not Certified in Infection Prevention 38.9 (35) Years of Work Experience as an Infection Preventionist < 1 year 11.4 (11) 1 – 2 years 7.2 (7) 3 – 4 years 16.5 (16) 5 – 10 years 26.8 (26) ≥ 11 years 38.1 (37) Work Setting Hospital 48.9 (44) Long-Term Care 18.9 (17) Healthcare System 12.2 (11) Behavioral Health 7.9 (7) Home Health 4.4 (4) Ambulatory Surgery Center 3.3 (3) Public Health 2.2 (2) Outpatient Clinic 2.2 (2) Hospital Bed Size N = 44 ≤ 50 beds 36.4 (16) 51 – 99 beds 4.5 (2) 100 – 199 beds 2.3 (1) 200 – 299 beds 6.8 (3) 300 - 399 beds 6.8 (3) 400 – 499 beds 4.5 (2) ≥ 500 beds 38.6 (17) Location of Employer Rural 38.9 (35) Urban 35.6 (32) Suburban 25.6 (23) U.S. Census Region West 27.8 (25) Midwest 25.6 (23) South 26.7 (24) Northeast 20.0 (18) Continued Challenges with Personal Protective Equipment (PPE) The participating IPs noted that they were experiencing ongoing challenges with PPE, even a year and a half into the pandemic. One of the challenges they faced involved needing to vet the PPE they were receiving because they were obtaining it from new vendors and the quality of the products was questionable. As one IP noted, “[We are receiving] lower quality supplies”. One IP asked the group, “Did anyone else get ‘dirty gloves’?” to which another participant responded, “No dirty gloves, but we got some gowns with ‘bugs’ on them.” Another IP provided a more detailed explanation of the PPE quality challenges they were facing:Multiple times we would have to bring in a new type of gloves and we would just get complaint after complaint that they would break, and gowns that would tear or you couldn't wear them more than a few minutes without being drenched in sweat, but we had no alternative. So yeah, the quality of the substitutes was not great. We had dollar store raincoats as isolation gowns for about a month. Many of the participating IPs talked about the “PPE fatigue” they were witnessing and hearing about from the healthcare staff, patients, and visitors at their agency. Many indicated that PPE compliance is low overall, for all types of PPE. As one explained, “PPE compliance has just gone down tremendously since the beginning of the pandemic.” Some IPs discussed how compliance varied, depending on which type of PPE it was. As one stated, “As long as people are wearing N95 masks, they keep their masks on. The eye protection is a little bit more of a challenge. And the minute you go back to surgical masks, I see them under noses.” As another described, “[Staff] want to reuse PPE from room to room, like the gowns…just not wanting to don and doff it, on and off.” The most commonly reported PPE noncompliance was related to the use of eye protection. As one IP stated, “It's almost taboo to not have your mask on, but we have struggled with eye protection [compliance]”. As another IP noted, “Eye protection has definitely been the biggest challenge for us as well. Folks just don't want to wear it.” Changes in Restrictions Caused Confusion and Pushback One of the challenges for IPs when the Delta variant emerged was the change in COVID-19-related restrictions. In some cases, it was the need to return to COVID-19 mitigation restrictions that had been lifted recently. For others, it was a loosening of restrictions just as a healthcare surge was at its worst. As IPs explained:These choices were made to deescalate, and we have guidance on how to do it safely. But then we were turning around and saying, “Oh just kidding. Go back to what you were doing. Masks, eye protection, everything all the time.” And that was very difficult to message. We pulled back from masking not in patient care areas, but in meetings and conference rooms. Our facility handled that okay, but it is disruptive to the infection prevention department. We were nervous. And once we started getting hit with Delta, it really felt like we had to react pretty fast and put those protocols back in place. CMS opened up visitation and they started lowering restrictions when we were at our worst. So that has been very confusing to staff and residents. Staff are just exhausted of the flip flopping back and forth with policies and procedures. We open things back up and then we have to close everything back down. It's just getting really frustrating for everyone. CDC had said that if you've been vaccinated, you can go back to basically living your normal lives, and you can hang out with folks as long as everyone in the room is vaccinated. And then months later it's like, “Nope just kidding. You can't do that. Everyone needs to go back to masks and social distance.” That was a great example of the flip flop. The rapidly changing guidance and sources of misinformation resulted in distrust and frustration among healthcare personnel. The IPs reported that healthcare staff contributed to the confusion by spreading misinformation among their colleagues, and some personnel began to push back against the COVID-19 restrictions. The following are examples of how IPs described their experiences with staff spreading misinformation or pushing back against restrictions:Staff have an idea that may not be correct or doesn't quite line up with science and then it kind of just spreads. You're up against that all the time. I don't want to say it's a battle, but that's kind of the best [description]. You are kind of fighting back and forth. What fatigues me the most is staff fighting policy, changes, restrictions, etc. Our staff are not only starting to distrust what we're telling them, but also what even the CDC is saying. We're the face of a lot of this information, so when regulations and rules change, you lose a bit of your credibility. Changes in the IPC Field After COVID-19 Vaccine Became Available Many IPs discussed the benefits and challenges experienced once the COVID-19 vaccine became available. One challenge many IPs reported related to a staff COVID-19 vaccine requirement policy that their facility or agency had implemented. Most indicated that the vaccine was controversial among at least some healthcare personnel, resulting in a small number of employees leaving. One IP said it created “a lot of friction between the employees of whether or not they're going to stay, are they gonna leave nursing all together”. Other IPs reported their employer decided to approve many of the requested vaccination exemption requests so that they would not lose staff. As one IP explained, “We knew that we had staff who were not vaccinated who had been working here for like 20 years, and they didn't want to lose their job, and so the administration decided to take a liberal approach [to vaccine exemptions].” Another IP indicated that being less strict about exemption requests aided in hiring of new healthcare personnel: “We've actually had a little bit of an increase in hiring and I feel that is because my administration decided to take a very liberal approach to accepting exceptions for the [vaccine] mandate.” Many of the IPs discussed frustration around vaccine hesitance among healthcare staff and the general public, indicating that they believed the hesitancy to get vaccinated had more to do with misinformation and politization of the pandemic and COVID-19 vaccine than with science. The following are examples of how IPs described their experiences with vaccine hesitancy:Many [of our staff] feel this pandemic was man made and it makes them be untrusting of the vaccine. There are staff that don't trust the [EUA-approved] vaccine. But when they themselves became positive, they sure consented quickly to the use of the [EUA-approved] treatments. They will believe Dr. Google or Dr. Facebook before believing our providers. We saw little or no impact on vaccination rates when Pfizer [COVID-19 vaccine] gained full [FDA] approval. They still believe that it's too fast, it's too new, we don't know anything. There was a group of anti-vaxxers in the community that were picketing across the street from the hospital because of our vaccine mandate. For me, I think about the impact of social media on trying to get people vaccinated. It's just something that I could have done without in my lifetime, combating the conspiracy theories and all of the misinformation out there. We've tried every way that we could think of to help the public have the information that they need to make a good decision [about vaccination], but there's so much misinformation out there, so much political aspect to the vaccination that we can't break through. There's still a lot of people out there that have stopped believing in science. The IPs discussed how healthcare staff began fighting or experiencing conflict over who was vaccinated versus not vaccinated. The CDC guidance that allowed vaccinated individuals to forego masking and social distancing as long as everyone in the group was vaccinated also created a lot of tension in their workplace. As IPs described it:The ability to unmask if everybody was vaccinated caused such an issue. We're too small to be fighting amongst ourselves, so we decided not to implement that. We used to work as a unit together, but now there's so much division at our facilities. At my facility, both of my directors of nursing were against getting the vaccine. Staff heard that they didn't get it and people drew lines. And then there were things like you wear your COVID vaccination T shirt on Friday, but then half the staff were not vaccinated and couldn't wear the T shirt and it has created such division between people. Another challenge was trying to get vaccine status in a meeting. That was very difficult. We couldn't really ask folks, so it was just up to them to be truthful if they had been vaccinated. No one wanted to be the only one in the room that wasn't vaccinated, to have to stand up and be like, “No, I'm not vaccinated, so everybody in this meeting has to wear a mask.” A common theme among the IPs was the sense of hope they felt and sensed among their healthcare colleagues when the COVID-19 vaccine first became available. There was a belief that the pandemic might finally be ending or that the vaccine might allow a return to a more normal pre-pandemic way of life. This sense of hope faded quickly into frustration when there was so much vaccine hesitance among healthcare personnel and the general public, and new viral variants resulted in more post-vaccination breakthrough infections than anticipated. The following quotes illustrate the sense of hope provided by the vaccine and what happened after Delta emerged:I think people thought we had this light at the end of the tunnel [when the vaccine came out]. The day the vaccine came out, it was the first time I had seen hope among our team members in so long. We kind of lived on that hope for a little bit, but it did not take long for that hope to turn to despair, because we are very rural and we have one of the lowest vaccination coverage rates in the country. Initially when the vaccine become available, many of the staff were relieved and hopeful for an end to the pandemic - but the hope unfortunately was short-lived. I had several staff members that volunteered at the vaccine clinics when the vaccine first came out. Even for me personally, I just loved a day when I was working in the vaccine clinic because there were so much positivity and hope. And unfortunately, that of course dwindled. Many IPs discussed how PPE compliance decreased after the COVID-19 vaccine became available. The consensus among IPs was that the vaccine provided staff a perceived level of protection against infection and resulted in staff not being willing to continue to wear PPE. As IPs explained:Many of the vaccinated staff gave themselves permission to not wear their mask. Since over 90% of our staff is vaccinated, people feel more comfortable not following PPE recommendations, and then we have to remind them why [PPE protocols are] still in place. Once folks are vaccinated, they thought they could unmask and they weren't doing it in secret. Healthcare Surge and Heavy Workloads Due to the Delta Variant IPs reported that their work duties were starting to lessen slightly in the late winter and spring of 2021, as more individuals became vaccinated and infection rates dropped nationwide. However, the emergence of the Delta variant in late spring of 2021 quickly resulted in new patient surges and the need to implement COVID-19 restrictions once again. These healthcare surges were primarily due to Delta-related COVID-19 infections, but also resulted from higher acuity patients being admitted because they had postponed medical services from earlier in the pandemic. Two major challenges during this time included extensive staffing and medical supply shortages, even compared to earlier pandemic waves. IPs discussed not having access to silicone catheters, linens, urine collection cups, and many other medical supplies. The medical supply shortages were due to supply chain issues, but infections among staff and the ease with finding higher paid positions elsewhere contributed to the staffing shortages. As IPs described:Staffing shortages are much worse at this time compared to prior to the Delta Variant. I work in long term care and staffing shortages are real. Bigger corporations can offer more money or offer bonuses. So we get a lot of people who apply and go through the hiring process, but then they find a higher paying job within a month or two and leave. Or you're competing with Starbucks down the road who can offer double the salary because they're short. With the first surge, we were not prepared for it, but I felt like we were able to manage in the moment. However, with Delta, the number of staff that were developing COVID was causing an enormous amount of strain. Our biggest struggle was absolutely having the staff to perform [COVID-19] testing. I know we were all dealing with PPE supply issues at the beginning of the pandemic, but it's like almost everything that we use to care for patients now is short. I agree about the shortages...surgical wraps, catheters, blood collection tubes, hub disinfectants, suction canisters...we have had ongoing supply issues. We are short on staffing and short [on beds]. We are surging into places of our hospital where we have never surged before. So, we've taken over conference rooms and turned them into patient care areas. I equate [supply shortages] to Apollo 13, that movie. They're trying to create that scrubber and make the [square peg fit into a round hole] and failure is not an option. You have to figure it out with what you have. We're constantly working a problem and have to come up with solutions creatively and you just sometimes want to bury your head in the sand because you hope your answer is right. And you're having to make these decisions very quickly because we've never had some of these issues with supplies. The healthcare surges caused by emergence of the Delta variant needed to be managed during times of supply and staffing shortages, a return to COVID-19 restrictions that had been loosened temporarily, an increase in disease severity compared to previously circulating strains, and the staff had not had any real reprieve or time to de-escalate from previous waves before Delta emerged. This was reported to be exhausting, frustrating, and emotionally challenging for healthcare personnel. The IPs described it like this:We thought we were prepared for Delta. We were wrong. It hit us very, very hard. One doctor said, “I felt like [the hospital] was the mass trauma unit” and he wasn't kidding, he wasn't exaggerating. He said, “I've never seen anything like this my entire career”. We had a massive surge in Delta cases and had to re-implement more restrictive protocols. And everyone was so, so frustrated and tired. [This surge is] something that will forever stand out. I've been doing infection prevention for over 35 years and never in my lifetime did I think I would see anything close to this. Delta ravaged our rural hospitals in a way that they had not seen. They were not prepared for it because they weren't hit with earlier [surges]. Our urban hospitals were more resilient. The healthcare surges caused by the Delta variant also resulted in renewed heavy workloads for IPs, who reported feeling overwhelmed and exhausted. The combination of ongoing pandemic response and returning to routine IPC work resulted in untenable working conditions. The relentless nature of the pandemic contributed to the IPs’ exhaustion as well, because they were not experiencing periods when they could relax and recover between healthcare surges. As the IPs described it:[When Delta hit] my role quickly evolved into a 24/7 role that is all consuming and equivalent to 2 full time jobs. It's hard to keep all the wheels on the car as we're hit with wave after wave after wave, and I think that's what Delta really drove home the most. On top of the regular IP work, [we are] dealing with any COVID changes. And then now getting ready for [the Joint Commission] regulatory readiness, so that feels like another job kind of added on to that. The number of calls that the department had to take was like a 600% increase and it was people wanting answers when we didn't have answers. I've had like a tenfold increase of responsibilities and tasks. Many IPs reported that they once again had to drop routine IPC duties, but they did not believe they did this in a strategic way. They described running from crisis to crisis without time to think through which duties could be safely dropped or delegated to another individual or team. This sometimes led to critical mistakes being made by non-IPs engaged in IPC work. As IPs described:I think that's what myself and my team are struggling with is the balance of solving the immediate fires. We're not preventing any fires from happening. We're not putting any fire mitigation strategies in place. We're just putting out the fires that keep popping up. I just went from one [pandemic crisis] to another. I didn't feel like an infection prevention and control nurse. I just felt like an infection control nurse. I can't focus on rounding. I can't do normal infection prevention stuff because there are so many employee health issues. Our supply chain managers, their intent is good, but [they make IPC mistakes]. Take for example, we ran out of kits that had sterile urine collection cups. So, they went and started putting new kits together, [but] they were using nonsterile cups in what are supposed to be sterile urine collection kits. So, even though their intent was good, that can cause greater problems. Some IPs described effective ways of easing the COVID-19 and IPC burden. Some IPs’ facilities used perioperative staff and quality nurses to help with contact tracing, IPC audits, or surveillance. IPs described practices that worked for their facility:We had staff and nursing leadership fill some of those roles that don't take an IP's expertise, like rounding on foleys. And we can leverage [IPC] champions and others for some of that boots-on-the-ground work…or conduct audits using clinical experts in those practices. I contracted out surveillance for the six federally reported types of infections. That was probably one of the best decisions I made because the IPs really would not have been able to keep up with it. We used some quality analysts…to actually do surveillance, so we could free up the IPs. One thing that has been helpful is that we hired three IP infection prevention associates. They have been instrumental in working with us and helping take some of the load off, so that we can do the actual prevention activities. Challenges Due to Contact Tracing A frequently discussed challenge for the IPs related to the time and effort needed to conduct contact tracing within their healthcare facility or agency. Many IPs reported that they were responsible for conducting contact tracing for patient and staff exposures, including community exposures when public health officials lacked the resources to do it. However, a few indicated that their facility had stopped contact tracing because the source of exposure was less clear. As one IP stated, “One of the reasons we stopped is that we couldn't determine if [staff] developed COVID from their employment or from out in the community.” The following quotes relate to the challenges IPs faced around contact tracing:IP was responsible for all contact tracing of both staff and patients, so it seemed like that was all I did for over 2 months. We had an outbreak and at the time the transmission rate was so high in our community that our public health system was just overwhelmed. So, it was basically you figure it out. You contact all these people or they're not going to get contacted. So unfortunately, that's what your focus becomes and the rest of the important parts of infection prevention get put aside. We were doing all of the contact tracing and it was incredibly tedious. I think that was part of our overwhelming fatigue. We could not keep up with it….getting 19, 20, 30 cases every day. The health department was no benefit. They just threw up their arms and said, “Community prevalence is so high that we're not doing contact tracing anymore.” So we are doing it, but it's just too much of a burden on our staff. Anger Towards Healthcare Staff & IPs Many IPs reported a very unpleasant shift in attitude among patients and visitors towards healthcare personnel after the Delta variant emerged. As the pandemic continued into year two, members of the general public became less willing to follow healthcare COVID-19-related restrictions or answer questions about their vaccination status, and their behavior even escalated into being angry, rude, and unsafe. This was particularly distressing to some IPs given that healthcare personnel were widely complimented and treated well earlier in the pandemic. The IPs described it as follows:It's like we went from healthcare hero to the worst person in the world. We are seeing increased violence in the workplace. People are angry about not being able to visit people, angry about “you must wear a mask when you come in”. Security is working overtime. In rural hospitals, [anger towards healthcare workers] tends to be worse given some of the [politization of the pandemic]. We've had nurses verbally assaulted over and over by family members. ICU nurses are being accused of being part of a conspiracy. It's so difficult for healthcare workers to have been the ones where people left lasagna on your front door and they had people cheering in the parking lot when night shift left, and then that ended during Delta. A month ago is probably like the lowest point I know for some of our staff. We had an overcrowded ED and Delta was surging, and we had a bunch of hall beds…just a really crowded situation. And in unison, the patients just started tackling [the staff] and yelling at them. The community is getting very angry with our staff for various reasons. Just asking vaccination status throws some over the edge. The IPs reported that they were also witnessing anger and unprofessional behavior from their healthcare staff towards each other and towards the IP. As IPs described:There's a lot of division right now. There's division in the country, but it's also in the facility. And you need to work as a team. It doesn't work if you're not a team. [My staff said,] “We don't need to wear that PPE anymore. You're just an infection preventionist.” You are just constantly challenged and that's an environment that we have not had to work in. In the beginning of the pandemic, we were heroes, where everyone was listening to every single word that came out of our mouths, but now you are challenged on every single thing that you say from wearing eye protection to maintaining proper mask wearing. Healthcare staff were just blatantly not listening to what we're saying about personal protective equipment and just challenging everything that we say. Everyone's tolerance to errors and triggers is much lower, so our issues with civility among staff is a challenge. Positive Changes That Have Resulted From the Pandemic The IPs discussed a number of positive changes that had developed during the second year of the pandemic. Availability of vaccine resulted in lower morbidity and mortality rates among the most vulnerable patients, which made a huge difference in staff morale, especially in long term care settings. As one IP said, “We did not see very many large outbreaks compared to the previous waves, and the long-term care residents were highly vaccinated and did much better. We saw a lot less deaths and hospitalizations, which was awesome.” One of the most frequently reported silver linings from the pandemic by IPs was the recognition within their administration, teammates, and colleagues about the importance of IPC. As IPs described:I feel like IP is finally being valued. This is my 37th year in infection prevention and control, and I have never been more appreciated than I have this past year and a half. I am thrilled with how much our staff appreciate Infection Prevention now, even though it should have been so before?;) [From a chat box during a focus group session] There's nothing like a pandemic to show the value of infection prevention and control. The IPs pointed out the better internal collaboration and external coordination of care across regions that has resulted from the pandemic. As IPs described:I've never felt more connected to our team and stakeholders. In our state, all of our health systems competed for everything [before the pandemic], but we all work together right now. We're all trying to help, and we do it by county, so we all meet together and that's really cool. That never happened. It's sad that it took a pandemic to do that, but hopefully we can keep those things going, because it really helps take care of our community overall. Our state has set up triage where if we don't have any room at our hospital, there's a phone tree that we can call. We will find that person a bed somewhere within the state, it doesn't have to be within our system. That has been a real positive that I've experienced. Discussion This study identified a number of continued challenges raised by previous focus group participants examining IP experiences during the COVID-19 pandemic.1 , 2 IPs reported ongoing high workloads with the return of voluminous COVID-related work after emergence of the Delta variant along with the expectation to continue usual IPC work. This was worsened by high-acuity patients frequently being admitted to their facilities due to postponements in care during earlier times of the pandemic, challenged further by ongoing staffing and supply shortages. Though the IPs reported that some work was shed from their usual responsibilities to accommodate pandemic response duties, they believed this was not always done in a strategic manner. It is essential that IPs’ duties are systematically evaluated and categorized by priority so that less critical work may be delegated to other healthcare staff or dropped temporarily when IPs are overwhelmed due to disaster response. Preliminary findings from these focus groups were used to aid in development of a tool to guide IPs and healthcare leaders in making decisions regarding prioritization of IPC duties. In response, APIC developed the IPC Acuity Scale for Crisis Situations6, intended to be used alongside a facility's risk assessment in order to help guide prioritization of IPC program work and maintain patient and staff safety. IPs and healthcare leaders should collaborate on how to use the IPC Acuity Scale for Crisis Situations or similar tool to determine the best use of IPs’ specialized knowledge and skills, while delegating or temporarily halting less essential IPC duties during the remainder of the COVID-19 pandemic or other disasters. As was seen during the early part of the pandemic, frequently changing IPC protocols in healthcare facilities remained a challenge for IPs. The stress of rapidly changing protocols reported by IPs in this study aligns with the experiences shared by healthcare workers. Vacillating between looser and more restrictive protocols creates anxiety and stress for IPs and front-line healthcare workers, and in turn can generate anger and distrust towards the IP. Nori et al.7 described dyssynchrony between IPC guidance in healthcare compared to public settings, resulting in confusion and tension between healthcare workers and leaders. Romeu-Labayen et al.8 identified a global theme of changing PPE protocols as a source of confusion and discomfort among healthcare workers, sometimes not coinciding with the actual availability of PPE. Gray et al.9 examined the experiences of clinical nurses providing care to COVID-19 patients and found that their major stressors related to uncertainty, inconsistent PPE protocols, and fear caused by frequently changing information. Robinson et al.10 reported that nurses in rural settings perceived their work environment to be chaotic due to frequently changing protocols, and this led to role frustration. Updating protocols as science evolves is not atypical for outbreaks of emerging pathogens and pandemics1 , 11 , 12, and this should be expected to continue as the U.S. moves out of the acute pandemic phase towards an endemic state with periodic outbreaks of COVID-19. The expectation of changing protocols as science evolves needs to become normalized among healthcare personnel to reduce frustration and stress. In addition to the tension and stress between IPs and healthcare personnel, IPs in this study reported witnessing escalating anger and frustration between healthcare workers and patients or visitors. This is likely due to multiple stressors, including frequently changing IPC protocols, diminished hope related to vaccine breakthrough and uptake, misunderstanding of IPC protocols for vaccinated and unvaccinated healthcare workers, increased workloads for all healthcare workers, staff shortages, and diminished healthcare resources such as PPE and medical supplies. It may also be related to pandemic fatigue and the desire among many to return to pre-pandemic healthcare protocols, and frustration that COVID-19 mitigation strategies are still in place. Despite the reasons, this escalating anger and violence towards healthcare personnel is not acceptable. IPs reported experiencing verbal assaults and sometimes unsafe situations towards and among healthcare workers. Hollingsworth and Schulte13 reported this hostile behavior towards healthcare personnel beginning as early as fall of 2021. Patients and visitors have responded with threats and violence towards healthcare staff when they disagreed with healthcare facility policy regarding COVID-19 protocols.13 IPs should partner with their human resource departments to engage healthcare personnel in workplace violence prevention training, such as that used with emergency department professionals.14 Examples include training on how to de-escalate situations and individuals, manage a violent individual, and/or implement self-defense maneuvers.14 , 15 IPs in this study discussed multiple challenges related to the COVID-19 vaccine, including opposition to vaccination requirement policies among some healthcare personnel, masking policies based on vaccination status, and difficulties in communicating with those who are vaccine hesitant. Many IPs witnessed an increase in PPE noncompliance after vaccination of healthcare personnel. This may have contributed to occupational exposures and illness seen among healthcare staff, especially after the Delta variant emerged and proved to have the ability to evade vaccine-induced immunity.16 Of all the vaccine-related challenges discussed by the IPs, the most difficult was the shift from hopefulness when the vaccine first became available to a sense of hopelessness when there were many more cases of vaccine breakthrough than anticipated. Hope was even associated with willingness to get vaccinated. Adam et al.17 surveyed nearly 12,000 U.S. adults and found that hope was positively associated with vaccine uptake and confidence. Vaccine hesitancy, liberalized vaccine exemptions to maintain healthcare worker staffing, reported low community vaccination rates in some areas, and vaccine breakthrough were identified as sources for hope diminishment for IPs. Although new variants, including Delta and Omicron, have been found to be associated with lower vaccine effectiveness compared to the ancestral strain, COVID-19 vaccine remains an essential protective measure to minimize the risk of severe illness, hospitalization, and death.16 , 18 , 19 Although IPs in this study reported feelings of frustration, being overwhelmed, fatigue, and hopelessness, IPs also reported positive changes that have emerged. Despite hopes for vaccine development being diminished by their inability to completely prevent COVID-19 infection or transmission, the ability of vaccination to greatly reduce the risk of hospitalization and death was viewed as a positive result. In addition, healthcare staff vaccination was found to be associated with lower infection rates among long-term care facility residents.20 Data from 12,364 nursing homes in the U.S. indicated that long-term care facilities with high rates of staff vaccination were associated with lower resident COVID-19 cases and deaths between June 13 through August 22, 2021.20 IPs and other healthcare leaders should continue to encourage healthcare personnel and the general public to stay up-to-date on COVID-19 vaccination to minimize the risk of severe illness and limit COVID-related healthcare surges. This study identified challenges IPs faced during the second year of the COVID-19 pandemic after the Delta variant emerged. Using qualitative methods via focus groups helped richly convey these experiences. However, limitations must also be noted. Only APIC members were eligible to participate; therefore, findings may not be generalizable to IPs who are not APIC members or to non-participating APIC members. Although the IP participants were reflective of APIC members as a whole in regard to their racial diversity, the sample did lack diversity. IPs from minority groups may have had different perceptions than those presented in this study. Conclusion The second year of the pandemic continued to challenge infection preventionists who have served at the frontlines during COVID-19 response. This study identified challenges that occurred in 2021 after the Delta variant emerged, including the need to vet personal protective equipment, fluctuating pandemic restrictions that caused confusion and pushback, staffing and medical supply shortages, hope turning to despair when there were more cases of COVID-19 vaccine breakthrough than anticipated, overwhelming workloads, and anger from the general public towards healthcare personnel and infection preventionists. Conversely, infection preventionists reported gratitude that mortality rates dropped among the vaccinated, they felt more valued by leadership, and noted increased internal collaboration and external coordination of care. The infection prevention and control field needs to address the newly identified pandemic response gaps. Acknowledgement The authors would like to thank the APIC COVID-19 Task Force (in alphabetical order): Rebecca Alvino, Pam Falk, Liz Garman, Jill Holdsworth, Kathleen McMullen, Silvia Quevedo, Terri Rebmann, Barbara Smith, and Lisa Tomlinson. Thanks also to Monica Alexander from APIC for aiding with focus group facilitation. ==== Refs References 1 Rebmann T Alvino RT Mazzara RL Sandcork J. Infection preventionists' experiences during the first nine months of the COVID-19 pandemic: Findings from focus groups conducted with Association of Professionals in Infection Control & Epidemiology (APIC) members Am J Infect Control 49 2021 1093 1098 34454681 2 Rebmann T Alvino RT Mazzara RL Sandcork J. Rural infection preventionists' experiences during the COVID-19 pandemic: Findings from focus groups conducted with association of professionals in infection control & epidemiology (APIC) members Am J Infect Control 49 2021 1099 1104 34454682 3 Edwards E Bennett G. Alba M. Fully vaccinated? You can ditch the mask, CDC says 2021 NBC https://www.nbcnews.com/health/health-news/cdc-plans-drop-mask-requirements-fully-vaccinated-people-n1267249 4 Maragakis L. Coronavirus second wave, third wave and beyhond: What causes a COVID surge. In: Medicine JH, editor. https://www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus/first-and-second-waves-of-coronavirus 2021. 5 Tomlinson, L. Infection Preventionists: Overview and Workforce Needs. Personal communication via report submitted to Presidential Advisory Council on Combating Antibiotic Resistant Bacteria (PACCARB). September 22, 2022. 6 Association for Professionals in Infection Control and Epidemiology. Infection Prevention and Control Acuity Scale for Crisis Situations. https://apic.org/ipc-acuity-scale-for-crisis-situations/2021. 7 Nori P Stevens M. Patel P. Rising from the pandemic ashes: Reflections on burnout and resiliency from the infection prevention and antimicrobial stewardship workforce Antimicrobial Stewardship & Healthcare Epidemiology 2 2022 E101 8 Romeu-Labayen M, Alvarez, B, Block, E, Cabral, JR, Diallo, M, Djellouli, N, et al. Protecting and feeling protected: HCWs’ experiences with personal protective equipment during the COVID-19 pandemic. In: Vindrola-Padros C, Johnson, G.A., editor. Caring on the Frontline during COVID-19. Palgrave Macmillan, Singapore2022. 9 Gray K Dorney P Hoffman L Crawford A. Nurses' pandemic lives: A mixed-methods study of experiences during COVID-19 Appl Nurs Res 60 2021 151437 10 Robinson KR Jensen GA Gierach M McClellan C Wolles B Bartelt S The lived experience of frontline nurses: COVID-19 in rural America Nurs Forum 57 2022 640 649 35445432 11 Rebmann T Wagner W. Infection preventionists' experience during the first months of the 2009 novel H1N1 influenza A pandemic Am J Infect Control 37 2009 e5 e16 20004810 12 Drehle DV. The new Ebola protocols. Time. https://time.com/3512672/the-new-ebola-protocols/2014. 13 Hollingsworth HaS G. Health workers once saluted as heroes now get threats. AP News 2021 AP News https://apnews.com/article/coronavirus-pandemic-business-health-missouri-omaha-b73e167eba4987cab9e58fdc92ce0b72 14 D'Ettorre G Pellicani V Mazzotta M Vullo A. Preventing and managing workplace violence against healthcare workers in Emergency Departments Acta Biomed 89 2018 28 36 15 Wirth T Peters C Nienhaus A Schablon A. Interventions for Workplace Violence Prevention in Emergency Departments: A Systematic Review Int J Environ Res Public Health 18 2021 16 Fowlkes A Gaglani M Groover K Thiese MS Tyner H Ellingson K Effectiveness of COVID-19 Vaccines in Preventing SARS-CoV-2 Infection Among Frontline Workers Before and During B.1.617.2 (Delta) Variant Predominance - Eight U.S. Locations, December 2020-August 2021 MMWR Morbidity and mortality weekly report 70 2021 1167 1169 34437521 17 Adam M Mohan D Forster S Chen S Gates J Yu F Hope as a predictor for COVID-19 vaccine uptake in the US: A cross-sectional survey of 11,955 adults Human Vaccines & Immunotherapeutics 18 2022 18 Andrews N Stowe J Kirsebom F Toffa S Rickeard T Gallagher E Covid-19 Vaccine Effectiveness against the Omicron (B.1.1.529) Variant N Engl J Med 386 2022 1532 1546 35249272 19 Pilishvili T Gierke R Fleming-Dutra KE Farrar JL Mohr NM Talan DA Effectiveness of mRNA Covid-19 Vaccine among U.S. Health Care Personnel N Engl J Med 385 2021 e90 34551224 20 McGarry BE Barnett ML Grabowski DC Gandhi AD. Nursing Home Staff Vaccination and Covid-19 Outcomes N Engl J Med 386 2022 397 398 34879189	36463974	PMC9714080	NO-CC CODE	2022-12-16 23:21:38	no	Am J Infect Control. 2022 Dec 1; doi: 10.1016/j.ajic.2022.11.023	utf-8	Am J Infect Control	2,022	10.1016/j.ajic.2022.11.023	oa_other
==== Front J Contextual Behav Sci J Contextual Behav Sci Journal of Contextual Behavioral Science 2212-1447 2212-1455 The Authors. Published by Elsevier Inc. on behalf of Association for Contextual Behavioral Science. S2212-1447(22)00123-5 10.1016/j.jcbs.2022.11.010 Article Magis – A magical adventure: Using a mobile game to deliver an ACT intervention for elementary schoolchildren in classroom settings Keinonen K. a∗ Lappalainen P. a Kotamäki-Viinikka S. b Lappalainen R. a a University of Jyväskylä, Jyväskylä, Finland b University of Vaasa, Vaasa, Finland ∗ Corresponding author. 1 12 2022 1 2023 1 12 2022 27 2633 12 7 2022 28 11 2022 30 11 2022 © 2022 The Authors 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Studies of the effects of the COVID-19 pandemic have shown that this health emergency has affected especially young people. Supporting the well-being of children is thus particularly urgent. However, the high prevalence of ill-being among children requires novel approaches to providing help. Health care resources are limited, and many children did not receive support even before the pandemic. The current study presents a novel approach to delivering brief interventions for school-aged children. A mobile game based on acceptance and commitment therapy was used to increase psychological flexibility and well-being among 10 to 12-year-old schoolchildren. A sample of 106 students played the game in four weekly sessions as part of normal teaching practice in school. The effectiveness of the brief game intervention was examined as a universal intervention among the whole sample and among subgroups created on the basis of baseline psychological flexibility (i.e., based on the need for an intervention). The results show that higher psychological flexibility was associated with less emotional and behavioral problems, higher health-related quality of life, mood, and school satisfaction, and less loneliness (r = 0.46–0.63). While a significant effect was not detected in the whole sample, the subsample of children with initially high psychological inflexibility benefitted from participating in the intervention (Cohen's d = 0.35). These preliminary findings suggest that the brief game-based intervention can increase psychological flexibility among children when the need for an intervention is considered. Further research is necessary to examine the stability of improvements in psychological flexibility. Keywords Acceptance and commitment therapy Therapeutic games Children School-based intervention Brief intervention ==== Body pmc1 Introduction A large portion of children and adolescents experience psychological problems. International research literature suggests that up to 20% of children and adolescents develop clinically significant depressive or anxiety symptoms by the age of 18 (Grist et al., 2019). Additionally, the global prevalence for psychological disorders has been estimated at 13.4% (Polanczyk et al., 2015). Despite these findings showing a need for mental health services, most children and adolescents receive no treatment (Kazdin, 2019). Alarmingly, in some reports, only approximately 50% of adolescents who experience severely impairing psychological disorders have received treatment (Merikangas et al., 2011). Furthermore, recent reports have suggested that the COVID-19 pandemic may have increased the need for psychological help among children and adolescents (e.g., Hafstad et al., 2021 ; Rogers et al., 2021). Large survey studies have found that the pandemic has harmed the quality of life and mental health of children and adolescents and that factors such as low socioeconomic status and limited living space can increase the risk of psychological impairments (Ravens-Sieberer et al., 2021). The high rates of child and adolescent mental health problems and low rates of young people who receive support call for new models and interventions to improve access and prevent the development of mental health disorders and improve well-being (Eyre & Thapar, 2014). Schools can identify children in need of support or treatment. However, developing new ways to offer early interventions is necessary to help them respond efficiently. This is particularly important as surveys indicate that many schools do not use evidence-based prevention programs to address behavioral and emotional diﬃculties in children (Gottfredson & Gottfredson, 2002; Ringwalt et al., 2009). Among the children and adolescents who do receive psychological help, up to 80% do so in school (Merikangas et al., 2011). In addition to psychological treatment, milder problems might also benefit from preventive interventions in nonclinical settings, particularly in schools and colleges (Kieling et al., 2011; Patel et al., 2007). Furthermore, the ability of school psychologists and other school-based professionals to meet the mental health needs of schoolchildren is limited (Forman, 2019). It is thus necessary to consider alternative methods, including teachers delivering a mental health prevention program (Domitrovich et al., 2019; Forman, 2019). One approach to increasing access to psychological support is to utilize technology-based support models (Hollis et al., 2015). Some research is available on previous universal programs to promote well-being. Large-scale programs delivered in schools have yielded mixed results. For instance, a large school-based universal intervention program targeted the resilience factors that reduce mental health problems in adolescents by having intervention schools provide 9 h of resilience-focused content as part of teaching activities and additional 9 h of resilience-focused content as part of other school activities (Dray et al., 2017). The study found no significant difference in mental health outcomes at follow-up between the intervention and the control group among the 12 to 16 years-old adolescents (Dray et al., 2017). Other universal, school-based interventions aimed at enhancing well-being have reported non-significant effects (e.g., Burckhardt et al., 2017; van der Gucht et al., 2017). However, school-based interventions where participants have been selected based on their need for an intervention have offered evidence of significant reductions in depression among high school students with elevated symptoms (Burckhardt et al., 2016). There is thus a need for more research examining preventive interventions that promote mental health and improve well-being in adolescents. It is possible that a ceiling effect in measures of symptoms and well-being can potentially cause difficulties in detecting the effects of universal prevention programs offered to young people many of whom do not experience symptoms. Thus, it may be important to pay attention to the variation and individual effects in school-based samples. One approach to developing programs that are effective and easy to disseminate is using e-health technologies, such as games, which can extend the reach of mental health prevention and treatment (Kazdin & Blase, 2011). Using technology and mobile devices (e.g., smartphones, tablets) to deliver preventive programs offers new and flexible ways to reach large numbers of children and adolescents (Edwards‐Hart & Chester, 2010). There is some evidence of technology-based interventions’ effects in alleviating symptoms among young people. For instance, a meta-analysis investigating seven internet-based interventions for children and adolescents aged between 7 and 25 years indicated that such interventions, which included several types of delivery methods, were effective in reducing anxiety and ineffective in reducing depression (Ye et al., 2014). However, other reviews have suggested that internet-delivered interventions can be beneficial for both depression and anxiety. Clarke et al. (2015) review reported that while the number of high-quality studies investigating mental health outcomes was small, there was support for the efficacy of computerized cognitive behavioral therapy in reducing depression and anxiety among adolescents. In addition, Välimäki et al. (2017) have similarly reported in their review that web-delivered interventions are beneficial in reducing depression and anxiety among 10–24 years old participants, though more research is needed to evaluate their long-term effects. Moreover, the acceptance and commitment therapy-based Youth Compass intervention has been found to be effective in alleviating depression (Lappalainen et al., 2021) and stress (Puolakanaho et al., 2019) among adolescents. In addition to internet-delivered interventions, mobile games hold great potential for enhancing motivation, interest, interaction, and engagement in children and adolescents (Koutromanos & Avraamidou, 2014). As games are originally a form of play or recreation, the term serious games is used to refer to games that are designed to achieve a purpose besides entertainment (Baranowski et al., 2016; Ritterfeld et al., 2009). Therapeutic mobile games are one form of serious game that can engage and motivate children and adolescents and offer them the opportunity to be exposed to contingency-based learning and acquire skills they can practice (Kato et al., 2008). The advantages of ease of use, accessibility, and interactivity in the gamification process may make therapeutic games suitable tools for promoting mental health among children and adolescents (David et al., 2020). Therapeutic online games can be used both to prevent and address mental health concerns or health-related behavioral changes. However, as serious games have been seldom investigated, the evidence on them is mainly qualitative or focused on playability and acceptability (David et al., 2020). A recent systematic review and meta-analysis, which included 34 controlled studies, examined the efficacy of serious games in reducing symptoms of mental disorders and promoting health-related behavioral change (David et al., 2020). It found a small main statistical effect in favor of the intervention group receiving serious game intervention compared to the control group. These results suggest that serious games are likely not yet ready for dissemination as a stand-alone treatment/prevention strategy or as an adjunct to treatment-as-usual interventions aimed at reducing symptoms or promoting mental health and health-related behavioral change in children and adolescents (David et al., 2020). These findings call for the further development of technology-based interventions to help young people who experience psychological problems. One potential approach to developing universal prevention programs for young people is acceptance and commitment therapy (ACT; Hayes et al., 2009), which has been shown to be effective in reducing psychological distress and behavioral problems and enhancing general well-being among children (Fang & Ding, 2020). ACT is a transdiagnostic third-wave cognitive behavioral therapy (CBT). Its goal is to increase psychological flexibility (i.e., the flexibility in responding to thoughts, feelings, and sensations), thus increasing the behavioral repertoire of an individual (Hayes et al., 2013). This is achieved through six core therapeutic processes: values clarification, committed action, acceptance, mindfulness, cognitive defusion, and self-as-context (Hayes et al., 2012). The construct of psychological flexibility is well suited for prevention purposes as ACT aims to enable living a meaningful life—not just reduce symptoms (Biglan et al., 2008). When applying ACT among adolescents and children, developmental adaptations are required to ensure age-appropriateness, such as increased behavioral activation, the inclusion of parents or peers, and tailored examples and explanations to support the understanding of abstract concepts (Halliburton & Cooper, 2015). It has also been suggested that children may benefit from the active use of metaphors and experiential exercises, which may allow them to grasp theoretical notions through experience (O'Brien et al., 2008). In the case of young people, ACT may remediate and prevent the onset of psychologically inflexible responses to inner events that lead to experiential avoidance strategies (Swain et al., 2015). The use of ACT among adults has produced evidence of positive outcomes for a wide range of mental and physical health conditions and lifestyle changes (A-Tjak et al., 2015; Ruiz, 2010). However, there are limited data on applying ACT among children and early adolescents, with most of it coming from small samples and case studies. One of the few available reviews and meta-analyses has shown that a very small number of studies have included younger children and that most have focused on adolescents over 13 years of age (Swain et al., 2015). According to a review by Halliburton and Cooper (2015), ACT may be effective for adolescents with chronic pain, anorexia, depression, obsessive–compulsive disorder, post-traumatic stress disorder, stress, disruptive behavior disorders, learning disorders, and autism spectrum disorders. Others have concluded that ACT produces significant improvements in the majority of self- and clinician-reported clinical outcomes across presenting problems. However, limited research is available on ACT's core mechanisms of change among children (Swain et al., 2015). Finally, a recent meta-analysis, which included 14 randomized controlled trials with participants aged 2–21, found that among young people, ACT is more effective than treatment-as-usual or no treatment for depression, anxiety, and other mental and behavioral problems. It is not, however, superior to traditional CBT (Fang & Ding, 2020). In conclusion, more research on the effectiveness and underlying mechanisms of change in ACT among young people, particularly younger children, is necessary to draw strong conclusions. More research is also needed on technology-based ACT interventions for children and adolescents. The current study sought to evaluate the use of ACT in school settings to provide engaging mental health prevention programs to children. Specifically, the study wished to examine the effectiveness of a mobile game that uses dialogues and problem-solving tasks in introducing ACT-based conversations and exercises in a classroom setting. We thus investigated whether a four-session ACT intervention, which included the mobile game and game-related written tasks, was effective in improving psychological flexibility and well-being in a nonclinical sample of 10 to 12-year-olds. Furthermore, we were interested in examining the association between experiential avoidance and well-being among school-aged children at a general level (before the intervention was provided) and in relation to the observed changes during the intervention. The study asked the following research questions:1) Is the level of psychological inflexibility among 4th–6th-grade schoolchildren associated with the level of emotional and behavioral problems and health-related quality of life (HRQoL) and with self-rated mood, school satisfaction, loneliness, or friendships? 2) Is the game-based ACT intervention effective in decreasing psychological inflexibility and improving well-being among children who report high inflexibility at baseline? Prior to the main analyses we examined the baseline level of the outcome variables and if there were baseline differences between the intervention groups and the control groups or between male and female participants that would require attention before performing the analyses. We hypothesized that psychological inflexibility would be associated with lower well-being and higher emotional and behavioral problems. Furthermore, we hypothesized that the intervention would be effective in decreasing inflexibility among those children who reported high inflexibility at baseline. Based on previous studies of universal, preventive interventions for well-being, we assumed that the intervention effect would be difficult to detect among children who do not report high inflexibility due to a floor effect on the measure of psychological inflexibility. Thus, we expected no intervention effect among children with already good flexibility skills (low inflexibility). 2 Methodology The study was conducted in the fall of 2020 in elementary schools (grades 4–6) in Vaasa, Finland. The study was reviewed and approved by the University of Jyväskylä ethics committee in June 2020. In May 2020, an invitation letter was sent to all Vaasa regional schools (N = 17), welcoming participation in the study. The letter included information about the study's aims (i.e., promoting children's mental health by teaching well-being skills in schools). In the autumn of 2020, six schools (i.e., principals) agreed to participate in the study. Finally, seven teachers volunteered, and their six classes were recruited (N = 123 children; see Fig. 1 ). In one case, two teachers co-taught their classes, which were thus treated as one class. Written permission to participate in the study was required from both children and parents. Children whose parents declined participation or did not return the consent form (n = 17) did not participate in the study but did play the game, as the game sessions were arranged during regular teaching practice. The six classes were randomly assigned to the treatment group (n = 3) and the control group (n = 3). The mean age of the children in the intervention group was 11.07 and 44% were female. In the control group, the mean age of the children was 11.26 and 57% were female. The treatment group received the game-based intervention immediately after the baseline measures, while the control group only played the game after the posttreatment questionnaires had been collected.Fig. 1 Flow of participants. Fig. 1 3 Intervention The game was used in the classroom, and game sessions were scheduled weekly during regular school hours. The game sessions were led by project workers involved in the research who were very familiar with the game and had playthrough directions in case any of the children got stuck. The teachers of the six participating classes were also present during the game sessions to assist the children. The game has four chapters that each take 30–45 min to complete (see Table 1 for chapter content). Each week, the class played through one chapter. In addition to the game content, the intervention included written tasks to be completed after each game session. The written tasks were designed to highlight important content and facilitate applying the presented material to everyday situations. The written assignments took 10–15 min to complete. For example, one of the assignments for Chapter 2 asked the player to write down which Value Rune they had chosen in the game world's Rune Grove and plan one action they could do that would reflect their chosen value.Table 1 A summary of game content by chapter. Table 1 Content Themes Dialogue examples Chapter 1: Acceptance, self-as-context, values and valued actions, defusion Fear, self-critical thoughts, peer pressure and comparing to others, planning your actions “How can you remember not to listen to your angry thoughts next time?“, “What kind of friend would you like to be?” Chapter 2: Acceptance, self-as-context, valued actions, defusion Bullying, anger and hate, fear “When you go along with your anger, you will often find yourself in trouble.“, “It says to try and locate the feeling in your body. Where do you feel it?” Chapter 3: Acceptance, defusion, values and valued actions Adjusting to change, identifying and noticing your feelings, difficult thoughts, thoughts about others “Now that you've chosen courage, you can choose to be courageous even when afraid.“, “When you feel like there are too many thoughts in your head, you can place them on leaves.” Chapter 4: Self-as-context Responding to your feelings, prosocial behavior “It says: ‘Just like the sky is a place for the weather, you are a place for your feelings.’“, “I'll drink from the potion and describe the feeling. You choose the right label.” Throughout the game: Tracking, changing perspective (deictic framing), normalizing thoughts and feelings, metaphors Helping others who struggle “If I were at the beach and you were on top of the mountain. If here were there and there were here, where would I be?“, “Water can boil or it can freeze. Just like you might laugh or tease. But water is water, be it frozen or boiling. And you are you, no matter what thought in your head you might be voicing.” 4 The game Magis – A Magical Adventure is a mobile game that models psychological flexibility using dialogue and plot. The game has been designed to improve psychological flexibility skills among 10 to 12-year-old children. It was developed by an expert group (XX,XX,XX) with more than 10-year experience of construction and development of digital interventions. The construction was done under the supervision of the last author (XX), an expert in ACT with more than 20 years of experience in teaching, supervising, and practicing ACT. Several prototypes and versions of the game were tested at school context during a period of 6 months. The content, semantics, design, feasibility, and acceptability of the game was evaluated (for example using observations and interviews) and the game was modified and refined based on the feedback. The game asks the player to engage in ACT-based conversations with different characters in the game world (see Fig. 2 ). These conversations model how to process difficult thoughts and emotions, identify personal values, take values-based action, and take another person's perspective (see Table 1 for a summary of the themes). By improving psychological flexibility and related skills, the game also aims to improve overall wellbeing of the players. The game is free and available for download from the Google Play store and the AppStore globally. The supported languages currently include Finnish, Swedish, and English.Fig. 2 Players engage in ACT-based conversations. The example above models how to respond to difficult thoughts. Fig. 2 5 Measures Three questionnaires were used to measure psychological inflexibility (Avoidance and Fusion Questionnaire for Youth, AFQ-Y8), emotional and behavioral symptoms (Strengths and Difficulties Questionnaire, SDQ), and Health-Related Quality of Life (HRQoL/KINDL). Participants also filled out brief self-rating visual analog scales (VAS) for mood, school satisfaction, loneliness, and friendships. Baseline questionnaires were completed during the week prior to the intervention in classrooms in schools. Post-treatment questionnaires were filled out in the week after the intervention also in the classroom environment. The measures were thus completed approximately five weeks apart. The children completed the questionnaires themselves, but the researchers and the teacher helped if necessary. The pen and paper questionnaires were checked to ensure all items had been answered. The few missing responses that were still identified, were replaced with the mean values of the questionnaire's items if at least 50% of the items had been filled in. This strategy was selected based on the recommendation of a statistician who was consulted on the matter of missing data. Thus, for the AFQ-Y8, mean values for items that were rated were used to calculate the total score if at least four out of eight items had been rated. For the SDQ and the KINDL, 50% of each of the subscales were required to have been rated. Most variables had 2-5 missing responses for individual items of the scale. The highest amount of missing item-level responses was the SDQ peer relationships subscale, where nine participants had failed to respond to all items at baseline. However, eight out of these nine participants had responded to at least 50% of the items for the subscale and their score was calculated using the mean value of the items they had rated. Avoidance and Fusion Questionnaire for Youth (AFQ-Y8; Greco et al., 2008) is a measure of psychological inflexibility for children and adolescents. The scale has eight items that focus on the processes of psychological inflexibility, cognitive fusion (e.g., “The bad things I think about myself must be true”), experiential avoidance (e.g., “I am afraid of my feelings”), and inaction or behavioral ineffectiveness in the presence of unwanted internal experiences (e.g., “I stop doing things that are important to me whenever I feel bad”). Each item is rated for how true it feels to the responder on a scale of 0–4, where 0 = not at all true and 4 = very true. The AFQ-Y8 scores are obtained by summing all eight items (min. 0, max. 32). Lower scores indicate better outcomes, that is, less inflexibility (Valdivia-Salas et al., 2017). The internal consistency of the AFQ-Y8 was acceptable in the current sample (α = 0.79, 8 items). The Strengths and Difficulties Questionnaire (SDQ; Goodman, 1997) is a measure of emotional and behavioral problems that focuses on five domains: emotional symptoms (5 items, e.g., “I worry a lot”), conduct problems (5 items, e.g., “I usually do as I am told”), hyperactivity/inattention (5 items, e.g., “I am easily distracted”), peer relationship problems (5 items, e.g., “I am usually on my own”), and prosocial behavior (5 items, e.g., “I usually share with others”). The current study used the self-report version of the scale, which has a slightly modified wording (Goodman et al., 1998). Each item is rated on a scale of 0–2, where 0 = not true, 1 = somewhat true, and 2 = certainly true. The total difficulties score is calculated by summing the score of all the domains except the prosocial one. The total score ranges between 0 and 40, with higher scores indicating more behavioral and emotional problems. The internal consistency of the SDQ in our sample was good (α = 0.82, 20 items). Health-Related Quality of Life (KINDL; Erhart et al., 2009) is a measure of health-related quality of life for healthy and ill children and adolescents. The Kid-KINDL version of the scale for children aged 7–13 was used in the current study. The scale consists of 24 items associated with six dimensions: physical well-being (4 items, e.g., “I felt ill”), emotional well-being (4 items, e.g., “I had fun and laughed a lot”), self-esteem (4 items, “I was proud of myself”), family (4 items, e.g., “I got on well with my parents”), friends (4 items, e.g. “I played with friends”), and everyday functioning (4 items, e.g., “Doing schoolwork was easy”). The additional disease module for ill children was not used in the current study. Each item is rated on a 5-level Likert scale, where 0 = never and 5 = all the time. Higher scores on each dimension of KINDL indicate a higher health-related quality of life. The internal consistency of the KINDL was good in the current sample (α = 0.85, 24 items). VAS scales were used to evaluate mood, school satisfaction, loneliness, and friendships. Regarding mood, participants were asked to rate “How have you felt in the past few days?” on a scale of 1–5 (1 = very bad, 5 = very good). For school satisfaction, participants were asked to rate “How do you like going to school at the moment?” on a scale of 1–5 (1 = not at all, 5 = very much). Concerning loneliness, participants were asked to rate “Do you feel lonely?” on a scale of 1–3 (1 = often, 3 = not at all). For friendships, participants were asked to rate “How many good friends do you have?” on a scale of 1–3 (1 = none, 3 = two or more). 6 Statistical analyses All statistical analyses were performed using the IBM SPSS Statistics software. First, baseline level of the measures and possible differences between the participants allocated in the intervention and control groups were analyzed using independent samples t-tests with the treatment group as a grouping variable. Possible differences between male and female participants were also examined using independent samples t-tests with gender as the grouping variable. The associations between the level of psychological inflexibility and the level of emotional and behavioral problems, HRQoL, self-rated mood, school satisfaction, loneliness, and friendships were examined using Pearson's correlation coefficients. The effect of the intervention was then analyzed using repeated measures multivariate analysis of variance (MANOVA) analysis, first for the psychological questionnaires and then for the VAS scales, with the treatment group as a grouping variable. In further analysis, a baseline psychological inflexibility grouping variable was created by dividing the participants into three groups based on the observed distribution of scores in the current data. The baseline inflexibility classification was then used as a covariate in further repeated measures MANOVA analyses. Finally, the relationship between baseline inflexibility classification and changes during the intervention was examined using parallel paired samples t-test analyses for the classes in both the intervention and the control group. 7 Results 7.1 Baseline psychological inflexibility, HRQoL, and well-being The children in the sample were not identified as having mental health problems, which was reflected in the mean levels of the well-being measures. However, a large variation was observed among the participants. The levels of psychological inflexibility (AFQ-Y8), Health-Related Quality of Life, HRQoL (KINDL), and well-being, including emotional and behavioral problems (SDQ), mood, school satisfaction, loneliness, and friendships (VAS scales), are presented in Table 2 .Table 2 Descriptive values for baseline experiential avoidance, emotional and behavioral problems, quality of life, and the VAS scales for mood, school satisfaction, loneliness, and friends satisfaction. Table 2 N Min. Max. Mean SD 95% CI AFQ-Y8 105 0 26 6.55 5.37 5.58; 7.68 SDQ 104 1 23 9.53 5.47 8.50; 10.64 KINDL 104 56 114 95.26 12.16 92.87; 97.64 Mood 106 1 5 3.99 0.97 3.82; 4.19 School 106 1 5 3.69 0.91 3.51; 3.87 Loneliness 106 1 3 2.68 0.50 2.60; 2.79 Friends 106 1.50 3.00 2.95 0.22 2.91; 3.00 There were no significant differences between participants in the intervention group and the control group on any of the measures. A significant difference was observed between girls and boys in self-rated school satisfaction—girls reported higher satisfaction (M = 3.90 for girls and M = 3.49 for boys, t (102) = 2.34, p = .021). No other gender differences were observed. The gender difference in one of the VAS scales was deemed a minor difference and it was not controlled for in for further analysis. 7.2 Association between psychological inflexibility and measures of well-being A high (r > 0.55) or close to high (r > 0.45) correlation was found between psychological inflexibility, Health-Related Quality of Life, and all the measures of well-being (SDQ and VAS scales) except for friendships (see Table 3 ). In all cases, the direction of the relationship was as expected. The findings suggest that psychological inflexibility was associated with higher psychosocial symptomatology and lower life satisfaction. Psychological inflexibility was also associated with poorer self-rated mood and school satisfaction and higher loneliness. Psychological flexibility was not associated with friendships, however.Table 3 Pearson correlation coefficients for the association between experiential avoidance, psychosocial symptoms, and quality of life at baseline. Table 3 AFQ-Y8 SDQ KINDL Mood School Lonelin. Friends AFQ-Y8 Psychological inflexibility 1 SDQ Emotional and behavioral problems .63a 1 KINDL Health-related quality of life −.54a −.72a 1 Mood VAS −.55a −.61a .78a 1 School VAS −.46a −.48a .63a .60a 1 Loneliness VAS −.46a −.55a .53a .59a .43a 1 Friends VAS −.09 −.20* .25* .27a .12 .38a 1 a Correlation is significant at the .01 level (2-tailed). *Correlation is significant at the .05 level (2-tailed). 7.3 Intervention effect: psychological inflexibility and well-being A repeated measures MANOVA was used to examine between-group differences in changes in psychological inflexibility (AFQ-Y8) and well-being during the intervention, as measured by the SDQ. There were no significant interactions between time and group for any of the psychological questionnaires. Among the self-rated VAS scales, there was a significant interaction for loneliness (F (1,102) = 5.77, p = .018). The descriptive values for the intervention and the control condition suggest that the intervention group experienced a small decrease in loneliness during the intervention, while the control group experienced an increase in loneliness. To further examine individual differences and if the large variation in baseline psychological inflexibility was associated with changes during the intervention that targeted flexibility skills, additional analyses were conducted. We expected that the effect of the intervention would be different among participants with a lower/higher need for psychological flexibility skills training. The participants were divided into three groups based on the distribution of the AFQ-Y8. Participants who scored 0–3 on AFQ-Y8 were allocated to group 1 (flexible kids, 35.2%). Participants who scored 3–7 points were allocated to group 2 (moderately inflexible kids, 32.4%). Finally, participants who scored 8–26 were allocated to group 3 (highly inflexible kids, 32.4%). The third group's level of psychological inflexibility (≥8 points) corresponds to the mean level of inflexibility reported in school-based samples (M = 8.93, SD = 6.32, N = 515; Greco et al., 2008). This suggests that our sample had less psychological inflexibility than that reported in larger samples in this age group. For the current analysis, the grouping was used as a covariate in a repeated measures analysis of variance (ANOVA) analysis. A significant interaction was thus found between time and the baseline level of psychological inflexibility (F (1,102) = 9.58, p = .003). However, the interaction between group (intervention/control) and time was not significant (F (1,102) = 2.33, p = .130). The descriptive values for pre- and post-measurements for the intervention group and the control group are presented in Table 4 .Table 4 Descriptive values for the pre- and post-measures of behavioral and emotional problems, quality of life, self-rated mood, school satisfaction, loneliness, and friendship satisfaction. Table 4 Pre-measurement Post-measurement Intervention group M (SD) Control group M (SD) Intervention group M (SD) SDQ 9.55 (5.53) 9.51 (5.47) 10.50 (6.48) 9.31 (5.08) KINDL 94.81 (12.40) 95.74 (11.99) 94.06 (14.53) 94.64 (12.34) Mood 3.94 (1.00) 4.08 (0.92) 4.06 (1.10) 4.05 (0.78) School 3.62 (0.97) 3.79 (0.86) 3.59 (1.13) 3.74 (0.96) Loneliness 2.71 (0.49) 2.67 (0.52) 2.78 (0.43) 2.54 (0.56) Friends 2.94 (0.27) 2.97 (0.16) 2.90 (0.32) 2.98 (0.14) As hypothesized, we expected to detect changes among children who reported high inflexibility. To examine how baseline inflexibility was associated with changes during the intervention, the intervention group and the control group were analyzed separately using paired-samples t-tests with the baseline inflexibility grouping. The results suggest that participants in the intervention group who were highly inflexible (group 3) at baseline experienced significant decreases in psychological inflexibility during the intervention (t (16) = 1.86, p = .041, d = 0.35). This effect was not observed in the control group. However, participants who were classified as flexible kids at baseline experienced a statistically significant increase in psychological flexibility (t (21) = −2.43, p = .012, d = 0.29). This finding suggests that participants with very low inflexibility may experience a measuring effect that influences how they evaluate their flexibility skills. This conclusion is supported by the findings of the control group. Similarly, participants in the control group who reported very low inflexibility at baseline (score 0–3) experienced an increase in inflexibility during the waiting-list period (t (14) = −2.21, p = .022, d = 0.15). The within-group changes are presented in Fig. 3 . In both the intervention and the control group, the level of inflexibility remains very low despite the statistically significant increases among participants who report very low inflexibility at baseline.Fig. 3 Changes in psychological inflexibility during the brief game-based intervention among flexible kids, moderately inflexible kids, and highly inflexible kids (nine points cutoff). Fig. 3 8 Discussion This study aimed to examine the effects of a brief, game-based intervention on the psychological flexibility skills of 10 to 12-year-old children (n = 106). The intervention was delivered in school settings, and the children represented a nonclinical, school-based sample who received the intervention as part of usual teaching activities. Large variation was observed in measures of psychological inflexibility and well-being. The results suggest that psychological inflexibility was associated with health-related quality of life, emotional and behavioral problems, self-rated mood, school satisfaction, and loneliness. The observed associations indicate that psychological flexibility skills may be an important intervention target among schoolchildren. As for the effects of the intervention, we did not detect an effect on psychological flexibility or health-related quality of life among the whole intervention group. However, the results suggest that the intervention was effective in alleviating self-rated loneliness in the intervention group. More research is necessary to understand the mechanism of change underlying the observed effect on loneliness. In further analyses, the intervention was found to have different effects on psychological inflexibility among children with varying levels of psychological inflexibility at baseline, i.e., among children with varying needs for the intervention. A significant interaction between time and baseline psychological inflexibility was found in further analyses that aimed to understand the development of flexibility skills during the intervention. Parallel analysis of flexible kids, moderately inflexible kids, and highly inflexible kids suggests that the intervention was effective in decreasing psychological inflexibility among children who had high inflexibility at baseline, but not among flexible kids or moderately inflexible kids (score 0–7 in the AFQ-Y8) before the intervention. This finding is in line with previous research on universal interventions for nonclinical samples that have failed to demonstrate significant effects among children who have not been identified as having low well-being (e.g., Burckhardt et al., 2017; van der Gucht et al., 2017). Two factors should be considered when interpreting these results. First, the available scales may not be sensitive enough to detect changes among participants who have high well-being. Second, it may be more important to demonstrate that universal interventions are effective among children who do experience emotional or behavioral problems than to increase already high well-being. Schools play a central role in supporting children and adolescents, but resources to identify risk factors and intervene in developing mental health problems are limited (Forman, 2019). Therefore, scholars have called for technology-based, universal interventions that can be offered to large groups of children and that are effective in alleviating symptoms for those who are at risk or who experience ill-being (e.g., Kazdin & Blase, 2011). The current study suggests that ACT-based mobile games could be one alternative to offering early interventions. Interestingly, the children who scored 0–3 on the AFQ-Y8 at baseline experienced an increase in psychological inflexibility in both the intervention group and the control group. This could reflect a measurement effect (i.e., measuring psychological inflexibility may influence self-ratings of the associated skills). It should be noted that previous reports of 5th–6th-grade schoolchildren samples have reported higher mean levels of psychological inflexibility than those found in the current sample (M = 8.93, SD = 6.32, N = 515 in Greco et al., 2008, vs. M = 6.55, SD = 5.37 in the current sample). Thus, the increase in inflexibility among children with very low scores could reflect more accurate self-ratings after the intervention. It is also possible that the Finnish version of the AFQ-Y8 is less sensitive than the original English scale. Future research should aim to increase sensitivity in the lower range of inflexibility so that the reliability of self-rated scales of psychological inflexibility for young people may be strengthened. This study has several limitations. First, the sample is relatively small; the subsample analysis should thus be interpreted with caution. Second, the data were collected during the COVID-19 pandemic, but the impact of the pandemic and related disruptions to school and home life cannot be accounted for. Third, the current design did not include a follow-up phase; the long-term effect of the intervention cannot thus be established. Finally, the classification for baseline inflexibility was rooted in the observed distribution of the current sample. For this reason, the results should be replicated to understand how higher/lower inflexibility moderates the impact of the intervention in larger samples. Previous research has concluded that technology-based interventions may be effective in alleviating symptoms of poor mental health among children and adolescents (Clarke et al., 2015; Välimäki et al., 2017). However, using serious games as a delivery method for interventions aimed at improving well-being has not yielded significant results. Further investigation has thus been called for (David, 2020). The current study suggests that a brief, game-based ACT intervention can be beneficial to children with existing risk factors for well-being (i.e., high psychological inflexibility). Furthermore, such an intervention may decrease loneliness. Future research should examine how game-based interventions that are motivating, easy to disseminate, and inexpensive for schools could be developed to serve both children with emotional or behavioral problems and children who do not currently exhibit symptoms. If risk factors and early development of mental health problems cannot be efficiently screened and responded to, universal interventions that target unidentified children may be needed to support young people facing difficulties. Data availability Data is available upon reasonable request. Declaration of competing interet Given their role as Editorial Board Members, Lappalainen P. and Lappalainen R. had no involvement in the peer-review of this article and had no access to information regarding its peer-review. All other authors have declared no conflicts of interest. ==== Refs References A-Tjak J.G. Davis M.L. Morina N. Powers M.B. Smits J.A. Emmelkamp P.M. 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Web-based interventions supporting adolescents and young people with depressive symptoms: Systematic review and meta-analysis JMIR Mhealth and Uhealth 5 12 2017 e180 Ye X. Bapuji S.B. Winters S.E. Struthers A. Raynard M. Metge C. …Sutherland K. Effectiveness of internet-based interventions for children, youth, and young adults with anxiety and/or depression: A systematic review and meta-analysis BMC Health Services Research 14 1 2014 1 9 24382312	36471822	PMC9714081	NO-CC CODE	2022-12-06 23:15:54	no	J Contextual Behav Sci. 2023 Jan 1; 27:26-33	utf-8	J Contextual Behav Sci	2,022	10.1016/j.jcbs.2022.11.010	oa_other
==== Front Actas Dermosifiliogr Actas Dermosifiliogr Actas Dermo-Sifiliograficas 0001-7310 1578-2190 AEDV. Published by Elsevier España, S.L.U. S0001-7310(22)01020-1 10.1016/j.ad.2022.05.033 Carta Científico-Clínica Prevalencia y características clínicas de pacientes diagnosticados de escabiosis durante la pandemia producida por el coronavirus de tipo 2 causante del síndrome respiratorio agudo (SARS-CoV-2) en un hospital de tercer nivel. Un estudio descriptivo [[Translated article]]Diagnosis and Clinical Characteristics of Scabies in a Tertiary Care Hospital During the SARS-CoV-2 Pandemic: A Descriptive StudyAguado Vázquez Á. ⁎ Gegúndez Hernández H. Melgosa Ramos FJ. Díaz Corpas T. Servicio de Dermatología, Hospital Universitario Doctor Peset, Valencia, Spain ⁎ Autor de correspondencia 1 12 2022 1 12 2022 22 1 2022 22 5 2022 © 2022 AEDV. Published by Elsevier España, S.L.U. 2022 AEDV Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmc	36464010	PMC9714123	NO-CC CODE	2022-12-02 23:24:33	no	Actas Dermosifiliogr. 2022 Dec 1; doi: 10.1016/j.ad.2022.05.033	utf-8	Actas Dermosifiliogr	2,022	10.1016/j.ad.2022.05.033	oa_other
==== Front Clinical Immunology Communications 2772-6134 2772-6134 The Authors. Published by Elsevier Inc. S2772-6134(22)00030-0 10.1016/j.clicom.2022.11.004 Article Differential peripheral blood mononuclear cell reactivity against SARS-CoV-2 proteins in naïve and previously infected subjects following COVID-19 vaccination Bispo Elizabete Cristina Iseke a Silva-Carvalho Amandda Évelin a Sousa Marielly Reis Resende a Neves Francisco de Assis Rocha b Carvalho Juliana Lott c Arganaraz Enrique Roberto d Saldanha-Araujo Felipe a⁎ a Laboratório de Hematologia e Células-Tronco, Faculdade de Ciências da Saúde, Universidade de Brasília, Campus Darcy Ribeiro, Av. L2 Norte, Brasília, DF 70.910-900, Brasil b Laboratório de Farmacologia Molecular, Faculdade de Ciências da Saúde, Universidade de Brasília, Av. L2 Norte, Brasília, DF 70.910-900, Brasil c Laboratório Interdisciplinar de Biociências, Faculdade de Medicina, Universidade de Brasília, Av. L2 Norte, Brasília, DF 70.910-900, Brasil d Laboratório de Virologia Molecular, Faculdade de Ciências da Saúde, Universidade de Brasília, Av. L2 Norte, Brasília, DF 70.910-900, Brasil ⁎ Corresponding author. 1 12 2022 12 2022 1 12 2022 2 172176 4 11 2022 29 11 2022 30 11 2022 © 2022 The Authors. Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. The decline in vaccine efficacy and the risk of reinfection by SARS-CoV-2 make new studies important to better characterize the immune response against the virus and its components. Here, we investigated the pattern of activation of T-cells and the expression of inflammatory factors by PBMCs obtained from naïve and previously infected subjects following COVID-19 vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins. PBMCs showed low levels of ACE2 and TMPRSS2 transcripts, which were not modulated by the exposure of these cells to SARS-CoV-2 proteins. Compared to S1 and RBD, N-RBD stimulation showed a greater ability to stimulate T-cell reactivity, according to CD25 and CD69 markers. Interestingly, T-cell reactivity was more pronounced in vaccinated subjects with prior SARS-CoV-2 infection than in vaccinated donors who never had been diagnosed with COVID-19. Finally, N-RBD stimulation promoted greater expression of IL-6 and IFN-γ in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. These data suggest that PBMCs from previously infected and vaccinated subjects are more reactive than those derived from just vaccinated donors. Moreover, the N-RBD together viral proteins showed a greater stimulatory capacity than S1 and RBD viral proteins. Keywords SARS-CoV-2 Vaccination Nucleocapsid T-cells ==== Body pmc1 Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused a devastating pandemic (https://covid19.who.int/). Virus entry begins with the viral S1 Spike subunit binding to the cellular angiotensin-converting enzyme (ACE) 2 receptor through the high-affinity receptor-binding domain (RBD). Subsequently, the S2 protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) promoting fusion with the host cell membrane [1]. The pathophysiology of COVID-19 is not fully understood at this time, but acute inflammation and disseminated intravascular coagulation appear as the main causes of mortality worldwide [2]. The immune response to viral infection involves both innate and adaptive immunity components participating in this process, and, depending on the viral load, the production of interferons (IFN) is essential for infection control [3,4]. In the pathophysiology of COVID-19, activated macrophages are the main source of pro-inflammatory cytokines, such as IL-1β, IL-6, IFN-γ, IL-8, and TNF-α, known as “cytokine storm syndrome” [5]. These cytokines activate the acute inflammatory response due to increased endothelial permeability and a chemotactic effect on neutrophils, monocytes, and cytotoxic T-cells. These inflammatory cellular infiltrates in the alveolar lumen subsequently release toxic molecules, leading to diffuse alveolar damage, pulmonary edema, and fibrin deposition into the alveolar space [6]. Despite recent advances in the characterization of COVID-19 pathophysiology, further studies are still needed to better characterize the immune response against the virus and its components. These studies become particularly important considering the advance in vaccination worldwide, and the risk of reinfection [7]. In the present study, we investigated the pattern of T-cell activation and the expression of inflammatory factors by peripheral blood mononuclear cells (PBMCs) from naïve and previously infected subjects following vaccination, after PBMCs stimulation with S1, RBD, and N-RBD SARS-CoV-2 proteins. 2 Methods 2.1 Sample collection and peripheral blood mononuclear cell isolation Peripheral blood samples were obtained from 19 donors at the School of Health Sciences of the University of Brasilia (UnB), including convalescent COVID-19 donors and individuals unexposed to SARS-CoV-2. All donors followed the vaccination protocol established in Brazil. PBMCs were isolated from whole blood by density gradient centrifugation according to the manufacturer's instructions (Histopaque, Sigma-Aldrich, USA). The study protocols were approved by the Institutional Ethics Committee. 2.2 SARS-CoV-2 recombinant proteins and PBMC stimulus The following SARS-CoV-2 proteins were used in this study: SARS-COV-2 Spike Protein (RBD, aa319–541, mFc Tag, Thermo Fisher); SARS-CoV-2 Spike Protein S1 (S1, aa11–682, hFc-His-Tag, Thermo Fisher), and SARS-CoV-2 Nucleoprotein/Spike Protein (N-RBD) (Thermo Fisher). PBMCs were stimulated with 0.5 μg/ml of each recombinant protein [8]. 2.3 Flow cytometry After PBMC stimulation with SARS-CoV-2 proteins for 24 h, the cells were harvested and stained with the following fluorochrome-conjugated antibodies: CD3-APC (BD Pharmingen), CD3-FITC (Invitrogen), CD25-APC (BD Pharmingen), CD25-PerCP (Invitrogen), CD69-PerCP (Invitrogen), CD69-FITC (Invitrogen), CD137-PE (Invitrogen). The expression of the T-cell activation markers was determined in a FACsCalibur Flow Cytometer (BD Bioscience, USA), using FlowJo software 10.0.7. Ten thousand events were recorded for each sample. 2.4 Real-Time PCR Following PBMC stimulation with the SARS-CoV-2 proteins for 72 h, RNA extraction was performed using TRI Reagent (Sigma-Aldrich), following the manufacturer's instructions. The total RNA yield and quality were determined using NanoDrop 1000 spectrophotometer (NanoDrop, USA). Total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermofisher, USA), and real-time PCR was performed using GoTaq Probe qPCR Master Mix (Promega Corp., USA) or GoTaq qPCR Master Mix (Promega Corp., USA), following the manufacturer's re- commendations. Transcriptional levels of ACE2 (Hs01085333_m1), TMPRSS2 (Hs Hs01122322_m1), and IL-6 (Hs00985639_m1) were determined, using TaqMan probes (ThermoFisher, USA). Specific primers were used to assess IFN-γ (F:5′-ACTGTCGCCAGCAGCTAAAA-3′; R: 5′- TATTGCAGGCAGGACAACCA-3′) and TNF-α (F: 5′-CACAGTGAAGTGCTGGCAAC-3′; R: 5′-GATCAAAGCTGTAGGCCCCA-3′) mRNA expression. The reactions were performed in duplicates, and the relative fold value was obtained by the 2 –DDCt method. 2.5 Data analysis and statistics FlowJo software 10.0.7. was used for Flow Cytometry data analysis (FlowJo LLC, USA). Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis and plotting. Statistical significance was calculated using Student's t-test analyses. The value of p < 0.05 was considered statistically significant. 3 Results 3.1 Volunteers’ characterization A total of 19 donors were recruited for this study (6 male and 13 female). All of them received at least 2 doses of vaccine against COVID-19. Among such individuals, 10 were convalescent from previously COVID-19 infection, and 9 were not infected by SARS-CoV-2. Convalescent patients had COVID-19 diagnosis confirmed by nasopharyngeal swab qRT-PCR. The average age of the volunteers is 27 years (16–60 years), and all convalescent COVID-19 donors had mild disease without hospitalization (Table 1 ).Table 1 Clinical characteristics of individuals enrolled in this study. Table 1Subject Age Gender SARS-COV2RT-PCR Time from COVID diagnosis (months) Vaccine (dose) Time from last vaccine dose (months) COVID-19 Symptoms 1 41 M + 24 Sinovac (2) Pfizer (2) 3 Yes 2 26 F + 3 Sinovac (2) Pfizer (2) 3 Yes 3 49 M + 27 Sinovac (2) Pfizer (1) 7 Yes 4 16 F + 7 Pfizer (2) 9 Yes 5 26 F + 7 Sinovac (2) Pfizer (2) 11 Yes 6 24 F + 6 AstraZeneca (2), Pfizer (1), Sinovac (1) 2 Yes 7 21 F + 8 AstraZeneca (2), Pfizer (1) 5 Yes 8 27 F + 2 Sinovac (2) Pfizer (2) 3 Yes 9 27 M + 18 Pfizer (2), Astrazeneca (1) 4 Yes 10 26 F + 7 Sinovac (2) Pfizer (2) 11 Yes 11 21 F – NA Pfizer (2), Jansen (1) 6 NA 12 23 F – NA AstraZeneca (2), Pfizer (1) 9 NA 13 23 F – NA Pfizer (3) 7 NA 14 21 F – NA Pfizer (2), Astrazeneca (1) 6 NA 15 60 M – NA Astrazeneca (2) 12 NA 16 24 F – NA AstraZeneca (2), Pfizer (1) 8 NA 17 19 M – NA AstraZeneca (2), Pfizer (1) 7 NA 18 33 F – NA AstraZeneca (2), Pfizer (2) 2 NA 19 22 M – NA Jansen (2) 8 NA NA: not aplicable. 3.2 Activation marker in PBMCs stimulated with SARS-CoV-2 proteins Stimulation of PBMCs with RBD protein did not change the expression of any evaluated lymphocyte activation markers (Fig. 1 a-d). In contrast, PBMC stimulation with S1 protein induced an increase in CD25 (p = 0.03) (Fig. 1e). Interestingly, stimulation of PBMCs with N-RBD protein also promoted an increase in the expression of CD25 (p = 0.002). Interestingly, between two groups of vaccinated individuals, only the previously infected subjects showed a statistically significant increase in CD25 expression (p = 0.002) (Fig. 1h). CD69 expression was also induced on T-cells after stimulation with N-RBD (p = 0.005). Both samples from naive and previously infected individuals showed an increased expression of this receptor (p = 0.03 and p = 0.04, respectively) (Fig. 1i).Fig. 1 Effects of SARS-CoV-2 S1, RBD and N-RBD proteins on T-cell activation. (A) Gating strategy. T-cells were characterized by size and complexity. CD3 lymphocytes were evaluated for the expression of CD69, CD137, and CD25. Expression of CD25 (B–D), CD69 (E–G) and CD137 (H–J) were determined on T-cells, after stimulation of PBMCs of vaccinated subjects with S1, RBD, and N-RBD proteins for 24 h. Samples and statistical analysis are represented by the blue color for unexposed COVID-19 donors (n = 5) and red color for individuals who had SARS-CoV-2 infection (n = 5). Statistical significance was calculated using Student's t-test analyses. The value of p < 0.05 was considered statistically significant. Fig 1 3.3 Expression levels of SARS-CoV-2 receptors and inflammatory factors in PBMCs stimulated with SARS-CoV-2 proteins PBMCs expressed very low levels of the ACE2 gene, with one sample showing no gene amplification. Furthermore, none of the SARS-CoV-2 proteins evaluated was able to stimulate the expression of ACE2 in PBMCs (Fig. 2 a-c). We identified a certain variability in the expression of TMPRSS2 among the samples of PBMCs evaluated, but, once again, the antigenic stimuli tested using the different SARS-CoV-2 proteins failed to exert a significant modulation on the transcriptional levels of this gene (Fig. 2 d-f).Fig. 2 ACE2, TMPRSS2, IL-6, IFN-γ, and TNF-α mRNA levels in PBMCs from vaccinated subjects, in response to S1, RBD, or N-RBD SARS-CoV-2 proteins. PBMCs from vaccinated subjects were stimulated with S1, RBD, and N-RBD for 72 h and, after this period, the transcriptional levels of (A–C) ACE2, (D–F) TMPRSS2, (G–I) IL-6, (J–L) IFN-γ, and (M–O) TNF-α were determined. To analyze ACE2 expression, the CT value of one PBMCs sample was arbitrarily defined as 40. Samples and statistical analysis are represented by the blue color for unexposed COVID-19 donors (n = 4) and red color for individuals who have had SARS-CoV-2 infection (n = 5). Statistical significance was calculated using Student's t-test analyses. The value of p < 0.05 was considered statistically significant. Fig 2 Interestingly, the stimulation of PBMCs with RBD or S1 protein promoted a reduction in the transcriptional levels of IL-6 (p = 0.01 and p = 0.006, respectively). In both cases, the reduction of IL-6 was shown to be statistically significant in samples from donors who did not have COVID-19 (p = 0.03 and p = 0.02, respectively). In contrast, PBMCs stimulated with N-RBD protein had increased transcriptional levels of IL-6 (p = 0.02) and IFN-γ (p = 0.02). Samples from donors who had COVID-19 sustained increased IFN-γ (p = 0.04), different from samples of participants with no COVID-19 history. Stimulation of PBMCs with RBD led to a reduction in TNF-α transcription only in samples from donors who did not have COVID-19 (p = 0.04). Finally, the stimulation of PBMCs with S1 protein also promoted a reduction in TNF-α (p = 0.03), which was only maintained in samples from donors who did not have COVID-19 (p = 0.02) (Fig. 2 g-o). 4 Discussion Recently, countries with broader access to vaccination have made significant advances in immunizing the population through the use of booster doses, given the reported decline in vaccine efficacy with time. However, this decline in vaccine efficacy and the risks associated with the emergence of new variants of SARS-CoV-2, keep the world on alert regarding the risks that still exist due to the COVID-19 pandemic [9]. In this context, a better understanding of the immune response of immunized patients becomes essential. In this study, after stimulating PBMCs of vaccinated patients with specific proteins of SARS-CoV-2, we demonstrated that the expression of ACE2 and TMPRSS2 is not modulated by the exposure of these cells to proteins S1 and RBD, and that the N-RBD protein stimulates greater T-cell reactivity, mainly in patients who were vaccinated and had previous SARS-CoV-2 infection. Remarkably, it has been demonstrated that the incubation of epithelial cells with S protein or RBD induces ACE2 expression, which can worsen the infection by providing more receptors to the virus [10]. Our results demonstrated a reduced expression of ACE2 mRNA levels in PBMCs, some of which did not even show gene amplification. Furthermore, incubating these cells with S1, RBD, or N-RBD did not modulate their ACE2 and TMPRSS2 mRNA levels. In accordance, little to no expression of ACE2 has been observed in most human PBMC samples, as assessed at the protein level [11]. Surprisingly, even in patients with COVID-19 there seems to be lower ACE2 levels in circulating blood cells compared to healthy individuals [12]. Despite this, even at low levels, the amount of ACE2 on the T-cell membrane is sufficient to mediate virus binding and entry into these cells [13]. The cellular immune response mediated by T-cells is essential for the control of the infection caused by a coronavirus. Individuals who recovered from the infection have been shown to have SARS-CoV-specific memory T-cells [14]. In this study, the stimulation of T-cells with S1 protein increased CD25 expression on T-cells. On the other hand, the stimulation of T-cells with the N-RBD protein stimulated the expression of CD25 and CD69 on T-cells from vaccinated patients, which suggests that the N-RBD segment showed a greater ability to stimulate the T-cell reactivity in our samples, compared to the RBD and S1. In agreement with our observations, it has been shown that convalescent patients from previously COVID-19 infection have specific T-cells against N and S proteins [15] and that N protein has an outstanding immunogenic potential in these patients [16]. It is also important to note that the increased expression of CD25 was observed in subjects who had previously COVID-19 infection, but not in vaccinated individuals who were not exposed to the virus. This finding indicates that possibly the vaccinated convalescent individuals have greater immunologic memory and reactivity against the N-RBD viral protein, compared with subjects vaccinated but not infected by SARS-CoV-2. Intriguingly, PBMC stimulation with either S1 or RBD did not stimulate IL-6 and TNF-α transcription in such cells. We actually found a reduction in the levels of these transcripts, which was maintained in vaccinated patients who did not have SARS-CoV-2 infection. We also did not observe changes in INF-γ levels after stimulating PBCMs with S1 and RBD proteins. On the other hand, stimulation with N-RBD promoted higher expression of IL-6 and IFN-γ in PBMCs, which reinforces the greater immunogenic potential of this protein in the vaccinated subjects. More importantly, the increase in the expression of activation markers, associated with the greater production of IFN-γ, indicates a greater capacity for viral protein recognition and protection against the virus in the vaccinated donors [17]. Our study has as main limitation the small number of samples. However, the results obtained consistently show that PBMC exposure to S1, RBD, and N-RBD proteins does not stimulate ACE2 and TMPRSS2 expression in these cells. Furthermore, N-RBD protein has greater immunogenic potential in vaccinated donors, and T-cells from vaccinated patients who had SARS-CoV-2 infection show greater reactivity when exposed to virus antigens, especially to N-RBD protein. In addition to contributing to the investigation of T-cell reactivity, in vitro studies with models of human immune cells and viral proteins are important for a better understanding of the immunogenic potential of SARS-CoV-2 in vaccinated patients and for the establishment of platforms that allow a better understanding of the inflammatory process involved in COVID-19. Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ) and Fundação de Apoio à Pesquisa do Distrito Federal. Ethics approval The present study was approved by the local Ethical Committee. Written informed consent was obtained from all subjects included in the study. Declaration of Competing Interest The authors declare no competing financial interests. Data Availability Data will be made available on request. ==== Refs References 1 Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.-.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. Cell. 181 2020 271–280.e8 2 Connors J.M. Levy J.H. Blood 135 2020 2033 2040 32339221 3 Koerber N. Priller A. Yazici S. Bauer T. Cheng C.-.C. Mijočević H. Wintersteller H. Jeske S. Vogel E. Feuerherd M. Tinnefeld K. Winter C. Ruland J. Gerhard M. Haller B. Christa C. Zelger O. Roggendorf H. Halle M. Erber J. Lingor P. Keppler O. Zehn D. Protzer U. Knolle P.A. Nat. Commun. 13 2022 153 35013191 4 Park A. Iwasaki A. Cell Host Microbe 27 2020 870 878 32464097 5 Fajgenbaum D.C. June C.H. Cytokine Storm N. Engl. J. Med. 383 2020 2255 2273 33264547 6 Xu Z. Shi L. Wang Y. Zhang J. Huang L. Zhang C. Liu S. Zhao P. Liu H. Zhu L. Tai Y. Bai C. Gao T. Song J. Xia P. Dong J. Zhao J. Wang F.-.S. Lancet Respir. Med. 8 2020 420 422 32085846 7 Gómez-Carballa A. Pardo-Seco J. Bello X. Martinón-Torres F. Salas A. Trends Genet. 37 2021 1069 1080 34556337 8 Naranbhai V. Nathan A. Kaseke C. Berrios C. Khatri A. Choi S. Getz M.A. Tano-Menka R. Ofoman O. Gayton A. Senjobe F. 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Wang P. Zhou C. Zhang R. Liang P. Guo H. Wang X. Qin C.-.F. Chen F. Dong C. Immunity 52 2020 971 977.e3 32413330 16 Grifoni A. Weiskopf D. Ramirez S.I. Mateus J. Dan J.M. Moderbacher C.R. Rawlings S.A. Sutherland A. Premkumar L. Jadi R.S. Marrama D. de Silva A.M. Frazier A. Carlin A.F. Greenbaum J.A. Peters B. Krammer F. Smith D.M. Crotty S. Sette A. Cell. 181 2020 1489 1501.e15 32473127 17 Stephens D.S. McElrath M.J. JAMA 324 2020 1279 1281 32915201	0	PMC9714124	NO-CC CODE	2022-12-05 23:15:31	no	2022 Dec 1; 2:172-176	utf-8	null	null	null	oa_other
==== Front Explor Res Clin Soc Pharm Explor Res Clin Soc Pharm Exploratory Research in Clinical and Social Pharmacy 2667-2766 The Authors. Published by Elsevier Inc. S2667-2766(22)00105-6 10.1016/j.rcsop.2022.100206 100206 Article Pharmacy students' experience of technology-enhanced learning during the COVID-19 pandemic Durand Emma a Kerr Aisling b Kavanagh Oisín c Crowley Erin a Buchanan Beth a Bermingham Margaret a⁎ a Pharmaceutical Care Research Group, School of Pharmacy, University College Cork, Cork, Ireland b School of Pharmacy and Life Sciences, Robert Gordan University, Aberdeen, Scotland, United Kingdom c School of Pharmacy, Newcastle University, Newcastle, United Kingdom ⁎ Corresponding author at: Lecturer in Clinical Pharmacy Practice, School of Pharmacy, University College Cork, Cork, Ireland. 1 12 2022 1 12 2022 1002066 10 2022 29 11 2022 29 11 2022 © 2022 The Authors. Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Background With the advent of the COVID-19 pandemic, pharmacy students and educators experienced an abrupt shift as programmes that were previously taught exclusively in-person were then predominantly taught online. This sudden change provided little time for students to prepare for the new learning environment. Objectives The study objective was to explore pharmacy students' experiences of technology-enhanced learning during the COVID-19 pandemic. Methods A cross-sectional survey was developed and distributed by email to all 3rd year (N = 76) and 4th year (N = 68) pharmacy students undertaking an MPharm programme in an Irish university. Results A total of 32 responses were collected, including 20 third year and 12 fourth year pharmacy students (response rates of 26.3% and 17.6%, respectively). The majority of respondents reported good or very good internet speed (71%) and stability (59%). Almost all were confident or very confident using Canvas (97%) prior to the onset of online learning. Respondents preferred engaging with other students in-person rather than online for coursework (68.8%) and learning new material (56.3%). Students favoured face-to-face delivery, with a recording of the session available online afterwards, for lectures (68.8%), workshops (50%) and tutorials (56.3%). Analysis of free-text comments indicates that respondents used recorded content to support exam revision and that a key drawback of online learning was social isolation. Implications Pharmacy students favoured a blended learning approach, with in-person learning being recorded to support study and revision. Students' experience of TEL during the pandemic should be considered in the development and ongoing review of pharmacy programmes. Keywords Technology enhanced learning COVID-19 Pharmacy education Survey Blended learning ==== Body pmcIntroduction The delivery of higher education has changed radically in recent years. With the emergence of successful online teaching applications, the traditional approach of didactic lectures is under scrutiny, and a greater emphasis is placed on new teaching methodologies that are grounded in pedagogical theory.1 Some authors have challenged the long-standing tradition of an educator in the classroom and teaching software has emerged to facilitate this. This new pedagogical approach may particularly suit independent learners1 but is perhaps less appropriate for learners who are lower in the constructivist hierarchy.2 In this context, the shift to online learning during the COVID-19 pandemic provided an ideal testbed to explore these issues, and to assess the role of Technology-Enhanced Learning (TEL). Technology-Enhanced Learning describes learning that is enhanced, supported, mediated or assessed using educational technologies.3 Teaching delivered in this way may be entirely digital or may be blended with traditional in-person learning.4 A variety of technologies are available as educational tools and have been used successfully for many years in pharmacy education.5 Recent reports of TEL use in pharmacy curricula include the use of TEL for lecture capture systems,6 wet lab simulation,7 feedback on pharmaceutical calculations assessment,8 and virtual objective structured clinical examinations.9 Advantages of the TEL approach include its flexibility, ease of use and variety.5 With the advent of the COVID-19 pandemic, pharmacy students and educators in many places experienced an abrupt shift as programmes that were previously taught exclusively face-to-face moved to online learning.10., 11., 12., 13. Many, but not all, undergraduate students are “digital natives”, meaning they are the first generation of learners that did not need to adapt to new digital technologies.14 However, previous work has shown that some students struggle with the use of technology for learning.15 Poorly developed information technology (IT) skills, computer anxiety, and finding TEL more time intensive than face-to-face learning have been cited as reasons that students may find TEL challenging.15 However digital poverty may also create a barrier for some students.16 Digital poverty concerns exclusion of an individual or group from aspects of daily life through not having appropriate devices, software or internet connectivity and can impact many facets of life, including education.17 The cost of internet services or devices, limited internet access, limited access to devices, and poorly developed IT skills have been described as factors that may create inequalities in access to, and benefit from, TEL.15 , 16 The sudden change from in-person to online learning at the beginning of the pandemic provided pharmacy educators with little time to prepare students for the new learning environment or to take account of the challenges they may experience.12 Studies published prior to the COVID-19 pandemic have outlined the benefits and effectiveness of TEL in pharmacy education,5 , 18 however we are still learning about the experiences of pharmacy students online learning during the pandemic.11., 12., 13. , 19 , 20 Therefore, the objective of this study was to assess pharmacy students' experience of TEL during the pandemic, with an emphasis on confidence, skills and concerns during that time. Methods Ethical considerations Ethical approval for this study was granted by the School of Pharmacy Research Ethics Committee in University College Cork, approval number 2021–009. All participants received information explaining the purpose and procedures of the study and provided informed consent. Study design A cross-sectional survey was developed to capture students' perceptions, attitudes and opinions of online learning during the academic year 2020/2021, during the COVID-19 pandemic. The questionnaire was designed based on previously published studies pertaining to students' perceptions of online learning21 , 22 and was informed by the researchers' own experiences of TEL. The research team consisted of a pharmacy student, four lecturers in clinical pharmacy practice and an instructional designer. A draft of the survey was reviewed by five pharmacy students. The students' feedback was considered, and modifications based on this feedback were made to the survey. The final survey was approved by all authors. Survey composition The survey consisted of 16 questions comprising multiple-choice statements, “select all that apply”, Likert scale and free-response questions. Data collected included students' experiences with TEL prior to the onset of the COVID-19 pandemic, students' perceptions pertaining to their experience of TEL during the 2020/21 academic year, challenges faced by students while engaging with online learning and recommendations for improving future TEL experiences. Participants could skip questions if they wished. A brief explanation of the term Technology-Enhanced Learning was provided as follows: “The term Technology-Enhanced Learning is used to describe learning that is enhanced, supported, mediated, or assessed using educational technologies. This can include the use of technology during face-to-face teaching, or the use of technology in blended and online teaching”. The questionnaire consisted of two sections. The first section contained 12 closed questions. Firstly, participants were asked to give their consent to complete the survey and were then asked to select their year of study. Questions 3–5 related to the internet connection available to participants during the academic year of 2020/21. Question 6 pertained to participants' confidence with online technologies prior to the onset of the pandemic. Question 7 inquired about participants preferences relating to instructional methods for educational activities. Participants preferences for future delivery of lectures, tutorials, workshops and practicals were examined in Questions 8–11. Participants' experiences of TEL were assessed in Question 12. The second section contained four open questions which were used to further explore participants opinions on TEL. It was estimated that the questionnaire would take 8–10 min to complete. A copy of the questionnaire is available in the Appendix. Sample characteristics and data collection methods All third year (n = 76) and fourth year (n = 68) pharmacy students undertaking the MPharm programme in the School of Pharmacy, University College Cork were invited to participate in the study. The survey was open for responses for a three-week period in November 2021. The survey was circulated by a member of the research team (MB) to students' institutional email addresses. A link to the online questionnaire, via Microsoft Forms, was provided in the email. One reminder email was sent to students within two weeks of initial contact. No honorarium was provided to participants. Statistical analysis Data were stored and analysed using Microsoft Excel. Descriptive statistics were calculated, and categorical data based on the Likert scale were analysed descriptively by calculating frequencies and percentages. As it was not compulsory to answer any survey question, other than the consent question, the percentages are calculated based the total number of responses to each question. The respondents' opinions and experiences were captured by utilising open text questions and were explored using thematic analysis. Results The email invite was sent to 76 third year and 68 fourth year pharmacy students. Thirty-two responses were received, 20 from third year students (response rate 26.3%) and 12 from fourth year students (response rate 17.6%), with overall response rate of 22.2%. For online learning, respondents most frequently accessed the internet using broadband supplied by a landline provider (93.8%) or using a mobile hotspot (78.1%). Very good or good internet speed was reported by 71.0% of respondents and very good or good internet stability was reported by 59.4% of respondents. Over half of the students (59%) felt very confident and 38% felt confident using the virtual learning environment Canvas prior to the onset of the pandemic (Fig. 1 ). However, 56% of respondents reported being unconfident or very unconfident using Microsoft Teams prior to the pandemic. Similarly, 57% reported being unconfident or very unconfident using Zoom.Fig. 1 Participant responses to question 6, “Prior to the onset of online learning, how would you describe your confidence using the following technologies for online learning?” Fig. 1 Respondents reported that they preferred in person learning for engagement with other students for coursework (68.8%) and for learning new material (56.3%), however they preferred online methods for engagement with academic staff (59.4%) and a mix of online and in person learning for remembering course material (40.6%), Fig. 2 . Third year (55%) and fourth year (67%) pharmacy students preferred engaging with the staff in the School of Pharmacy using a blended approach and both third year (75%) and fourth year (58%) students preferred engaging with other students for coursework in person. Half of third year (50%) and two-thirds of fourth year (67%) students preferred learning new material in person. Just under half (45%) of third year students found that they retained new course material better by using a mixture of both online and in person teaching methods in comparison to fourth year students (50%) who had improved retention of new content when delivered in-person.Fig. 2 Participant responses to question 7, “What is your preferred learning method for each of the following?” Fig. 2 In terms of delivery of teaching in the future, the majority of the respondents (68.8%) prefer lectures to be delivered in person, with the recording available afterwards. Respondents prefer live workshops, with 34.4% preferring workshops delivered live in person and 50% preferring workshops delivered live in person, with the recordings available afterwards (Fig. 3 ). Similarly, 56.3% of respondents prefer tutorials to be delivered in person with the recording available afterwards. The majority of respondents (78.1%) prefer laboratory practicals to be delivered in person (Fig. 4 ).Fig. 3 Participant responses to (A) question 8 “How would you prefer lectures to be delivered in the future” and (B) question 9 “How would you prefer workshops to be delivered in the future?” Respondents chose one answer option from the list provided. Fig. 3 Fig. 4 Participant responses to (C) question 10 “How would you prefer tutorials to be delivered in the future” and (D) question 11 “How would you prefer practicals to be delivered in the future?” Respondents chose one answer option from the list provided. Fig. 4 Almost all respondents (97%) slightly agreed or strongly agreed that online learning aided in the development of their technology skills. The majority of respondents (94%) agreed that Canvas was the easy to use, while 88% agreed that Microsoft Teams and 65% agreed that Zoom was easy to use. Most respondents (72%) found recorded lectures easy to watch and 65% of respondents felt that videos and images made learning more interesting however, fewer (53%) agreed that videos and images aided their understanding of laboratory content. Overall, 50% of students agreed that online learning helped them to develop their understanding of the course material, however 44% of respondents disagreed with this statement (Fig. 5 ).Fig. 5 Participant responses to question 12, “Please rate your agreement with the following statements as they relate to your learning in the academic year 2020/21.” Respondents answered on a Likert scale from Strongly disagree to Strongly agree. Fig. 5 Twenty-six (82%) respondents provided replies to the open questions. Three major themes emerged from the data. Themes and sub-themes are outlined, with supporting quotes, in Table 1 .Table 1 Themes and subthemes identified in thematic analysis of open-text questions, with quotation(s) to support each subtheme. Participant number is indicated after each supporting quote. Table 1Theme Subtheme Quotation Positives of TEL approach Revision “Having recordings of the lectures [was] very beneficial when it came to revision” P3 “…a very useful tool for revision and study” P5 Time management/Flexibility “Lectures could be watched at any time” P32 Control of learning, Note taking and enhanced learning “I found huge value in having access to recorded material. I could work through it at my own pace, take far better notes” P5 “…found it helpful…to repeat sections of the lecture you found difficult you comprehend during the first sitting” P14 Drawbacks of TEL approach Time management “Workload built up very quickly as lecturers may post two lectures in a slot that was timetabled for one… Impossible to stick to timetable.” P11 “When the lectures were recorded, I found that it could take a very long time to watch them … dedicating too much time to each lecture and not getting through all of the material” P31 Social interaction “It is important that the in-person aspect is not forgotten about, I felt quite isolated from my peers over the last year and a half and definitely depend on them both socially and academically” P11 “Very difficult sitting in front of a laptop all day and isolated from social interaction, therefore attention to the learning materials was limited” P15 Practicalities of online material “Have the lectures live streamed and the recordings made available afterwards” P18 “I would have broken up the lecture recordings into shorter more bite-sized videos” P6 “The [laboratory] practicals that were held online [were] less beneficial than… if they were held in laboratories” P14 Internet connectivity “My internet connection was bad and the stream kept cutting out… I felt that I had missed out on some of the tutorials even though I had attended… it was out of my control” P23 Future preferences for learning activities using TEL Blended/hybrid approach “Keep recording lectures to listen back to them after the lecture” P17 “I like the hybrid of in person and online learning, both live and pre-recorded” P29 In-person learning “Online lectures are absolutely no replacement for in person lectures… while recordings are great for revision, they are no comparison to having a lecturer standing in front of you explaining and teaching” P3 Discussion This study used a cross sectional survey of pharmacy students to explore their experiences of TEL during the COVID-19 pandemic. The majority of students favoured a hybrid learning approach, in the form of face-to-face delivery of lectures, tutorials and workshops with a recording of the session available online afterwards for revision purposes. Students preferred learning new material and engaging with their peers for coursework in-person, however, they reported that they retained course material better when delivered online. Students reported that online learning aided the development of their technology skills but not their skills pertaining directly to the pharmacy programme. Drawbacks associated with TEL included difficulties associated with poor internet connection, time-management challenges, and potential social isolation. The students who participated in this survey were in third and fourth year of a pharmacy programme when the survey was conducted in late 2021. The group of third year students had experienced just one full semester of in-person university education prior to the onset of the pandemic and consequent switch to online learning. Elements of TEL such as computer-aided learning programmes and shared online documents for groupwork were used in the programme prior to the pandemic, however at their early stage in the programme, the students included in this study were minimally exposed to these techniques. Therefore, the onset of online learning in March 2020 represented a significant change in the learning environment of these students. Almost half of third year students stated that they retain new course material better by using a mixture of both online and in person teaching methods, however 50% of fourth year students reported improved retention of new content when delivered in-person. This may reflect that third-year students, with less experience of in-person learning in university, adapted to TEL and online learning to a greater extent than their more experienced fourth year colleagues. The results presented here indicate that in-person learning is the preferred learning environment among the surveyed students. This aligns with a survey of medical students in the United Kingdom (UK), where students reported that they did not find online teaching to be engaging or enjoyable, and that it provided limited opportunities for students to ask questions of lecturers.23 The in-person approach also allows educators the opportunity to gauge student understanding from non-verbal cues.22 , 24 In the current study students preferred in-person learning new material and engaging with their peers in-person. This reflects Essilfie and colleagues who found that online learning could not replicate the benefits and dynamic interactions of face-to-face learning.25 Students favoured the in-person delivery of lectures, tutorials, workshops and laboratory practicals, and reported that recorded material is beneficial for revision purposes. Over 40% of students reported that they remembered the course material better when delivered online in comparison to other methods of instruction. This accords with a study by Yu and colleagues who found that in an assessment of online team-based learning, students stated that online delivery did not aid their retention of knowledge.10 Furthermore, Yu and colleagues reported that in the online learning setting students felt less engaged with the course instructor and with their classmates compared to in-person learning.12 Faculty members were also surveyed by Yu and colleagues and their responses were strongly aligned with those of their students.12 Lean and colleagues have reported that a conventional in-person classroom approach is superior to online learning for gaining a thorough understanding of pharmacy programme content.24 Therefore, evidence suggests that a blended learning approach may be most beneficial for delivery of the pharmacy curriculum in the future.10., 11., 12. , 21 , 23 , 26., 27., 28. Almost all respondents agreed that online learning helped them to develop their technology skills however, most also perceived that online learning did not support the development of skills relating directly to the pharmacy programme. This finding is consistent with Chuang and colleagues who reported that the continued development of soft skills such as oral communication, empathy, reflective practice and problem solving proved challenging through online platforms during the pandemic.19 In a nationwide survey of medical students in the UK conducted during the pandemic, 82% of respondents reported that they couldn't learn practical skills through online learning.23 A systematic review published in 2014 found that when “traditional” learning was compared to online learning for health professions students, there was disagreement among studies examining students' skills acquisition from online learning, with just 6 of the 15 included studies demonstrating significantly greater skill acquisition among students assigned to online learning.29 Kolb's experiential learning theory suggests that learners must integrate formal education with lived experience and ongoing reflection, in order to develop skills.30 The switch to online learning, necessitated by the pandemic, may have led to teaching being delivered online that was not specifically designed for the online medium and this may have limited students' capacity for reflection and development. The internet connection available to students is an essential factor for online learning. Although most students in the current study reported that they had an internet connection with good speed and stability available to them for online learning, it is necessary to consider the needs of the almost 30% who reported low internet speed and 40% who reported lack of stable internet. The quality of a student's internet connection will directly impact on their online learning experience. Students with a stable internet connection may have healthier attitudes towards TEL than those who do not.31 In the current study, a number of students reported that technical difficulties during online workshops and tutorials posed as an issue for them. This reflects other reports in the literature that found that one of the major challenges associated with TEL include technological difficulties.10 , 12 These problems are intensified by digital poverty. Summers and colleagues demonstrated that in a UK university, economic disadvantage influenced student engagement during the pandemic, and that the pattern of engagement with learning materials changed between the pre-pandemic to peri-pandemic period.17 Pre-pandemic, students from disadvantaged backgrounds tended to access course materials and view recorded lectures more frequently than those from more affluent backgrounds. During the pandemic, differences in recorded lecture views reversed with students from the most disadvantaged backgrounds watching significantly fewer recorded lectures than those from more affluent backgrounds.17 Elsewhere, in a study of digital teaching innovations among nursing students in a UK university, an association was identified between ethnicity and understanding technology.16 McAllister and colleagues noted the importance of considering digital poverty when developing technology enhanced learning interventions but also highlighted that as healthcare becomes more digitised, healthcare educators must prepare their students for an increasingly electronic and technology-driven workplace.16 In addition to issues concerning poor internet connection, respondents found it challenging to manage their time appropriately when recorded lectures were not released according to the official programme timetable or when recorded lectures were longer than the allocated timetable slot. Students expressed that it was difficult to remain engaged with longer recorded lectures. According to Lean and colleagues, the face-to-face delivery of lectures may overcome these barriers.24 In-person lectures allow students to remain focused for extended periods as they are more motivated to learn when given the opportunity to engage with the lecturer and to discuss ideas and concepts with their peers.24 , 25 Research comparing longform didactic lecture recordings and shorter chunked recordings, showed greater student engagement with chunked videos that were between 3- and 17-min duration than with 60-min videos.32 Furthermore, students achieving higher grades had greater engagement with the chunked recordings.32 Humphries and Clark concluded that among digital natives, chunked lectures may improve student attention, assist with time management and increase engagement.32 Almost 70% of respondents reported that they prefer in-person learning for engagement with other students for coursework. Participating students reported feelings of social isolation associated with online learning during the pandemic. Irish university students, when interviewed by RTE, the Irish public broadcaster in November 2020, described the transition to online learning during the pandemic as “lonely”, “overwhelming” and “chaotic”.33 A survey of nursing students, conducted during the pandemic, found that online learning lacked feeling and was impersonal.31 Elsewhere, a survey of physicians undertaking orthopaedic surgery education and conducted during the pandemic, found that the development of meaningful relationships with classmates was more likely to occur during face-to-face encounters.25 As the current study was conducted during the pandemic, the isolation that many students felt at that time may have influenced their perception of TEL. It is important that future studies assess perception of TEL at a timepoint when students have a greater opportunity to partake in a combination of in-person and online learning. This study was limited to a single School of Pharmacy in Ireland and to two study-year levels, leading to a small sample size which limits the generalisability of the results. That the response rate was low must also be acknowledged. This may be attributable to study time-pressures, the fact that some eligible students were on placement at the time of survey dissemination and potentially survey fatigue, given the number of surveys circulated to students by the School of Pharmacy and the University. Nonetheless, the survey instrument was well designed and tested, and the results align with the finding of work published by several other educators during the pandemic. These findings should be interpreted in the context of the study being conducted during the pandemic. Conclusion Pharmacy students favour a blended learning approach, with in-person learning being recorded to support study and revision. Students' experience of TEL during the COVID-19 pandemic should be considered in the development and ongoing review of pharmacy programmes. Funding No funding was received for this work. Data availability The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by ED and MB. The first draft of the manuscript was written by ED and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose. Appendix A Supplementary data Supplementary material Image 1 Acknowledgements The authors wish to thank all the respondents for giving their valuable time to participate in this study. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.rcsop.2022.100206. ==== Refs References 1. Kang L.O. Brian S. Ricca B. Constructivism in pharmacy school Curr Pharm Teach Learn 2 2 2010 Mar 126 130 2. Kurt S. Constructivist learning theory Educ Technol February 2021 Available from: https://educationaltechnology.net/constructivist-learning-theory/ 3. University College Dublin Technology enhanced learning [Internet] [cited 2022 Nov 29]. Available from: https://www.ucd.ie/teaching/resources/technologyenhancedlearning/ 4. Nicoll P. MacRury S. van Woerden H.C. Smyth K. Evaluation of technology-enhanced learning programs for health care professionals: systematic review J Med Internet Res 20 4 2018 Apr 11 e131 5. Salter S.M. Karia A. Sanfilippo F.M. Clifford R.M. 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Pew Research Center Millennials in Adulthood: Detached from Institutions, Networked with Friends [Internet] 2014 Pew Research Center Mar [cited 2022 Nov 3]. Available from: https://www.pewresearch.org/social-trends/2014/03/07/millennials-in-adulthood/ 15. Childs S. Blenkinsopp E. Hall A. Walton G. Effective e-learning for health professionals and students-barriers and their solutions. A systematic review of the literature-findings from the HeXL project Health Inf Libr J 22 s2 2005 Dec 20 32 16. McAllister N. Tavener-Smith T. Williams J. Decoding medical terminology: implementing digital teaching innovations to support nursing students' academic and clinical practice Teach Learn Nurs 2022 Oct S1557308722001056 17. Summers R. Higson H. Moores E. The impact of disadvantage on higher education engagement during different delivery modes: a pre- versus peri-pandemic comparison of learning analytics data Assess Eval High Educ 2022 Jan 9 1 11 18. Ruehter V. Lindsey C. Graham M. Garavalia L. Use of online modules to enhance knowledge and skills application during an introductory pharmacy practice experience Am J Pharm Educ 76 4 2012 May 10 69 22611278 19. Chuang S. Trevaskis N. Mak V. The effects of the COVID-19 pandemic on pharmacy education, staff and students in an Australian setting Pharm Educ 2021 Jan 15 87 90 20. Gavazva E. Grekova D. Students' perceptions and impact of the COVID-19 pandemic on the pharmaceutical education in Bulgaria: a pilot project Pharm Educ 22 1 2022 Jun 11 569 572 21. Hamilton L.A. Suda K.J. Heidel R.E. McDonough S.L.K. Hunt M.E. Franks A.S. The role of online learning in pharmacy education: a nationwide survey of student pharmacists Curr Pharm Teach Learn 12 6 2020 Jun 614 625 32482262 22. Alsoufi A. Alsuyihili A. Msherghi A. Impact of the COVID-19 pandemic on medical education: medical students' knowledge, attitudes, and practices regarding electronic learning Kotozaki Y, editor PLoS One 15 11 2020 Nov 25 e0242905 23. Dost S. Hossain A. Shehab M. Abdelwahed A. Al-Nusair L. Perceptions of medical students towards online teaching during the COVID-19 pandemic: a national cross-sectional survey of 2721 UK medical students BMJ Open 10 11 2020 Nov e042378 24. Lean Q.Y. Ming L.C. Wong Y.Y. Neoh C.F. Farooqui M. Muhsain S.N.F. Online versus classroom learning in pharmacy education: students' preference and readiness Pharm Educ 20 1 2020 19 27 25. Essilfie A.A. Hurley E.T. Strauss E.J. Alaia M.J. Resident, fellow, and attending perception of E-learning during the COVID-19 pandemic and implications on future orthopaedic education J Am Acad Orthop Surg 28 19 2020 Oct 1 e860 e864 32732495 26. Al-Balas M. Al-Balas H.I. Jaber H.M. Distance learning in clinical medical education amid COVID-19 pandemic in Jordan: current situation, challenges, and perspectives BMC Med Educ 20 1 2020 Dec 341 33008392 27. Olum R. Atulinda L. Kigozi E. Medical education and E-learning during COVID-19 pandemic: awareness, attitudes, preferences, and barriers among undergraduate medicine and nursing students at Makerere University Uganda J Med Educ Curric Dev 7 2020 Jan 238212052097321 28. Alqudah N.M. Jammal H.M. Saleh O. Khader Y. Obeidat N. Alqudah J. Perception and experience of academic Jordanian ophthalmologists with e-learning for undergraduate course during the COVID-19 pandemic Ann Med Surg (Lond) 59 2020 Nov 44 47 32934810 29. George P.P. Papachristou N. Belisario J.M. Online elearning for undergraduates in health professions: a systematic review of the impact on knowledge, skills, attitudes and satisfaction J Glob Health 4 1 2014 010406 30. Kolb D.A. Experiential Learning: Experience as the Source of Learning and Development 2nd ed. 2015 Pearson Education Upper Saddle River, New Jersey 31. Oducado R.M.F. Soriano G.P. Shifting the education paradigm amid the COVID-19 pandemic: nursing students' attitude to e-learning Afr J Nurs Midwifery 23 1 2022 1 14 32. Humphries B. Clark D. An examination of student preference for traditional didactic or chunking teaching strategies in an online learning environment Res Learn 2021 29 Available from: https://journal.alt.ac.uk/index.php/rlt/article/view/2405 33. Students feel ‘overwhelmed and overlooked’ during Covid [internet] 2020 RTE News [cited 2022 Nov 29]. Available from: https://www.rte.ie/news/2020/1105/1176247-student-life-pandemic/	36471895	PMC9714125	NO-CC CODE	2022-12-09 23:15:08	no	Explor Res Clin Soc Pharm. 2023 Mar 1; 9:100206	utf-8	Explor Res Clin Soc Pharm	2,022	10.1016/j.rcsop.2022.100206	oa_other
==== Front Adv Ophthalmol Pract Res Adv Ophthalmol Pract Res Advances in Ophthalmology Practice and Research 2667-3762 Published by Elsevier Inc. on behalf of Zhejiang University Press. S2667-3762(22)00073-7 10.1016/j.aopr.2022.11.001 Review COVID-19 and ocular complications: A review of ocular manifestations, diagnostic tools, and prevention strategies Dong Jilian a Chen Ruida bc Zhao Hanhe c Zhu Yirui c∗ a School of Public Health, Nanjing Medical University, Nanjing, China b Eye Department, Affiliated Dongyang Hospital, Wenzhou Medical University, Dongyang, China c Eye Center, Affiliated Second Hospital, School of Medicine, Zhejiang University, Hangzhou, China ∗ Corresponding author. Eye Center, Affiliated Second Hospital, School of Medicine, Zhejiang University, 1 Xihu avenue, Hangzhou, 310009, China. 1 12 2022 1 12 2022 30 7 2022 18 10 2022 13 11 2022 © 2022 Published by Elsevier Inc. on behalf of Zhejiang University Press. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the severe Corona Virus Disease 2019 (COVID-19) outbreak that started in December 2019 in China and caused enormous health and economic problems worldwide. SARS-CoV-2 can attack the respiratory system by attaching to angiotensin-converting enzyme 2 receptors located on human cells. There is clinical evidence for the close relationship between ocular manifestations and SARS-CoV-2 infection. In fact, the eye could be a new route of infection, and finding ways to protect the eyes could further reduce the risk of infection. This review aims to sum up the ocular complications of COVID-19, the possible pathogenesis, and preventive strategies to protect ophthalmology practitioners and patients by reviewing the currently available literature on the topic. ==== Body pmc1 Introduction The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was responsible for the severe Corona Virus Disease 2019 (COVID-19) outbreak that started in December 2019 in Wuhan, Hubei Province, China.1 Due to its strong infectivity, the epidemic quickly spread around the world, and the virus was officially named SARS-CoV-2 based on the results of nucleic acid sequencing of patients’ lower respiratory tract samples. As of 3 October 2022, more than 617 million people have been infected by COVID-19, and the cumulative number of deaths has exceeded 6.53 million globally.2 The most common symptoms of COVID-19 are fever, dry cough, and shortness of breath.3 It mainly causes lung and immune system damage, but it also causes secondary damage to other organs that varies based on co-existing diseases in the patients.4 Studies have shown that SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) receptors.5 As these receptors are also present in the eye, this may be a possible transmission route and an organ of infection. The study of eye complications related to COVID-19 and effective ways of prevention and treatment may be useful for the control of this pandemic. This article reviews the ocular complications associated with COVID-19 and provides suggestions for healthcare practitioners with regards to protecting themselves from the virus. 2 Characteristics of the novel coronavirus and its variants SARS-CoV-2 has exhibited the ability for mutation over a period of time.6 Four variants have been identified between December 20, 2020, and May 2021, namely, the Alpha (b.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) variants, which are associated with increasing levels of infectiousness and disease severity. In late December 2020, according to the results of whole genome sequencing of patients who tested positive for SARS-CoV-2, the Alpha variant (B.1.1.7) was first reported in the United Kingdom.7 A preliminary matching case-control study showed that there was no significant difference in the hospitalization risk or mortality of the B.1.1.7 variant compared with the other variants. However, subsequent studies have shown that patients infected with this variant exhibit more serious symptoms and have a higher risk of death.8, 9, 10 The Delta variant was first discovered in India in December 2020 and caused the second wave of SARS-CoV-2 infection there in April 2021.11 First detected in the United States in March 2021, SARS-COV-2 Delta variant was the dominant strain in the United States in 2021. Studies have shown that the fatality rate of the Delta variant may be related to differences in population characteristics.12 On November 11, 2021, a new variant was discovered in the Republic of Botswana in Africa, and since then, it has also been reported in many parts of South Africa. The World Health Organization designated the mutant as a variable of concern on November 26, 2021, and named it the Omicron variant (B.1.1.529).13 The Omicron variant has undergone more significant mutations than the previous variants.14 The Omicron variant is more infectious and causes milder clinical symptoms, but the risk of secondary infection is six times higher than that with the Delta variant.15 , 16 Globally, the Omicron variant has replaced the Delta variant as the world's leading novel coronavirus variant.17 3 Virus transmission SARS-CoV-2 invades the human body by binding to ACE2 receptors on cells.5 Therefore, tissues that express ACE2 receptors are likely to be invaded by the virus. ACE2 receptors have been identified in a wide range of human tissues, including the lungs, small intestines, brain, kidneys, and blood vessels.18 Liulin et al. have detected the expression of ACE2 receptors in the conjunctiva and corneal tissue of the human eye, so it can be inferred that the SARS-CoV-2 may invade the human body through the eyes.19 Zhou Y et al. studied 67 patients with confirmed SARS-CoV-2 infection at the People's Hospital of Wuhan University in China.20 SARS-CoV-2 was detected in the conjunctival sac of three of the patients, but they did not exhibit any ocular symptoms. The research so far indicates that it is almost impossible for COVID-19 to spread through the conjunctiva. Of 72 patients diagnosed with COVID-19 at Tongji Hospital in Wuhan, the virus was detected in the eye secretion of only one person. The researchers concluded that the surface of the eye may be a potential route of infection of SARS-CoV-2, but the probability of infection through this route is extremely low for the general population.21 Thus, it seems that while SARS-CoV-2 may be present in tears or conjunctival sacs, it is unclear whether it can be transmitted via the eyes. 4 Ocular manifestations of COVID-19 Coronavirus disease (COVID)-19 is associated with ophthalmic manifestations during and after recovery from the disease and may be sight-threatening. Other more serious eye complications associated with COVID-19, such as epiphora, reactivation of quiescent anterior uveitis, anterior sclero-uveitis, cotton wool spots, retinal hemorrhages, retinal artery/vein occlusion, ophthalmic artery occlusion, panuveitis, papillophlebitis, central serous retinopathy, presumed fungal endophthalmitis, and multifocal chorioretinitis, have been reported in clinical cases.22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 1. Anterior segment manifestations of COVID-19 A research study showed that out of 38 COVID-19 patients in Wuhan, Hubei Province in China, 12 had eye abnormalities, which mainly included conjunctival congestion, tear overflow, and conjunctival edema, among other signs. Only one of them had conjunctivitis as the first symptom, and most eye complications occurred in patients with severe infection.34 Another study included 72 COVID-19 patients from Tongji Hospital in Wuhan City in China: only 2 showed symptoms of conjunctivitis, and SARS-CoV-2 was detected in the eye secretion of one patient.35 In a meta-analysis of three studies (1167 patients), the frequency of conjunctivitis was higher in patients with severe COVID-19 at admission (3 vs. 0.7% with an odds ratio of 3.4).36 Another study, which included a total of 16 studies of 2347 confirmed COVID-19 cases, found that the pooled data showed 11.64% of COVID-19 patients had ocular surface manifestations. Ocular pain (31.2%), discharge (19.2%), redness (10.8%), and follicular conjunctivitis (7.7%) were the main features.37 These findings indicates that the likelihood of SARS-CoV-2 infection being transmitted through the ocular surface is extremely low; nonetheless, nosocomial transmission of SARS-CoV-2 infection through the eyes after occupational exposure is a potential route. Sanjay et al. reported the case of a COVID-19 positive patient presenting with anterior sclero-uveitis and demonstrated that the acute form of this condition could precede the SARS-CoV-2 infection.32 They suggested that in all hitherto first episodes of ocular inflammation, COVID-19 should be considered to be diagnosis as a possible cause because this virus is preceded by ocular inflammation.2. Posterior segment manifestations of COVID-19 Studies have shown that COVID-19 may make patients more vulnerable to thrombotic disease that affects both venous and arterial circulation.38 , 39 Further, thrombotic disease in these patients is characterized by excessive inflammation, platelet activation, endothelial dysfunction, and stasis. However, due to the complex and interacting vascular risk factors in most patients, the relationship between thrombotic disease and COVID-19 is not clear yet.40 The increased risk of retinal vascular obstruction caused by COVID-19 has yet to be studied. It has been reported that 12 patients with confirmed COVID-19 showed hyper-reflective lesions at the level of the ganglion cell and inner plexiform layers that were more prominent in the papillomacular bundle of both eyes.41 Hyper-reflective features in the inner retina on optical coherence tomography (OCT) images in all 12 patients. The results of OCT angiography and complex analysis of ganglion cells were normal. Fundus examination in four cases revealed the presence of subtle cotton wool spots and microhemorrhages along the retinal arcade. Despite this, all the patients had normal visual acuity and pupillary reflexes and no symptoms or signs of intraocular inflammation. Nevertheless, this results led to some controversy. Frederick T Collison et al. identified the OCT findings reported by Marinho and colleagues represent normal retinal anatomy.42 One case presenting with vitritis had bilateral redness in the eyes, and examination showed a yellowish macular lesion 12 days after the onset of COVID symptoms. OCT displayed hyper-reflective lesions at several sites and fluorescence fundus angiography revealed hyper-fluorescence.43 Moreover, some acute retinal necrosis cases during or after COVID-19 have been reported. Some reactivated cases of varicella zoster virus presented as acute retinal necrosis.44, 45, 46, 47, 48 There are few case reports that detail the reactivation of chorioretinal disease after SARS-CoV-2 infection or COVID-19 vaccination.49, 50, 51 The inflammatory effect of COVID-19 is considered to be the mechanism of action on highly vascularized choroid tissue, resulting in chorioretinal inflammation.51 3. Neuro-ophthalmic manifestations of COVID-19 Numerous efferent complications have been reported in association with SARS-CoV-2 infection, including isolated cranial nerve palsies with diplopia, Miller Fisher syndrome and nystagmus or saccadic intrusions in association with either brainstem infarction or hemorrhagic acute necrotizing encephalopathy. Proposed mechanisms include direct neural invasion into the peripheral or central nervous system, parainfectious inflammation provoked by the immunogenicity of novel antigens presented by the SARS-CoV-2 virus, and secondary hypercoagulability and endothelial dysfunction resulting in stroke. Neuro-ophthalmic symptoms have rarely been reported following SARS-CoV-2 vaccination, but evidence for causation is lacking and the benefits of inoculation continue to far outweigh any perceived risks.52 4. Orbital manifestations of COVID-19 Rhino-orbital cerebral mucormycosis is the most common orbital involvement in patients with COVID-19, reported in several case series.53, 54, 55 Mucormycosis induced by COVID-19 is more common in patients with diabetes mellitus and who are suffering from critical or severe COVID-19.56 Immunologic changes in diabetic patients are a potential risk factor for such fungal conditions. The main mechanism is the spread from the colonization of nasal mucosa. This disease is a life-threatening infection that can occur in patients with COVID-19, because of their compromised immune system and decreased lymphocytes, due to the disease itself, decompensated pulmonary function, and treatment with corticosteroids.57 5. Post-vaccination ocular complications COVID-19 vaccines can cause transient local post-vaccination reactions. Different types of ocular complications have been reported after COVID-19 vaccination, including herpetic keratitis, facial nerve palsy, abducens nerve palsy, new-onset Graves' disease, episcleritis, anterior scleritis, anterior uveitis, multifocal choroiditis, reactivation of Vogt-Koyanagi-Harada disease, multiple evanescent white dot syndrome, acute macular neuroretinopathy, paracentral acute middle maculopathy, thrombosis, and central serious retinopathy. These complications could be related to SARS-CoV-2 vaccines’ capacity to induce autoimmune manifestations or thromboembolic events.58 5 Ophthalmic detection of COVID-19 Specimens such as nasopharyngeal or oropharyngeal swab, bronchoalveolar lavage fluid, sputum, bronchial aspirate, and blood are generally recommended for early screening or diagnosis of COVID-19.59 , 60 Studies show that higher viral loads of SARS-CoV-2 are typically detected in the lower respiratory tract,61 and the highest detection rates were reported in bronchoalveolar lavage fluid, sputum, and nasal swabs.62 However, from the viewpoint of comprehensive analysis, convenience of sampling, and patient comfort, nasopharyngeal swabs and throat swabs are more suitable for large-scale nucleic acid determination. For the diagnosis of ocular SARS-CoV-2 infection, tear samples and conjunctival swabs may be useful samples for obtaining clinical evidence.1. Tear samples In a recent case series of a prospective interventional study, the tear sample from only 1 of 30 confirmed cases of COVID-19 was positive (according to PCR) for SARS-CoV-2.63 Another study tested both tear samples and nasopharyngeal swabs and showed that the nasopharyngeal swabs were positive and the tear samples were negative. Thus, the hypothesis that nasolacrimal ducts act as a transmission route for the virus may be incorrect. Within the study time frame, only 1 in 17 patients had eye symptoms, but SARS-CoV-2 was not detected in the tear samples of any of the patients.64 This result indicates that the possibility of the virus spreading through tears is really low.2. Conjunctival swab samples In a prospective study series on 30 COVID-19 patients, only 1 out of 30 conjunctival swab samples were positive for SARS-CoV-2.63 In another study on 67 patients with COVID-19 (63 cases with confirmed COVID-19 and 4 cases of suspected COVID-19), three conjunctival swabs were positive (PCR).20 However, none of the three patients with positive conjunctival swab samples exhibited conjunctivitis. The initial manifestation was conjunctivitis in only one patient, but the conjunctival swab sample was negative for SARS-CoV-2 (PCR). This patient is an anesthesiologist, and he was probably infected because he did not wear protective glasses during the operation. However, the symptoms of conjunctivitis were mild. Based on the findings of this study, it seems unlikely that SARS-CoV-2 can be transmitted through the conjunctival route. However, subsequent study has shown that a small number of patients with confirmed SARS-CoV-2 infection did have conjunctivitis.21 Despite this, only 2 of 72 patients with confirmed COVID had conjunctivitis, and the conjunctival sac secretion was positive according to the virus nucleic acid test in only one of these two patients in Wuhan province. Similarly, in Zhejiang province, only 1 out of 30 patients with COVID-19 had conjunctivitis and a positive result for conjunctival sac secretion.20 Thus, it appears that although there is clinical evidence of the transmission of SARS-CoV-2 through the conjunctival route, the overall positivity rate for ocular infection is low. One study included 21 patients with confirmed novel coronavirus infection from the First Affiliated Hospital of Zhejiang University, but viral RNA was detected in the tears and conjunctival secretions of only one patient. The researchers also collected sputum samples of patients to use as the controls, and 55 of the 60 samples were positive.63 The authors believe that this result shows, to some extent, that COVID-19 patients who do not have conjunctivitis do not transmit the novel coronavirus through their tears and conjunctival secretions.3. Nasolacrimal duct samples The eyes are directly exposed to the external environment and are also connected to the respiratory tract through the nasolacrimal duct and nasal cavity. Some researchers have speculated that the virus can enter the eyes through droplets and reach the respiratory tract through the nasolacrimal duct, where it can bind to ACE2 receptors and cause SARS-CoV-2 infection.65 A study on rhesus monkeys showed that monkeys inoculated via conjunctival fluid developed mild interstitial pneumonia.66 By determining the virus load in different swabs within 1–7 days after vaccination, the researchers found that in monkeys inoculated through the conjunctival route, the virus was detected only in the conjunctival swab sample obtained on the first day and was not detected later. This indicates that the virus may have caused the infection on first entering the conjunctiva and then later migrated to the respiratory tract.4. Intraocular fluid samples Bilgic et al. reported that they successfully isolated the SARS-CoV-2 in three patients with endogenous endophthalmitis from vitreous samples.67 In another study by Koo et al., six (19.4%) patients demonstrated detectable SARS-CoV-2 RNA in aqueous samples, fortunately, none of these individuals had any systemic symptoms.68 However, Maya Hada and colleagues detected the presence of SARS-CoV-2 in the aqueous and vitreous humor of seven ocular trauma COVID-19 patients.69 SARS-CoV-2 was not detected by polymerase chain reaction in aqueous or vitreous humor in this pilot study. Similarly, Srinivasan Sanjay and colleagues were unable to isolate the SARS-CoV-2 virus in either aqueous or vitreous samples.70 This may be explained by the fact that SARS-CoV-2 is not detected in a very low percentage of ocular samples in patients who have COVID-19. Therefore, whether current tests are sensitive enough to detect SARS-CoV-2 through intraocular fluid sampling is debatable. 6 Reducing the risk of COVID-19 in the ophthalmology department In order to control the spread of SARS-CoV-2 infection and reduce the risk of infection among ophthalmological clinicians and patients, medical staff should work closely with local infection control teams to carry out risk assessment and undertake appropriate strategies for infection control in the real-world clinical environment. SARS-CoV-2 is excreted through respiratory secretions. Inhalation of respiratory droplets and direct contact with contaminated surfaces are the main routes of transmission. The incubation period after infection is 1–14 days, and both symptomatic and asymptomatic COVID-19 patients can spread the virus.71 The virus is transmitted through exhaled droplets that are 5 microns or larger in diameter, such as the droplets produced when coughing, sneezing, singing, and speaking.72 It can also spread via inhalation of aerosols that circulate in relatively closed environments for a long time. One study found that surfaces contaminated by SARS-CoV-2 can also lead to spread of the virus, and the rate of reduction of surface virus concentration is related to the properties of the surface. The estimated half-life of SARS-CoV-2 is approximately 6.8 h on plastic surfaces and 5.6 h on stainless steel surfaces. However, the virus is not stable on cardboard, and it is especially unstable on copper.73 Therefore, choosing the right material for surfaces in the clinic may help to effectively reduce the risk of viral infection. Considering the relationship between COVID-19 and eye complications, conjunctivitis may be the first clinical manifestation of COVID-19.74 Although the incidence rate is not high, we believe that indicators of ocular abnormalities, such as conjunctivitis, may help in the diagnosis of this infection. In addition, medical workers should inform patients of the possibility of SARS-CoV-2 transmission through the eyes. Irrespective of whether the patient has eye symptoms, healthcare workers should advise patients to minimize or avoid touching their eyes, nose, and mouth to prevent the spread of the virus. Further, instruments that come into contact with the surface of the eye should be disinfected with ethanol or be replaced with reliable disposable alternatives where possible. In addition, the American Academy of Ophthalmology recommends that framed glasses be used instead of contact lenses as much as possible. Glasses provide a physical barrier for eye mucosal tissue and reduce the possibility of SARS-CoV-2 transmission through the eyes.75 Further, to lower the risk of nosocomial SARS-CoV-2 infection, all healthcare professionals should wear protective goggles. In Hong Kong, the pandemic was managed via three main levels of control measures: (1) administrative control, such as reducing the number of patients, suspending selective clinical services, and classifying patients; (2) environmental control, such as providing fresh air, regularly and frequently disinfecting surfaces that medical staff often come into contact with, and the use of staff video conferencing, as opposed to face-to-face meetings, as much as possible; (3) qualified use of personal protection equipment and strict disinfection of individuals.76 British scholars believe that during the pandemic, clinical areas should be redesigned, personal protective equipment should be worn, and sufficient social distancing should be maintained. At the same time, new working methods should be considered, such as simplifying services, reducing patient backlog, and telemedicine.77 One study found that surgical masks provided an average aerosol filtration rate of 96% for bacteria and 90% for viruses. The filtering effect of homemade masks varies according to the material used to make the mask-from 60% to 94% for bacteria, and from 49% to 86% for viruses.78 In one study, 492 medical workers from Zhongnan Hospital of Wuhan University were divided into two groups. The first group was mainly responsible for the respiratory medicine department, the intensive care unit, and the infectious diseases department (main isolation area); they wore N95 masks and regularly disinfected themselves and cleaned their hands. The second group included medical staff from other departments; this was a mask-free group that only occasionally disinfected themselves and cleaned their hands. None of the 278 staff members (56 doctors and 222 nurses) in the N95 group were infected, but 10 of the 213 staff members (77 doctors and 136 nurses) in the mask-free group were confirmed to be infected.79 In a pragmatic, clustered randomized clinical trial involving 2862 medical staff, no significant difference was found in the incidence of influenza between medical staff who used N95 respirators (8.2%) and laboratory doctors who used medical masks (7.2%).80 7 Conclusion Based on clinical evidence of eye complications caused by SARS-CoV-2, more reliable research should be performed to confirm its potential to infect the eye and its specific pathogenesis. Some of the topics for future research are the mechanism of eye complications associated with COVID-19, the relationship between the virus and thrombotic diseases, treatment strategies for eye complications associated with COVID-19, and the feasibility of using tear samples or conjunctival swabs for diagnosis. A larger-scale, crowd-based research with standardized investigation methods is required to answer these questions. In particular, elucidating the relationship between SARS-CoV-2 and the eye not only is conducive to guiding strategies to control the pandemic, but also provides insights into the feasibility of using ocular samples, such as tear samples, for the diagnosis of COVID-19. At present, many parts of the world are experiencing the fourth wave of the COVID-19 pandemic, and it has become clear that we must be prepared to coexist with SARS-CoV-2 for the foreseeable future. Given this, it is important that eye care practitioners pay attention to practical protection measures to minimize the risk of infection in patients and practitioners until more effective anti-viral therapies or vaccines are created. Author contributions Conception and design of study: Y.Z, J.D; Data collection: J.D and H.Z; Analysis and interpretation of results:Y.Z, J.D and R.C; Drafting the manuscript: Y.Z, J.D; All authors reviewed the results and approved the final version of the manuscript. Funding No funding was received for this work. Intellectual property We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. Research ethics Not applicable. Authorship The International Committee of Medical Journal Editors (ICMJE) recommends that authorship be based on the following four criteria:1. Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; AND 2. Drafting the work or revising it critically for important intellectual content; AND 3. Final approval of the version to be published; AND 4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All listed authors meet the ICMJE criteria. We attest that all authors contributed significantly to the creation of this manuscript, each having fulfilled criteria as established by the ICMJE. One or more listed authors do(es) not meet the ICMJE criteria. We confirm that the manuscript has been read and approved by all named authors. We confirm that the order of authors listed in the manuscript has been approved by all named authors. Contact with the editorial office This author submitted this manuscript using his/her account in Editorial Manager. We understand that this Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). That person is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that the email address shown below is accessible by the Corresponding Author, is the address to which Corresponding Author's Editorial Manager account is linked, and has been configured to accept email from the editorial office of Advances in Ophthalmology Practice and Research: We understand that this author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). That person is responsible for communicating with the other authors, including the Corresponding Author, about progress, submissions of revisions and final approval of proofs. Declaration of compeing interest No conflict of interest exists. Acknowledgments Thanks to all the peer reviewers for their opinions and suggestions. ==== Refs References 1 Zhu N. Zhang D. Wang W. A novel coronavirus from patients with pneumonia in China N Engl J Med 382 8 2019 727 733 10.1056/NEJMoa2001017 Feb 20 2020 2 WHO Coronavirus (COVID-19) Dashboard https://covid19.who.int/ 2022 3 Wiersinga W.J. Rhodes A. Cheng A.C. Peacock S.J. Prescott H.C. Pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (COVID-19): a review JAMA 324 8 2020 782 793 10.1001/jama.2020.12839 Aug 25 32648899 4 Jungang C.L.X. 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==== Front Obesity Pillars 2667-3681 2667-3681 The Authors. Published by Elsevier Inc. on behalf of Obesity Medicine Association. S2667-3681(22)00037-7 10.1016/j.obpill.2022.100046 100046 Article A comparison between weight loss outcomes with anti-obesity medications before and during Covid-19 pandemic at a tertiary weight management center De la Rosa Alan a1 Ghusn Wissam a1 Sacoto Daniel a Campos Alejandro a Cifuentes Lizeth a Feris Fauzi a Busebee Bradley a Calderon Gerardo a Acosta Andres a Hurtado Maria D. ab∗ a Precision Medicine for Obesity Program, Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, MN, USA b Division of Endocrinology, Diabetes, Metabolism & Nutrition, Mayo Clinic, Jacksonville, FL, USA ∗ Corresponding author. Precision Medicine for Obesity Program, Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, 4500 San Pablo Rd S Jacksonville, FL, 32224, USA 1 Alan De la Rosa and Wissam Ghusn contributed equally to this manuscript. 1 12 2022 1 12 2022 10004617 10 2022 28 11 2022 28 11 2022 © 2022 The Authors. Published by Elsevier Inc. on behalf of Obesity Medicine Association. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Background /Objectives: Obesity is a risk factor for COVID-19 infection severity and mortality. Anti-obesity medications (AOM) are effective for weight loss. However, weight loss outcomes with AOM during the COVID-19 pandemic are yet to be described. Subjects /Methods: Between January 1, 2016, and June 30, 2021, a total of 966 patients were prescribed long-term FDA-approved AOMs at the Mayo Clinic. From these patients, 711 patients did not meet inclusion criteria. A total of 255 patients were included. Interventions/methods We performed a retrospective systematic review of electronic medical records and included patients who started a long-term FDA-approved AOM. We excluded patients with history of bariatric procedure, AOM prescription with lorcaserin, orlistat, semaglutide (approved for weight loss after the pandemic), or phentermine (short-term AOM), those taking ≥2 AOMs, <3 months of prescribed AOM, and/or pregnancy. Analysis was divided by 1)preCOVID-19: those who started an AOM before COVID-19 restrictions, 2)COVID-19: those who started an AOM during first quarter of 2020 after the establishment of COVID-19 restrictions. Our primary endpoint was the total body weight loss percentage (%TBWL) at 3, 6, and 12 months after AOM initiation. Results There was a statistical difference in TBWL% between the preCOVID-19 and COVID-19 group: 5.3 ± 3.5% vs 4 ± 3.0% (95% CI -2.4 to −0.2; p = 0.02) and 9.7 ± 7.2% vs 6.2 ± 4.7% (95% CI -5.7 to −1.3; p = 0.002) at 3 and 12 months, respectively. At 6 months, the TBWL% was 7.1 for the preCOVID-19 group compared to 6.2% for the COVID-19 (95% CI -2.5 to 0.7; p = 0.25). Conclusion With the possible exception of liraglutide, this study shows that weight loss outcomes to AOMs were inferior when prescribed during the routine clinical practice throughout COVID-19 pandemic, compared to the outcomes observed prior to the COVID-19 pandemic. Keywords Anti-obesity medications COVID-19 Obesity Pandemic ==== Body pmc1 Introduction Obesity is a chronic and relapsing disease, with a rising prevalence and a high economic burden [1]. It is estimated that 42.5% of U.S. adults have obesity (i.e., body-mass index [BMI] ≥30 kg/m2), including 9.0% with severe obesity (i.e., BMI ≥40 kg/m2) [[2], [3], [4]]. Recent studies demonstrate an attributed annual medical costs of individuals with obesity exceed $2000, which poses a substantial financial burden on the healthcare system [5]. Obesity is also associated with higher rates of mortality and worse outcomes due to associated weight-related comorbidities such as dyslipidemia, type 2 diabetes mellitus, hypertension, and some forms of cancer [6]. The spread of COVID-19 imposed many governmental restrictions putting billions of people into a lockdown which may have increased their engagement in obesogenic prone behaviors [7]. Closure of fitness facilities, stay-at home policies, and increased frequency of unhealthy snacking may also have minimized the effectiveness of weight management programs leading to a sustained weight gain even after lockdown restrictions were lifted [8,9]. Literature studies have reported an average mean weight gain of 1.5–3 kg in the general population, with greater weight gain in males and individuals with overweight and obesity [[10], [11], [12], [13]]. Weight management guidelines recommend an intensive and multicomponent approach for weight loss in patients living with obesity and overweight. This approach has traditionally relied on a multidisciplinary team and on-site, in-person clinical care [14]. In view of the social distancing mandates, COVID-19 presented a challenge to multidisciplinary weight management programs, where telehealth became a popular alternative to in-person visits [15]. Telemedicine has demonstrated a safe and successful intervention to populations of difficult access [16]. Current evidence has proven the efficacy of telemedicine for short-term follow-up and its impact on patients with obesity [17]. The use of anti-obesity medications (AOMs) is a reliable and efficacious intervention for weight control, aiming at improving quality of life and preventing the progression of weight-related comorbidities [18,19]. The Food and Drug Administration (FDA) has approved AOMs for long-term use for individuals with BMI ≥30 kg/m2 or ≥ 27 kg/m2 with weight-related comorbidities. These AOMs include: Phentermine-topiramate [PHEN-TOP], naltrexone-bupropion [NBSR], orlistat, liraglutide, and semaglutide [[20], [21], [22], [23], [24]]. To date, most of the weight loss outcomes are derived from a focused testing of AOMs under strict control settings of randomized clinical trials (RCT). Few real-world studies often report different weight loss outcomes compared to previous RCTs [2,18,19,21,[25], [26], [27]]. Furthermore, the impact of limited healthcare access, confinement policies, and COVID-19 infection in individuals with overweight and obesity managed with AOM for weight control remains unknown [28]. Although the field of obesity medicine is rapidly advancing prior and during the pandemic, we aim to examine in the real-world weight-loss outcomes of FDA approved long-term AOM before and after the COVID-19 pandemic. 2 Methods 2.1 Design and eligibility criteria We performed a systematic review of electronic medical record (EMR) of patients from out-patient clinics (e.g., weight management clinic) from all the Mayo Clinic Health System sites. Informed consent was waived by the Institutional Review Boards (IRB) committee due to its minimal-risk nature. We included all patients who started a long-term AOM (PHEN-TOP, NBSR, and liraglutide) from January 1st, 2016, until June 30th, 2021. We abstracted our cohort population from Mayo Clinic Healthcare data through its medical record tool (MDE- Mayo Data Explorer). We screened patients using the electronic health record. Inclusion criteria included: 1) patients with a BMI ≥30 kg/m2 or ≥27 kg/m2 with at least one weight-related comorbidity; 2) patients prescribed an approved long-term AOM; 3) ≥3 months prescription of a long-term AOM. We excluded patients with a history of bariatric surgery, prior endoscopic procedure for weight loss (e.g., balloon, sleeve gastroplasty, transoral outlet reduction [TORe]), those taking ≥2 AOMs, history of previous or current malignancy, history of clinical trials for experimental weight loss interventions, pregnancy, and AOM prescription for lorcaserin (due to its discontinuation in 2020), orlistat (due to a restricted number of patients on this medication), semaglutide (due to its approval for weight loss after the pandemic), or phentermine (short-term AOM). This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline. 2.2 Data collection We searched patient's comorbidities information based on ICD-10 codes. We abstracted from the EMR information about demographics, anthropometrics, including laboratory clinical values and weight in kg at 3, 6, and 12 months. We collected data from in-person and/or virtual encounters. For the 3- month data abstraction, we gave a timeframe of ±30 days after starting the AOM and for the 6- and 12- months data abstraction, we gave a timeframe of ±45 days after starting the AOM. We included patients prescribed phentermine-topiramate, naltrexone-bupropion, and liraglutide regardless of the dose achieved. We collected provider, dietitian, and psychology visits after the first day of AOM was started until AOM was suspended. We divided our cohort in 1) preCOVID-19: AOM prescribed from January 1st, 2016, to December 31st, 2020, i.e., 3 months prior to the establishment of COVID-19 restrictions and 2) COVID-19: AOM prescribed during and after COVID-19 restrictions were established. 2.3 Weight management program The Mayo Clinic weight management program involves a multidisciplinary team that includes obesity medicine physicians, registered dietitians, advanced practice providers (physician assistants and nurse practitioners), and behavioral bariatric psychologists. Upon initial evaluation, patients are encouraged but not obligated to meet with a dietitian and the behavioral psychology team. All patients are encouraged but not obligated to participate in a standardized behavioral program. The general recommendations are to (1) reduce dietary intake to 1200–1500 calories per day for women and 1500–1800 calories per day for men, (2) achieve a goal of 10,000 steps or more per day and 150 min or more of moderate intensity activity per week, and (3) limit the consumption of liquid calories (e.g., sodas, juices, alcohol). Calorie restriction and counseling on activity might vary widely based on weight-related comorbidities and functional capacity. Some patients are prescribed AOMs. Patients were encouraged to return for follow-up visits 4–6 weeks after starting the medication and every 3 months thereafter. During each visit, providers recorded information on body weight and weight-related comorbidities, gathered information on medication adherence based on patients’ report and pharmacy data on prescriptions filled, and reported side effects of the AOMs. 2.4 Study endpoints The primary endpoint was the total body weight loss percentage (%TBWL) at 3, 6, and 12 months after AOM initiation. %TBWL was calculated using the following formula: [100*(weight at first visit – weight at each time point)]/weight at the first visit. Secondary endpoints included: (1) TBWL% at 3-, 6-, and 12-months by AOM type; (2) percentage of patients who achieved a TBWL of ≥5%, ≥10%, ≥15%, and ≥20%; (3) comparison between telemedicine (virtual) healthcare and in-person follow-up, and (4) metabolic changes at last follow up including: lipids [total cholesterol, low-density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides], fasting glucose, glycated hemoglobin (HbA1c), and systolic and diastolic blood pressures (SBP and DBP, respectively). 2.5 Statistical analysis Baseline anthropometric and demographics were normally distributed and are summarized as mean ± standard deviation (SD). For continuous variables not normally distributed, data are summarized as median and interquartile ranges (IQR). Categorical data are presented as frequencies and percentages. We used two tail t-test to analyze the association of %TBWL at 3, 6, and 12 months, compared to baseline between the COVID-19 and preCOVID-19 groups. We performed a multiple regression analysis to obtain the effect between AOMs and %TBWL adjusted by BMI, age, and sex at baseline, and COVID-19 status, and follow up visits with providers, dietitians, and psychologists. Results were based on parameter estimates (PE) with 95% confidence intervals (95% CI) and significance values. Statistical significance was set at 2-sided p < 0.05. We used JMP®, Version 14.3.0 (SAS Institute Inc., Cary, NC, 1989–2019) to perform the statistical analysis. 3 Results 3.1 Patient selection Between January 1, 2016, and June 30, 2021, a total of 966 patients were prescribed long-term FDA-approved AOMs at the Mayo Clinic. As shown in Fig. 1 , 711 patients did not meet inclusion criteria. A total of 255 patients were included. From these, 116 completed 12 months of AOM use, 87 (51%) in the preCOVID-19 group and 36 (40%) in the COVID-19 group (p = 0.12). The overall reasons for discontinuation included: lost to follow-up (n = 80), AOM discontinued due to cost or insurance denial (n = 21), added/switched to another AOM (n = 7), side effects (n = 13), achieved goals (n = 4), bariatric/endoscopic procedure (n = 4), stopped by another medical condition (n = 2), and pregnancy (n = 1).Fig. 1 Follow-up flowchart. Fig. 1 3.2 Baseline characteristics From the 255 patients who met inclusion and exclusion criteria, 75.6% were female with a mean age of 48.2 ± 12.9 years and mean BMI of 41.9 ± 8.5 kg/m2. Most patients, 55.3%, had obesity class 3 (BMI≥40 kg/m2) and were primarily White (96.1%). A total of 169 patients were identified in the preCOVID-19 group and 86 in the COVID-19 group. Similarly, there was an increased number of patients with obesity class 3 in the preCOVD-19 in comparison with the COVID-19 group: 59.8% vs. 46.5%; p = 0.04. There were no other differences in demographic and anthropometric baseline characteristics between COVID-19 and preCOVID-19 groups (Table 1 ).Table 1 Continuous data are summarized as mean and standard deviation (SD) or median and interquartile ranges (IQR). Categorical data are presented as frequencies and percentages. Significant p values are indicated in bold. Table 1 All patients preCOVID-19 COVID-19 p value A. Baseline demographic information N (%) 255 (100) 169 (66.2) 86 (33.7) Age, years (SD) 48.2 (12.9) 49.1 (13.1) 46.6 (12.5) 0.13 Sex, Female (%) 193 (75.6) 126 (74.6) 67 (77.9) 0.55 Race, White (%) 245 (96.1) 167 (98.8) 78 (90.7) 0.0008 Baseline clinical information Weight, kg (SD) 119.6 (29.3) 118 (23.8) 122.7 (37.9) 0.29 BMI, kg/m2(SD) 41.86 (8.5) 41.7 (7.4) 42.3 (10.2) 0.63 Overweight, n (%) 11 (4.3) 6 (3.6) 5 (5.8) 0.41 Obesity Class 1, n (%) 53 (20.8) 33 (19.5) 20 (23.3) 0.49 Obesity Class 2, n (%) 50 (19.6) 29 (17.2) 21 (24.4) 0.17 Obesity Class 3, n (%) 141 (55.3) 101 (59.8) 40 (46.5) 0.04 SBP, mmHg (SD) 129 (15) 129 (15) 129 (15) 0.90 DBP, mmHg (SD) 78 (22) 78 (10) 78 (12) 0.80 Glucose, mg/dL (SD) 122 (52) 129 (61) 109 (29) 0.03 HbA1c, % (SD) 7.0 (1.7) 7.1 (1.8) 6.5 (1.2) 0.11 Total Cholesterol, mg/dL (SD) 179 (49) 175 (40) 186 (61) 0.32 Total Triglycerides, mg/dL (SD) 151 (78) 160 (84) 133 (63) 0.06 LDL-cholesterol, mg/dl (SD) 103 (40) 96 (32) 115 (51) 0.04 HDL-cholesterol, mg/dl (SD) 50 (14) 48 (14) 53 (13) 0.08 B. Comorbidities Dyslipidemia, n (%) 153 (60) 106 (62.7) 47 (54.7) 0.35 Diabetes mellitus, n (%) 80 (31.4) 58 (34.3) 22 (25.6) 0.15 Prediabetes, n (%) 23 (9.1) 13 (7.7) 10 (11.6) 0.36 Hypertension, n (%) 123 (48.2) 81 (47.9) 42 (48.8) 0.69 GERD, n (%) 66 (25.9) 44 (26) 22 (25.6) 0.93 Obstructive sleep apnea, n (%) 92 (36.1) 63 (37.3) 29 (33.7) 0.58 Degenerative joint disease, n (%) 83 (32.5) 67 (39.6) 16 (18.6) 0.0009 NAFLD, n (%) 19 (7.5) 13 (7.7) 6 (7.0) 0.89 C. Medication Phentermine/topiramate, n (%) 124 (48.6) 92 (54.4) 32 (37.2) 0.01 Naltrexone/bupropion, n (%) 59 (23.1) 31 (18.3) 28 (32.6) 0.01 Liraglutide, n (%) 72 (28.2) 46 (27.2) 26 (30.2) 0.66 All p values < 0.05 were considered significant. BMI, body mass index; DBP, diastolic blood pressure; GERD, gastroesophageal reflux disease; HbA1c, Hemoglobin A1c; HDL, High-density lipoprotein; LDL, low-density lipoprotein; NAFLD, nonalcoholic fatty liver disease; SBP, systolic blood pressure. Diagnosis of obesity comorbidities was based on diagnoses listed by clinicians. Dyslipidemia was the predominant comorbidity found in the study cohort (60%), followed by hypertension (48.2%) and obstructive sleep apnea (OSA) (36.1%). In term of comorbidities, no significant difference was found except for degenerative joint disease, which was more predominant in the preCOVID-19 group compared to the COVID-19 group: 39.6% vs. 18.6%, p = 0.009. Mean values of glucose, HbA1c, LDL-cholesterol, HDL-cholesterol, total cholesterol, and triglycerides at baseline are presented in Table 1. There were no differences in laboratory results between both groups, except for glucose and LDL-cholesterol. Pre-COVID-19 had a median glucose of 129 ± 69 mg/dl in comparison with the COVID-19 group 109 ± 29 mg/dl (p = 0.03). LDL-cholesterol in the COVID-19 group was higher in comparison with the preCOVID-19 group: 115 ± 51 vs. 96 ± 32 mg/dl, p = 0.04. 3.2.1 Prescribed antiobesity medications In the entire cohort, the most frequent prescribed medication was phentermine-topiramate (48.6%) followed by liraglutide (28.2%), and bupropion-naltrexone (23.1%). Phentermine-topiramate was prescribed at 7.5–46 mg daily in 77.1% of patients, while 6% received 11.25–69 mg, and 16.9% received 15–92 mg. For liraglutide, a weight loss dose ≥1.8 mg (high dose), was achieved in 90.3% of patients. When stratified by our two groups, 84.6% patients in the COVID-19 group achieved a dose >1.8 mg, whereas 93.5% patients in the preCOVID-19 group achieved the same dose range (p = 0.21). All patients taking bupropion/naltrexone received 16–180 mg twice daily. 3.3 Changes in total body weight loss outcomes The entire cohort had a mean of 8.6 ± 6.7 %TBWL at one year. The COVID-19 group lost significantly less TBWL% compared to the preCOVID-19 group at 3 and 12 months (Fig. 2 A). At 3 months, the COVID-19 group lost 0.65% less weight compared to the preCOVID-19 group [% TBWL 4.7% vs. 5.35%; 95% CI -2.4 to −0.2; p = 0.02]. At 12 months, COVID-19 group lost 3.6% less weight compared to preCOVID-19 group [% TBWL 6.16% vs 9.66%; 95% CI -5.7 to −1.3; p = 0.02] (Table 2 A). There was no significant difference in %TBWL at 6 months (TBWL 7.07% for pre-COVID-19 vs 6.15% for COVID-19 group; 95% CI -2.5 to 0.7; p = 0.25) for the preCOVID-19 group (see Fig. 3).Fig. 2 A. Total body weight loss at 3, 6 and 12 months in COVID-19 and PreCOVID-19 patients. B-D. Total body weight loss at 3, 6 and 12 months in COVID-19 and PreCOVID-19 patients by medication. E-F. Percentage of patients who achieved 5, 10, 15 and 20% TBWL at 6 and 12 months. Figures A–D are represented in mean and standard error. Fig. 2 Fig. 3 A. Percentage of patients who achieved 5, 10, 15 and 20% TBWL at AOM termination for all the medications. B-D. Percentage of patients who achieved 5, 10, 15 and 20% TBWL at AOM termination by medications. Fig. 3 Table 2 Multidisciplinary provider visits. Continuous data are summarized as median and interquartile ranges (IQR). Table 2 preCOVID-19 COVID-19 p value N = 169 N = 86 Number of visits with a physician, (IQR) 2 (2–3) 2 (1.75–3) 0.02 Patients with virtual physician visit, n (%) 0 (0) 30 (34.8) <0.0001 Patients with ≥1 dietitian visit, n (%) 77 (45.6) 23 (26.7) 0.004 Total no. of patients with virtual dietitian visit n (%) 1 (0.6) 11 (12.8) <0.0001 Patients with ≥1 psychologist visit, n (%) 61 (36.1) 15 (17.4) 0.002 Total no. of patients with virtual psychologist visit, n (%) 0 (0) 9 (10.4) <0.0001 Significant p values are indicated in bold. All p values < 0.05 were considered significant. 3.4 Secondary end points 3.4.1 TBWL% at 3-, 6-, and 12-months analysis by AOM type In patients prescribed phentermine-topiramate, we found no significant differences in % TBWL at 3, 6, or 12 months between COVID-19 and preCOVID-19 groups (Fig. 2B). There was a trend for higher weight loss in the preCOVID-19 group at 12 months, but the trend did not achieve significance. Patients prescribed naltrexone-bupropion during COVID-19 had a significantly lesser TBWL% at all time points in comparison with those in the preCOVID-19 group (Fig. 2C). In patients prescribed liraglutide, no significant differences in % TBWL were observed at 3, 6, or 12 months between COVID-19 and pre-COVID-19 group (Fig. 2D). There was a trend for greater weight loss in patients in the COVID-19 group. We performed a subanalysis in the liraglutide group by dose (≤1.8 mg low dose vs. ≥2.4 mg high dose). In the high dose subanalysis at 12 months, preCOVID-19 achieved a −13.9% vs −7.8% TBWL in the COVID-19 group (p = 0.66). In the low dose subanalysis at 12 months, preCOVID-19 achieved a −3.62% vs −6.0% TBWL in the COVID-19 group (p = 0.67) (Fig. 1S). 3.4.2 Proportion of patients achieving ≥5–20% TBWL The percentages of patients achieving a TBWL of ≥5%, ≥10%, ≥15%, and 20% were significantly higher in the preCOVID-19 group in comparison to COVID-19 group (Fig. 2) When displayed by medication, phentermine-topiramate and naltrexone-bupropion led to a greater proportion of patients achieving a TBWL >5%, 15%, and 20% in the preCOVID-19 group compared to the COVID-19 group. No differences were observed between groups in patients prescribed liraglutide. 3.4.3 Multidisciplinary weight management program and telemedicine (Table 2) There was a difference in the number of provider visits (in-person or via telemedicine) between groups: 3 (2–3) for preCOVID-19 vs. 2 (1.75–3) for COVID-19, p = 0.02. No participants in the preCOVID-19 group had an appointment via telemedicine, while 36.6% of participants in the COVID-19 group had at least 1 or more virtual encounter (p < 0.0001). The proportion of patients with at least one dietitian encounter was higher in the preCOVID-19 group compared to COVID-19 group: 45.6% vs. 26.7%, p = 0.004. When stratified by group, 92.3% of the virtual dietitian visits happened in the COVID-19 group (p=<0.0001). Similarly, the proportion of patients with at least one bariatric psychology visit was higher in the preCOVID-19 vs. COVID-19 group: 36.1% vs. 17.4%, p = 0.002. Telemedicine visit modality was predominantly in the COVID-19 group (p=<0.0001). 3.4.4 Baseline metabolic changes (Table 2S) At last follow-up visit, there were no significant difference between groups in terms of clinical improvements in glucose, hemoglobin A1c, SBP, DBP or in the lipid panel (total cholesterol, HDL-cholesterol, LDL-cholesterol, or triglycerides) when compared between groups. 3.4.5 Multiple regression analysis To assess what variables predict %TBWL at 12 months, we performed regression analysis. Variables considered included: starting an AOM during COVID-19 pandemic, BMI at baseline, and number of visits with a physician, dietitian, and psychologist. Multiple regression analysis showed that the following variables predicted a lower %TBWL at 12 months: starting an AOM in the COVID-19 cohort (PE [95% CI]: −1.45; p = 0.03) and a lower number of visits with a physician (PE [95% CI]: 1.83; p = 0.003). When adding the type of medication to the variables in the multiple regression analysis, the only variable that predicted a lower TBWL% was lower number of visits with a physician (PE [95% CI]: −1.30; p = 0.03). 4 Discussion The COVID-19 pandemic affected individuals’ eating and lifestyle habits with multiple studies reporting an increase in weight [29]. Most of the research to date has focused on weight gain and negative health consequences of COVID-19 in the general population. However, obesity treatment strategies, as AOM and its weight loss outcomes, have not been assessed. Therefore, in this study, we report weight loss outcomes to AOM in a real-world setting during COVID-19 pandemic. This retrospective study of adult population revealed that the use of approved long-term AOM during the COVID-19 pandemic had a different impact on weight loss outcome when compared to a population prescribed before the pandemic. We report a significant lower %TBWL of about 0.65% and 3.6% at 3- and 12-months follow-up respectively, for those who were prescribed an AOM during the COVID 19 pandemic in comparison with those who were prescribed an AOM before the pandemic. The %TBWL achieved among patients prescribed an AOM before the COVID-19 pandemic showed similar results to other studies evaluating the efficacy of AOM ranging from 2.9% to 6.8% [25,26,[30], [31], [32], [33], [34]]. Our results showed a weight loss response very similar of about 5.3% at 3 months and 9.8% at 12-month follow-up. In our study, the AOM response by medication during COVID-19 pandemic represented important differences to previously reported data [25]. Phentermine-topiramate showed the greatest %TBWL with 11.4% and 8.0% before and during the COVID-19 pandemic at 12 months, respectively. These overall robust response of phentermine-topiramate is clinically less in the COVID-19 group when compared to other studies, where the highest %TBWL at 12 months can reach about 12% at 1 year [25]. The weight loss response of naltrexone-bupropion before and during the COVID-19 pandemic at 12 months was 6.7 vs 3.7% TBWL, respectively, with significantly less %TBWL in all timepoints in the COVID-19 group, when compared to the preCOVID-19 group and published studies [33]. Interestingly, only naltrexone-bupropion achieved significant differences when compared between both groups. The etiology behind this seen difference is not well understood. The weight loss response to liraglutide at 12 months follow-up before and during the COVID-19 pandemic was 4.8 vs. 7.4 %TBWL, respectively. Previously reported data have reported a weight loss as high as 7.4 kg and a 8% TBWL at 12 months [24,32]. The discrepancy in the results (greater weight loss during the pandemic) is explained by our secondary analysis. When the liraglutide group response was divided into ≤1.8 mg vs ≥ 2.4 mg dose, the preCOVID-19 group achieved a greater weight loss at ≥2.4 mg whereas the COVID-19 group achieved greater weight loss at doses ≤1.8 mg. We do not have any explanation as what this difference may be related to. The sample is small, and no further conclusions can be drawn from the current data. The proportion of patients achieving weight loss outcomes of >5%, >10%, >15%, and >20% before the pandemic was 77.7%, 44.7%. 17.7% and 11.8% and is similar to those observed in multicenter clinical studies [25]. In our cohort, categorical weight loss outcomes (5–20% TBWL) during the COVID-19 pandemic showed a lower proportion of patients achieving the same outcomes, with significant differences in those who achieved >10% and >15% TBWL. While significant weight loss was observed in both groups, only modest improvements were observed in the individual's metabolic parameters. It is reported that for certain comorbidities (e.g., hypertension, dyslipidemia, and diabetes type 2) modest weight loss of 5–10% is required to prevent their progression or development [35,36]. Whereas other comorbidities (OSA or NAFLD) require a greater weight loss to translate into a clinical improvement [37]. We hypothesized that prescribing an AOM will lead to a significantly less weight loss during the pandemic due to a limited standard medical care and changes in social behaviors due to the pandemic. The importance of these findings relies on its contribution towards a better understanding of the impact a stressful situation and how contributory factors such as this one can influence obesity therapies and their weight loss outcomes. To date, no former studies have approached the study of weight loss outcomes of AOM during the COVID-19 pandemic or other similar related situation, hence the importance of our findings. A probable aspect that may have impacted the weight loss outcome response to AOM is possibly related to the difference regarding the frequency and type of visit (e.g., telemedicine vs. in-person) of provider, dietitian, and psychology visits. It is known that a weight loss multidisciplinary intervention is associated with a greater and more clinically significant and sustained weight loss compared with standard of care [[38], [39], [40]]. Although we showed that the pandemic was a independent predictor of weight loss on multiple regression analysis, after taking into consideration the type of medication, the number of visits with an obesity medicine provider was the strongest predictor of response. Visits with additional members of the multidisciplinary team did not independently affect the differences observed in weight loss outcomes before and during the pandemic in this report. It is important to note that with the advent of technologies, telemedicine has become a popular alternative to conventional in-person visits to achieve significant and sustained weight loss. Consequently, depending on the patient propensity to obesity, telemedicine could be geared towards prevention of weight gain rather than weight loss in patients at high risk of weight gain during high stress situations such as the COVID-19 pandemic [29,41,42]. This study has several limitations. First, as this is a retrospective study, not all variables were available for all patients at all time points. Furthermore, we rely on chart documentation which sometimes is not accurate and comprehensive. Consequently, we could not report data on relevant outcomes such as changes in the prevalence of weight-related metabolic risk factors (prediabetes, diabetes, HTN, HLD) or changes in the number of medications used for these diseases. Similarly, due to this, data reported on metabolic outcomes do not account for potential implementation or adjustment of drugs altering these parameters during the period of using AOM. Second, demographically, our cohort is predominantly female and White which limits the generalization of these data to other populations. Third, weight and visits data were abstracted through in-person and virtual (self-report) follow-up. This is especially relevant for the COVID-19 group, where a telemedicine medical setting was preferentially opted, altering the regularity of standard medical care, therefore EMR records [43]. The retrospective nature of this report does not allow to determine the accuracy of EMR recording, therefore the impact of this factor on weight loss outcomes remains undetermined. Fourth, due to limited number of prescriptions, some AOM were left out, including well proven medications such as orlistat due to no prescriptions during COVID pandemic and semaglutide due to being approved during the pandemic and not having a control in the pre-pandemic era. One could argue that we could have used patients prescribed semaglutide at diabetes doses, however, the doses for weight loss are higher (2.4 mg weekly versus 1mg weekly, as of the submission of this manuscript). Finally, another limitation includes the lack of assessment of the social and mental health factors that may contribute to the difference observed (e.g., access to exercise facilities, stress, anxiety, depression). Studying these factors may help explain the decrease in weight loss outcomes of the COVID-19 group. However, this was limited due to the retrospective nature of our study. With the possible exception of liraglutide, this study shows that weight loss outcomes to AOMs were inferior when prescribed during the routine clinical practice throughout COVID-19 pandemic, compared to the outcomes observed prior to the COVID-19 pandemic. Further studies are needed to understand whether this observation is due to changes in care delivery during the pandemic or due to individual factors such as stress, decreased physical activity, remote working, among others. Author contribution Concept and design: De la Rosa, Hurtado, Acosta. Acquisition, analysis, or interpretation of data: De la Rosa, Ghusn, Sacoto, Campos, Cifuentes, Busebee, Hurtado, Acosta. Drafting of the manuscript: De la Rosa, Ghusn, Sacoto, Campos, Cifuentes, Hurtado, Acosta. Critical revision of the manuscript for important intellectual content: De la Rosa, Ghusn, Sacoto, Campos, Cifuentes, Acosta, Hurtado. Statistical analysis: De la Rosa, Campos, Cifuentes, Ghusn, Acosta, Hurtado. Administrative, technical, or material support: Acosta, Hurtado. Ethical review The submission represents original work. The submission does not involve any human test subjects or volunteers. Source of funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Data availability statement Deidentified data will be made available for the journal upon request. Declaration of competing interest The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Abbreviations AOM anti-obesity medications PHEN-TOP phentermine-topiramate NBSR naltrexone-bupropion sustained release BMI body-mass index %TBWL total body weight loss percentage FDA Food and Drug Administration RCT randomized clinical trials EMR electronic medical record IRB Institutional Review Boards PE parameter estimates TORe transoral outlet reduction MDE Mayo Data Explorer STROBE Strengthening the Reporting of Observational Studies in Epidemiology LDL low-density lipoprotein HDL high-density lipoprotein HbA1c glycated hemoglobin OSA obstructive sleep apnea NFLD non-alcoholic fatty liver disease SBP systolic blood pressure DBP diastolic blood pressure SD standard deviation IQR interquartile ranges Appendix A Supplementary data The following is the Supplementary data to this article:Multimedia component 1 Multimedia component 1 Acknowledgment All authors had full access to all the data and statistical analyses. Dr. Hurtado had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.obpill.2022.100046. ==== Refs References 1 Li Q. Prevalence and healthcare costs of obesity-related comorbidities: evidence from an electronic medical records system in the United States J Med Econ 18 12 2015 1020 1028 26134917 2 Gadde K.M. Atkins K.D. The limits and challenges of antiobesity pharmacotherapy Expet Opin Pharmacother 21 11 2020 1319 1328 3 Ogden C.L. Trends in obesity prevalence by race and hispanic origin—1999-2000 to 2017-2018 JAMA 324 12 2020 1208 1210 32857101 4 Wang Y. Has the prevalence of overweight, obesity and central obesity levelled off in the United States? Trends, patterns, disparities, and future projections for the obesity epidemic Int J Epidemiol 49 3 2020 810 823 32016289 5 Cawley J. 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Weight loss outcomes with telemedicine during COVID-19 Obesity 2021 191 192	0	PMC9714128	NO-CC CODE	2022-12-10 23:15:24	no	2022 Dec 1; 4:100046	utf-8	null	null	null	oa_other
==== Front Appl Soft Comput Appl Soft Comput Applied Soft Computing 1568-4946 1872-9681 Elsevier B.V. S1568-4946(22)00940-1 10.1016/j.asoc.2022.109891 109891 Article Risk assessment approach for analyzing risk factors to overcome pandemic using interval-valued q-rung orthopair fuzzy decision making method Seker Sukran a Bağlan Fatma Betül b Aydin Nezir a Deveci Muhammet cd Ding Weiping e⁎ a Department of Industrial Engineering, Yildiz Technical University, Besiktas, 34349, Istanbul, Turkey b Department of Industrial Engineering, Istanbul Esenyurt University, Esenyurt, 34510, Istanbul, Turkey c Department of Industrial Engineering, Turkish Naval Academy, National Defence University, 34940 Tuzla, Istanbul, Turkey d The Bartlett School of Sustainable Construction, University College London, London WC1E 6BT, UK e School of Information Science and Technology, Nantong University, Nantong 226019, China ⁎ Corresponding author. 1 12 2022 1 12 2022 10989131 5 2022 29 10 2022 25 11 2022 © 2022 Elsevier B.V. All rights reserved. 2022 Elsevier B.V. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. The process of developing and implementing sustainable strategies to prevent spread of COVID-19 for society typically requires integrating all social, technological, economic, governmental aspects in a systematic way. Since the clear understanding of risk factors contribute to the success of the strategies applied against COVID-19, a risk assessment procedure is applied in this study to properly evaluate risk factors cause to spread of pandemic as a multi-complex decision problem. Therefore, due to the evaluation of risk factors, which often involves uncertain information, the model is constructed based on interval-valued q-rung orthopair fuzzy-COmplex PRoportional ASsessment (IVq-ROF-COPRAS) method. While the developed framework is efficient to enhance the quality of decisions by implementing more realistic, precise, and effective application procedure under uncertain environment, it has capability to help governments for developing comprehensive strategies and responses. According to the results of the proposed risk analysis model, the top three risk factors are “The Approach that Prioritizes the Economy in Policies”, “Insufficient Process Control in Normalization” and “Lack of Epidemic Management Culture in Individuals and Businesses”. Lastly, to show applicability and efficiency of the model sensitivity and comparative analysis were conducted at the end of the study. Keywords Risk assessment Fuzzy sets Interval valued q-ROFSs COPRAS ==== Body pmc1 Introduction COVID-19 first announced in Wuhan, China on December 19, 2019, and the disease was spread to other countries in a short time with the impact of globalization. COVID-19 was reported with the definition of “unspecified lung disease” by WHO and declared as a global pandemic on January 30, 2020 [1]. Currently (15 May 2022), the number of confirmed cases worldwide has reached 521 million and the number of deaths has reached 6.3 million [2]. WHO explained that the spread of the virus can be ended with a successful system for early diagnosis, isolation, rapid treatment, and monitoring of contacted individuals [1]. To reduce the mobility of the virus, while country borders and cities are partially or completely closed all over the world, education and office works turned to be conducted from home, using masks, and applying hygiene rules have been implemented. Among these measures, the most recommended measures have been the use of masks, cleaning of contact surfaces with disinfectant and social distance [3]. While some countries such as China have taken more drastic measures, some countries such as Sweden have followed softer strategies by trying the herd immunity method. Most of the countries, on the other hand, have started to take measures quickly since the WHO declared the pandemic and has started to work to get over this process with minimal damage. In February, flights from China were cancelled, citizens come from other countries were quarantined for 14 days and education activities were turned to distance learning. In addition, places where citizens can be found collectively (cafes, gyms, theaters, etc.) were closed, and as of April, intercity travel and curfews began to be implemented on weekends [4]. As given in Table 1, with the measures taken in the first phase of the epidemic, some countries have successfully come to the fore among European countries with its fast and effective measures against the COVID-19 pandemic [5]. As a result of the different measures taken by different countries, the pandemic process also varied from country to country. While the center of the pandemic was initially China, it was replaced by other countries over time. As presented in Table 2, the United States, Brazil, and India are among the countries with a poor pandemic progress. Considering Turkey’s struggle against COVID-19, the “Pandemic Influenza National Preparation Plan” was successfully implemented in June 2020, as a result, the number of possible cases and deaths decreased with this plan. With the revival of social life, second wave of pandemic process existed as of November 30, 2020, and the number of daily cases and deaths started to increase [6]. While the restrictions continued until March 1, 2021, the course of the pandemic started to move positively and with the vaccination-controlled normalization process started again. In this stage, four groups of countries with high, very high, medium and low-level risks are identified, and restrictions have been gradually lifted accordingly [7].Table 1 Measures taken in the first phase of the epidemic [5]. Country Emergence of first case School closure Closing public places Restriction of public events Extensive travel and transport restrictions Italy 31st of January 10th of March (After 39 days) 12nd Of March (After 41 Days) 10th Of March (After 39 Days) 10th of March (After 39 Days) Spain 31st of January 11st of March (After 40 Days) 14th of March (After 43 Days) 11th of March (After 40 Days) 14th of March (After 43 Days) England 31st of January 18th of March (After 47 Days) 20th of March (After 49 Days) 13rd of March (After 42 Days) 24th of March (After 53 Days) Germany 27th of January 17th of March (After 50 Days) 16th of March (After 49 Days) 13rd of March (After 46 Days) 17th of March (After 40 Days) France 24th of January 13rd of March (After 49 Days) 14th of March (After 50 Days) 13rd of March (After 48 Days) 17th of March (After 53 Days) Turkey 10th of March 12nd of March (After 2 Days) 15th of March (After 5 Days) 12nd of March (After 2 Days) 13rd of March (After 3 Days) However, as a result of both the socialization brought about by normalization and the mutations of the virus has undergone, the number of cases has reached its peak in the whole of the process since the first day. Because of the rapid decrease in the number of cases, thanks to the full closure and vaccination campaigns, all restrictions other than wearing masks and social distance have been lifted across the country as of July 1, 2021 [8]. The course of the pandemic, which was stable for a while, started to show a remarkable negative increase as of July 26, 2021. During this process, the minimum and maximum daily case numbers for Turkey are given in Table 3. Ongoing restrictions, increasing number of cases, and ongoing vaccinations not producing a complete solution create uncertainties in the course of the epidemic and the management of the process.Table 2 Number of cases and deaths of countries [2]. Country Number of cases (Ranking) Number of deaths (Rank) United States of America 37.5 million (1) 626 thousand (1) India 32.0 million (2) 429 thousand (3) Brazil 20.5 million (3) 573 thousand (2) Russia 6.68 million (4) 174 thousand (4) France 6.56 million (5) 113 thousand (11) United Kingdom 6.39 million (6) 131 thousand (7) Turkey 6.16 million (7) 54 thousand (18) Argentina 5.12 million (8) 110 thousand (12) Colombia 4.88 million (9) 124 thousand (9) Spain 4.76 million (10) 83 thousand (15) Since the COVID-19 pandemic has negatively affected all countries socially and economically, governments have explored various strategies to prevent the spread of the epidemic [10]. The pandemic we are in will only be terminated by handling and coping with the risk factors in front of it. Thus, these risk factors need to be understood correctly, and a clear understanding of the risk levels will contribute to the success of the strategies applied against COVID-19. Hence, the clear understanding of risk factors is important to contribute to the success of the strategies that prevent the spread of COVID-19 pandemic. A new risk assessment model is applied in this study to properly evaluate risk factors determined based on social, technological, government and economic aspects under uncertain information. Due to limited knowledge or data obtained in a short period of time for COVID-19, there may be hesitation and uncertainty with respect to experts’ evaluation information. According to the complexity of problem, this research adopts IVq-ROF-COPRAS to determine the most important risk factors based on people, technology, government and economic conditions to deal with COVID-19 pandemic by considering maximizing and minimizing attributes simultaneously.Table 3 The minimum and maximum daily case numbers for Turkey [9]. Date Daily number of cases 11nd of April 2020 13.976 4th of December 2020 32.736 14rd of April 2021 62.797 16st of April 2021 63.082 01st of February 2022 102.601 08st of February 2022 111.096 Thus, since interval-valued q-rung fuzzy concept is very flexible to capture the uncertainty and incompleteness of information, interval-valued q-rung orthopair fuzzy-COmplex PRoportional ASsessment (IVq-ROF-COPRAS) method is established. In addition to its logical concepts to make risk assessment more effectively, this method has been selected for the purpose of providing more flexibility to professionals in stating their judgments about the ambiguity and imprecision of the considered problem than other fuzzy extensions. Thus, considering four important attributes available in traditional occupational health and safety (OHS) for risk evaluation, the risk factors cause COVID-19 pandemic spread in a society are evaluated to develop strategies by government and health agencies. The main contributions of this paper are as follows. • Risk assessment for the pandemic is a crucial undertaking to increase consciousness and conduct safety measures for controlling the disease. Managing epidemics need critical strategic decisions to handle due to its vital consequences on people and society. Therefore, an extensive risk analysis framework is conducted. • For this aim, a new risk assessment model considering uncertainty and incompleteness of information in decision making process has been presented to determine the risk levels of the risk factors that cause spreading of COVID-19 pandemics in a society. The methodology is constructed based on interval-valued q-rung orthopair fuzzy-COmplex PRoportional ASsessment (IVq-ROF-COPRAS) method. Thus, the constructed model is appropriate for enhancing the quality of decisions by offering capability to represent complex and vague information. • To the best knowledge of authors, in the literature there are only a few studies to evaluate and rank risk factors for the spread of the COVID-19 epidemic in general, specifically using Fuzzy MCDM models [11], [12], [13]. Thus, there are very few studies in the literature that can help governments in this regard. By the study, the most important risk factors are determined and highlighted for managers and governments to fight against COVID-19 pandemic. Consequently, the contribution of this study to the existing literature is to develop a decision support model to evaluate the risk factors causing the spread of the pandemic for the development of strategies against the COVID-19 pandemic. Using technique and information in this study decision makers will be able to create better strategies to prevent pandemics. • The framework is capable of determining risk factors against COVID-19 in any country given that the region-specific data is updated. Furthermore, the proposed model may be used or developed to fight other pandemics and different types of disasters. The study continues as follows: In Section 2, a literature review that examines the Multi-Criteria-Decision Making (MCDM) applications related to COVID-19 and the applications that make use of MCDM methods in risk analysis is presented. Section 3 is the preliminary section in which information about the methods used in the study is presented. Section 4 includes the methodology of the study, Section 5 includes the real case study is performed and sensitivity and comparative analyses are conducted to demonstrate the efficiency of the proposed method, and finally Section 6 includes the results of the implementation and the controversial analysis of the results. 2 Literature review Although the cause and exact solution are not known, the number of studies related to an epidemic as serious as COVID-19 has increased continuously in a short time. The epidemic did not only affect health globally, but also had an impact on many areas from economy to sports, from tourism to politics and brought life to a standstill. In this process, while scientists tried to overcome the disease, social scientists investigated the economic, social and political effects of the work [14]. The literature review in this study consists of two parts. In the first part, MCDM practices on the COVID-19 pandemic were summarized, and in the second part, risk analysis practices made by MCDM methods were summarized. Shrestha et al. [15] examined the influence of COVID-19 in potential global health based on calculated pandemic vulnerability index (PVI) using the Technique for order performance by similarity to ideal solution (TOPSIS) method. The objective was to develop and implement strategies in required countries to help ease emerging burden. The results showed that in Africa; more vulnerable countries were South Africa and Egypt, in Europe; Russia, Germany, and Italy; in Asia and Oceania; India, Iran, Pakistan, Saudi Arabia, and Turkey, and for the Americas; Brazil, USA, Chile, Mexico, and Peru. Maqbool and Khan [16] divided the risk factors in the execution of the measures taken against the COVID-19 in India into 10 titles and analyzed them with a decision-making trial and evaluation laboratory (DEMATEL) method. As a result of the analysis, while lack of resources was found to be the most influential barrier, it was found that the successful implementation of the measures depends on medical equipment and financial resources. Yüksel et al. [17] applied the DEMATEL to analyze which factors were prioritized in determining the most optimal economic recovery package for COVID-19 and concluded that the interest-free loan option was the most important factor. Albahri et al. [18] established a joined Analytic hierarchy Process (AHP) and VIse KriterijumsaOptimiz acija I Kompromisno Resenje (VIKOR) methodology to compare Artificial Intelligence (AI) practices used in the discovery and categorization of COVID-19 examination illustrations in terms of assessment and benchmarking. AHP was used to assess the evaluation factors, and VIKOR was applied for benchmarking AI classification techniques. Ocampo and Yamagishi [19] applied the fuzzy-DEMATEL in determining the most important criteria in the transition of the Philippine government for COVID-19 normalization protocols and determined the exact cause groups as: compliance with the standards, limited mobility of people, 50% capacity, non-operating places such as hotels, industries being closed, and collective meetings were prohibited. Results showed that compliance of community well-being standards, restricted movement of people, postponement of physical courses, the prevention of mass meetings, non-operation of type IV businesses, and non-operation of resorts or like businesses are the most critical properties for developing strategies in the governments to distribute resources and apply measures for developing mitigation efforts. Altuntaş and Gök [20] used DEMATEL to analyze the effect of quarantine decisions on domestic tourism. The objective was to diminish the negative effect of a pandemic on the hospitality industry. The results showed that Istanbul has significant influence on Turkey’s rest. The results also showed that the DEMATEL method produced appropriate solutions for quarantine decisions in a pandemic to prepare the hospitality industry and the method applied also is convenient for similar pandemic. Alkan and Kahraman [13] developed two q-ROF TOPSIS methodologies to evaluate the strategies implemented by governments against the COVID-19. The goal of the research is to identify and analyze the risks or obstacles to control the negative consequences of COVID-19 systematically that may arise from the uncertainties. As a result of the riskanalysis, the risk factors with the highest risk level are identified and measures related to these risk factors to deal with COVID-19 are determined systematically. In almost all risk assessment techniques, the factors that create the risk are determined, the risks are graded and measures are determined in order of importance. In addition to classical risk assessment techniques, the risk levels of risk factors can be listed with MCDM Techniques, which are used to determine the degree of importance of certain alternatives under certain criteria [21]. However, due to risk factors often involves uncertain information, fuzzy MCDM applications were effectively used in the literature [21], [22]. For example, Yazdani et al. [22] developed a fuzzy COPRAS methodology to perform risk analysis of critical infrastructures and compared it with Risk Analysis and Management for Critical Asset Protection (RAMCAP), one of the traditional risk analysis methods, to analyze the accuracy of the results. As a result of the comparison, it was determined that the proposed methodology has the capability to provide more accurate results for ranking the risk of critical infrastructures. Jin et al. [23] used IV-q-ROF, stating that the traditional Failure Mode and Effect Analysis (FMEA) methodology is insufficient in the risk assessment process of tool changing manipulators. To ensure experts’ assessments more reliable, a stable review and process is applied and experts’ opinions are integrated by IVq-ROF weighted average (IVq-ROFWG) operator and IVq-ROF weighted Maclaurin symmetric mean (IVq-ROFWMSM) operator. IVq-ROF-ARAS method applied and sufficient, adequate and precise outcomes were obtained in positioning failure modes. Wan and Zhou [24] combines CRITIC-WASPAS under uncertainty for group decision making. To deal with the uncertainty Interval-valued q-rung orthogonal sets were used. The objective was to evaluate early warning management of hypertension risk diseases. Since the proposed method was adaptable and effective, the results were consistent with decision-makers’ opinions. Cheng et al. [25] used VIKOR (VlseKriterijumska Optimizacija Kompromisno Resenje) approach under q-rung orthopair fuzzy set (q-ROFSs) to identify and assess Enterprise Risk Management (ERM) in the universal appeal under the sustainability platform includes 29 sub-criteria considering social, environmental, technological, and economic issues. The results demonstrated that technological appropriateness was the significant risk aspect followed by technical improvement, work-related protection and well-being, merchandise and services concern, (advantage) anti-corruption labor force applies, and industrial practicality. The results of the study revealed that the suggested technique was effective and successful to evaluate risk factors of sustainable ERM in the Small and Mid-Size Enterprises. The pandemic we are in will only be terminated by handling and coping with the risk factors in front of it. Thus, these risk factors need to be understood correctly, and a clear understanding of the risk levels will contribute to the success of the strategies applied against COVID-19. According to the complexity of problem, this research adopts IVq-ROF-COPRAS to determine the most important risk factors based on people, technology, government and economic conditions to deal with COVID-19 pandemic by considering maximizing and minimizing attributes simultaneously. Due to uncertain and vague information of decision makers (DMs) derived from the information obtained in a short period of time for COVID-19, the model is implemented under an uncertain environment. Thus, since DMs can express their judgement more accurately in IVq-ROF format, the proposed risk assessment methodology become more robust to evaluate risk factors affecting the spread of COVID-19. Different from the literature, this study presents a useful method for risk assessment with simplified calculations, which provide reasonable and practical solutions to DMs. 3 Preliminaries 3.1 Interval-valued Q-rung orthopair fuzzy sets Q-rung orthopair fuzzy sets (q-ROFSs) first launched by Yager [26]. q-ROFSs are characterized with degree of membership and non-membership. In this method, q-ROFSs, the summation of the qth power of the membership and non-membership values should be at one. Some definitions for q-ROFSs and IVq-ROFSs are as follows: Definition 1 A q-ROFS Q~ in a finite universe of discourse X is specified as following by Yager [26]. (1) Q~=x,μQ~x,vQ~(x)\|xϵX where μQ~:X→0,1 and vQ~:X→0,1 are the degree of membership and non-membership, respectively. q-ROFS must satisfy the condition of (2) 0≤(μQ~x)q+(vQ~x)q≤1(q≥1) The degree of indeterminacy: (3) πQ~x=1−μQ~xq−vQ~xqqwhere q≥1 q-ROFSs offers more wide scale for DMs [27], [28] to prompt ambiguous info comparing to Pythagorean fuzzy sets (PFSs) and intuitionistic fuzzy sets (IFSs).1̑-gul-regular20 20The differences IFNs, PFNs, and q-ROFNs are seen in Fig. 1. It is clearly seen that the IFS and PFS can be deduce under the restricted domains μ∈0,1,ϑ∈[0,1], μ+ϑ<1 and μ2+ϑ2<1 by setting q = 1 and q = 2, respectively. In the idea of q-ROFSs, we see that μ+ϑ ≰ 1 and μ2+ϑ2 ≰ 1 but μq+ϑq ≤ 1 by setting 2< q <∞ [29], [30]. Some preliminaries used through this paper for q-ROFSs and IVq-ROFSs are shown in the following. These operations for q-ROFSs are preferred in this study, since they broadly employed by the scholars to solve real-world MCDM problems in the literature. In many real-life problems, it is complicated for the DMs to express the degree of membership and non-membership with a single value. To solve this problem, Joshi et al. [32] introduced IVq-ROFS notion whose main feature is to express membership and non-membership function rates in intervals instead of exact values.Fig. 1 Geometric space range of IFS, PFS, and q-ROFS [31]. Definition 2 [33] Let X be a non-empty and limited set, a IVq-ROFS P~ on X is given as: (4) P~=\|x,μP~x,vP~x\|xϵX where the function μ~P~x=μP~Lx,μP~Ux:X→0,1 and v~P~x=vP~Lx,vP~Ux:X→0,1 are intervals representing the membership and non-membership degrees of the element x ∈ X to the set P~, correspondingly, which satisfies the condition of (5) μP~Uxq+vP~Uxq≤1,q≥1 The degree of indeterminacy is expressed as: (6) πp~x=πp~Lx,πp~Ux=1−μP~Uxq+(vP~Ux)qq,1−μP~Lxq+(vP~Lx)qq While q=1, IVq-ROFS reduces to interval-valued intuitionistic fuzzy set (IVIFS), q = 2, IVq-ROFS reduces to interval-valued pythagorean fuzzy set (IVPFS). Definition 3 [33] For a IVq-ROFN p~=μp~L,μp~U,vp~L,vp~U, the score function s(p~) and the accuracy function Hp~ of p~ are calculated as follows: (7) s(p~)=14(1+(μp~L)q−(vp~L)q)+(1+(μp~U)q−(vp~U)q),sp~∈0,1. (8) H(p~)=(μp~L)q+(vp~L)q+(μp~U)q+(vp~U)q2,Hp~∈0,1. Definition 4 [34] p~1=μp~1L,μp~1U,vp~1L,vp~1Uandp~2=μp~2L,μp~2U,vp~2L,vp~2U are two IVq-ROFNs. Some arithmetic operations of the IVq-ROFNs are defined as follows and the result of each operation is an IVq-ROFN: p~1⊕p~2= (9) μp~1Lq+μp~2Lq−μp~1Lqμp~2Lqq,μp~1Uq+μp~2Uq−μp~1Uqμp~2Uqq,vp~1Lvp~2L,vp~1Uvp~2U (10) p~1⊗p~2=μp~1Lμp~2L,μp~1Uμp~2U,vp~1Lq+vp~2Lq−vp~1Lqvp~2Lqq,vp~1Uq+vp~2Uq−vp~1Uqvp~2Uqq (11) λp~=1−(1−μLq)λq,1−(1−μUq)λq,vLλ,vUλ,λ>0; (12) p~λ=μp~Lλ,μp~Uλ,1−(1−vp~Lqq)λ,1−(1−vp~Uqq)λ,λ>0 Definition 5 [35] Interval-valued q-rung orthopair fuzzy weighted geometric (IVq-ROFWG) operator: Suppose P~i=μP~iL,μP~iU,vP~iL,vP~iU (i = 1,2,…,n) is a collection IVq-ROFNs then Interval-valued q-rung orthopair fuzzy weighted geometric (IVq-ROFWG) operator is defined as follows and the result is an IVq-ROFN : IVq-ROFWG(P~1,P~2,…,P~n)= (13) ∏i=1n(μP~iL)wi,∏i=1n(μP~iU)wi1−∏i=1n(1−vP~iL)wiq,1−∏i=1n(1−vP~iU)wiq where wj=(w1,w2,…,wn)T thereby satisfying wj∈[0,1] and ∑j=1nwj=1 A comparative examination of the proposed approach with other state-of-the-art methods under both q-ROFS and IVq-ROFS context considering theoretical and numerical factors. A large amount of uncertainties may occur in risk analysis, IVq-ROFSs have an distinctive benefit in coping with the uncertainty and vagueness of DMs’ assessment information and provide a efficient aggregation operators such as IVq-ROFWG and q-rung interval-valued orthopair fuzzy weighted Hamy mean (q-RIVOFWHM) operator. IVq-ROFWG is well suited for calculating the relative weights of four main attributes available in proposed risk assessment process. IVq-ROF COPRAS method easy-use and comprehensible steps, which can produce rational, satisfactory and relatively accurate results in ranking of risk factors (see Table 4). Table 4 Characteristic investigation of proposed and different models under q-ROFS and IVq-ROFS context. Proposed method [34] [35] [29] [30] Context Data IVq-ROFNs IVq-ROFNs IVq-ROFNs q-ROFNs q-ROFNs Decision-making type Group decision-making Group decision-making Group decision-making Group decision-making Group decision-making Attributes’ weight calculation IVq-ROFWG operator q-rung interval-valued orthopair fuzzy weighted Hamy mean (q-RIVOFWHM) operator Gini index SV method Aggregation operator IVq-ROFWG Hamy mean (HM) operator, weighted HM (WHM) operator, dual HM (DHM) operator, and the dual-weighted HM (WDHM) IVq-ROFWA, Vq-ROFOWA, GIVq-ROFHA Muirhead mean q-ROFGMSM Prioritization methods COPRAS q-RIVOFWHM IVq-ROFWA, IVq-ROFOWA,GIVq-ROFWA, GIVq-ROFOWA, IVq-ROFHA, and GIVq-ROFHA TODIM VIKOR 3.2 COPRAS method The main steps of COPRAS are summarized as follows (Zavadskas et al. [36]): Step 1: Definition of the problem: Considering the problem, determine the sets of alternatives and measures. Step 2: Construction of the decision-matrix: Specialists assess the determined alternatives taking into consideration the criteria. Accordingly, construct the decision-matrix as: (14) xijnxm=x11x12⋯x1m⋮⋱⋮xn1xn2⋯xnm where xij represents the performance value of ith {i ∈ 1,2,…, n} alternative associated with the criterion j, j ∈{1,2,…, m}. Step 3: Normalize decision-matrix. The normalized matrix is created by employing normalization process as in Eq. (15). (15) nij=xij∑i=1nxij where i ∈{1,2,…, n} and j ∈{1,2,…, m} Step 4: Establish the weighted normalizes-matrix. This matrix is created by performing Eq. (16). (16) rij=wjnij where i ∈{1,2,…, n}, j ∈{1,2,…, m} and ∑j=1nwj=1. Step 5: For each alternative, compute the summation of maximizing and minimizing criteria: Alternatives are rated considering the maximizing Pi(benefit) and minimizing Ri, (cost) features. The summation of both types of criteria are calculated by employing Eqs. (17), (18) correspondingly. k represents the number of criteria to be maximized while there are n criteria; (17) Pi=∑i=1krij (18) Ri=∑i=k+1nrij Step 6: Calculate Rimin, the minimal value of Ri: (19) Rimin=miniRi Step 7: Obtain the relative weights associated with each alternative (Qi) by performing Eqs. (20)–(21): (20) Qi=Pi+Rimin∑i=1nRiRi∑i=1nRiminRii∈1,2,…,n Qi can be simplified as: (21) Qi=Pi+∑i=1nRiRi∑i=1n1/Rii∈1,2,…,n Step 8: Obtain the alternative with the highest value of Qi: (22) Qmax=maxiQii∈1,2,…,n Step 9: For each alternative, compute the utility-degree −Ui, which is used to rank the alternatives and obtained by applying the Eq. (23): (23) Ui=QiQmax The priority of the alternatives is determined by the value of Ui. In other words, the greater Ui the more priority. 4 Proposed methodology: IVq-ROF COPRAS method The proposed methodology, which is used in risk assessment model for the risk factors to cope with the COVID-19 pandemic faced. The Interagency Security Committee (ISC) defines risk “as a function of threat, vulnerability, and impact values. The purpose of risk management is to create a protection scheme that reduces vulnerabilities and potential impacts against threats, thereby reducing the risk to an acceptable level. Various mathematical models are available to calculate risk and show the effect of increased protective measures on the risk equation [37]. The framework of the methodology followed in this study is shown in Fig. 2. Considering the complication of the decision-making process and the uncertain information during decision making process, this study implements IVq-ROF-COPRAS to define the most significant risk factors to cope with COVID-19 pandemic spread by considering maximizing and minimizing criteria concurrently. COPRAS, as one of the well-known and easy to apply method [38], [39], is preferred in assessing the risks. Because of IVq-ROFSs’s ability to handle a wider range of uncertainty of information in the real-world decision-making processes and providing freedom degree to DMs’ assessments, an extension of COPRAS, which is named as IVq-ROF COPRAS, is developed as a risk assessment framework for assessing risk factors against COVID-19 pandemic spread, in the following steps:Fig. 2 Framework of the methodology proposed. Step 1. Create a DM group to determine risk factors cause of spreading COVID-19 and determine attributes with respect to risk assessment to evaluate risk factors. The members of this group determine the risk factors cause spreading of COVID- 19 pandemic. Step 2. Determine the weights of attributes: The DMs opinions regarded to attributes are represented in the form of IVq-ROFNs based on linguistic expressions given in Table 5. The aggregation operator IV q−ROFWG, which is given in Eq. (13), is applied once the weights given by experts are assigned. wjk=aij,bij,cij,dij represents the kth (k=1,2,…,K) DM’s weights assigned to jth (j=1,2,n) attribute. Step 3. Create the decision matrix X, which is the IVq-ROF evaluation matrix: In the X, xˇijk=〈aijk,bijk,cijk,dijk〉 designates the IVq-ROF ranking value of ith (i=1,2,…m) risk factors taking into account the attribute j, (j=1,2,n) for the kth (k=1,2,…r) DM. Once judgements are provided by the DMs linguistically to evaluate risk factors for overcoming COVID-19 pandemic, they are transformed into IVq-ROFNs via linguistic measures provided in Table 6. (24) X~=x~ijknxm=x~11kx~12k⋯x~1mk⋮⋱⋮x~n1kx~n2k⋯x~nmk where x~ijk≥0 and x~ijk=〈aijk,bijk,cijk,dijk〉 and 0≤(bijk)q+(dijk)q≤1.Table 5 Linguistic expressions and correspondence IVq-ROFNs to evaluate attributes [40]. Linguistic expression μU μU vL vU Extremely High Important (EHI) 0.80 0.95 0.00 0.15 Very High Important (VHI) 0.70 0.80 0.15 0.25 High Important (HI) 0.55 0.70 0.25 0.40 Medium Important (MI) 0.45 0.55 0.40 0.55 Low Important (LI) 0.30 0.45 0.55 0.70 Very Low Important (VLI) 0.20 0.30 0.70 0.80 Extremely Low Important (ELI) 0.00 0.20 0.80 0.95 Step 4. Create the aggregated IVq-ROF decision-matrix considering the opinions of DMs: In this step, previously created individual decision-matrices are combined in this matrix. To determine the overall expert opinions, IVq-ROFWG operator is conducted, which is provided in Eq. (13) and known as aggregation operator. Accordingly, X~A matrix is constructed.Table 6 Linguistic expressions and correspondence IVq-ROFNs to evaluate risk factors [33]. Linguistic expression μL μU vL vU Extremely High (EH) 0.90 0.95 0.10 0.15 Very High (VH) 0.80 0.85 0.20 0.25 High (H) 0.70 0.75 0.30 0.35 Medium High (MH) 0.60 0.65 0.40 0.45 Medium (M) 0.50 0.55 0.50 0.55 Medium Low (ML) 0.40 0.45 0.60 0.65 Low (L) 0.30 0.35 0.70 0.75 Very Low (VL) 0.20 0.25 0.80 0.85 Extremely Low (EL) 0.10 0.15 0.90 0.95 Step 5. Normalization of the aggregated IVq-ROF decision matrix: The matrix created in the previous step is normalized by using Equations given in Eqs. (25)–(26). (25) N~ij=n~ij (26) n~ij=cij,dij,aij,bijfor cost attributeaij,bij,cij,dijfor benefit attribute Step 6. Determine the IVq-ROF weighted normalized matrix: The weights of criteria are multiplied by the normalized values. (27) R~=r~ijnxm (28) r~ij=w~j⊗n~ij Step 7. Compute sum of weights, which is gathered through Eq. (29), for cost (minimized or non-beneficial) attributes: (29) Pi=∑i=1kr~ij Step 8. Compute sum of weights, which is gathered through Eq. (30), for beneficial-(maximized) attributes. (30) Ri=∑i=k+1nr~ij Step 9. In this step, the relative weight of each risk factor (Qi) is calculated: The priorities (relative weights) of risk factors are determined via Equations given in Eqs. (31)–(32). The score functions of Pi and Ri are shown by s(Pi) and s(Ri) and they are calculated using Eq. (7) respectively. (31) Qi=s(Pi)+s(Rimin∑i=1ns(Ri))/s((Ri)∑i=1ns(Rimin))/s(Ri) (32) Qi=s(Pi)+∑i=1ns(Ri)/s(Ri)∑i=1n1/s(Ri) where Rimin shows the min. value of Ri. Step 10. Select the highest Qi and the risk factor that belongs to by using Equation given in Eq. (33). (33) Qmax=maxiQi Step 11. Using the Equation given in (34) calculate the utilities, which represents the priority of the risk factors: (34) Ui=QiQmax 5 Application 5.1 Problem definition Although it has been almost 2 years since the declaration of a global pandemic on January 30, 2020, and vaccination continues to be widespread, COVID-19 pandemic, which has affected the whole world continued to appear. While some countries have successfully dealt with the pandemic, the situation is still bad in many countries such as in America, India, and Brazil. For this aim, in this part of the study, it is aimed to prioritize the risk factors that prevent countries to overcome the COVID-19 pandemic according to risk levels and in turn presenting strategic precautions that can be applied. 5.2 Factors preventing societies from overcoming the covid-19 pandemic Risk factors in dealing with the COVID-19 pandemic spread are represented under four factors as in Fig. 3 considering the literature and opinions of expert on the field. The hierarchical structure consists of two levels under target objective. The first and second level are the main and sub-risk factors that can cause spreading of COVID-19. Accordingly, in this paper, the risk factors cause COVID-19 pandemic spread in society are ranked for developing strategies by governments.Fig. 3 Risk factors cause COVID-19 pandemic spread. 5.2.1 People Insufficient Isolation at Home (R1) Once the virus-carrying people are identified, they must be quarantined. The people who contact with them must be also identified after they subjected to the necessary tests and quarantined according to the test result. At the beginning of the pandemic, all individuals at risk of COVID-19 were quarantined for 14 days in designated areas outside the hospital [41]. Further, the units where they can be quarantined outside the hospital for individuals who stated that they could not provide isolation at home were determined on the basis of provinces, but in the following process, implementation was insufficient. People who have tested positive for COVID-19 stayed at home by stating that they could provide isolation [42]. However, most of these individuals could not meet sufficient distance and cleaning measures with family members, and as a result of this contact, all households were infected. Lack of Pandemic Management Culture in Individuals and Businesses (R2) The increase of COVID-19 cases is closely related to the society’s compliance with measures such as social distance, hand hygiene and use of masks. Precautions and policies were not sufficient to prevent the spread of COVID-19 in their workplace for employers [43]. In addition, it was not enough to close the coffee shops or restaurants etc., where people could hang out in groups. Individuals continued to contact each other at home, on beaches, wedding ceremonies etc. instead of reducing contact with each other to the required level. Furthermore, many citizens do not maintain social distance and wear masks properly. As a result, most people got infected with COVID-19 and they cause to spread of virus throughout the community. Applying only the measures taken by government the pandemic will increase the number of cases and deaths if the necessary measures are not taken individually [37]. Lack of Health Literacy (R3) During the pandemic process, communication network established between the society and responsible institutions to share information for COVID-19 pandemic. Thus, thanks to communication network, individuals should clearly know the importance of hygiene and distance measures and the possible consequences that may arise as a result of the implementation or non-application of these measures. In the current pandemic process, due to limited and inadequate health literacy, negative information bias leads to catastrophic thinking in the society, while positive information bias leads to unrealistic optimism [44]. In addition, for example, while citizens agree for vaccination, individuals who refuse to be vaccinated endanger not only their own health but also the public health based on adequate. 5.2.2 Technology Efficiency of Tracking Applications (R4) Through the Tracking Applications, the user codes that are checked at the entrance to the public areas are strictly followed by the officials. However, it is a matter of debate that the public uses the application regularly and correctly to manage the application data correctly. It is known that thousands of people are infected every day and, since people do not use the application sufficiently before it turns out to be positive, it cannot be determined with whom they exceeded the social distance, and the application cannot achieve its purpose. For this reason, the promotion and operation of the Tracking Applications should be made more widely and effectively [45]. Patients who want to get a vaccine appointment over the phone face problems such as line preoccupation, Tracking Applications is not functional in some areas, and most patients over the age of 60 are having hard times using the internet. In the mobile application, it is emphasized that technical problems caused the delay and sometimes inability to vaccinate [46]. Lack of Medical Technologies in diagnosis and treatment (R5) A strong health technology infrastructure is one of the factors that facilitates the fight against pandemic. Many countries have assigned and supported experts in their fields for treatment and vaccination studies. Behind the success of countries such as Germany, South Korea, and China, which are among the successful examples of combating pandemics, is the high number of laboratories and high testing capacity [47]. If samples are taken from the contact persons for 5–7 days and are negative, the isolation periods are terminated on the 7th day. However, this period is insufficient considering the situations where people who have contact with one of their families live in the same house with positive patients during the isolation process. Because there is no 100% reliable method for diagnosis yet swab tests can give ‘false negative’ results [48]. In addition, it is known that a significant portion of the cases experience the process asymptomatically and these people are sometimes not tested because they do not show symptoms. These people cause the disease to spread without realizing that they are sick [49]. In some countries, the fact that a successful vaccine has not been developed, there is no COVID-19 treatment standard in practice, and the tests used are not yet 100% reliable, show that medical technologies are inadequate. Lack of Infrastructure in Carrying out Mandatory Activities (R6) Technological infrastructure is an important element for activities that should be carried out in educational institutions, businesses, etc. in combating the pandemic. Due to the lack of this infrastructure, many business employees could not switch to work from home and multiple cases have emerged in businesses. In addition, the disrupted educational activities have also created negative psychological effects on the people’s adoption of the pandemic culture. 5.2.3 Government Lack of Strict Restrictions (R7) During the coronavirus epidemic process, many countries had to take restrictive measures in unprecedented levels. The aim of the governments’ objective was to apply restrictions such as social distancing, curfew, travel etc. to reduce the spread of the virus [50]. However, public measures taken to control individuals’ compliance with the precautions have been insufficient. The uncertainty in business life and the suspension of the activities in many workplaces and schools accelerated the mobility and this caused the spread of virus in society [51]. Lack of Process Control in Normalization (R8) The purpose of the normalization process should be to maintain life as close to normal as possible while keeping the number of diseases and deaths in the society as low as possible. WHO recommends that restrictions on travel and curfews be gradually lifted. Following the steps to be taken to reduce restrictions, their effects should be controlled. Experts state that the number of tests should be increased and contact tracking should be done strictly to control the epidemic rather than lifting the restrictions. WHO recommends allowing activities gradually during the normalization period. However, before decision taken predictive models should be developed, tested, and the use of public spaces should be categorized. On the other hand, the normalization process in some countries was not realized as stated, and priority was given to normalization in areas such as “tourism”, where at the measures will be very difficult to apply. Based on the statistics, it can be concluded that there was a serious decrease in the number of cases and patients during the curfews. The sudden reopening of the public areas, which enabled the crowds to coexist together, caused people who were closed at home for a period to perceive that the “virus danger has passed” or decreased and behave irresponsibly [45]. Flow of Information and Communication/ Trust in Institutions (R9) King states that “information flow, trust in government and trust in institutions are important” to ensure people comply with restrictive measures [52]. The uncertainty of the messages given by the government or health authorities can cause confusion and fear in the public, which may make people to behave uncontrolled because they tend to take measures to secure themselves. For example, in the first days of the epidemic, people emptied the shelves in markets, and cash withdrawal queues were formed in front of ATMs. The closure that will start at 24:00 in Turkey was announced only two hours in advance, which caused a similar confusion. This type of confusion causes the disease to spread. Thus, citizens should be warned about fake news and false information, and information should be shared with the public in a timely and accurate manner. Another of the most important elements in this process is the communication. Proper communication between health authorities, government, leaders, and public will encourage individuals to be conscious about the pandemic and take suitable actions to prevent its spread [53]. 5.2.4 Economy Insufficient Economic Resources Against Economic Anxiety (R10) The strength of people to comply with their calls to stay at home is proportional to their jobs and economic situations. Poverty has become the key in determining the more sensitive segments of societies to restrictions [37]. While individuals with high income are trying to protect themselves from the epidemic by living in isolation in their homes in the outside world, those with low income and those who must work outside are more likely to get the disease [53]. The government, tried to help people in economic difficulties by announcing support packages. Various aid campaigns (such as “We Are Enough for Us”, “Pay the Bill For Others”) have been carried out for those who are unemployed or who are in poor financial situation after the decision of staying at home, and a large participation in them has been achieved. However, it is very difficult for governments as well as individuals to bear the economic difficulties caused by restrictions. For this reason, sometimes the economic resources required to implement public health and social measures may be insufficient [54], [55], [56]. Lack of Medical Facilities and Staff (R11) The COVID-19 pandemic has increased the need for medical supply, healthcare facilities and healthcare professionals to a much higher than before [57]. With the onset of the pandemic, it is known that despite the rapid construction of emergency hospitals and the increase in healthcare worker employment, polyclinics are now evacuated and converted into COVID-19 services, and intensive care occupancy rates have reached high values in some provinces [58], [59], [60]. Approach in Policies that Prioritize the Economy (R12) While taking precautions during the pandemic process, first, gathering of big groups such as stadiums and concerts should be prevented. If this measure is insufficient afterwards, measures such as medium-sized weddings, funerals, and if it continues, measures should be taken to prevent 10 people from coming together. The opposite should be followed when the measures are loosened [55]. However, in this process, the first loosened environments in some countries have been environments where thousands of people can coexist. This situation indicates that the pandemic management process has an approach that prioritizes the economy. 5.3 Attribute evaluation Four main attributes available in risk assessment process are determined as evaluation criteria to evaluate risk factors shown in Fig. 3. The attributes and related explanations are shown in Fig. 4. Due to the nature of the COPRAS method, while “Likelihood of threat”, “Vulnerability” and “Impact or Severity” are determined as cost attributes, “Precaution” is determined as benefit attribute. While the cost attribute should be as low as possible, the benefit attribute should be as high as possible maximized. Since q-ROFSs can be used to describe larger and more complex fuzzy information [61], [62] we developed a new risk assessment approach based on IVq-ROFS including maximization of the chance and outcomes of positive trials and minimization of the chance and outcomes of undesirable consequences. Accordingly, four attributes are evaluated by DMs using Table 5 and then, the importance weights of attributes are determined using IVq-ROFWG operator, which is given in Eq. (13).Fig. 4 Proposed risk assessment model. First of all, the DM group (DM1, DM2, DM3, DM4 and DM5) consist of academicians in training and research hospitals and the member of medical and public health agencies are invited. Five DMs evaluated risk factors based on four important attributes using linguistic variables presented in Table 6. For example, DM1 rated the probability of “Insufficient Isolation at Home” risk occur as medium (M), the impact and vulnerability of this risk as medium high (MH) and high (H), respectively. The measures taken for this risk in society is evaluated as medium (M) by DM1. The evaluations made by other DMs are reported in Table 7. The decision matrices are aggregated in one decision matrix using IVq-ROFWG operator. The results are given in Table 8.Table 7 The evaluations of DMs for risk factors considering risk factors. DM1 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Likelihood M MH M MH MH M MH H M MH MH H Impact MH H M H H M MH VH MH H H VH Vulnerability H H M H VH M M H MH MH H H Precautions M MH MH MH H M MH VH M H MH VH DM2 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Likelihood MH H MH MH H MH MH H MH H VH H Impact MH VH M H MH M MH VH H VH H H Vulnerability H VH MH VH H M MH VH MH MH H H Precautions VH MH H H VH MH MH VH MH H H VH DM3 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Likelihood M VH H MH H H H VH MH H VH VH Impact MH VH M H MH M MH VH MH VH EH H Vulnerability H VH H VH H H H VH H MH VH VH Precautions H H H VH VH H MH H H VH H VH DM4 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Likelihood MH MH L M L M MH M MH M M MH Impact MH H M MH M M M M M MH M MH Vulnerability M H ML MH ML M M M M M M MH Precautions MH MH L M L M MH M MH M M MH DM5 R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Likelihood M M M MH ML M M MH MH MH M MH Impact L MH ML MH ML M MH M MH M MH MH Vulnerability L MH ML MH ML M MH M MH M MH MH Precautions M M M MH ML M M MH MH MH M MH Once the aggregated decision matrix is normalized using Eqs. (25)–(26) the weights of attributes are multiplied by normalized decision matrix using Eqs. (27)–(28). The results are reported in Table 9.Table 8 Aggregated decision matrix. Risk Likelihood Impact Vulnerability Precautions μ− μ+ v− v+ μ− μ+ v− v+ μ− μ+ v− v+ μ− μ+ v− v+ R1 0.538 0.588 0.470 0.519 0.522 0.574 0.536 0.582 0.552 0.605 0.535 0.580 0.609 0.660 0.433 0.479 R2 0.632 0.683 0.405 0.452 0.716 0.766 0.316 0.362 0.716 0.766 0.316 0.362 0.597 0.647 0.419 0.460 R3 0.501 0.553 0.551 0.598 0.478 0.528 0.527 0.577 0.507 0.558 0.526 0.574 0.536 0.588 0.538 0.502 R4 0.579 0.629 0.429 0.478 0.658 0.708 0.354 0.402 0.694 0.745 0.343 0.389 0.632 0.683 0.405 0.404 R5 0.512 0.565 0.559 0.605 0.550 0.601 0.485 0.532 0.575 0.627 0.505 0.550 0.557 0.611 0.554 0.529 R6 0.555 0.605 0.463 0.512 0.500 0.550 0.500 0.550 0.535 0.585 0.480 0.529 0.555 0.605 0.463 0.500 R7 0.597 0.647 0.419 0.467 0.579 0.629 0.429 0.478 0.575 0.626 0.443 0.492 0.579 0.629 0.429 0.471 R8 0.652 0.702 0.393 0.439 0.663 0.714 0.418 0.462 0.645 0.697 0.421 0.465 0.669 0.720 0.390 0.409 R9 0.579 0.629 0.429 0.478 0.597 0.647 0.419 0.467 0.597 0.647 0.419 0.467 0.597 0.647 0.419 0.460 R10 0.615 0.666 0.408 0.456 0.669 0.720 0.390 0.434 0.558 0.608 0.451 0.500 0.652 0.702 0.393 0.415 R11 0.626 0.677 0.430 0.476 0.667 0.718 0.393 0.438 0.652 0.702 0.393 0.439 0.593 0.644 0.435 0.448 R12 0.676 0.726 0.349 0.396 0.676 0.726 0.349 0.396 0.676 0.726 0.349 0.396 0.713 0.764 0.336 0.364 Once the score functions of Pi and Ri are calculated, the relative weight of each risk factor Q is calculated. The priorities of risk factors are calculated using Equations given in Eqs. (31)–(32). The risk factors with the highest value is determined using Eq. (34) and the results are given in Table 10.Table 9 Weighted normalized matrix. Likelihood Impact Vulnerability Precautions μ− μ+ v− v+ μ− μ+ v− v+ μ− μ+ v− v+ μ− μ+ v− v+ R1 0.342 0.446 0.451 0.493 0.358 0.463 0.517 0.549 0.389 0.505 0.518 0.548 0.346 0.455 0.609 0.639 R2 0.402 0.518 0.391 0.438 0.491 0.618 0.308 0.360 0.504 0.639 0.308 0.357 0.335 0.437 0.597 0.626 R3 0.318 0.419 0.528 0.561 0.328 0.426 0.509 0.545 0.357 0.466 0.510 0.543 0.431 0.477 0.536 0.569 R4 0.368 0.477 0.412 0.459 0.451 0.571 0.343 0.392 0.488 0.621 0.333 0.378 0.324 0.383 0.632 0.661 R5 0.326 0.429 0.536 0.567 0.377 0.485 0.468 0.504 0.404 0.523 0.489 0.521 0.443 0.503 0.557 0.592 R6 0.353 0.459 0.445 0.487 0.343 0.443 0.483 0.520 0.376 0.488 0.465 0.502 0.371 0.475 0.555 0.586 R7 0.379 0.491 0.403 0.450 0.396 0.507 0.415 0.456 0.405 0.522 0.430 0.468 0.343 0.448 0.579 0.609 R8 0.414 0.533 0.379 0.428 0.454 0.576 0.405 0.442 0.454 0.581 0.408 0.445 0.312 0.389 0.669 0.697 R9 0.368 0.477 0.412 0.459 0.409 0.521 0.405 0.446 0.420 0.540 0.406 0.446 0.335 0.437 0.597 0.626 R10 0.391 0.505 0.393 0.441 0.459 0.581 0.377 0.418 0.392 0.507 0.437 0.476 0.315 0.394 0.652 0.680 R11 0.398 0.513 0.414 0.457 0.457 0.579 0.380 0.421 0.459 0.586 0.382 0.421 0.348 0.426 0.593 0.623 R12 0.430 0.551 0.338 0.398 0.463 0.585 0.338 0.386 0.476 0.606 0.339 0.384 0.269 0.345 0.713 0.739 Table 10 The relative weights and ranking of risk factors. Risk factor Q U Rank R1 2.447 0.890 9 R2 2.599 0.945 3 R3 2.364 0.859 12 R4 2.590 0.941 4 R5 2.403 0.873 10 R6 2.393 0.870 11 R7 2.451 0.891 8 R8 2.634 0.958 2 R9 2.477 0.900 7 R10 2.568 0.934 5 R11 2.447 0.922 6 R12 2.568 1.000 1 5.4 Results In normalization period “The Approach that Prioritizes the Economy in Policies”, “Insufficient Process Control in Normalization” and “Lack of Epidemic Management Culture in Individuals and Businesses” are detected as the most major factors to deal with COVID-19. It has been observed that these factors are frequently emphasized in the literature scanned at the beginning of the study and in the expert opinions. For this reason, it is possible to say that the results of the study reflect the truth. It would be more accurate to consider the factors of “The Approach that Prioritizes the Economy in Policies” and “Insufficient Process Control in Normalization” together. These two factors are interrelated. WHO recommends the gradual lifting of restrictions such as travel and curfews. Modeling should be done and tested when deciding which measures should be mitigated first, and the use of public spaces as lower risk activities should be prioritized [45]. Thus, the effects of the alleviating of restrictions should be monitored in the social environment after 15 days, and decisions should be reconsidered at the end of this 15-day process. In the normalization processes initiated in June 2020, March 2021 and July 2021, contrary to these recommendations, adequate control could not be achieved, and a rapid normalization process was experienced. First of all, the tourism sector returned to normal and public areas such as beaches, shopping malls, cafes, where the number of people and contact were high, were opened to public. This situation shows that there is an approach that prioritizes the economy in policies. The economic resources required to implement public health and social measures may be insufficient from time to time. Thus, it is possible to say that it is usual for the policies implemented to prioritize the economy at a certain level, and it would be more accurate to make strategy proposals specific to this factor by economists who are experts in the subject. However, whatever the reason may lie on the basis of the strategies applied, the practices must be ensured in order and control. Currently, pandemic restrictions have been lifted in countries, except for masks and social distance, and it is seen that the number of cases is increasing rapidly. It seems inevitable that the restrictions will be re-enacted in the future. However, serious strategies should be implemented to prevent the number of cases, which are predicted to decrease with the restrictions, from increasing as in previous times with the resumption of normalization. “Lack of Epidemic Management Culture in Individuals and Businesses” is actually interrelated with other factors. The increase in cases is closely related to the public’s compliance with measures such as social distance, hand cleaning and mask use, which are the measures that are not strictly complied by the public. Recall the factors given in the application section and in line with the results, it can be said that the factors on the basis of “Human” and “Economy” rather than the factors on the basis of “Technology” and “Government”, are the most important factors to cope with the pandemic. Suggestions for what the authorities should consider to overcome these risk factors in the strategies to be implemented in the future are listed as follows: • The normalization process should be applied gradually and in a controlled manner, spreading over a long period of time. Entry/exit measures of environments where people can be found in groups should be increased. Businesses and individuals should be checked frequently for compliance with the measures taken by the government. • In order to comply with the maximum number of people determined within the framework of social distance rules for areas such as cafes, buses, shopping malls, and beaches, surveillance should be more frequent. However, due to these restrictions, updates should be made in fields such as bus times to prevent people from experiencing disability. Otherwise, the life of the people becomes difficult when the usage is restricted while the service remains at the same level. • Due to the lack of knowledge of individuals on this subject, clear, understandable, and detailed information should be provided to all households on how to provide isolation at the beginning of quarantine to infected individuals and households in order to prevent the spread of the virus. • Increasing the trust of individuals towards institutions and information flow is an important step to ensure compliance with the measures taken. For this reason, efforts should be made to convince the public that public spaces, political/social groups, and people who are central in these groups comply with the measures taken. 5.5 Sensitivity and comparison analysis To validate the robustness of the proposed risk assessment model in determining risk factors to cope with COVID-19, a sensitivity analysis is performed. To apply sensitivity analysis, the rung q, as the most significant feature of the proposed IVq-ROF concept, is changed slightly and the ranking results for risk factors are investigated. In this study, q is selected as 5 to handle the uncertain information better in the base case. The effect of changing q in the ranking of risk factors are shown in Fig. 5. Sensitivity analyses confirmed that the proposed model is stable and efficient due to slight changes in the ranking results. To verify effectiveness and feasibility of the approach a comparison analysis is conducted. To do comparison analysis the results of proposed approach are compared with Interval valued q-rung orthopair fuzzy hybrid averaging (IVq-ROFHA) operator proposed by Ju et al. [35], IVIF TOPSIS conducted by Budak et al. [63] and IVPF-COPRAS introduced by Seker [64]. The comparison results are given in Fig. 6. The comparative analysis results reveal that there is high consistency between proposed approach and existing approaches since the ranking order of the 12 risk factors are parallel. The differences are derived from the use of different fuzzy extensions in the whole process. The results of sensitivity and comparison analyses demonstrates that the proposed risk assessment model to cope with COVID-19 is stable and practical for DMs. In addition, the proposed model produces feasible results since the results coincides with the ones shown in existing approaches.Fig. 5 Sensitivity analysis results. Fig. 6 Comparative analysis results. 6 Conclusion In the global pandemic announced by WHO on January 30, 2020, the number of confirmed cases worldwide has reached 521 million and the number of deaths has reached 6.3 million by May 16, 2022 [2]. Almost all countries of the world have adopted various strategies to apply to their own societies. Many measures have been taken since the beginning of the pandemic but the situation is not very promising at the moment. The virus has once again started to increase its effect rapidly. With the increasing of daily number of cases, many countries started to experience the 4th wave concern in the pandemic. Despite the various measures implemented since the beginning of the pandemic, the fact that the number of cases reaches peak points from time to time is an indication that the measures applied are insufficient. Based on the results gathered from this study, to be able to take clearer steps in the measures and strategies implemented, it is necessary to clearly understand the reasons that hinder in this fight. In line with this reason, the risk factors that cause the spread of the epidemic were determined based on an extensive literature research and expert judgments. Subsequently, the risk levels of these factors were determined by considering certain criteria using IVq-ROF- COPRAS method. According to the results of the proposed risk analysis to determine risk factors against COVID-19 in countries, the top three factors were “The Approach that Prioritizes the Economy in Policies”, “Insufficient Process Control in Normalization” and “Lack of Epidemic Management Culture in Individuals and Businesses”. It has been observed that these factors are frequently emphasized in the literature, in the expert opinions, and in the news sources as the reasons for the inability to overcome the pandemic. For this reason, it is possible to say that the results of the study reflect the fact. To check the validity and robustness of the developed methodology, the analysis was also carried out with two existing methods available in the literature, and a comparative analysis and sensitivity analysis were carried out. As a result of these analyses, the proposed methodology produces consistent and effective results. For future work, the proposed methodology can be improved for the new risk analysis problems faced in real life under uncertain environments. CRediT authorship contribution statement Sukran Seker: Conceptualization, Methodology, Formal analysis, Writing – review & editing. Fatma Betül Bağlan: Writing – original draft, Writing – review & editing, Visualization. Nezir Aydin: Software, Investigation, Data curation, Writing – review & editing. Muhammet Deveci: Validation, Investigation, Data curation, Writing – review & editing. Weiping Ding: Supervision, Project administration, Writing – review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Soft Comput. 92 2020 106322	36471784	PMC9714129	NO-CC CODE	2022-12-13 23:17:36	no	Appl Soft Comput. 2023 Jan 1; 132:109891	utf-8	Appl Soft Comput	2,022	10.1016/j.asoc.2022.109891	oa_other
==== Front Neurologia (Engl Ed) Neurologia (Engl Ed) Neurologia (Barcelona, Spain) 2173-5808 Sociedad Española de Neurología. Published by Elsevier España, S.L.U. S2173-5808(22)00185-7 10.1016/j.nrleng.2021.12.005 Letter to the Editor Functional neurological disorders in post COVID-19 patients. Case series Trastornos neurológicos funcionales en pacientes post-COVID-19. Serie de casosAlbu S. abc⁎ Guven Z. ad Vallés M. abc Kumru H. abc a Fundación Institut Guttmann, Institut Universitari de Neurorehabilitació adscrit a la UAB, Badalona, Barcelona, Spain b Universidad Autónoma de Barcelona, Cerdanyola del Vallès, Barcelona, Spain c Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Barcelona, Spain d Faculty of Arts and Science, Department of Biology, Uludag University, Nilüfer-Bursa, Turkey ⁎ Corresponding author. 1 12 2022 1 12 2022 © 2022 Sociedad Española de Neurología. Published by Elsevier España, S.L.U. 2022 Sociedad Española de Neurología Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcSince the beginning of the COVID-19 pandemic, neurological manifestations have been reported in association with SARS-CoV-2 virus infection, including encephalopathy, encephalitis, stroke, neuropathies, and Guillain-Barré syndrome1. Functional neurological disorders (FND), including involuntary movements, tremor, ataxia, motor and sensory deficits, have rarely been reported in patients with post–COVID-19 syndrome2, 3 or after vaccination against SARS-CoV-24, 5, 6. In this article, we report 2 clinical cases of patients with FNDs after COVID-19 who were referred to Institut Guttmann for rehabilitation. Patients gave written informed consent for the publication of videos. A third patient requested compensation for participating in the study; this request was declined. Patient 1 The first patient was a 37-year-old nursing assistant, under treatment for anxiety disorder. He was admitted due to bilateral pneumonia secondary to SARS-CoV-2 infection, and presented respiratory insufficiency requiring non-invasive oxygen therapy and treatment with dexamethasone, remdesivir, levofloxacin, and enoxaparin. Two weeks after discharge, he reported hyperaesthesia of the feet, loss of muscle mass in the bilateral tibialis anterior and gastrocnemius muscles; he also reported that his legs “collapsed” when walking, and used a crutch for safety reasons. Suspecting acute inflammatory polyneuropathy, we requested biochemical and autoimmune studies and an analysis of infections, which yielded negative results. Electromyography findings were normal. He attended our centre 6 months later. reporting loss of strength in the legs, difficulty walking, and hyperaesthesia of the feet. A neurological examination revealed slightly decreased strength in the lower limbs (4/5 muscle strength); incongruent gait pattern and lower walking speed, wide-based gait, and reduced arm sway (video 1a); and normal tandem gait (video 1b). Clinical symptoms in a subsequent examination were inconsistent, now presenting a hemiplegic pattern with a slightly flexed arm with reduced sway, and leg rigidity (video 1c). After 2 months of rehabilitation, gait fully recovered but hyperaesthesia of the feet persisted. Patient 2 The second patient was a 25-year-old nurse under treatment for major depression with suicidal ideation. She was self-isolating at home due to mild COVID-19. Eight days later, she experienced ascending paraesthesia/hypoaesthesia in the left hemibody up to the C5 dermatome, and loss of strength in the left limbs. Given suspicion of myelitis, we requested serum and CSF biochemical and autoimmunity studies and an analysis of infections, which yielded negative results. The brain and cervical-thoracic spinal MRI scan showed normal results. The patient subsequently reported tremor in the left hand and worsening of gait, needing a crutch to prevent falls. Reassessment with a brain MRI scan and motor and sensory evoked potentials in all 4 limbs revealed normal results. She attended our centre 10 months later due to persistent paresis and left hemihypoaesthesia; joint pain; fatigue; headache; attentional, memory, and naming difficulties; and symptoms of anxiety and depression. The neurological examination revealed a hemiplegic gait pattern, requiring the use of a crutch, with the leg in external rotation with support of the inner edge of the foot and dragging behind the body axis, with significant overexertion and excessive fatigue, which suggested functional paresis (video 2a); variable muscle strength in the arm with incongruent and inconsistent symptoms: lack of voluntary movements and resistance during the examination, with “remitting weakness” (video 2b); kinetic tremor and “signs of coactivation” of agonist/antagonist muscles opposing resistance during the examination, which suggests functional tremor (video 2b). The patient required some help to perform basic activities of daily living. The neuropsychological assessment revealed attentional difficulties, memory and executive dysfunction, and major depressive disorder. After 3 months of rehabilitation, symptoms did not improve. FNDs account for approximately 5% to 10% of primary consultations in neurology7. Diagnosis of FNDs is based on the presence of alterations in mobility and sensitivity, which have a significant psychosocial impact but are not compatible with known neurological disorders8. Their diagnosis in patients with post–COVID-19 syndrome may be challenging due to the novelty of the infection and the limited understanding of its neurological symptoms. Motor FNDs are characterised by sudden onset, variable motor deficit and muscle tone with distraction, knee buckling, overexertion, fatigue, or excessively slow movements and gait. Sensory FNDs are characterised by a loss of sensitivity in a delimited area, which is incompatible with physiological sensory innervation or clinical/topographical distribution determined by neurological lesions. The pre-existent psychopathology and the stress resulting from SARS-CoV-2 infection and the resulting isolation may be risk factors for the development of FND9. The prognosis of FNDs is generally poor, especially in patients with long-lasting symptoms, associated psychological or behavioural disorders, or possible secondary gains; therefore, the literature recommends early diagnosis, informing the patient about their disorder, and the identification of appropriate psychological and rehabilitation interventions10. Appendix A Supplementary data The following are Supplementary data to this article: Appendix A Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.nrl.2021.12.002. ==== Refs References 1 Varatharaj A. Thomas N. Ellul M.A. Davies N.W.S. Pollak T.A. Tenorio E.L. Neurological and neuropsychiatric complications of COVID-19 in 153 patients: a UK-wide surveillance study Lancet Psychiatry. 7 2020 875 882 32593341 2 Garg A. Goyal S. Comellas A.P. Post-acute COVID-19 functional movement disorder SAGE Open Med Case Rep. 9 2021 2050313X211039377 3 Piscitelli D. Perin C. Tremolizzo L. Peroni F. Cerri C.G. Cornaggia C.M. Functional movement disorders in a patient with COVID-19 Neurol Sci. 41 2020 2343 2344 32661885 4 Fasano A. Daniele A. Functional disorders after COVID-19 vaccine fuel vaccination hesitancy J Neurol Neurosurg Psychiatry. 2021 10.1136/jnnp-2021-327000 5 Ercoli T. Lutzoni L. Orofino G. Muroni A. Defazio G. Functional neurological disorder after COVID-19 vaccination Neurol Sci. 42 2021 3989 3990 34324120 6 Butler M. Coebergh J. Safavi F. Carson A. Hallett M. Michael B. Functional neurological disorder after SARS-CoV-2 vaccines: two case reports and discussion of potential public health implications J Neuropsychiatry Clin Neurosci. 33 2021 345 348 10.1176/appi.neuropsych.21050116 34261345 7 Carson A. Lehn A. Epidemiology Hallett M. Stone J. Carson A. Handb Clin Neurol 139 2016 Elsevier 47 60 8 Stone J. LaFrance W.C. Jr. Levenson J.L. Sharpe M. Issues for DSM-5: conversion disorder Am J Psychiatry. 167 2010 626 627 20516161 9 Keynejad R.C. Carson A.J. David A.S. Nicholson T.R. Functional neurological disorder: psychiatry’s blind spot Lancet Psychiatry. 4 2017 e2 e3 10 Espay A.J. Aybek S. Carson A. Edwards M.J. Goldstein L.H. Hallett M. Current concepts in diagnosis and treatment of functional neurological disorders JAMA Neurol. 75 2018 1132 1141 29868890	36464223	PMC9714180	NO-CC CODE	2022-12-12 23:20:30	no	Neurologia (Engl Ed). 2022 Dec 1; doi: 10.1016/j.nrleng.2021.12.005	utf-8	Neurologia (Engl Ed)	2,022	10.1016/j.nrleng.2021.12.005	oa_other
==== Front Financ Res Lett Financ Res Lett Finance Research Letters 1544-6123 1544-6131 Elsevier Inc. S1544-6123(22)00738-3 10.1016/j.frl.2022.103562 103562 Article Firms' exposures on COVID-19 and stock price crash risk: Evidence from China Kong Xiaowei a Jin Yifan b Liu Lihua c⁎ Xu Jialu d a School of Economics and Management, Dongguan University of Technology, China b School of Management, Jinan University, China c School of Accounting, Guangdong University of Foreign Studies, China d Department of Sustainability, Wake Forest University, United States ⁎ Corresponding author. 1 12 2022 3 2023 1 12 2022 52 103562103562 15 11 2022 26 11 2022 30 11 2022 © 2022 Elsevier Inc. All rights reserved. 2022 Elsevier Inc. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. This study examines the impact of firms’ exposures on COVID-19 sentiment on the stock price crash risk. We show the exposure on COVID-19 sentiment related to the medical, travelling and uncertain aspects significantly increases the stock price crash risk, while the exposure on COVID-19 sentiment related to vaccines significantly decreases the risk of stock price crash. Furthermore, our findings are stronger for non-state-owned firms and firms with low information transparency. Overall, we provide timely policy implication for economic impacts of the COVID-19 on the stock market. Keywords COVID-19 Sentiment Stock price crash risk China ==== Body pmc1 Introduction The COVID-19 is a global pandemic considered by World Health Organization (WHO). By the end of June 2022, there are close to 570 million reported cases and over 6 million deaths, according to the Johns Hopkins University Coronavirus Resource Centre (https://coronavirus.jhu.edu/). Therefore, the sentiment in stock market can be easily affected by different news of COVID-19 (Lwin et al. 2020) and then affect the stock price dynamics as well as the traders’ behavior (John and Li 2021). Examining the effect of investors sentiment incurred by the epidemic on stock price crash in an emerging market during the COVID-19 period motivates our research. Now the COVID-19 is not over so evaluating its impact specifically on economics can help policymakers to respond with various policies for a speedy recovery of economics. In this study, we empirically investigate the impact of the exposure of COVID-19 sentiment on the stock price crash risk. Consistent with Lwin et al. (2020), Cevik et al. (2022), and Akram and Haider (2022), we use the news about COVID-19 to measure the sentiment. Specifically, we use the comprehensive indices constructed by Narayan et al. (2021) which capture the news about COVID-19 from different aspects. Due to the sentiment brought by COVID-19 news, including medical, travelling, and uncertain aspects, the tolerance for other bad news of managers and investors declines, as the total tolerance of bad news is limited. In this condition, more bad news is hidden to maintain the positive attitude of firms from investors. Therefore, we believe the sentiment related to general news of COVID-19, and the detailed aspects of medical, travelling, and uncertain can lead to an increase in stock price crash risk. While vaccines can help us drive COVID-19 out, so the sentiment exposed to vaccines is opposed to the sentiment exposed to other news of COVID-19, which is consistent with the point of Cevik et al. (2022). Hence, sentiment exposed to vaccines can reduce the risk of stock price crash. The results support our hypotheses. Specifically, we find heterogeneous impacts of the exposure of COVID-19 sentiment on the stock price crash risk. On the whole, the exposure of COVID-19 sentiment significantly increases the stock price crash risk using the aggregate COVID-19 index. And the exposure of COVID-19 sentiment related to the COVID-19, medical, travelling, and uncertain aspects significantly results in the increase of the stock price crash risk. But the exposure of COVID-19 sentiment related to vaccines significantly decreases the risk of stock price crash. We further find results above are milder in the SOEs and firms with the high information transparency. The study contributes to the literature in the following ways. Firstly, it contributes to the ongoing discussion on the real effect on economics of COVID-19 (Cevik et al. 2022; John and Li 2021; Kong et al., 2022a; Kong et al., 2022b). Prior studies concentrate more on the whole effects of COVID-19 using the number of new confirmed cases or deaths, which ignore more detailed information brought by COVID-19. Secondly, this study is also related to the board literature on the economic consequences of sentiment. Based on the development of society, we capture the impacts of sentiment related to the macro environment (COVID-19) on stock price crash risk. Thirdly, it adds more evidence to the determinants of the stock price crash risk (Chen et al. 2001; Callen and Fang 2015; Wu et al., 2022; Wen et al. 2019). Although several studies have explored the role of investors’ sentiment on the stock price crash, the role of heterogeneous investor sentiment incurred by COVID-19 in the crash risk has received little attention. Our results provide new evidence to support the bad news hoarding theory. 2 Prior literature and hypotheses development There is a series of academic studies using the bad news hoarding theory to explain stock crash risk. Managers tend to delay or hide negative information to avoid disclosing unfavorable information to the market (Chang et al. 2017; Graham et al. 2005). However, the stock price crash risk increases when bad news accumulates, and the crash happens when bad news reaches a limited threshold level (Callen and Fang, 2015). Wen et al. (2019) provide evidence that firms with higher retail investor attention have a lower future stock price crash risk. Xu et al. (2021) find the decrease of investors’ ability to find information online leads to the increase of the crash risk, as firms are more likely to hide adverse information. A broad wave of sentiment will disproportionately affect the price of stocks (Baker and Wurgler, 2006). Stambaugh et al. (2012) find sentiment is one of the reasons of anomalies. Specifically, Ding et al. (2019) divide the investor sentiment into long- and short-run components, finding the opposing impacts of long- and short-run sentiment on stock returns. Similar to our topic, Fu et al. (2021) using the traditional sentiment index, find a significant positive relationship between firm-specific investor sentiment and stock price crash risk. Unlike prior literature, we investigate the impact of sentiment resulted from the change of COVID-19, which is a focus of society. Existing studies show that the COVID-19 epidemic can affect the capital market significantly, such as the analyst's forecasts (Zhang et al., 2022), trading and returns (Bing and Ma, 2021), and individual investor sentiment (Sun et al., 2021). More specifically, apart from the whole sentiment under the environment of COVID-19, we capture the more detailed sentiment using various fields of news about COVID-19, including the medical, travelling, uncertain and vaccines’ aspects. News of COVID-19 pandemic, including the number of patients, the use of masks, the condition of travelling and uncertainty, can easily influence the sentiment of people. While the ability of tolerance for bad news is limited, so in this condition, despite the news of COVID-19, both managers and investors are all hoping for positive information from stock market. To ensure the accessibility of financing, managers of firms are more likely to hide negative information, which increases the risk of stock price crash. Based on the analysis above, we assume the sentiment exposed to the overall news of COVID-19 or medical, travelling, and uncertain aspects of COVID-19, can increase the stock price crash risk. On the contrary, the news of vaccines can bring people positive sentiments (Cevik et al. 2022). Vaccines present the hope for removing viruses. According to the reports of Chinese National Health Commission (NHC), there are stronger impacts of vaccines on the population of 18-59-year-old, who are also the main workforce of firms and more active in stock market. Thus, we develop the hypothesis that exposures to vaccines of COVID-19 sentiment can reduce the stock price crash risk. 3 Data and variables 3.1 Data sources To capture the exposure on COVID-19 sentiment, we collect the time-series dataset computed by Narayan et al. (2021) from ResearchGate (https://www.researchgate.net/). Considering the accessibility of indices relating to COVID-19, our sample includes Chinese listed firms from the first quarter of 2020 to the third quarter of 2021. We extract financial data from the China Stock Market & Accounting Research database (CSMAR). We remove firms in financial service industry and firms with missing observations of key financial variables. To avoid the influence of outliers, all continuous variables are winsorized at the upper and lower 1% levels. 3.2 Variables Independent Variable. To measure the exposure of COVID-19, we use the indices constructed by Narayan et al. (2021) which can reflect different types of news including an aggregate COVID-19 index (LA_COVID), a COVID-19 index (LCOVID), a medical index (LMedical), a travel index (LTravel), an uncertainty index (LUncertain) and a vaccine index (LVaccine). Specifically, using the ProQuest TDM Python algorithm to count the number of times each word in different dictionaries presented different topics appear in popular newspapers. Then, adjusting proxies of different types of news about COVID-19 based on the results after running a heteroskedasticity-consistent ordinary least squares (OLS) regression of the times from each detailed topic of words on day-of-the-week dummy variables. More detailed information of calculating the proxies of COVID-19 news, including the lists of newspapers, the dictionary of words for each aspect of COVID-19 news and the equation used to adjust the proxies are shown in the paper of Narayan et al. (2021) . Dependent Variable. Following Kim et al. (2011), we compute the negative conditional return skewness (NCSKEW) to measure the crash likelihood used in baseline regression. We use Model (2) to estimate the firm-specific daily return Wi,d which equals to the natural log of one plus the residual returned from Model (1). Then we use Wi,d to compute NCSKEW quarterly based on Model (3). In Model (1) to Model (3), the subscripts i, d and q respectively represent different stocks, the time of each day and each quarter, variables R and Rmkt represent the daily return and the corresponding value-weighted market index respectively. And n is the number of market days for stock i during quarter q. The larger the value of NCSKEW, the higher likelihood of stock price crash.(1) Ri,d=αi,d+β1Rmkti,d−2+β2Rmkti,d−1+β3Rmkti,d+β4Rmkti,d+1+β5Rmkti,d+2+εi,d (2) Wi,d=ln(1+εi,d) (3) NCSKEWi,q=ln−[n(n−1)32∑Wi,d3](n−1)(n−2)(∑Wi,d2)32 In robustness checks, we use the down-to-up volatility (DUVOL) to weigh the crash risk using Model (4) referring to Chen et al. (2001). We firstly calculate the average Wi,d in quarter q of firm i, recorded as Wavei,q. Then we count the number of days when Wi,d is higher than Wavei,q for each firm in each quarter, labelled as nup. Also, ndown is the the number of days when Wi,d is lower than Wavei,q for firm i during quarter q. The risk of stock price crash is higher if the value of DUVOL is larger.(4) DUVOLi,q=ln(nup−1)∑downWi,d(ndown−1)∑upWi,d Control Variables. According to the existing literature on the determinants of stock price crash (Fu et al., 2021; Kong et al., 2021a; Kong et al., 2021b; Wen et al., 2019; Xu et al., 2021), we also introduce a vector of control variables, including profitability (LROA), the book-to-market equity ratio (LBM), firm size (LSize), leverage (LLev), the volatility (Lsd) and mean value (LR) of stock prices in a quarter. LROA is the return on assets, defined as the ratio of net income to total average assets of a firm last quarter. LBM is the ratio of the book value of equity to the market value of equity of a firm last quarter. We measure firm size (LSize) using the natural logarithm of a firm's circulation market values last quarter. LLev is the ratio of total debt to total assets of a firm last quarter. Using standard deviation of the daily return of each firm last quarter to calculate the volatility (Lsd). LR is the mean value of daily return of firm i in quarter q-1. 4 Empirical results 4.1 Descriptive statistics Table 1 shows the summary statistics of key variables and the number of observations is 26,404. All independent variables and control variables are lagged by one quarter to avoid the effect of reverse causality. The mean value of NCSKEW is -0.318, while the mean value of DUVOL is -0.255.Table 1 Summary statistics. Table 1 N Mean SD Min Max NCSKEW 26,404 -0.318 0.872 -2.303 2.339 DUVOL 26,404 -0.255 0.698 -1.833 1.625 LA_COVID 26,404 47.355 10.149 26.596 64.493 LCOVID 26,404 43.462 9.134 26.279 60.476 LMedical 26,404 44.846 9.085 26.869 60.466 LTravel 26,404 25.018 8.823 17.816 43.519 LUncertain 26,404 52.242 10.180 31.951 69.054 LVaccine 26,404 32.406 15.621 15.810 56.022 LROA 26,404 0.026 0.045 -0.148 0.184 LBM 26,404 0.648 0.267 0.103 1.247 LSize 26,404 22.243 1.133 20.084 25.619 LLev 26,404 0.408 0.202 0.053 0.884 Lsd 26,404 0.031 0.027 0.009 0.238 LR 26,404 0.002 0.007 -0.006 0.054 Notes: This table reports the summary statistics of variables in this paper. 4.2 Baseline results To identify the impact of the exposures on COVID-19 sentiment on the stock price crash risk, we use Model (5) as follows:(5) Crashi,q=αi,q+β1Sentimenti,q−1+β2Controlsi,q−1+β3FirmFixedEffecti,q+εi,q where Crash represents the stock price crash risk, we use NCSKEW to measure it. Sentiment represents the sentiment affected by different news of COVID-19. Controls represent a series of control variables. To further alleviate omitted variable bias, we include the firm fixed effects. The results of baseline regression are shown in Table 2 . The coefficient of LUncertain is 0.007, and coefficients of LA_COVID, LCOVID, LMedical, LTravel are 0.006, which are all significant, indicating the sentiment affected by total COVID-19 news, COVID-19 news, medical news, travelling news, uncertain news significantly increases the stock price crash risk. While, LVaccine is significantly negative in Column (6) of Table 2, illustrating the sentiment affected by vaccines decreases the stock price crash risk. Therefore, the results of the baseline regression indicate that the negative sentiment exposed to the overall news of COVID-19 can increase the stock price crash risk while positive sentiment exposures to vaccines of COVID-19 can reduce the stock price crash risk.Table 2 COVID-19 and stock price crash risk. Table 2 (1) (2) (3) (4) (5) (6) NCSKEW NCSKEW NCSKEW NCSKEW NCSKEW NCSKEW LA_COVID 0.006* (11.39) LCOVID 0.006* (11.40) LMedical 0.006* (10.19) LTravel 0.006* (8.31) LUncertain 0.007* (13.45) LVaccine -0.003* (-6.84) LROA -0.740* -0.777* -0.779* -0.668* -0.731* -1.130* (-4.36) (-4.59) (-4.60) (-3.85) (-4.32) (-6.80) LBM -1.674* -1.643* -1.677* -1.520* -1.641* -1.404* (-12.32) (-12.15) (-12.31) (-11.19) (-12.16) (-10.14) LSize 0.154* 0.168* 0.154* 0.255* 0.163* 0.299* (4.47) (4.92) (4.46) (7.34) (4.79) (8.07) Llev -0.192 -0.185 -0.201 -0.073 -0.175 -0.097 (-1.55) (-1.49) (-1.61) (-0.58) (-1.42) (-0.79) Lsd -0.632 -0.476 -0.481 -1.482*** -0.793* -0.541 (-1.52) (-1.15) (-1.16) (-3.48) (-1.91) (-1.30) LR 6.810* 6.216* 6.498* 8.941* 6.891* 6.113* (5.16) (4.70) (4.92) (6.73) (5.23) (4.56) Constant -2.816* -3.165* -2.808* -5.060* -3.132* -5.897* (-3.37) (-3.81) (-3.35) (-5.96) (-3.78) (-6.58) Observations 26,404 26,404 26,404 26,404 26,404 26,404 R-squared 0.201 0.201 0.201 0.200 0.203 0.199 Firm FE YES YES YES YES YES YES Notes: The t statistics reported in parentheses are based on all standard errors clustered at the firm level. , , and indicate two-tailed significance at the 10%, 5%, and 1% levels, respectively. 4.3 Robustness checks To further verify the impacts of firms’ exposures on COVID-19 on the risk of crash, we use the alternative proxy of crash risk (DUVOL) to repeat our baseline regression. The results of running Model (5) replacing NCSKEW with DUVOL as the dependent variable are provided in Table 3 . Consistent with baseline regression, LA_COVID, LCOVID, LMedical, LTravel and LUncertain are positive at 1% significant level and the value of corresponding coefficient is similar with the baseline result. The coefficient of LVaccine is -0.002 and significant at 1% level, which is consistent with the result using NCSKEW to measure Crash.Table 3 Robust tests. Table 3 (1) (2) (3) (4) (5) (6) DUVOL DUVOL DUVOL DUVOL DUVOL DUVOL LA_COVID 0.006* (14.82) LCOVID 0.006* (14.77) LMedical 0.006* (13.28) LTravel 0.005* (10.19) LUncertain 0.007* (17.61) LVaccine -0.002* (-7.66) LROA -0.454* -0.497* -0.494* -0.415* -0.445* -0.840* (-3.32) (-3.65) (-3.61) (-2.99) (-3.27) (-6.22) LBM -1.631* -1.598* -1.634* -1.479* -1.596* -1.386* (-14.40) (-14.19) (-14.39) (-12.98) (-14.23) (-11.88) LSize 0.167* 0.182* 0.167* 0.267* 0.177* 0.301* (5.64) (6.20) (5.62) (8.84) (6.04) (9.41) Llev -0.071 -0.063 -0.079 0.042 -0.053 0.012 (-0.70) (-0.62) (-0.78) (0.41) (-0.52) (0.11) Lsd 0.656 0.818 0.813 -0.124 0.488 0.764** (1.98) (2.47) (2.45) (-0.36) (1.48) (2.29) LR 1.898* 1.290 1.572 3.912*** 1.983* 1.325 (1.77) (1.20) (1.46) (3.60) (1.85) (1.22) Constant -3.175* -3.546* -3.163* -5.369* -3.505* -5.987* (-4.43) (-4.97) (-4.40) (-7.30) (-4.94) (-7.74) Observations 26,404 26,404 26,404 26,404 26,404 26,404 R-squared 0.237 0.237 0.236 0.234 0.240 0.232 Firm FE YES YES YES YES YES YES Notes: The t statistics reported in parentheses are based on all standard errors clustered at the firm level. , , and indicate two-tailed significance at the 10%, 5%, and 1% levels, respectively. To sum up, the sentiment exposure to COVID-19, medical, travelling, and uncertain news or aggregate news of COVID-19 can significantly increase the stock price crash risk, while sentiment exposed to news of vaccines leads to the decrease in crash risk. Thus, the results in robustness checks again support our hypotheses. 4.4 Cross-sectional analysis 4.4.1 Effects of state-owned enterprises (SOEs) SOEs generally have close connections with local governments and take some social functions in China. Therefore, SOEs can obtain long-term loans with low credit worthiness requirements (Lin et al. 2020). Accordingly, SOEs may face less financial constraints than private firms during the COVID-19 epidemic. In this subsection, we thus explore the moderation effect of SOEs on our main findings. Specifically, we divide the sample into SOEs and private firms, then regress again based on Model (5). Column (1) to (6) of Table 4 shows the variable of Sentiment isn't significant except LTravel in the sample of SOEs. But for private firms in Column (7) to (12) of Table 4, the variable of Sentiment is significant positive. These results show the impact of exposures on COVID-19 sentiment on the stock price crash risk is milder in SOEs.Table 4 The effects of SOEs. Table 4 Results based on SOEs Results based on private firms (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) LA_COVID 0.001 0.008* (1.12) (13.20) LCOVID 0.001 0.009* (0.60) (13.54) LMedical 0.001 0.009* (0.63) (12.14) LTravel 0.004* 0.007* (2.99) (7.83) LUncertain 0.001 0.010* (1.56) (15.18) LVaccine -0.000 -0.004* (-0.07) (-7.90) LROA -1.793* -1.834* -1.830* -1.568* -1.770* -1.874* -0.522* -0.570* -0.562* -0.487 -0.532* -1.069* (-4.30) (-4.40) (-4.40) (-3.73) (-4.26) (-4.62) (-2.62) (-2.88) (-2.83) (-2.38) (-2.67) (-5.50) LBM -1.796* -1.785* -1.788* -1.759* -1.797* -1.775* -1.649* -1.603* -1.661* -1.433* -1.588* -1.230* (-7.54) (-7.49) (-7.50) (-7.39) (-7.56) (-7.28) (-9.46) (-9.28) (-9.49) (-8.26) (-9.24) (-7.04) LSize 0.333* 0.338* 0.336* 0.382* 0.334* 0.342* 0.099 0.118* 0.097 0.236* 0.114* 0.321* (4.44) (4.51) (4.46) (4.92) (4.46) (4.26) (2.35) (2.85) (2.29) (5.61) (2.77) (7.14) LLev 0.710* 0.713* 0.711* 0.813* 0.713* 0.716* -0.407* -0.396 -0.419*** -0.273* -0.382** -0.265* (2.70) (2.70) (2.70) (3.05) (2.71) (2.70) (-2.63) (-2.58) (-2.71) (-1.78) (-2.49) (-1.74) Lsd 1.539 1.588 1.590 0.337 1.466 1.598 -1.172 -0.968 -0.990 -1.948* -1.351* -1.091 (1.51) (1.56) (1.57) (0.32) (1.44) (1.57) (-2.42) (-2.00) (-2.04) (-3.91) (-2.79) (-2.25) LR 7.828* 7.822* 7.828* 10.256* 7.833* 7.931* 6.505* 5.658* 6.110* 8.464* 6.557* 5.455* (2.75) (2.74) (2.75) (3.67) (2.76) (2.75) (4.10) (3.57) (3.85) (5.27) (4.14) (3.38) Constant -6.908* -7.010* -6.958* -8.118* -6.960* -7.089* -1.699* -2.175 -1.642 -4.653* -2.188 -6.345* (-3.80) (-3.86) (-3.82) (-4.30) (-3.84) (-3.65) (-1.68) (-2.18) (-1.62) (-4.56) (-2.20) (-5.91) Observations 8,025 8,025 8,025 8,025 8,025 8,025 16,135 16,135 16,135 16,135 16,135 16,135 R-squared 0.193 0.193 0.193 0.194 0.193 0.193 0.214 0.215 0.213 0.209 0.217 0.210 Firm FE YES YES YES YES YES YES YES YES YES YES YES YES Notes: The t statistics reported in parentheses are based on all standard errors clustered at the firm level. , , and indicate two-tailed significance at the 10%, 5%, and 1% levels, respectively. 4.4.2 Information transparency Bad-news-hoarding activities finally lead to the stock price crash (Chang et al. 2017; Cevik et al. 2022; Ding et al. 2019). We set the high transparency sample with the rating of A or B and low transparency sample with the rating of C or D according to the information transparency rating issued by Stock Exchange. While less transparency provides firms with more chances to hide bad news. So we predict that the impacts of firms’ exposures on COVID-19 sentiment on stock price crash risk are stronger for firms with low transparency which are more likely to hide bad news. Then, we follow Model (5) to identify heterogeneous results. The results are presented in Table 5 . As for the variable measuring sentiment, the coefficient of low information transparency sample in Column (7) to (12) of Table 5 is more than twice as large as the coefficient of high information transparency sample in Column (1) to (6) of Table 5. These results support our hypothesis, that is, the impact of the sentiment on the stock price crash risk is larger in the low information transparency sample.Table 5 Heterogeneous results by information transparency. Table 5 Results based on firms with high information transparency Results based on firms with low information transparency (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) LA_COVID 0.004* 0.010* (6.29) (7.78) LCOVID 0.003* 0.011* (5.49) (7.88) LMedical 0.004* 0.010* (5.74) (7.35) LTravel 0.002* 0.008* (2.79) (4.37) LUncertain 0.004* 0.011* (6.90) (8.66) LVaccine 0.001 -0.003 (1.46) (-2.34) LROA -1.341* -1.406* -1.381* -1.434* -1.337* -1.619* -1.248* -1.233* -1.222* -0.927 -1.301* -0.808 (-6.04) (-6.36) (-6.24) (-6.18) (-6.04) (-7.66) (-3.37) (-3.34) (-3.30) (-2.47) (-3.52) (-2.11) LBM -1.638* -1.611* -1.637* -1.555* -1.620* -1.639* -1.309* -1.308* -1.282* -1.315* -1.357* -1.166* (-9.38) (-9.24) (-9.37) (-8.86) (-9.31) (-8.99) (-3.39) (-3.39) (-3.32) (-3.28) (-3.53) (-2.95) LSize 0.237* 0.251* 0.238* 0.302* 0.243* 0.253* 0.336* 0.338* 0.341* 0.388* 0.322* 0.402* (5.22) (5.55) (5.22) (6.60) (5.39) (5.11) (2.98) (3.00) (3.02) (3.41) (2.86) (3.57) LLev 0.220 0.223 0.216 0.279 0.223 0.217 0.312 0.315 0.305 0.385 0.321 0.346 (1.27) (1.29) (1.25) (1.59) (1.29) (1.25) (1.04) (1.05) (1.02) (1.32) (1.07) (1.19) Lsd -0.319 -0.170 -0.122 -1.119 -0.550 -0.091 -0.588 -0.169 0.003 -2.626 -1.246 1.288 (-0.46) (-0.25) (-0.18) (-1.52) (-0.80) (-0.13) (-0.37) (-0.11) (0.00) (-1.45) (-0.78) (0.82) LR 8.868* 8.612* 8.601* 10.524* 9.117* 9.419* 6.702 6.068 5.933 10.925** 7.276* 6.113 (5.20) (5.03) (5.03) (6.14) (5.35) (5.44) (1.61) (1.46) (1.42) (2.57) (1.75) (1.45) Constant -4.750* -5.069* -4.766* -6.154* -4.931* -4.959* -7.424* -7.486* -7.563* -8.290* -7.177* -8.496* (-4.31) (-4.61) (-4.31) (-5.50) (-4.50) (-4.14) (-2.73) (-2.75) (-2.78) (-3.03) (-2.65) (-3.12) Observations 18,698 18,698 18,698 18,698 18,698 18,698 3,394 3,394 3,394 3,394 3,394 3,394 R-squared 0.216 0.215 0.215 0.214 0.216 0.214 0.288 0.288 0.286 0.278 0.292 0.274 Firm FE YES YES YES YES YES YES YES YES YES YES YES YES Notes: The t statistics reported in parentheses are based on all standard errors clustered at the firm level. , , and ** indicate two-tailed significance at the 10%, 5%, and 1% levels, respectively. 5 Conclusion Using the evidence from the COVID-19 pandemic, we find the sentiment affected by medical, travelling, and uncertain news about COVID-19 significantly increases the stock price crash risk. On the contrary, the exposure of COVID-19 sentiment related to vaccines significantly decreases the risk of the stock price crash. We further find the results above are milder in the SOEs and firms with the high information transparency. Our findings not only enrich the existing studies on the economic consequences of the COVID-19 epidemic and the influence factors of stock price crash risk but also provide some implications for managers. Our cross-sectional analysis shows that the positive effect of sentiment affected by COVID-19 on stock price crash can be mitigated by information transparency to some extent. Therefore, the managers could allocate more resources to improve their information quality to avoid crash risks. Author statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. ==== Refs References Akram V Haider S. A dynamic nexus between COVID-19 sentiment, clean energy stocks, technology stocks, and oil prices: global evidence[J] Energy Res. Lett. 3 2022 32625 Early View Baker M Wurgler J. Investor sentiment and the cross-section of stock returns[J] J. Finance 61 4 2006 1645 1680 Bing T Ma H. COVID-19 pandemic effect on trading and returns: Evidence from the Chinese stock market[J] Econ. Anal. Policy 71 2021 384 396 35719893 Chen J Hong H Stein J C Forecasting crashes: Trading volume, past returns, and conditional skewness in stock prices[J] J. Financ. Econ. 61 3 2001 345 381 Callen J L Fang X. Religion and stock price crash risk[J] J. Financ. Quantit. 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==== Front Heart Rhythm Heart Rhythm Heart Rhythm 1547-5271 1556-3871 Published by Elsevier Inc. on behalf of Heart Rhythm Society. S1547-5271(22)02682-0 10.1016/j.hrthm.2022.11.020 Article A Comparison of Data Quality and Monitoring Completion Rates Between Clinic and Self-Applied ECG Patches Goergen Jack A. MD a Peigh Graham MD, MSc a Hsu Mike PhD b Wilk Alan BS b Nayak Tanvi BA a Crosson Lori PhD b Lenane Judith RN, MHA b Knight Bradley P. MD a Passman Rod MD, MSCE a# a Northwestern University, Feinberg School of Medicine, Chicago, IL b iRhythm Technologies, San Francisco, CA # Corresponding Author: Rod Passman, MD, MSCE, Division of Cardiology, Northwestern Memorial Hospital, 251 E Huron St. Rm. 8-503., Chicago, IL 60611. , 1 12 2022 1 12 2022 24 8 2022 28 10 2022 22 11 2022 © 2022 Published by Elsevier Inc. on behalf of Heart Rhythm Society. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Background Since the onset of the COVID-19 pandemic, direct-to-patient, self-applied ECG patch use has substantially increased. There are limited data comparing clinic versus self-applied ECG patches. Objectives To compare rates of ECG patch return, percentages of time patches yielded analyzable data (analyzable time), and percentages of prescribed time ECG patches were worn between clinic and self-applied ECG patches prior to and during COVID-19. Methods A retrospective analysis of patients prescribed an ECG patch during “pre-COVID” (3/1/2019-3/1/2020) and “COVID” (4/1/2020-4/1/2021) years was conducted. ECG patch return rates, mean percentages of analyzable time, and mean percentages of prescribed wear time were compared between clinic and self-applied groups. Results Of 29,093 ECG patch prescriptions (19% COVID self-applied), the COVID self-applied group had a lower return rate (90.8%) than both clinic-applied groups (COVID; 97.1%; pre-COVID; 98.1%; p<0.001). Among the 28,048 ECG patches (17.5% self-applied) returned for analysis, the COVID self-applied group demonstrated a lower mean percentage of analyzable time (95.9 + 8.2%) than both clinic-applied groups (COVID: 96.6 + 6.6%; pre-COVID 96.6 + 7.4%; p<0.001). There were no differences in mean percentage of prescribed wear time between groups (pre-COVID clinic-applied: 96.7 + 34.3%; COVID clinic-applied 97.4 + 39.8%; COVID self-applied 98.1 + 52.1%, p=0.09). Conclusions Self-applied ECG patches were returned at a lower rate and had a statistically lower percentage of analyzable time than clinic-applied patches. However, there were no differences between groups in mean percentages of prescribed wear time, and mean percentages of analyzable time were >95% in all groups. Key Words Remote Telemedicine Diagnosis Rhythm monitor Arrhythmia ==== Body pmcIntroduction The use of telemedicine, with its associated reduction in in-person office visits, has increased dramatically since the start of the COVID-19 pandemic. Ambulatory ECG monitoring patches are a widely used diagnostic tool due to their ease of use and reliability in arrhythmia detection.1, 2, 3 Prior to the COVID-19 pandemic, ECG patches were predominantly applied in a clinic setting by trained medical technicians. However, COVID-19 led to the widespread adoption of mailing ECG patches directly to patients for self-application in an attempt to maintain social distancing and prevent unnecessary exposures. Prior research has validated the arrhythmia detection capability of ECG patches by comparing metrics to Holter monitors, the traditional standard for ambulatory ECG monitoring.1 , 4 However, in these validation studies, ECG patches were applied to patients in clinics by trained technicians. To date, the performance metrics surrounding self-applied ECG patch use are largely unknown. Because the self-application process includes multiple new steps subject to variability, including the mailing of patches to patients, patients effectively applying the patch, and patients activating the patch, an examination of the metrics surrounding this process would better inform current and future practice. The goal of the present study is to compare the rate of patch return, the mean percentage of analyzable time (defined as the percentage of time the patch was worn that yielded analyzable data), and the mean percentage of prescribed time that the patch was worn between cohorts of patients who had clinic or self-applied ECG patches prior to and during the COVID-19 pandemic within a single healthcare system. Methods Patients All patients >18 years old prescribed an iRhythm Zio XT ECG (San Francisco, CA) patch by a Northwestern Memorial Healthcare (Illinois, USA) provider between 03/01/2019-03/01/2020 and 04/01/2020–04/01/2021 were eligible for inclusion in this study. Patients with an ECG patch prescription between 03/01/2019-03/01/2020 were designated as the “pre-COVID” cohort and those prescribed an ECG patch between 04/01/2020–04/01/2021 were designated as the “COVID” cohort. Patients were further categorized according to whether their ECG patch was applied by a technician in clinic or self-applied at home (Figure 1 ).Figure 1 Flow of patient inclusion., Caption: Consort diagram detailing exclusion criteria and the allocation of 29,093 patients prescribed an ECG patch by a Northwestern Medicine provider between 03/01/2019 - 04/01/2021 into cohorts based on type of patch application and time period., Footnote: Data were unavailable if patients returned the ECG patch without collection of data. In the pre-COVID clinic-applied, COVID clinic-applied, and COVID self-applied cohorts, this included 33 ECG patches, 41 ECG patches, and 102 ECG patches, respectively. Additionally, 6 ECG patches in the COVID clinic-applied cohort and 1 ECG patch in the COVID self-applied cohort were successfully returned with data available but not included in the final analysis because the ECG report was not available at the time of data extraction for this study., Abbreviations: Abbreviations: COVID: Novel Coronavirus Disease 2019; ECG: electrocardiogram. The ECG patch and its application The iRhythm Zio XT patch is a small (2 in x 5 in), adhesive continuous ECG monitor. In the self-application process, after a patch is prescribed, iRhythm mails a patch and an instruction manual regarding self-application to the patient. The patient must receive the patch in the mail, and prepare their skin by shaving, abrading, and cleaning the patch-site according to instructions provided. Then, the patient must remove the adhesive covering, apply the device, activate the device, wear the patch, and return the patch in the mail after the prescribed wear time. When ECG patches are applied in clinic, patients present to clinic for a visit with a trained technician who shaves, abrades, and cleans the patient’s skin, then applies and activates the patch in a manner similar to the self-application process. During this visit, the technician also describes precautions the patient should follow while the patch is worn. In both clinic and self-applied groups, patients are provided with an addressed and postage-paid box to return the patch. At the conclusion of the prescribed wear time, all patients are instructed to mail the patch to iRhythm for analysis (Figure 2 ). When the patch is received in the mail by iRhythm, arrhythmia adjudication occurs via iRhythm’s FDA-approved arrhythmia processing algorithm, and the results are confirmed by a certified cardiographic technician.5 The rhythm data processed by the algorithm must be above a specified signal-to-noise ratio for the processing algorithm to accurately distinguish various rhythms. Analysis results and rhythm tracings are then sent to the ordering institution for physician over-read.Figure 2 Process of self vs. clinic-application of ECG patches., Caption: Patients in the self-application group receive an ECG monitor and instruction manual in the mail, apply, activate, and wear the patch, and then return the patch in the mail after their completed wear time. Patients in the clinic-application group schedule an appointment, a technician applies the patch, the patient receives instructions, and then returns the patch in the mail after their completed wear time., Abbreviations: COVID: Novel Coronavirus Disease 2019; ECG: electrocardiogram. Data acquisition Data were collected from the prospectively maintained iRhythm deidentified commercial electronic health record (EHR) of demographic and ECG rhythm data. This study was deemed exempt by the Institutional Review Board at Northwestern University. Study endpoints The primary metrics analyzed in this study included the ECG patch return rate, the mean percentage of analyzable time, and the mean percentage of ECG patch wear time. The return rate was calculated as “(patches returned to iRhythm with data available/prescribed patches) * 100”. ECG patches could be returned to iRhythm but not have data available for various reasons, including: a patient failed to activate the patch, the device failed to activate, the device was applied incorrectly, or the device detached from the patient. The mean percentage of analyzable time reflects the quality of data collected by the ECG patch. The arrhythmia adjudication algorithm designates data above the necessary signal-to-noise ratio as ‘analyzable.’ The percentage of analyzable time was calculated as “(time that data were above the necessary signal-to-noise ratio)/(patient wear time) * 100”. The mean percentage of analyzable time was then calculated for each cohort. The percentage of prescribed wear time was calculated as “(actual patient wear time/prescribed wear time) * 100”. The mean percentage of prescribed wear time was then calculated for each cohort. It was possible for patients to wear the patch longer than the prescribed wear time, which resulted in a wear time percentage greater than 100%. ECG patches that were not returned were not included in the calculation of the wear time or the analyzable time metrics. Statistical analysis Patient demographics and prescription durations were compared between groups using ANOVA for continuous data and chi-square for categorical data. Differences in the ECG patch return rates, mean percentages of analyzable time, and mean percentages of prescribed wear time were compared between groups using ANOVA. Post-hoc analyses were performed to assess for specific between-group differences. Kruskal-Wallis tests and Cox proportional hazard modeling were performed as appropriate to investigate if demographic or clinical variables (age, gender, prescription duration) had significant effects on the between-group differences observed. Cox regression analysis was then utilized to determine if differences in study endpoints between cohorts were still significant after controlling for the confounding effects of clinical and demographic variables. Post hoc analysis was performed to assess for specific between-group differences. A threshold of p<0.05 was considered statistically significant. Numerical results are reported as mean ± standard deviation, median (interquartile range), or number (%). All analyses were performed using SAS (Version 9.4, 2013, North Carolina, USA). Results In total, 29,093 patients (13,178 (45%) pre-COVID clinic-applied, 10,520 (36%) COVID clinic-applied, 5,395 (19%) COVID self-applied) were prescribed an ECG patch over the two time periods. Of these, 28,048 patients (59.3 + 17.7 years, and 55.6% female) wore and returned an ECG patch with data available for analysis (Figure 1). The most common indications for ECG monitoring were palpitations (28.6%) and atrial fibrillation (19.1%) (Table 1 ). The median monitoring duration of the 28,048 patches returned for analysis was 14 days (3 – 14 days) and 54.3% of all prescriptions in the study were for 14 days. The mean duration of time between device registration and activation in the self-application group was 8.1 + 12.2 days.Table 1 Patient demographics and monitoring indications Overall n = 28,048 Pre-COVID clinic-applied n = 12,930 COVID clinic-applied n = 10,218 COVID self-applied n = 4,900 p Age (years) 59.3 + 17.7 59.5 + 17.6 59.8 + 17.9 57.9 + 17.5 < 0.001 Male gender 12,457 (44.4%) 5,883 (45.5%) 4,369 (42.8%) 2,205 (45.0%) < 0.001 Indications Palpitations 8,032 (28.6%) 3,694 (28.6%) 2,995 (29.3%) 1,343 (27.4%) < 0.001 Atrial fibrillation 5,362 (19.1%) 2,531 (19.6%) 1,747 (17.1%) 1,084 (22.1%) < 0.001 Other 14,654 (52.2%) 6,705 (51.9%) 5,476 (53.6%) 2,473 (50.5%) < 0.001 Prescription duration (days) 14 (3-14) 14 (3-14) 14 (3-14) 14 (7-14) < 0.001 Footnote: Age is displayed as mean +/- standard deviation and prescription duration is displayed as median (IQR). Abbreviations: COVID – Novel Coronavirus Disease 2019, ECG – electrocardiogram. ECG patch return rate In total, 28,048 (96.4%) ECG patches were returned to iRhythm with data available for analysis. The ECG patch return rates in the pre-COVID clinic-applied, COVID clinic-applied, and COVID self-applied cohorts were 98.1%, 97.1% and 90.8%, respectively (p<0.001, Figure 3 ). The COVID clinic-applied return rate was significantly lower than the pre-COVID clinic-applied return rate (p<0.001). Furthermore, the self-applied return rate was significantly lower than both the pre-COVID and COVID clinic-applied return rates (p for both comparisons <0.001, Figure 3).Figure 3 Comparison of clinic versus self-application of ECG patches., Caption: 28,048 patients returned their ECG patch with data available for analysis. The COVID self-applied group had a lower rate of patch return than both clinic-applied groups. Both the clinic and self-application groups yielded mean percentages of analyzable time >95% and wore the patch for >95% of the prescribed monitoring duration. There were significant differences in the mean percentages of analyzable time, but no differences in the mean percentages of prescribed wear time between groups., Footnote: ECG patch return rates were calculated from a total of 29,093 ECG patch prescriptions (45% pre-COVID clinic-applied; 36% COVID clinic-applied; 19% COVID self-applied) in the study period. The mean percentage of analyzable time and mean percentage of prescribed wear time metrics were calculated from a total of 28,048 ECG patches returned with data available., Abbreviations: COVID: Novel Coronavirus Disease 2019; ECG: electrocardiogram. In univariate analysis, age and prescription duration were found to be significant predictors of return rate (p for both variables <0.001) whereas gender was not (p=0.51) (Table 2 ). After using Cox proportional hazards modelling to account for the potential confounding effects of age and prescription duration, there remained significant differences between cohorts in return rate (p<0.001). Post hoc analysis demonstrated significant differences between each comparison of the pre-COVID clinic-applied, COVID clinic-applied, and COVID self-applied cohorts (Table 3 ).Table 2 Univariate analysis for ECG patch return rate and mean percentage of analyzable time Study endpoint Variable p ECG patch return rate Age < 0.001 Gender 0.51 Prescription duration < 0.001 Mean percentage of analyzable time Age < 0.001 Gender < 0.001 Prescription duration < 0.001 Abbreviations: ECG – electrocardiogram. Table 3 Cox regression analysis for ECG patch return rate and mean percentage of analyzable time Study endpoint Post hoc comparisons p ECG patch return rate Pre-COVID clinic-applied vs. COVID self-applied < 0.001 COVID clinic-applied vs. COVID self-applied < 0.001 Pre-COVID clinic-applied vs. COVID clinic-applied < 0.001 Mean percentage of analyzable time Pre-COVID clinic-applied vs. COVID self-applied < 0.001 COVID clinic-applied vs. COVID self-applied < 0.001 Pre-COVID clinic-applied vs. COVID clinic-applied < 0.001 Abbreviations: COVID – Novel Coronavirus Disease 2019, ECG – electrocardiogram. Mean percentage of analyzable time Among the 28,048 patients who returned their ECG patch, the mean percentage of analyzable time was 96.5% + 7.2%. While there was no significant difference in mean percentages of analyzable time between the two clinic-applied cohorts (pre-COVID clinic-applied: 96.6% + 6.6%; COVID clinic-applied: 96.6% +7.4%, p=0.93), the mean percentage of analyzable time was significantly lower in the self-applied cohort (95.9% + 8.2%) compared with the clinic-applied cohorts (p<0.001 for both comparisons, Figure 3). In univariate analysis, age, gender, and prescription duration were each found to be significant predictors of mean percentage of analyzable time (p for each variable <0.001, Table 2). After using Cox proportional hazards modelling to account for the potential confounding effects of age, gender, and prescription duration, there were still significant differences between cohorts in mean percentage of analyzable time (p<0.001). Post hoc analysis demonstrated significant differences between each comparison of the pre-COVID clinic-applied, COVID clinic-applied, and COVID self-applied cohorts (Table 3). Mean percentage of prescribed wear time Of all patients who returned their ECG patch, the mean percentage of wear time was 97.2% + 39.9% of the prescribed duration. There were no significant differences in mean percentages of prescribed wear time between the three cohorts (pre-COVID clinic-applied: 96.7% + 34.3%; COVID clinic-applied: 97.4% + 39.8%; COVID self-applied: 98.1% + 52.1%; p = 0.09, Figure 3). Discussion The results of the current study compare return rates and performance metrics of clinic and self-applied ECG patches. Compared with clinic-application, self-application was associated with a lower ECG patch return rate and a lower mean percentage of analyzable time. Conversely, there were no significant differences in the mean percentage of prescribed wear time between groups. These results may help inform decisions regarding current and future practices surrounding ECG patch application and use. Existing data on self-application Most of the prior research demonstrating the efficacy and reliability of ECG patches has involved clinic-application of patches by trained technicians.1 , 4 , 6 To date, limited data exist on the performance metrics of self-applied ECG patches. In a sub-cohort of the Multi-Ethnic Study of Atherosclerosis (MESA), the mean percentage of prescribed wear time and the mean percentage of analyzable time were compared between 15 patients in a self-applied group and 15 patients in a clinic-applied group. Similar to our study, results from the MESA cohort demonstrate no significant difference in the mean percentage of prescribed wear time between the two groups. However, results from the MESA cohort differ from the present analysis by demonstrating no significant difference in the mean percentage of analyzable time between groups.7 The current study expands on prior work by investigating metrics of self-applied ECG patches in a large cohort and by presenting data on ECG patch return rates. ECG patch return rate In the current analysis, patients returned ECG patches at a significantly lower rate during the COVID pandemic, with the lowest rate observed among the self-application group. These differences remained significant after controlling for the potential confounding variables included in this study. The differences in return rates between the pre-COVID and COVID time periods are potentially explained by COVID-specific factors, including interrupted mail pickup and patient social distancing during the beginning of the COVID-19 pandemic. Among other reasons, the lower return rate observed in the self-application cohort may have been secondary to unsuccessful mailing of the patch to patients, difficulty with the self-application process, or issues with mailing the patch back to iRhythm. Despite these differences that can be, at least partially, attributed to the specific time-period of the study, providers should be aware of the differences in return rates seen. Outreach to patients who fail to return the ECG patch could prove to be a valuable method for improving return rates. In this study, patients in the clinic-applied cohort who failed to return their patch were contacted by a hospital employee via telephone. For patients in the self-applied cohort, there were multiple outreach processes that developed during the pandemic to contact patients who failed to return their patch, including automated end-of-wear reminder phone calls, smartphone app alerts, text messages, and phone calls from iRhythm employees. These processes were updated throughout the study period, with more outreach efforts occurring towards the end of the study period than at the start of the pandemic. Continued improvement in these outreach processes could lead to an increase in ECG patch return rates. Mean percentage of analyzable time Statistically significant differences in the mean percentages of analyzable time between clinic and self-application cohorts were also found in the present study. These significant differences remained after controlling for potential confounding effects of the variables included in this study. The decrease in mean percentage of analyzable time seen in the self-application cohort may be related to suboptimal application of the patch by patients. However, despite a statistically significant result, the absolute differences in the mean percentage of analyzable time between groups were low (96.6% vs. 96.6% vs. 95.9%) and each cohort averaged >23 hours of analyzable time per day. Based on a 14-day wear time prescription (the most common duration prescribed in this study), the 0.7% decrease in analyzable time in the self-applied cohort equates to ∼10 fewer minutes of rhythm monitoring per day. As ECG patches have previously been validated for arrhythmia detection despite variable rates of analyzable time, it is likely that the mean percentages of analyzable time achieved in each cohort were sufficient for the detection of most arrhythmias.1, 2, 3 Mean percentage of prescribed wear time Pre-COVID clinic-applied, COVID clinic-applied, and COVID self-applied patients all wore their patches for a significant proportion (>95%) of their prescribed wear time, and there were no significant differences in the mean percentages of prescribed wear time between groups. This demonstrates that the method of ECG patch application is not associated with wear time in our sample. Utility of self-application Direct-to-patient, self-application of ECG patches may impact practice within healthcare systems, and there are many potential applications for this process. Among others, these include improved primary and secondary prevention efforts, expanded access to healthcare, and community-based research applications.8, 9, 10, 11, 12 In aggregate, the findings from this study provide valuable information to providers and researchers who utilize self-application of ECG patches. These results may help inform decisions regarding whether to apply ECG patches in the clinic or home, and they should also enable improved patient counseling prior to ECG patch prescription. Specifically, it is important for patients who are selected for self-application of ECG patches to be counseled on the importance of returning their patch, based on the lower rate of self-applied patch return demonstrated in the present analysis. Among those who do return their patches, it is encouraging that both clinic and self-applied patients achieve adequate durations of continuous rhythm monitoring for the detection of most arrhythmias. Indeed, it is likely that certain clinical scenarios will favor one application method over the other, and the flexibility to choose between methods should help providers adapt to an increasingly remote healthcare landscape. Limitations It is important to view the results of this study in the appropriate context. Self-application of ECG patches at our institution was initiated at the beginning of the COVID-19 pandemic, and within one year, thousands of patients were part of the self-application process. Therefore, the metrics surrounding self-application in this study do not reflect a longstanding process, but one that was rapidly adopted and began during a tumultuous and unpredictable time. It is likely that the metrics surrounding self-application would differ if examined further from the onset of the pandemic. Second, these results reflect the performance metrics of one device in one healthcare system and geographical region. There may be variability across vendors, institutions, and regions, and future studies should replicate this analysis with different ECG patch vendors in different healthcare systems and regions. Third, because percentage of prescribed wear time was calculated as “(actual patient wear time / prescribed wear time) * 100” it is possible that select patients had >100% wear time, which may have impacted analysis. Fourth, in this study, the most common indications for ECG monitoring were reported in the results section to provide readers with context surrounding the patient population. However, there were over 100 unique indications, and these indications are based on user-entered responses and not clinical diagnoses. As these indications were not strictly defined, and because there were a multitude of different indications entered for patients in this study, indication for ECG monitoring was not included as a potential confounding variable in our analyses. Lastly, there are likely other variables that were not included in our analysis that impact the study endpoints, and future research into these factors could better inform which patients would be best suited for clinic vs. self-application. Conclusions In an attempt to promote physical distancing, COVID-19 prompted a rapid adoption of mailing ECG patches to patients for self-application. Prior to this study, the performance metrics of self-applied ECG patches were largely unknown. The findings presented in the current study demonstrate a lower rate of patch return among patients in the self-applied cohort. However, there were no differences between groups in the mean percentage of prescribed wear time, and despite statistically significant differences, the mean percentage of analyzable time was >95% in both clinic and self-application groups. The results of the present study may help inform current and future decision-making for providers, researchers, and healthcare organizations engaged in arrhythmia detection. Sources offunding: This research did not receive grants, contracts, or other financial support from funding agencies in the public, commercial, or not-for-profit sectors. Disclosures: Rod S. Passman receives research support from AHA (#18SFRN34250013), research support and speaker fees from Medtronic, research support from Abbott, and royalties from UpToDate; Bradley Knight receives honoraria for speaking or consulting from Abbott, Biosense Webster, Biotronik, Boston Scientific, CVRx, Medtronic, and Philips; Mike Hsu, Alan Wilk, Lori Crosson, and Judith Lenane are employees of iRhythm Technologies; The remaining authors have no disclosures to report. ==== Refs References 1 Barrett P.M. Komatireddy R. Haaser S. Comparison of 24-hour Holter monitoring with 14-day novel adhesive patch electrocardiographic monitoring Am J Med 127 2014 95 e11 7 2 Heckbert S.R. Austin T.R. Jensen P.N. Yield and consistency of arrhythmia detection with patch electrocardiographic monitoring: The Multi-Ethnic Study of Atherosclerosis J Electrocardiol 51 2018 997 1002 30497763 3 Schreiber D. Sattar A. Drigalla D. Higgins S. Ambulatory cardiac monitoring for discharged emergency department patients with possible cardiac arrhythmias West J Emerg Med 15 2014 194 198 24672611 4 Rosenberg M.A. Samuel M. Thosani A. Zimetbaum P.J. Use of a noninvasive continuous monitoring device in the management of atrial fibrillation: a pilot study Pacing Clin Electrophysiol 36 2013 328 333 23240827 5 Eysenck W. Freemantle N. Sulke N. A randomized trial evaluating the accuracy of AF detection by four external ambulatory ECG monitors compared to permanent pacemaker AF detection J Interv Card Electrophysiol 57 2020 361 369 30741360 6 Turakhia M.P. Hoang D.D. Zimetbaum P. Diagnostic utility of a novel leadless arrhythmia monitoring device Am J Cardiol 112 2013 520 524 23672988 7 Zhang M.J. Roetker N.S. Folsom A.R. Alonso A. Heckbert S.R. Chen L.Y. Feasibility of using a leadless patch monitor in community cohort studies: The Multi-ethnic Study of Atherosclerosis Pacing Clin Electrophysiol 41 2018 1389 1390 30225944 8 Alkhouli M. Alqahtani F. Aljohani S. Alvi M. Holmes D.R. Burden of Atrial Fibrillation-Associated Ischemic Stroke in the United States JACC Clin Electrophysiol 4 2018 618 625 29798789 9 Gladstone D.J. Spring M. Dorian P. Atrial fibrillation in patients with cryptogenic stroke N Engl J Med 370 2014 2467 2477 24963566 10 Healey J.S. Connolly S.J. Gold M.R. Subclinical atrial fibrillation and the risk of stroke N Engl J Med 366 2012 120 129 22236222 11 Kaura A. Sztriha L. Chan F.K. Early prolonged ambulatory cardiac monitoring in stroke (EPACS): an open-label randomised controlled trial Eur J Med Res 24 2019 25 31349792 12 Steinhubl S.R. Waalen J. Edwards A.M. Effect of a Home-Based Wearable Continuous ECG Monitoring Patch on Detection of Undiagnosed Atrial Fibrillation: The mSToPS Randomized Clinical Trial JAMA 320 2018 146 155 29998336	36464126	PMC9714183	NO-CC CODE	2022-12-02 23:24:55	no	Heart Rhythm. 2022 Dec 1; doi: 10.1016/j.hrthm.2022.11.020	utf-8	Heart Rhythm	2,022	10.1016/j.hrthm.2022.11.020	oa_other
==== Front Environ Adv Environ Adv Environmental Advances 2666-7657 The Authors. Published by Elsevier Ltd. S2666-7657(22)00161-2 10.1016/j.envadv.2022.100326 100326 Article Monitoring of COVID-19 in wastewater across the Eastern Upper Peninsula of Michigan Jarvie Michelle M. a1 Reed-Lukomski Moriah a1 Southwell Benjamin a Wright Derek b Nguyen Thu N.T. a⁎ a School of Science and Medicine, Lake Superior State University, 650 W. Easterday Ave., Sault Ste, Marie, MI 49783, USA b School of Natural Resources and Environment, Lake Superior State University, 650 W. Easterday Ave., Sault Ste. Marie, MI 49783, USA ⁎ Corresponding author. 1 These authors contributed equally. 1 12 2022 4 2023 1 12 2022 11 100326100326 12 9 2022 1 11 2022 29 11 2022 © 2022 The Authors. Published by Elsevier Ltd. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Wastewater-based epidemiology is being used as a tool to monitor the spread of COVID-19 and provide an early warning for the presence or increase of clinical cases in a community. The majority of wastewater-based epidemiology for COVID-19 tracking has been utilized in sewersheds that service populations in the tens-to-hundreds of thousands. Few studies have been conducted to assess the usefulness of wastewater in predicting COVID-19 clinical cases specifically in rural areas. This study collected samples from 16 locations across the Eastern Upper Peninsula of Michigan from June to December 2021. Sampling locations included 12 rural municipalities, a Tribal housing community and casino, a public university, three municipalities that also contained a prison, and a small island with heavy tourist traffic. Samples were analyzed for SARS-CoV-2 N1, N2, and variant gene copies using reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR). Wastewater N1 and N2 gene copies and clinical case counts were correlated to determine if wastewater results were predictive of clinical cases. Significant correlation between N1 and N2 gene copies and clinical cases was found for all sites (⍴= 0.89 to 0.48). N1 and N2 wastewater results were predictive of clinical case trends within 0-7 days. The Delta variant was detected in the Pickford and St. Ignace samples more than 12-days prior to the first reported Delta clinical cases in their respective counties. Locations with low correlation could be attributed to their high rates of tourism. This is further supported by the high correlation seen in the public university, which is a closed population. Long-term wastewater monitoring over a large, rural geographic area is useful for informing the public of potential outbreaks in the community regardless of asymptomatic cases and access to clinical testing. Graphical abstract Image, graphical abstract Keywords SARS-CoV-2 Wastewater surveillance Wastewater-based epidemiology Early warning Rural populations ==== Body pmc1 Introduction COVID-19, a disease caused by the SARS-CoV-2 virus, has swept the globe since early 2020 after first being discovered in Wuhan, China at the end of 2019. Declared an official global pandemic on March 11, 2020, the World Health Organization (WHO) (World Health Organization, 2021) reported just under 280 million cases and nearly 5.4 million deaths worldwide by the end of 2021. One of the major challenges facing public health officials during the pandemic has been finding a reliable and timely surveillance system to monitor the spread of infections. The primary methodology for surveillance has focused on clinical testing of nasal swabs using reverse transcription quantitative PCR (RT-qPCR). However, due to the highly contagious nature of COVID-19, the capacity of medical facilities and other testing sites to perform these tests has often been overwhelmed, leading to a lag between the demand for testing and the reporting of positive cases (Ai et al., 2021; Graham et al., 2021; Nagarkar et al., 2021; Zhu et al., 2021). Furthermore, asymptomatic carriers often are not tested but still can spread the disease in the community undetected and unreported (Ai et al., 2021; Barua et al., 2022; Zhu et al., 2021). Wastewater-based epidemiology (WBE) is a potential tool to fill in the gaps created by the lag in testing and reporting, as well as to address the challenge of capturing asymptomatic spreaders. WBE has been used to track the presence of diseases like polio (World Health Organization, 2003) and enteroviruses (Bisseux et al., 2018; Brinkman et al., 2017), as well as microbial resistance (Hendrickson et al., 2019; Hutinel et al., 2019), and illicit drugs (Choi et al., 2018; Huizer et al., 2021; Sulej-Suchomska et al., 2020). Studies have shown that 60-70% of infected people shed the COVID-19 virus in their stool, even in asymptomatic and pre-symptomatic persons, making wastewater an ideal target for further study in the battle against the disease (Markt et al., 2021; Zhu et al., 2021). Many countries across the globe are utilizing wastewater surveillance as a barometer for COVID-19 community infection dynamics (Ahmed et al., 2022a; Ai et al., 2021; Galani et al., 2022; Karthikeyan et al., 2021; Markt et al., 2021). Some studies have focused on the potential of WBE as an "early warning" of increases in positive clinical cases and admittances to hospitals, a way to forecast an impending rise in infected individuals (Barua et al., 2022; Zhu et al., 2021). Peccia et al. (2020) reported a 2-8 day lag time between viral detection in sewer sludge and an increase in positive clinical cases (Table 1 ). Lag time here is defined as the increase in positive clinical cases lagging behind an increase in sewer viral load by 2-8 days, indicating that sewer increases are predictive of an uptick in clinical cases (Peccia et al., 2020). Kaplan et al. (2021) found a narrower lag time of 3-5 days, and Nagarkar et al. (2021) found a wider gap in which viral concentrations in wastewater spiked 1-2 weeks prior to an increase in positive clinical cases, and yet others fall right in the middle at a four-day lag time (Betancourt et al., 2021; Markt et al., 2021). Still, others have found the opposite; positive results in wastewater increase after positive clinical tests by up to a week, which is termed lead time (Fahrenfeld et al., 2021). These highly varied results indicate an incomplete understanding of the dynamics of detecting COVID-19 in wastewater, and further studies that examine the relationship between viral detection in wastewater and positive clinical cases will help assess the utility of wastewater surveillance. Factors such as viral shedding characteristics (Wolfel et al., 2020) and sewer system characteristics such as flow rate, in-sewer travel time, and sampling time of day are suspected to play an important role in wastewater viral load (Ahmed et al., 2022b; Nagarkar et al., 2021; Zhu et al., 2021).Table 1 Existing studies that show wastewater/clinical case relationship. Lag refers to increased clinical cases lagging behind increased viral load in wastewater, indicating wastewater as predictive. Lead refers to clinical case increases leading wastewater increases, therefore not indicating a predictive relationship. Table 1Study Lag/Lead Time Peccia et al., 2020 2-8 Day Lag Kaplan et al., 2021 3-5 Day Lag Betancourt et al., 2021, and Markt et al., 2021 4-Day Lag Nagarkar et al., 2021 1-2 Week Lag Fahrenfeld et al., 2021 1 Week Lead Most WBE studies related to COVID-19 tracking have focused on a limited number of sewersheds over short periods of time, typically a few months (Shah et al., 2021). As the pandemic persists, the addition of more long-term (six months or longer) monitoring studies provide much-needed data to assess the prevalence and predictability of COVID-19 in communities. Additionally, the majority of existing data was taken in sewersheds with populations in the tens-to-hundreds of thousands, leaving some question of whether the current models are valid in more rural areas (Shah et al., 2021). People in rural areas typically have reduced access to healthcare and testing sites (Cuadros et al., 2021; Melvin et al., 2020), are 1.2-1.6 times more likely to have a COVID-19 risk factor (Bradford et al., 2021), and are less likely to be vaccinated than urban residents (Sun and Monnat, 2021). These factors result in less research focus than in more-populated regions (Ali, et al., 2021; Bradford et al., 2021; Datz, 2021; Mueller et al., 2021). In addition to detecting asymptomatic virus shedders, wastewater monitoring can also help account for those that may have COVID-19 but do not get tested or enter the medical system, and thus may be particularly useful in disease monitoring in rural areas. The following study examined the relationship between wastewater surveillance and clinical COVID-19 cases in rural communities of Upper Michigan. 2 Materials and methods 2.1 Study location Lake Superior State University (LSSU) is part of a larger state-wide network (MiNET) of partners working on long-term wastewater analysis across 41 counties and 270 testing sites. LSSU has collected wastewater samples from 16 sites across the Eastern Upper Peninsula (EUP) of Michigan (Fig. 1 ), covering five counties, 12 rural towns/cities, a Tribal housing community and casino, a public university, three small cities with prisons, and a 3.8 square-mile island, Mackinac Island, that sees over a million tourists a year (Table 2 ).Fig. 1 Regional map of Eastern Upper Peninsula, Michigan highlighting sample site locations. Fig 1 Table 2 Collection site locations and their corresponding population, type of wastewater facility, county, and zip code association for the purposes of this study. Kincheloe, Munising, and Newberry include prison population numbers. These three sites are considered one zip code with a combined population of 3130, and were aggregated for analysis of case counts. The Bay Mills Resort wastewater sample is captured on-site, but eventually flows into the Brimley WWTP. Table 2Site Population WW Facility County Zip Code Bay Mills Indian Community 3,130 Sand Filter Chippewa 49715 Bay Mills Resort and Casino 3,130* Outfall Pump** Chippewa 49715 Brimley 3,130* WWTP** Chippewa 49715 Brevort Township 713 WWTP Mackinac 49760 Cedarville 1,362 WWTP Mackinac 49719 De Tour 766 WWTP Chippewa 49725 Kincheloe 6,609 WWTP Chippewa 49788 Lake Superior State University 280 Dorm Confluence Chippewa N/A Mackinac Island 492 WWTP Mackinac 49757 Manistique 6,469 WWTP Schoolcraft 49854 Munising 5,025 WWTP Alger 49862 Newberry 5,409 WWTP Luce 49868 Pickford 1,851 WWTP Chippewa 49774 Rudyard 1,946 WWTP Chippewa 49780 Sault Ste. Marie 19,668 WWTP Chippewa 49783 St. Ignace 4,020 WWTP Mackinac 49781 2.2 Wastewater sampling Wastewater grab samples (250 mL) were collected from 15 wastewater influent streams once per week beginning June 14, 2021. Grab samples from a 16th site, the campus of Lake Superior State University, were collected once per week at a manhole confluence of two student dormitories beginning on August 11, 2021, one week before students returned to campus for the fall semester. Samples were collected Monday through Thursday, but each collection site collected their sample on the same day every week unless holidays or special circumstances required the facility to sample on a different day. All samples were refrigerated or kept on ice until processed (up to 48 hours). 2.3 Viral concentration A standard operating procedure developed by Flood et al. (2021a) based on Flood et al. (2021b) and adapted from Borchardt et al. (2017) was followed for viral concentration, RNA extraction, and ddPCR detection. A deviation from the SOP was employed utilizing a refrigerated orbital shaker (VWR 5000IR) in place of a magnetic stir bar to mix samples (Markt et al., 2021). For each sample, 100 mL of raw sewer water was mixed with 8% (w/vol) molecular biology grade polyethylene glycol (PEG) 8000 (Fisher Scientific) and 0.2 M NaCl (w/v) (Fisher Scientific). Samples were mixed for two hours at 230 rpm and 4°C and were then centrifuged at 4200 x g for 45 minutes at 4°C. The supernatant was removed using a sterile serological pipet and the pellet was resuspended in the residual liquid. 2.4 RNA extraction Viral ribonucleic acid (RNA) was extracted from concentrated samples using the Qiagen QIAmp Viral RNA Minikit following the manufacturer's custom protocol for the QIACube Connect (Qiagen, Germany). A total of 200 µL of concentrate were used per sample for RNA extraction, resulting in 80 µL final elution volume. Pseudomonas phage Phi6 (105 GC/mL) was spiked into the viral lysis buffer solution during RNA extraction for estimation of process recovery efficiency. Recovery efficiency was measured by comparing Phi6 concentration in a raw (pre-PEG) RNA extract to the PEG processed RNA extract. This process was used solely to evaluate the method and detect potential PCR inhibition and was not used to adjust COVID-19 RNA concentrations. Extracted RNA was stored at -80°C until further analysis. 2.5 Virus detection and quantification Bio-Rad's One-step RT-ddPCR Advanced kit was used in conjunction with the Bio-Rad Automated Droplet Generator and the QX200 ddPCR system to quantify N1, N2, and Phi6 RNA (Bio-Rad, USA). The N1 and N2 genes were targeted because primers and probes designed on the divergent regions of the N gene will ensure the specific detection of SARS-CoV-2 (Zymo Research, 2021). The N genes (N, N1, N2, and N3) are the most commonly targeted in wastewater research thus far, making results more readily comparable to other studies (Shah, 2021). Each reaction contained a final concentration of 1x Supermix (Bio-Rad, USA), 20 U/µl reverse transcriptase (RT) (Bio-Rad, USA), 15 nM DTT (Bio-Rad, USA), 900 nmol of each primer (BioSearch Tech, supplied to MiNET by Michigan State University), 250 nmol of each probe (BioSearch Tech, supplied to MiNET by Michigan State University), 1 µL of nuclease-free water, and 5.5 µL of template RNA. The final reaction volume was 22 µL. Quality control samples on each plate included a non-template control, extraction control, and processing blank. Samples, controls, and blanks were analyzed in triplicate. Droplets were generated in the Bio-Rad Automated Droplet Generator (ADG) by mixing 20 µL of reaction volume with 70 µL of droplet generator oil (Bio-Rad, USA), resulting in a reaction mixture-oil emulsion of 40 µL containing up to 20,000 droplets. The droplets were transferred, via the ADG, to a 96-well PCR plate that was then heat-sealed with foil and put in a Bio-Rad C1000 deep-well thermocycler for PCR amplification under the following conditions: 25°C for 3 minutes, 50°C for 60 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 30 seconds and 55°C for 1 minute, 98°C for 10 minutes, and hold at 4°C. After thermocycling, the plate was transferred to the Bio-Rad QX200 Droplet Reader for concentration determination via spectrophotometric detection of fluorescent probe signal in gene-target positive droplets. Amplitude thresholding was performed manually for each analysis using the QuantaSoft (BioRad) software. Lower limit of detection, N1 and N2 gene copies, and Phi6 gene copies for each sample were then determined. The limit of detection was calculated for each sample based on the assay's theoretical detection limit (3 positive droplets) and the concentration factor of the processing method (Flood et al., 2021a). The following formula was used to calculate the gene copies per 100 ml of sample volume for each of the viral targets (Flood et al., 2021a):VirusGCper100mL=GCperreactionVr×Ve×VfVcVi×100 Where: Vi = Initial volume of sample concentration in mL Vf = Final volume of sample after concentration in mL Vr = Final volume of RNA template used for PCR reaction in µL Ve = Final volume of RNA eluted from RNA extraction in µL Vc = Volume of concentrated sample used for RNA extraction in µL 2.6 Water quality measurements Conductivity, total suspended solids, and turbidity were measured for each sample. Calibration followed the manufacturers’ recommendations, consistent with Standard Methods (2510B, 2540D, and 2130B, respectively) (Baird and Bridgewater, 2017). 2.7 Statistical analysis 2.7.1 Statistical representation of SARS-CoV-2 N1 and N2 gene copies For statistical analysis, the SARS-CoV-2 N1 and N2 gene copies for the samples were added together for trend analysis against clinical cases. For samples where N1 and N2 were not detected, gene copies were represented as ½ of the detection limit. Of the 16 sampling locations, 15 could be compared to daily clinical cases reported for the United States zip code they resided in. Both Bay Mills sites and the Brimley site are located within the same zip code; thus, these results were combined for further statistical analysis. LSSU separately reported weekly clinical cases, and this data was used for statistical comparison to LSSU N1 and N2 results. Consolidating data in this manner resulted in a total of 13 sites where gene copies could be compared to clinical cases. 2.7.2 COVID-19 clinical cases The number of new daily positive clinical cases (confirmed plus probable) was obtained from the Michigan Disease Surveillance System (MDSS) for each of the 13 zip codes. The clinical cases included cases in correctional facilities for 3 of the 13 zip codes (Table 1). The date assigned to each case was based on referral date (the date the case was reported into the MDSS) except for clinical case numbers for the LSSU dormitories contributing to the confluence, which were obtained from University reporting. The clinical cases were normalized based on population and expressed as the number of clinical cases per 100,000 people, and the 7-day moving average was calculated. Residential populations for sampling sites are reported in Table 1. 2.7.3 Correlation calculation Correlations between SARS-CoV-2 N1 and N2 gene copies in wastewater and COVID-19 clinical cases were determined using the Spearman correlation coefficient (⍴), where the best fit was interpreted to estimate the lag time (0-7 days). Correlations thus determined were designated as very high (⍴ > 0.90), high (⍴ = 0.70-0.90), moderate (⍴ 0.50-0.70), low (⍴ > 0.30-0.50), or negligible (⍴ < 0.30) (Mukaka, 2012). For LSSU, correlation between SARS-CoV-2 N1 and N2 gene copies in wastewater and COVID-19 clinical cases (sum of the previous week) were calculated using both Pearson and Spearman correlation (Mukaka, 2012). This method was utilized due to the small population, small number of clinical cases, and multiple weeks of zero reported clinical cases in this subpopulation. 3 Results and discussion 3.1 Correlation of SARS-CoV-2 N1 and N1 Gene Copies to COVID-19 clinical cases The samples in this study were collected from June 14, 2021, to December 15, 2021. The Spearman's rank correlation between SARS-CoV-2 and clinical cases ranged from ⍴ = 0.89 to 0.48. Table 3 shows the summary of correlation values for each location. The p-values ranged from 6.1 × 10−11 to 2.8 × 10−2, with all sampling locations showing significant correlation to clinical cases during the study period (Table 3).Table 3 Statistical summary of correlating SARS-CoV-2 N1 and N2 gene copies with COVID-19 clinical cases in the corresponding zip code. The term “Lag” refers to when wastewater surveillance is predictive of clinical cases. Data shaded in green shows locations with significant and high correlations. Data shaded in blue shows locations with significant and moderate correlations. Unshaded data shows locations with significant but low correlation. Table 3Location Day of Best Fit High, Moderate, or Low Correlation Correlation Sault Sainte Marie 0-Day Lag High 0.89 Rudyard 0-Day Lag High 0.74 Kincheloe 0-Day Lag High 0.74 Munising 0-Day Lag High 0.71 De Tour 0-Day Lag High 0.71 Manistique 4-Day Lag Moderate 0.69 Newberry 5-Day Lag Moderate 0.64 Brevort Township 6-Day Lag Moderate 0.59 Cedarville 1-Day Lag Moderate 0.57 St. Ignace 0-Day Lag Moderate 0.55 Pickford 1-Day Lag Moderate 0.52 BMIC/Brimley 7-Day Lag Low 0.49 Mackinac Island 7-Day Lag Low 0.48 The Sault Sainte Marie, Rudyard, Kincheloe, Munising, and De Tour (Fig. 2 A-E) locations all had high correlation (⍴= 0.89, 0.74, 0.74, 0.71, and 0.71 respectively) with a 0-Day lag time in clinical cases (Table 3). The Manistique, Newberry, Brevort Township, Cedarville, St. Ignace, and Pickford locations (Fig. 3 A-F) all had moderate correlation (⍴= 0.69, 0.64, 0.59, 0.57, 0.55, and 0.52 respectively). The lag time estimated for these sites ranged from 0-6 days, with the shortest lag time (0 days) at the St. Ignace location and the longest lag time (6 days) at the Brevort Township location (Table 2).Fig. 2 Locations where SARS-CoV-2 N1 and N2 gene concentration in wastewater samples had high correlation with COVID-19 cases including (A) Sault Sainte Marie, (B) Rudyard, (C) Kincheloe, (D) Munising, and (E) DeTour. Fig 2 Fig. 3 Locations where SARS-CoV-2 N1 and N2 gene concentration in wastewater samples had moderate correlation with COVID-19 cases including (A) Manistique, (B) Newberry, (C) Brevort Township, (D) Cedarville, (E) St. Ignace, and (F) Pickford. Fig 3 The Bay Mills/Brimley and Mackinac Island locations (Fig. 4 A-B) had low correlation (⍴= 0.49 and 0.48 respectively) to an estimated 7-day lag time in clinical cases (Table 3). The low correlations seen at the Brimley location are likely explained by the multiple sampling sites contributing to the Brimley area data. Additionally, casino wastewater inputs were dominated by tourists, possibly contributing to the low correlation. The low correlations seen at the Mackinac Island location could be similarly explained by the high tourism seen in this area given that there are only 492 residents on the island, and there were 1.3 million visitors over the study period (Mackinac Island Tourism Bureau, personal communication, February 17, 2022). Tourists carrying COVID-19 to a destination may take some time for it to spread to the local population, cause symptoms, and lead to registering as a positive case for the zip code. With wastewater surveillance, SARS-CoV-2 can be detected when the infected individual is in the area, regardless of whether or not they reside in the area or display symptoms.Fig. 4 Locations where SARS-CoV-2 N1 and N2 gene concentration in wastewater samples had low correlation with COVID-19 cases including (A) Bay Mills and Brimley and (B) Mackinac Island. Fig 4 A similar confounding factor of tourism is the prevalence of “snowbirds,” or seasonal residents that overwinter in warmer areas such as Florida and New Mexico. Positive cases in snowbirds were reported in their county of permanent residence, but the wastewater data would not reflect this if they contracted COVID-19 at their overwintering residence. The LSSU location (Fig. 5 ) showed very high correlation (r = 0.92) when calculated using the Pearson correlation coefficient and non-significant correlation (p-value > 0.05) when calculated using the Spearman correlation coefficient. The high Pearson correlation for the LSSU sampling location is likely explained by the closed population that the sampling location confluence represents. The confluence collects from dormitories which limits the likelihood of SARS-CoV-2 gene copies coming from anyone not residing in the dormitories. Betancourt et al. (2021) and Karthikeyan et al. (2021) found similar high, predictive correlations between college dormitory sewer surveillance and clinical testing of students.Fig. 5 Comparison between SARS-CoV2 gene copies and clinical cases for the LSSU sampling location. Fig 5 A moderate correlation was found between the strength of the significant Spearman correlation and population size (⍴= 0.58, p-value 0.038) for all sites. No correlation was found between the population size and the lag days associated with each location. 3.2 Phi6 recovery Phi6 recovery ranged from 33% to 111% and averaged 78% (±2, n=51). This recovery efficiency is comparable to Ai et al. (2021) who achieved an average recovery of 53.6% using a bacteriophage (MS2) as a recovery control and ddPCR quantification. Kantor et al. (2021) point out, however, that direct extraction (pre-PEG) and concentration extraction (PEG) results may not be comparable because they measure different fractions of the intact and non-intact viral signals. Despite this, recovery efficiency is still a valuable tool to verify method performance across sample types and over time (Kantor et al., 2021). PCR inhibition occasionally affected the Cedarville and Munising samples. When this occurred, the PEG concentrate was diluted (Schrader et al., 2012). RNA was extracted from the diluted concentrate, and the ddPCR process was rerun for the inhibited sample. In all cases, this process resolved inhibition. 3.3 Variant testing Variant testing kits (GT Molecular) for wastewater became available in August of 2021 and proved to be useful in tracking the spread of the highly contagious Delta variant. The Delta variant was detected in wastewater in the two largest study counties (Chippewa and Mackinac) prior to the first clinical cases attributed to the Delta variant (Table 4 ). In the case of Mackinac County, the Delta variant was detected in a wastewater sample (St. Ignace) nearly a month before the first attributed Mackinac County resident case. St. Ignace is both a tourist destination and the only road connection to the Lower Peninsula of Michigan, it is possible that the viral load was shed by travelers who are residents of other counties or states, as stated previously. In the case of tourists, any associated positive clinical test results would have been reported in their home county, even if they were tested in or temporarily residing in Mackinac County at the time of illness. This explanation is further supported by negative N1/N2 wastewater results for the following two weeks at the St. Ignace location, followed by weekly positive N1/N2 results starting August 5, 2021, when the Delta variant was spreading throughout the region.Table 4 Date of the first Delta variant detection in wastewater and the first clinical Delta variant case in each county. Clinical cases are reported based on the person's county of residence, but it is unknown whether the person was residing in the county at the time of positive test results. The first clinical Delta case in the Upper Peninsula region was on 7/20/2021 in Marquette County. Clinical dates were provided by the Chippewa and Luce/Mackinac/Alger/Schoolcraft Health Departments. Table 4County Delta in Wastewater Delta in Clinical Cases Alger 8/17/2021 8/6/2021 Chippewa 8/5/2021 8/17/2021 Luce 8/24/2021 7/29/2021 Mackinac 7/15/2021 8/12/2021 Schoolcraft 11/9/2021 7/28/2021 It is also of interest that the first positive N1/N2 results in wastewater from Munising, Newberry, and St. Ignace coincided with the first Delta-positive detection in wastewater from those sites. This is likely due to the increased transmissibility of the delta variant relative to previous variants ( Kang et al., 2022; Mikszewski et al., 2022; Zhan et al., 2022). This coincidence may also be a result of the Delta variant having an increased fecal shedding rate in some people and populations (Prasek et al., 2022). Another potential explanation for the increase in N1/N2 gene copies in wastewater results at roughly the same time across all sites (late August) was the return to school for preschool-12th grade students. Outbreaks in school settings have been well documented, and can potentially contribute to significant increases in community spread (Aiano et al., 2021; Stein-Zamir et al., 2020). Other studies have found that little transmission occurs in K-12 school settings (Boutzoukas et al., 2022; Falk et al., 2021; Xu et al., 2020). However, in rural areas, a large fraction of households discharge their wastewater to on-site wastewater treatment systems. The return to school might therefore result in a potential increase in viral loading of the municipal system, even if community transmission rates remained constant. Unfortunately, the nearly simultaneous arrival of the Delta variant with the return to school, precludes differentiation between these possible explanations. 4 Conclusions These results demonstrate the utility of wastewater surveillance in rural areas. Despite significant portions of the population residing outside of the municipal sewersheds, wastewater surveillance was generally predictive of clinical positive cases with estimated lag times (0-6 days) consistent with those reported in previous studies (Betancourt et al. 2021; Kaplan et al., 2021; Markt et al., 2021; Peccia et al., 2020). In instances where a weak or no correlation with clinical cases was observed, it is likely that issues with clinical case reporting related to transient and seasonal populations result in an inaccurate assessment of local disease prevalence. In these localities, wastewater monitoring data may be significantly more useful to public health specialists than clinical case data, permitting timely implementation of control measures. Additionally, our results demonstrate the utility of wastewater surveillance in monitoring the emergence of new viral variants. Wastewater surveillance detected the emergence of the delta variant in advance of clinical case detection. A key limitation in this regard was that our study did not include sequencing, and thus relied on the commercial availability of variant test kits. Future wastewater surveillance studies should consider including viral sequencing when novel variants are suspected and validated test kits are unavailable, as rapid detection supports an effective public health response. By partnering with the local health departments, health care practitioners and members of the public can be informed of potential outbreaks across a large geographic region prior to spikes in clinical cases. CRediT authorship contribution statement Michelle M. Jarvie: Conceptualization, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Moriah Reed-Lukomski: Conceptualization, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Benjamin Southwell: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Derek Wright: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Thu N.T. Nguyen: Conceptualization, Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michelle M. Jarvie, Moriah Reed, Benjamin Southwell, Derek Wright, Thu N.T. Nguyen reports financial support was provided by Michigan Department of Health and Human Services Data availability Data will be made available on request. 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Theodoratou E. What is the evidence for transmission of COVID-19 by children in schools? A living systematic review J. Glob. Health 10 2 2020 10.7189/jogh.10.021104 Zhan Y. Yin H. Yin J.-Y. B.1.617.2 (Delta) Variant of SARS-CoV-2: features, transmission and potential strategies Int. J. Biol. Sci. 18 5 2022 1844 1851 10.7150/ijbs.66881 35342345 Zhu Y. Oishi W. Maruo C. Saito M. Chen R. Kitajima M. Sano D. Early warning of COVID-19 via wastewater-based epidemiology: potential and bottlenecks Sci. Total Environ. 767 9 2021 10.1016/j.scitotenv.2021.145124 Zymo Research. (2021, January 15). Viral targets: what makes a good COVID-19 RT-PCR test, https://www.zymoresearch.com/blogs/blog/what-makes-a-good-covid-19-rt-pcr-test.	36471702	PMC9714184	NO-CC CODE	2022-12-05 23:15:31	no	Environ Adv. 2023 Apr 1; 11:100326	utf-8	Environ Adv	2,022	10.1016/j.envadv.2022.100326	oa_other
==== Front J Pathol Inform J Pathol Inform Journal of Pathology Informatics 2229-5089 2153-3539 The Authors. Published by Elsevier Inc. on behalf of Association for Pathology Informatics. S2153-3539(22)00756-8 10.1016/j.jpi.2022.100162 100162 Article Maintaining informatics training learning outcomes with a COVID-19 era shift to a fully online flipped course Maness Heather T.D. a Hakimjavadi Hesamedin bc Chamala Srikar bc⁎ a UFIT Center for Instructional Technology and Training, University of Florida, Gainesville, FL, USA b Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, USA c Department of Pathology and Laboratory Medicine, Children’s Hospital, Los Angeles, CA, USA ⁎ Corresponding author at: Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027, USA. 1 12 2022 2023 1 12 2022 14 100162100162 8 6 2022 26 11 2022 27 11 2022 © 2022 The Authors 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. The emergence of the coronavirus disease 2019 pandemic forced us to adapt our recently developed informatics training serving a variety of students as well as faculty and staff. The successful flipped classroom course series (a hybrid-format with both asynchronous online learning and in-person synchronous components) was shifted to a fully online format with the synchronous portion now held via web-based video conference. We repeated our participant survey at the end of each of the 3 one-credit courses to compare student satisfaction and learning outcomes achievement to the original offering. The responses were overall very positive again with a slight response distribution improvement in some measures of satisfaction. Likewise, students reported similar achievement of the learning outcomes across all courses with some Unix coourse objectives receiving higher competency agreement in the new, fully online version. Overall, the fully online version of the course series was equally successful, if not more so, than the original version with a physical classroom session each week. Given that participants also had strong agreement with a new question that they would prefer online class meetings instead of in a classroom, even if there wasn’t a global pandemic (citing a variety of logistical reasons such as “convenience of screen sharing,” parking issues, and job-related time constraints), the fully online version of the informatics training will be retained. Keywords Competency Continuing education Flipped classroom Graduate medical education Informatics Pathology Online learning Residency training ==== Body pmcBackground Shortly after developing a successful hybrid-format (both online learning and in-person components) course specifically se series, named Informatics for Pathology Practice and Research (previously published1), the emergence of coronavirus disease 2019 (COVID-19) forced us (and every other education provider in the world2) to pivot to a fully online format. We were fortunate that the program structure was already a flipped classroom design where students prepared for the in-person class session each week by asynchronously completing the assigned reading material and some activities in the online environment (via Canvas Learning Management System, Instructure, Inc., “Canvas”). Once the pandemic began, the synchronous portion of the flipped classroom (previously held physically on campus) was then easily transitioned to a synchronous, web-based video conference platform (via Zoom Video Communications, Inc. “Zoom”) to continue to provide the collaborative peer learning opportunities3 and instructor/teaching assistant (TA) support during the active learning components of the course. Zoom was familiar to the instructor and some students since it was an established vendor provider at the institution and was previously integrated into the course as an office hours tool. Prior to the pandemic, there were minimal examples of virtual flipped classroom use in the literature, for any discipline.4 , 5 Previous work focused mostly on physical flipped classrooms, often with asynchronous online learning components. Some synchronous online course design research has been conducted, but there was scant evidence for also using a flipped classroom model in those studies. It is further limited when looking for evidence of specific flipped classroom design principles, argued as necessary for meaningful comparisions.6 Yet, this strategy has now been proposed as a viable model, specifically in pathology training.2 , 7 Furthermore, herein we provide rare comparison data for the physical flipped classroom model to a virtual flipped classroom design, where synchronous activity time was also held in an online environment. In particular, with our shift to the fully online flipped classroom model, we wanted to gauge if the learning outcomes would still be successfully met by most students and if the design was still satisfactory for our broad participants of students, faculty, and staff. The Informatics for Pathology Practice and Research program began as a series of 3 one-credit courses offered sequentially for 5 weeks each, during a singular semester. It was designed to meet the modern training needs in anatomical/clinical and experimental pathology8 , 9 serving undergraduates, graduate students, medical students, post-doctoral fellows, residents, staff, and faculty. The series focuses on the Unix Operating System (Unix), Python Programming (Python), and Advanced Data Analysis and Visualization (ADAnV) with Pandas and Matplotlib. As popularity of the program has grown, it was renamed to Programming for Biomedical Research & Clinical Practice to expand outreach to other biomedical professionals and students across the health science colleges—Medicine, Pharmacy, Public Health & Health Professions, and Veterinary Medicine. Although this program was originally designed to serve the needs of the pathology department, this program is applicable (as evident from our participant growth in various disciplines) to all researchers, clinicians, and other healthcare professionals interested in building fundamental computational skills necessary for pursuing specialized and advanced informatics training in the areas of clinical bioinformatics, biomedical informatics, artificial intelligence, digital pathology imaging, etc. It is assumed students have no prior knowledge of programming or advanced computing. Methods The original 40-item questionnaire (previous publication on the IPPR course series) was modified to a 31-item version to investigate the research question: How does the online version of the pathology informatics training program compare to the previous physical classroom version? Since this study was a continuation of the previous study,1 it was classified within the same institutional review board approved protocol (study #201602565). The course instructor was the same person (Chamala) but the TA was a different graduate student than the original TA. Beyond using Zoom for the synchronous meetings instead of the physical classroom environment, the only notable change to the Spring 2021 training program was a shift in the course order to ending with Unix instead of beginning with it (Table 1 ).Table 1 Comparison of registrants to respondents for the 2 different offering styles. Table 1Flipped style Title Durationa Registrants Respondents Physical (2018) Unix Operating System First 5 weeks 21 20 Python Programming Second 5 weeks 22 19 Advanced Data Analysis & Visualization Third 5 weeks 22 15 Online (2021) Python Programming First 5 weeks 34 17 Advanced Data Analysis & Visualization Second 5 weeks 33 7 Unix Operating System Third 5 weeks 32 8 a All 3 courses are held during single semester that runs for 15 weeks. Data visualization and statistical analyses were performed using R (version 4.01). Descriptive measures (inclusive median ± interquartile range) were calculated for all questions for all groups of participants. Given the non-parametric nature of the data, Mann–Whitney–Wilcoxon tests were used to examine differences between the 2 course formats for students’ evaluation of each prompt related to satisfaction and objective achievement. For each comparison, we reported statistics (Mann–Whitney U), p value, and confidence interval. Differences with a p-value smaller than .05 were considered significant. Results Course participants and survey respondents Thirty-four individuals enrolled in one or more of the courses in the Spring 2021 semester which was higher than the initial course offerings (Table 1). Unique respondents (N = 20) to our latest surveys included undergraduates (n = 2), graduate students (n = 9), medical students (n = 3), a medical and graduate student (n = 1), post-doctoral students (n = 2), a medical fellow (n = 1), and faculty (n = 2). For this semester, respondents came from a variety of colleges compared to the original offering. While most respondents were still from the College of Medicine (n = 8), we also had respondents from the colleges of Public Health and Health Professions (n = 3), Pharmacy (n = 2), Engineering (n = 2), and Veterinary Medicine (n = 1). There were also 2 respondents who chose not to disclose their affiliation on the questionnaire. Prior experience Programming. The majority of respondents had no prior experience with Unix (n = 6) or Python (n = 9) before the course series. The rest chose “a little” (n = 6) or “a moderate amount” (n = 2) to describe their experience, except for 1 student who chose “a lot” for Unix and another who also reported that for Python experience. Online learning environment. Only 1 respondent had not previously taken a fully online course (either asynchronously or one with synchronous sessions). Therefore, the online learning environment was more familiar to most students than in the original offering where over half did not have previous experience with online learning. Course satisfaction comparison Once again, in this offering of the course series the responses were overall very positive. There was no median difference between the 2 semesters when aggregating all satisfaction measures (Fig. 1 and Table 2 ). When analyzing each of the 3 satisfaction metrics, there was no significant median difference for overall, interaction, or recommendation but there was a significantly higher agreement response distribution for recommending the online version of the course to a friend (Fig. 1 and Table 2).Fig. 1 Satisfaction and learning objective ratings comparisons by format. Distribution and median comparison (* = P < .05) of respondents’ agreement levels (5-point Likert-type scale) for course satisfaction and learning objective metrics in the new, fully online format versus the original physical-presence for synchronous sessions version. Fig. 1 Table 2 Course satisfaction metrics statistical comparison (fully online:physical format). Table 2Course Satisfaction (median comparison) U (CI) P value Total combined—Online vs Physical (4:4) 8774.5 (-0.07, 0.22) 0.281 Courses combined—Overall (4:4) 1087.0 (-0.06, 0.41) 0.113 Courses combined—Interaction (3.5:4) 700.5 (-0.44, 0.02) 0.066 Courses combined—Recommend course (4:4) 1120.5 (, 0.46) 0.046* I-Unix Overall (4:4) 82.5 (-0.42, 0.47) 0.914 Interaction (4.5:4) 62.5 (-0.61, 0.25) 0.340 Recommend course (4:4.5) 94.5 (-0.23, 0.63) 0.308 All 3 combined (4:4) 721.5 (-0.25, 0.29) 0.888 II-Python Overall (4:3) 224.5 (0.03, 0.66) 0.028* Interaction (3:4) 117.0 (-0.58, 0.10) 0.117 Recommend course (4:3) 219.5 (-0.01, 0.64) 0.060 All 3 combined (4:3) 1725.5 (-0.03, 0.39) 0.080 III-ADAnV Overall (4:4) 90.0 (-0.14, 0.68) 0.115 Interaction (4:4) 66.5 (-0.38, 0.55) 0.682 Recommend course (4:4) 89.5 (-0.15, 0.68) 0.161 All 3 combined (4:4) 744.0 (-0.00, 0.29) 0.040* Per course, median comparisons between the formats of the aggregated satisfaction measures showed no significant difference in the Unix course, a slight satisfaction improvement in the Python Programming course but not statistically different, and a slight response distribution improvement for satisfaction in the new, fully online Advanced Data Analysis and Visualization course while maintaining the same median score (Fig. 2 and Table 2). Independently, the only satisfaction metric to have a statistically significant difference was a higher overall satisfaction agreement for the online Python Programming course (Fig. 2 and Table 2). The median comparison between the 2 semesters showed no statistically significant difference in the level of interaction they had with others (classmates and instructor/TA) for any of the courses in the program (Fig. 2 and Table 2). Likewise, there was no statistically significant difference in recommending a specific course to a friend (Fig. 2 and Table 2).Fig. 2 Course satisfaction ratings. Distribution and median comparison (* = P < .05) of respondents’ agreement levels (5-point Likert-type scale) for each course offering with the 3 course satisfaction elements (overall, interaction, and recommendation) and when combined. Fig. 2 Achievement of the course objectives comparison Participants’ self-assessed rating of their achievement of the learning objectives for each course in the new online format, was mostly similar to the ratings by previous students, with the only statistically significant differences occuring in the Unix course (Fig. 3 and Table 3 ). There was an increase in the agreement distribution for overall learning objective achievement in the new online Unix course that was moved to the end of the series. Impact stemmed specifically from the increase in agreement distribution for the third objective (3. Create/interpret Unix programs) as well as the median and distribution in achievement of the fourth objective (4. Make informed decisions on informatics server needs in a biomedical setting).Fig. 3 Course learning objective achievement ratings. Distribution and median comparison (* = P < .05) of respondents’ agreement level (5-point Likert-type scale) that they achieved competency of the learning objectives in the new online format versus the original physical-presence for synchronous sessions. Fig. 3 Table 3 Course learning objectives statistical comparison (fully online:physical format). Table 3Course Objective (median comparison) U (CI) P value Total combined objectives—Online vs. Physical (4:4) 15606.0 (-0.05, 0.20) 0.195 I-Unix 1. Navigate through Unix/Linux environments (4.5:4) 86.0 (-0.39, 0.51) 0.758 2. Use high performance computing servers (4:3.5) 115.5 (-0.00, 0.74) 0.059 3. Create/interpret programs in Unix (4.5:4) 123.0 (0.12, 0.79) 0.023* 4. Determine biomedical informatics server needs (4:3) 131.0 (0.28, 0.85) 0.006* All 4 Combined I-Unix Objectives (4:4) 1800.0 (0.19, 0.58) 0.0004* II-Python 1. Think computationally about problem-solving (4:4) 147.5 (-0.36, 0.39) 0.926 2. Discuss software programming concepts (4:4) 125.0 (-0.49, 0.25) 0.474 3. Create high quality programs in Python (3:3) 154.5 (-0.31, 0.43) 0.731 4. Interpret Python programs written by others (4:4) 139.5 (-0.40, 0.34) 0.863 5. Determine biomedical programming needs (3:4) 132.5 (-0.44, 0.30) 0.672 All 5 Combined II-Python Objectives (4:4) 3510.5 (-0.20, 0.15) 0.734 III-ADAnV 1. Extract info. from Big Data using Python (4:4) 56.5 (-0.43, 0.54) 0.772 2. Extract info. from Big Data using Pandas (4:4) 52.5 (-0.49, 0.49) 1.000 3. Create data visualizations using Python (4:4) 53.5 (-0.47, 0.50) 0.968 All 3 Combined III-ADAnV Objectives (4:4) 489.0 (-0.26, 0.32) 0.796 Preference for synchronous online over a physical classroom Participants were also asked to rate their level of agreement with the statement: “Even if there wasn't a global COVID-19 pandemic, I prefer to have synchronous class meetings at specific times online, instead of in a classroom (considering travel time savings, parking, learning/interaction preferences, technology availability, etc.).” All but 3 respondents agreed or strongly agreed with the statement (n = 19; median = 4; mean 4.16, SD 1.01). One of the two neutral responses noted that while they slightly prefer in-person learning, to make the travel logistics worthwhile, they try to create their class schedule so they can take multiple classes back-to-back. The only respondent who strongly disagreed also cited a preference for in-person classes and found coding to be “a difficult topic to learn via Zoom, especially at a beginner level.” Those that agreed with the statement provided a variety of reasons including the “convenience of screen sharing,” job-related time constraints requiring the need for flexibility in pacing and participation, as well as not having a car. A few of these respondents did also note that they may have learned more from others if they were face-to-face in a physical classroom. Yet, 1 person noted that there was no need for “a live portion” at all. Conclusions The increase in self-reported achievement of 2 of the learning objectives in the Unix course was a surprising finding. One hypothesis we have for why that occurred is related to the change in the course sequencing. Based on the initial student feedback from the first offering, instead of starting the course series with Unix, it was moved to the end of the course series so students could better appreciate the application use cases for Unix. Thus, this shift to strengthen the relatedness of Unix to the other course topics may have also increased student perception of learning objective achievement. Likewise, the placement of Python Programming as the first course in the series may have contributed to participants’ higher overall satisfaction ratings and the higher combined satisfaction metric for Advanced Data Analysis and Visualization in its new mid-point presentation. It may also be pertinent that the Unix learning objectives with improved median scores are both of the higher order cognitive skills, using Bloom’s Taxonomy,10 in the set. There is an established need for experimental research on flipped classroom long-term effect on higher level outcomes, such as behavior,11 but more research is also warranted on the impact on lower level versus higher level thinking skills with consideration of learning environment modality. Regardless of the potential course order role in learning achievement reporting, the fully online format certainly did not decrease perceptions of learning objective achievement or satisfaction ratings, and may be a contributor for increased learning objective achievement and satisfaction. There is great variance in flipped classroom course designs, so it is important to consider the context of this study when considering the greater pedagogical implications. While previous research has shown favorable effects of flipped classroom design on outcomes when skill laboratories were not included in control groups11, 12, 13 and participant preference for flipped over traditional lecture formats,12 , 13 there were variances in effect sizes. Moreover, these meta-analyses did not consider online synchronous sessions as a variable, which has been previously reported as a detriment to quality and effectiveness of pandemic-learning.14 This contrast to our findings could be related to differences in topic or technologies, but another explanation is it is because of the difference in intentionality and preparation time for our fully online version. Key elements of our flipped classroom design include pre-class videos and graded quizzes in a highly organized learning management system, weekly real-world assignments and feedback, and a diverse learning community with students in a variety of degree programs as well as continuing education learners. The work to create the online student experience and facilitate active learning sessions was done before the pandemic-forced fully online semester, likely leading to a higher quality experience. Additionally, since it is not well-understood yet if the general increased effect from flipped classroom designs is associated more so, or equally, from the changes in student preparation for class or the in-class activities themselves,12 active learning experiences during synchronous class time should also be considered in further research. Our activities were mainly groupwork-encouraged assignments with individual submission and Question/Answer sessions. Future studies should compare outcomes amongst different techniques, such as game-based learning15 and team-based learning,16 with consideration for the proportion of class time utilizing each strategy and variances in student compliance with pre-class requirements and in-class participation. There are several logistical benefits that were identified with the transition to a fully online course design. Not only did students appreciate the time savings from not needing to travel to a campus classroom for the synchronous session, but the instructor also valued the time savings from the elimination of travel time and parking limitations since he has an off-campus office at a health science center community site. Additionally, the fully online version offers the ability to expand the learner reach to those outside of the college of medicine (those in colleges that are physically distant from the Health Science Center) as well as the potential to expand to interested learners outside of our institution. It also creates more opportunities to expand the expert teachers contributing to people beyond our institution/location, which has been previously reported as positively contributing to training program designs.17 Lastly, some participants noted their appreciation for the ability to quickly share their screen during class discussion. This is an added benefit since it provides additional experience with using their own equipment during bioinformatics analysis tasks17 and likely enhances their confidence in completely these tasks independently. Overall, the fully online version of the course series was equally successful, if not more so, than the original version with a physical classroom session each week. While acknowledging the response and recall bias our study is limited by, our conclusions are evidenced by the participant preference for fully online format and consistently high student ratings in satisfaction metrics for all courses with occasional higher ratings in the online version, such as recommending the course to a friend. Further support comes from the data showing there were no significant differences in the achievement of the student learning outcomes in Python Programming and Advanced Data Analysis and Visualization and improvement in the Unix course. Given that conducting the pathology informatics training program was also logistically easier in the fully online environment and being exclusively online provides flexibility for broader participation across and beyond the institution, we will likely continue to offer the flipped classroom format of the course series using video conferencing for our weekly synchronous meetings. Funding The authors received no financial support for the research, authorship, and/or publication of this article. ORCID iD Srikar Chamala https://orcid.org/0000-0001-6367-7615. Declaration of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Srikar chamala reports was provided by University of Southern California. Acknowledgements None. ==== Refs References 1 Maness H.T.D. Behar-Horenstein L.S. Clare-Salzler M. Chamala S. Informatics training for pathology practice and research in the digital era Acad Pathol. 7 2020 1 11 10.1177/2374289520911179 2 Koch L.K. Chang O.H. Dintzis S.M. Medical education in pathology: general concepts and strategies for implementation Arch Pathol Lab Med. 2021 10.5858/arpa.2020-0463-RA 3 Bower M. Richards D. 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Digital slides as an effective tool for programmed death ligand 1 combined positive score assessment and training: Lessons learned from the “Programmed death ligand 1 key learning program in Head-and-Neck squamous cell carcinoma.” J Pathol Inform. 12 1 2021 10.4103/JPI.JPI_63_20	36471780	PMC9714185	NO-CC CODE	2022-12-16 23:18:12	no	J Pathol Inform. 2023 Dec 1; 14:100162	utf-8	J Pathol Inform	2,022	10.1016/j.jpi.2022.100162	oa_other
==== Front J Comput Appl Math J Comput Appl Math Journal of Computational and Applied Mathematics 0377-0427 1879-1778 Elsevier B.V. S0377-0427(22)00567-2 10.1016/j.cam.2022.114969 114969 Article A novel fractional order model of SARS-CoV-2 and Cholera disease with real data Özköse Fatma a⁎ Habbireeh Rafla bc Şenel M. Tamer a a Erciyes University, Department of Mathematics, Faculty of Science, Kayseri, Türkiye b Institute of Science, Erciyes University, Kayseri 38039, Türkiye c Department of Mathematics, Faculty of Science, Misurata University, Misurata, Libya ⁎ Corresponding author. 1 12 2022 1 12 2022 11496916 8 2022 21 10 2022 © 2022 Elsevier B.V. All rights reserved. 2022 Elsevier B.V. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. This study presents a novel approach to investigating COVID-19 and Cholera disease. In this situation, a fractional-order model is created to investigate the COVID-19 and Cholera outbreaks in Congo. The existence, uniqueness, positivity, and boundedness of the solution are studied. The equilibrium points and their stability conditions are achieved. Subsequently, the basic reproduction number (the virus transmission coefficient) is calculated that simply refers to the number of people, to whom an infected person can make infected, as R0=6.7442389e−10 by using the next generation matrix method. Next, the sensitivity analysis of the parameters is performed according to R0. To determine the values of the parameters in the model, the least squares curve fitting method is beneficial. A total of 22 parameter values in the model are estimated by using real Cholera data from Congo. Finally, to find out the dynamic behavior of the system, numerical simulations are presented. The outcome of the study indicates that the severity of the Cholera epidemic cases will decrease with the decrease in cases of COVID-19, through the implementation and follow-up of safety measures that have been taken to reduce COVID-19 cases. Keywords SARS-CoV-2 and Cholera disease Fractional-order derivative Stability analysis Real data Parameter estimation Sensitivity analysis ==== Body pmc1 Introduction Cholera is a bacterial disease, caused by infection of the intestine with the toxigenic bacterium Vibrio cholera serogroup O1 or O139. It is usually spread through contaminated water. Cholera causes severe diarrhea and dehydration. If not treated, it can be fatal within hours, even in previously healthy people. The amount of Vibro cholera bacteria that causes Cholera is 103−105 Vibro cholera cells and it may be less than this. During the 19th century, cholera spread throughout the world from its original reservoir in the Ganges delta in India. Then, six pandemics of disease erupted, claiming the lives of millions of people on all continents. The current (seventh) pandemic broke out in South Asia in 1961 and reached Africa in 1971 and the Americas in 1991. Cholera is now endemic in many countries and it has been virtually eliminated in industrialized countries by modern sanitation and water treatment, but cholera is still present in Africa, Southeast Asia, and Haiti. The risk of a cholera epidemic increases when poverty, war, or natural disasters force people to live in crowded conditions without adequate sanitation. The incubation period of cholera is less than 24 h to 5 days and infection is frequently asymptomatic. Cholera is easily treatable, and death from severe dehydration can be prevented by using a simple and inexpensive rehydration solution. Researchers estimate that there are between 1.3 and 4.0 million cholera cases annually, and cholera causes between 21,000 and 143,000 deaths worldwide (WHO). Recently, the outbreak of the cholera epidemic, which coincides with the outbreak of the Coronavirus, represents a double blow to the countries that suffer from the two epidemics together, especially in light of the dire conditions in which these countries are living Such as malnutrition, constant wars, and contaminated drinking water. One of the effective ways to fight cholera is to take the vaccine. The vaccine contains completely dead or weakened cholera cells with a group of cholera toxins, to support immunity. The vaccine shows very good results and provides a high level of protection of up to 85%, and its effect lasts for 6 months for everyone who has received the vaccination over the age of two years. The protection level drops to 50% after 3 years. In epidemic control studies are important to predict the course of the epidemic in the future, to know the main ways to limit its spread, and to give necessary warnings before it becomes a disaster with many deaths. There are several contributions from researchers in many disciplines that address the prediction, study, and development of important approaches to cholera and COVID-19 epidemics. In the past, many mathematical models have been used to model many epidemics. These models have proven to be practical and extremely effective tools for treating, predicting, and eliminating disease, so much so that to this day many diseases are modeled mathematically to figure out how to eliminate them [1], [2]. Although the use of integer-order equations is successful to some extent, the results obtained with fractional-order equations are still better at representing real phenomena in terms of reality. Among the advantages of mathematical modeling by FDEs are some of the qualities found only in this type of equation, such as repeating operations, as well as nonlocal properties, properties that make the mathematical model’s status as of right now consistent with previous and subsequent states. This event can be successfully mirrored using FDEs. Also, they are naturally related to systems with memory which exists in many epidemic diseases and they have hereditary properties. In addition, FDEs help us to reduce the possible errors that may arise from parameters that have to be neglected while modeling. The memory effect of dynamic behavior as well as non-local ownership in addition to being more realistic when talking about biological and medical phenomena, as well as models that depend on this type of equations do not depend only on the current state but are also related to previous historical cases. In recent years, fractional differential equations have proven to be a reliable and well-organized mathematical tool for the study of many scientific and engineering processes. Research in fractional differential equations is multidisciplinary and is used in various fields, such as control systems, elasticity, electric drives, circuits, continuum mechanics, quantum mechanics, fluid mechanics, signal analysis, biomathematics, biomedicine, social systems, bioengineering, management, financial systems, traffic flow, turbulence, complex systems, pollution control, and more. Naik et al. [3] proposed a fractional order model of Viral kinetics for primary infection of HIV-1 in presence of immune control with treatment. Chen et al. [4] proposed and analyzed a partial differential equation model to ascertain the effect of human diffusion, and bacteria convection in Cholera transmission, they also investigated the various factors that determine the spatial spread of Cholera. Berhe [5] presented a study of the optimal control model of Cholera. Bakare and Hoskova-Mayerova [6], developed an ideal Cholera epidemic control model, their study showed that the four control measures considered have the potential to control and eliminate Cholera in asymptomatic populations. Monje et al. [7] studied a prolonged Cholera outbreak caused by drinking contaminated stream water, in the Kyangwali refugee settlement, Hoima District, Western Uganda. Moussouni et al. [8] modeled an optimal control problem based on the models of William Ogilvy Kermack et Anderson Gray McKendrick, called the SEIR model, modified by adding compact-ments suitable for their study. Özköse et al. [9] investigated the fractional-order model to study the COVID-19’s spread with and without the Omicron variant and the effects of the virus on heart attack patients. There is a lot of research to this day that deals with the use of FDE in formulating a mathematical model from various fields of science [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Despite their obvious differences, COVID-19 and Cholera have a lot in common as they can be at least partly controlled through vaccination and both spread easily in a crowded and unsanitary environment. Therefore, ensuring adequate shelter and strengthening WASH practices and infrastructure are vital to reducing infection. These commonalities explain why COVID-19 mitigation measures such as reduced travel and increased awareness regarding personal hygiene have led to a decline in Cholera cases. This study aims to know the extent of the Cholera epidemic and COVID-19 and to clarify the relationship between them in addition to predicting the future by clarifying the course of the disease, to give the necessary awareness and take the necessary measures to eliminate these epidemics, as well as to study the effects of vaccination campaigns on the population. The sections of the paper are as follows: in Section 2 presents the main definitions of fractional calculus. In Section 3, fractional model has been presented. Section 4 demonstrates the model solution’s existence and uniqueness. The positivity and boundedness of the solution as well as the stability of the equilibrium points of the suggested model are provided in Section 5. The basic reproduction number (R0) has also been determined. Sensitivity analysis has been examined as a function of the parameters to investigate the extent of their effects on R0 in Section 6. The method of parameter estimation is examined in Section 7. Section 8 presents the suggested model’s numerical approach. In Section 9, the Adams–Bashforth-Multon method is used to find numerical solutions to our model for the fitted parameter values shown in Table 1. Finally, we have summarized the current work in Section 10. 2 Preliminaries The key definitions of fractional calculus utilized in this study are stated below. Definition 1 [23] The fractional integral of order ϑ>0, of the function g(t), t>0 is presented by Iϑg(t)=∫0t(t−s)ϑ−1Γ(ϑ)g(s)ds. and the fractional derivative of order ϑ∈(n−1,n) of g(t), t>0 is defined by Dϑg(t)=In−ϑDng(t)(D=ddt),whereΓ(.)isGammafunction,ϑ>0. Definition 2 [23] Given a function g:(0,∞)→R, the Caputo fractional derivative of order ϑ>0 is given by (1) 0CDtϑg(t)=1Γ(n−ϑ)∫0td/dτng(τ)(t−τ)ϑ−n+1dτ,0≤n−1<ϑ<n,n=[ϑ],n∈N,ddtng(t),ϑ=n,n∈N. Definition 3 [23] Given a function g(t), the Laplace transform (LT) of the Caputo derivative of order ϑ>0 is given by (2) L[0CDtϑg(t)]=sϑg(s)−∑v=0n−1gv(0)sϑ−v−1. Definition 4 [23] The Laplace transform (LT) of the function g(t)=tϑ1−1Eϑ,ϑ1(±ωtϑ) is defined as (3) L[tϑ1−1Eϑ,ϑ1(±ωtϑ)]=sϑ−ϑ1sϑ±ω, where Eϑ,ϑ1 is Mittag-Leffler function. Theorem 1 [24], [25] Take into account the fractional-order system below: (4) dϑxdtϑ=f(x),x0=x0, with ϑ∈ (0,1] and x ∈Rn . The zeros of the function fX∗=0 are the equilibrium points of the system (4) and these equilibrium points: (1) Asymptomatically stable ⟺ the eigenvalues λi , of the Jacobian matrix J(X∗) satisfy that \|arg(λi)\|>ϑπ2 . ∀i , i=1,2,…,n . (2) Stable ⟺ it is Asymptomatically stable or the eigenvalues λi , i=1,2,…,n of J(X∗) that satisfy \|arg(λi)\|=ϑπ2 if have the same geometric and algebraic multiplicity. (3) Unstable ⟺ ∃i , such that the corresponding eigenvalue λi of J(X∗) satisfy \|arg(λi)\|<ϑπ2 , i=1,2,…,n . 3 Mathematical modelling To predict the severity and course of epidemics and to express them in the future, the representation of these epidemics with mathematical models is a very effective way to do. It is also possible to use mathematical models to develop treatments for many diseases. Numerous mathematical models have been developed to depict the COVID-19 disease and demonstrate how it negatively affects many other chronic diseases. Some of these models involved the vaccine and others involved the quarantine to stop the disease’s spread, also some of them depict the side effects of COVID-19 on other diseases as chronic diseases like diabetes [1] and heart attacks [9]. This study introduces a novel model to investigate the relationship between COVID-19 and Cholera. To assess the transmission of COVID-19 and Cholera, we consider eight sub-populations as susceptible individuals (S), asymptomatic individuals (A), exposed individuals (E), COVID-infected individuals without Cholera (I), Cholera-infected individuals without COVID-19 (C), infected individuals with both COVID-19 and Cholera (D), vaccinated individuals (V), recovered individuals (R) and the pathogen population (B). In fractional systems, dimensionally consistency is a very important tool, in which the units of measurement from the left- and right-hand sides of the equations are coherent. This consistency can be provided by modifying the parameters involved in the right-hand side of the equations, e.g. raising them to power ϑ. That’s why dimensional compatibility has been considered to better reveal the effect of fractional order in the proposed fractional order system. In this context, the proposed fractional-order model is as follows: 0CDtϑS=Λϑ−(μϑ+β1ϑNA+β2ϑNI+θ2ϑ+ψ2ϑ+δ3ϑNIC)S−βϑ(1−Pϑ)BN(B+KBϑ)S+(θ1ϑ+ψ1ϑ)V, 0CDtϑA=β1ϑN(1−ɛAϑ)AS−βϑ(1−Pϑ)BN(B+KBϑ)A−μϑA−γ1ϑA, 0CDtϑI=(β1ϑNɛAϑA+β2ϑNI)S−βϑ(1−Pϑ)BN(B+KBϑ)I−(d1ϑ+r1ϑ+μϑ)I, (5) 0CDtϑC=βϑ(1−Pϑ)BN(B+KBϑ)S−(d2ϑ+r2ϑ+δ2ϑNI+μϑ)C, 0CDtϑD=βϑ(1−Pϑ)BN(B+KBϑ)(A+I)+δ2ϑNCI+δ3ϑNSIC−(r3ϑ+d3ϑ+μϑ)D, 0CDtϑV=−(θ1ϑ+ψ1ϑ+μϑ)V+(θ2ϑ+ψ2ϑ)S, 0CDtϑR=r1ϑI+r2ϑC+r3ϑD+γ1ϑA−μϑR, 0CDtϑB=ɛϑC+(bBϑ−τϑ)B, with initial conditions: S(0)=S0≥0,A(0)=A0≥0,I(0)=I0≥0,C(0)=C0≥0,D(0)=D0≥0,V(0)=V0≥0, (6) R(0)=R0≥0,B(0)=B0≥0. Table 1 provides the biological meanings of the parameters in model (5): Table 1 Parameter values used for numerical analysis. Par. Meaning Value Source Λϑ Constant rate of the total population 4.25855e＋03 Calculated μϑ The natural death rate 4.49135e−05 Calculated δ2ϑ The rate of getting Cholera disease of COVID-19 infected individuals 0.3545062 Fitted δ3ϑ Infected rate by both Cholera and COVID-19 simultaneously 0.4300694 Fitted β1ϑ Rate of disease transmission through contact with A class 0.25005 Fitted β2ϑ Rate of disease transmission through contact with I class 0.2489494 Fitted γ1ϑ The rate of persons recovered from class A 0.5883616 Fitted r1ϑ The rate of persons recovered from class I 0.7578841 Fitted r2ϑ The rate of persons recovered from Class C 0.0725658 Fitted r3ϑ The rate of persons recovered from both I and C classes, 0.4293024 Fitted θ1ϑ COVID-19 vaccination Expiry Rate 0.49999 Fitted θ2ϑ The rate of susceptible population is vaccinated of COVID-19 1.701694 Fitted ψ1ϑ Cholera vaccination Expiry Rate 0.599986 Fitted ψ2ϑ The rate of susceptible population is vaccinated of Cholera 1.818662 Fitted ɛϑ Contribution of infected individuals to the population of Vibrio bacteria 4.747966e−04 Fitted ɛAϑ Testing rate of COVID-19 0.35005 Fitted bBϑ Birth rate of Vibrio bacteria 0.153465 Fitted τϑ Death rate of Vibrio bacteria 0.5143757 Fitted d1ϑ Death rate of COVID-19 0.5722392 Fitted d2ϑ Death rate of Cholera 0.04687007 Fitted d3ϑ Death rate of both Cholera and COVID-19 0.25005 Fitted βϑ Rates of ingesting Vibrio cholera from the contaminated aquatic reservoir or 5.1948989e−04 Fitted indirect transmission rate of cholera Pϑ Fraction of the compliance of hygienic, ingestion of cholera bacterium and 0.8999596 Fitted contact with cholera patients of the susceptible and recovered cohort KBϑ Concentration of Vibrio cholera in food and water 0.779666 Fitted that yield 50% chance of acquiring cholera disease Table 2 Real cases for 30 weeks in 2021, CONGO (http://www.plateformecholera.info). The first column contains the real cases of Cholera in CONGO, the second column contains predicted values from the proposed system’s simulations for the infectious class, and the last column contains the absolute errors among real data and the predicted values of model (5). Real cases Predicted values Absolute error 72 72 0 52 78 0.260315e＋02 81 84.5661 0.35661e＋01 80 91.6471 0.1164e＋02 63 99.3207 0.3632e＋02 45 107.6368 0.6263e＋02 31 116.6491 0.8564e＋02 25 126.4160 0.10141e＋03 53 137.47 0.840006e＋02 31 148.4716 0.11747e＋03 31 160.9029 0.12990e＋03 62 174.3751 0.11237e＋03 154 188 0.3479e＋02 186 204.7980 0.1879e＋02 281 221.9455 0.5905e＋02 270 240.5288 0.2947e＋02 200 260.6679 0.6066e＋02 228 282.4933 0.5449e＋02 317 306.1462 0.1085e＋02 291 331.7794 0.4077e＋02 249 359.5589 0.11055e＋03 335 389.6644 0.5466e＋02 376 422.2904 0.4629e＋02 544 457.6483 0.8635e＋02 541 495.9667 0.4503e＋02 637 537.4933 0.9950e＋02 640 582.4971 0.575002e＋02 649 631.2688 0.1773e＋02 639 684.1241 0.4512e＋02 742 741.4050 0.5950 4 Existence and uniqueness Take into account the system (5) with the initial conditions (ICs): S(0)=S0,A(0)=A0,I(0)=I0,C(0)=O0,D(0)=D0,V(0)=V0,R(0)=R0,B(0)=B0. System (5) can be written in the following form: (7) 0CDtϑXt=B1Xt+StB2Xt+AtB3X(t)+ItB4Xt+ϕ, X0=X0, where X(t)=S(t)A(t)I(t)C(t)D(t)V(t)R(t)B(t),X0=S(0)A(0)I(0)C(0)D(0)V(0)R(0)B(0), B1=−A10000θ1ϑ+ψ1ϑ000−A200000000−A300000000−A400000000−A5000θ2ϑ+ψ2ϑ0000−A600γ1ϑr1ϑr2ϑr3ϑ0−μϑ0000ɛϑ000bBϑ−τϑ, B2=0−β1ϑN−β2ϑN00...−βϑ(1−Pϑ)N(B+KBϑ)0β1ϑN(1−ɛAϑ)000...000000...βϑ(1−Pϑ)N(B+KBϑ)00δ3ϑNC00...000000...000000...000000...000000...0,B3=00...−βϑ(1−Pϑ)N(B+KBϑ)00...0β1ϑNɛAϑ0...000...000...βϑ(1−Pϑ)N(B+KBϑ)00...000...000...0, B4=0000...00000...0β2ϑN000...−βϑ(1−Pϑ)N(B+KBϑ)000−δ2ϑN...0000δ2ϑN...βϑ(1−Pϑ)N(B+KBϑ)0000...00000...00000...0,ϕ=Λϑ0000000, where A1=(μϑ+θ2ϑ+ψ2ϑ),A2=(μϑ+γ1ϑ),A3=(d1ϑ+r1ϑ+μϑ),A4=(d2ϑ+r2ϑ+μϑ),A5=(d3ϑ+r3ϑ+μϑ), A6=(θ1ϑ+ψ1ϑ+μϑ). It is worth noting that Eq. (7) is not unique and can be written in more than one way and it has been chosen based on the ease of proving the existence and uniqueness of the presented model. Definition 5 Let C∗[0,τ∗] be the class of continuous column vector X(t) whose components S,A,I,C,D,V,R,B∈C∗[0,τ∗] are the class of continuous functions on the interval [0,τ∗]. The norm of X∈C∗[0,τ∗] is given by ‖X‖=supt∣e−NtS(t)∣+supt∣e−NtA(t)∣+supt∣e−NtI(t)∣+supt∣e−NtC(t)∣+supt∣e−NtD(t)∣+supt∣e−NtV(t)∣+supt∣e−NtR(t)∣+supt∣e−NtB(t)∣, where N is a natural number and when t>σ≥m, Cσ∗[0,τ∗] and Cσ[0,τ∗] are written. Definition 6 X∈C∗[0,τ∗] is a solution of IVP (7) if (1) (t,X(t))∈D,t∈[0,τ∗] where D=[0,τ∗]×K, K={(S,A,I,C,D,V,R,B)∈R+8:\|S\|≤p,\|A\|≤r,\|I\|≤w,\|C\|≤q\|D\|≤l,\|V\|≤m,\|R\|≤h,\|B\|≤j}, p,r,w,q,l,m,h,j∈R+are constants. (2) X(t) satisfies (7). Theorem 2 The IVP (7) has a unique solution X1∈C∗[0,τ∗] . Proof Eq. (7) can be represented in following form as using the properties of fractional calculus: I1−ϑddtX1(t)=B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ. Operating by Iϑ we obtain (8) X1(t)=X1(0)+Iϑ(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). Now let G:C∗[0,τ∗]→C∗[0,τ∗] defined by (9) GX1(t)=X1(0)+Iϑ(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). Then e−Nt(GX1−GX2)=e−NtIϑ(B1(X1(t)−X2(t))+S(t)B2(X1(t)−X2(t))+A(t)B3(X1(t)−X2(t))+I(t)B4(X1(t)−X2(t))),≤\|1Γ(ϑ)∫0t(t−s)ϑ−1e−N(t−s)e−Ns(X1(t)−X2(t))ds\|(B1+pB2+rB3+wB4),≤(B1+pB2+rB3+wB4)\|1Γ(ϑ)∫0t(u)ϑ−1e−N(u)\|‖X1−X2‖,≤(B1+pB2+rB3+wB4)\|γ(ϑ,Nt)Γ(ϑ)\|Nϑ‖X1−X2‖, where γ(ϑ,Nt) is the Lower-Incomplete gamma function and s=t−u. We have N as an arbitrary constant, so we suppose that Nϑ≥\|γ(ϑ,Nt)Γ(ϑ)\|B1+pB2+rB3+wB4, then we get ‖GX1−GX2‖≤‖X1−X2‖. Operator F in (9) has a fixed point. Thus (8) has a unique solution X1∈C∗[0,τ∗]. From (8) we have X1(t)=X1(0)+tϑΓ(ϑ+1)(B1X1(0)+S(0)B2X1(0)+A(0)B3X1(0)+I(0)B4X1(0)+ϕ)+Iϑ+1B1X1′(t)+S′(t)B2X1(t)+S(t)B2X1′(t)+A′(t)B3X1(t)+A(t)B3X1′(t)+I′(t)B4X1(t)+I(t)B4X1′(t), e−NtX1′=e−NttϑΓ(ϑ)(B1X1(0)+S(0)B2X1(0)+A(0)B3X1(0)+I(0)B4X1(0)+ϕ)+IϑB1X1′(t)+S′(t)B2X1(t)+S(t)B2X1′(t)+A′(t)B3X1(t)+A(t)B3X1′(t)+I′(t)B4X1(t)+I(t)B4X1′(t). Supposing that X1′∈Cσ∗[0,τ∗]. From (8) we get dX1dt=ddtIϑ(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). Operating by I1−ϑ we get I1−ϑdX1dt=I1−ϑddtIϑ(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). 0CDtϑX1t=(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). and X1(0)=(X1)0+Iϑ(B1X1(t)+S(t)B2X1(t)+A(t)B3X1(t)+I(t)B4X1(t)+ϕ). Therefore (8) is the same as IVP (7). 5 Equilibria and stability To find equilibrium points, system (5) is written as: Λϑ−(μϑ+β1ϑNA+β2ϑNI+θ2ϑ+ψ2ϑ+δ3ϑNIC)S−βϑ(1−Pϑ)BN(B+KBϑ)S+(θ1ϑ+ψ1ϑ)V=0, β1ϑN(1−ɛAϑ)AS−βϑ(1−Pϑ)BN(B+KBϑ)A−μϑA−γ1ϑA=0,(β1ϑNɛAϑA+β2ϑNI)S−βϑ(1−Pϑ)BN(B+KBϑ)I−(d1ϑ+r1ϑ+μϑ)I=0, (10) βϑ(1−Pϑ)BN(B+KBϑ)S−(d2ϑ+r2ϑ+δ2ϑNI+μϑ)C=0, βϑ(1−Pϑ)BN(B+KBϑ)(A+I)+δ2ϑNCI+δ3ϑNSIC−(r3ϑ+d3ϑ+μϑ)D=0,−(θ1ϑ+ψ1ϑ+μϑ)V+(θ2ϑ+ψ2ϑ)S=0, r1ϑI+r2ϑC+r3ϑD+γ1ϑA−μϑR=0, ɛϑC+(bBϑ−τϑ)B=0. Solving system (10), we get the disease-free equilibrium point DFE¯=S¯,0,0,0,0,V¯,0,0, where S¯=Λϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ),V¯=Λϑ(θ2ϑ+ψ2ϑ)μϑ(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ), and endemic equilibrium point EE^=(S∗,A∗,I∗,C∗,D∗,V∗,R∗,B∗), in which B∗=16(243(e32−e4)e6e713+223e513e8+2e1−2KBϑβ1ϑN(δ2ϑNɛϑμϑ(−2(−1+Pϑ)βϑN+3(γ1ϑ+μϑ))τϑψ2ϑ+bBϑ(β1ϑNθ1ϑ)((3r1ϑr2ϑ+3d1ϑ(d2ϑ+r2ϑ)+d2ϑ(3r1ϑ+2βϑN))−3(δ2ϑNψ2ϑɛAϑ)(γ1ϑμϑ−3μ2ϑ))−e2+β1ϑNδ2ϑNɛϑμϑ(βϑN−PϑβϑN+γ1ϑ+μϑ)τϑψ2ϑ+bBϑβ1ϑN((β1ϑNθ1ϑ)(r1ϑr2ϑ+d1ϑ(d2ϑ+r2ϑ)+r2ϑβϑN+d2ϑ(r1ϑ+βϑN−PϑβϑN))−(δ2ϑNψ2ϑ)(γ1ϑɛϑμϑ−ɛϑμ2ϑ)), S∗=−B∗(βϑN+γ1ϑ+μϑ−βϑNPϑ)+KBϑ(γ1ϑ+μϑ)β1ϑN(ɛAϑ−1)(B∗+KBϑ), A∗=−(B∗+KBϑ)(ɛAϑ−1)B∗ɛϑ(βϑN(1−Pϑ)+(1+KBϑ)(γ1ϑ+μϑ))((B∗(bBϑ−τϑ)ɛϑ)(−d2ϑ−r2ϑ−μϑ)+b2)×(ɛϑ(βϑNB∗(1−Pϑ)B∗+KBϑ+d1ϑ+μϑ+r1ϑ)B∗δ2ϑN(bBϑ−τϑ)+b2), I∗=b2(ɛϑ(βϑNB∗(1−Pϑ)B∗+KBϑ+d1ϑ+μϑ+r1ϑ)B∗δ2ϑN(bBϑ−τϑ)+β2ϑNɛϑ((B∗βϑN)(1−Pϑ)+(γ1ϑ+μϑ)(B∗+KBϑ))β1ϑNB∗δ2ϑN(ɛAϑ−1)(B∗+KBϑ)(bBϑ−τϑ)), C∗=−B∗(bBϑ−τϑ)ɛ1ϑ, V∗=(θ2ϑ+ψ2ϑ)((βϑNB∗)(1−Pϑ)−(γ1ϑ+μϑ)(B∗+KBϑ))β1ϑN(ɛAϑ−1)(B∗+KBϑ)(θ1ϑ+μϑ+ψ1ϑ), D∗=1−d3ϑ−μϑ−r3ϑ(b2−1B∗+KBϑB∗(1−Pϑ)βϑN(−(ɛAϑ−1)(B∗+KBϑ)ɛAϑ(βϑNB∗(1−Pϑ)+(γ1ϑ+μϑ)(B∗+KBϑ))(ɛϑβϑNB∗(1−Pϑ)B∗+KBϑ+d1ϑ+μϑ+r1ϑ−(B∗(bBϑ−τϑ)ɛϑ)(d2ϑ+r2ϑ+μϑ)B∗δ2ϑN(bBϑ−τϑ)+b2δ2ϑN(bBϑ−τϑ)+β2ϑNɛϑ(B∗+KBϑ)δ2ϑN(b2B∗δ2ϑN−B∗(bBϑ−τϑ)ɛϑ)(d2ϑ+r2ϑ+μϑ)))+ɛϑ(βϑB∗(1−Pϑ)(βϑNB∗(1−Pϑ)+(B∗+KBϑ))(γ1ϑ+μϑ)1NB∗δ2ϑ(bBϑ−τϑ)β1ϑ(B∗+KBϑ)2(ɛAϑ−1) βϑNB∗(1−Pϑ)(βϑNB∗(1−Pϑ)+(γ1ϑ+μϑ)(B∗+KBϑ))B∗δ2ϑN(bBϑ−τϑ))−δ3ϑN(βϑNB∗(1−Pϑ)+(γ1ϑ+μϑ)(B∗+KBϑ))B∗δ2ϑNβ1ϑN(B∗+KBϑ)(ɛAϑ−1)(b2B∗δ2ϑN−B∗(bBϑ−τϑ)ɛϑ)(d2ϑ+r2ϑ+μϑ))−B∗(bBϑ−τϑ)ɛϑ)(d2ϑ+r2ϑ+μϑ)), R1∗=−1μϑ(γ1ϑ(ɛAϑ−1)(B∗+KBϑ)ɛAϑ(βϑNB∗(1−Pϑ)+(B∗+KBϑ)(γ1ϑ+μϑ))(ɛϑβϑNB∗(1−Pϑ)B∗+KBϑ+d1ϑ+μϑ+r1ϑB1ϑδ2ϑN(bBϑ−τϑ))×(b2−B∗(bBϑ−τϑ)ɛϑ(d2ϑ+r2ϑ+μϑ))+b1β2ϑNɛϑβ1ϑNB∗δ2ϑN(ɛAϑ−1)(B∗+KBϑ)(bBϑ−τϑ)−r3ϑ−d3ϑ−μϑ−r3ϑb2−βϑB∗(1−Pϑ)N(B∗+KBϑ))(−(ɛAϑ−1)(B∗+KBϑ)ɛAϑ(βϑNB∗(1−Pϑ)+(B∗+KBϑ)(γ1ϑ+μϑ))(ɛAϑβϑNB∗(1−Pϑ)B∗+KBϑ+d1ϑ+μϑ+r1ϑb1B∗δ2ϑN(bBϑ−τϑ)+β2ϑNɛϑ(B∗+KBϑ)b1b2B∗(bBϑ−τϑ)δ2ϑN+ɛϑb1B∗δ2ϑN(bBϑ−τϑ))−δ3ϑN(B∗+KBϑ)b2b1δ2ϑN−B∗(bBϑ−τϑ)ɛϑ(d2ϑ+r2ϑ+μϑ)−r1ϑɛϑb1B∗δ2ϑN(bBϑ−τϑ)+B∗r2ϑɛϑ), e1=β1ϑNbBϑKBϑ(d2ϑμϑ(3θ1ϑ(β2ϑN−β1ϑN)+(ɛAϑ−1)(−3β2ϑNγ1ϑ+3β1ϑNd1ϑ−2βϑN(Pϑ−1)(β1ϑN−β2ϑN)+3β1ϑNr1ϑ))+d2ϑθ1ϑ(−3β2ϑNγ1ϑ(ɛAϑ−1)+3β1ϑNɛAϑ(d1ϑ+r1ϑ)+2βϑN(Pϑ−1)(β2ϑN(ɛAϑ−1)−β1ϑNɛAϑ))+μϑ(3γ1ϑδ2ϑNδ3ϑNɛAϑ−3β1ϑNθ1ϑ(d1ϑ+r1ϑ)+β2ϑδ2ϑN(Pϑ−1)2ɛAϑ−2βϑN(Pϑ−1)(δ2ϑNɛAϑ(γ1ϑ+δ3ϑN)−β1ϑNθ1ϑ))+μϑr2ϑ(3θ1ϑ1N(β2ϑ−β1ϑ)+(ɛAϑ−1)(−3β2ϑNγ1ϑ+3β1ϑNd1ϑ−2βϑN(Pϑ−1)1N(β1ϑ−β2ϑ)+3β1ϑNr1ϑ))+θ1ϑr2ϑ(−3β2ϑNγ1ϑ(ɛAϑ−1)+3β1ϑNɛAϑ(d1ϑ+r1ϑ)+2βϑN(Pϑ−1)(β2ϑN(ɛAϑ−1)−β1ϑNɛAϑ))+δ2ϑNθ1ϑɛAϑ(3β1ϑNΛϑ(ɛϑ−1)+3γ1ϑδ3ϑN+β2ϑ(Pϑ−1)2−2βϑN(Pϑ−1)(γ1ϑ+δ3ϑN)))+βϑN(Pϑ−1)ɛ1ϑ(θ1ϑ+μϑ)(βϑN(Pϑ−1)−γ1ϑ)(β2ϑNγ1ϑ+β1ϑN(−d1ϑ)+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ), e2=β1ϑNbBϑ(d2ϑ(−β2ϑN(ɛAϑ−1)(γ1ϑ+μϑ)(θ1ϑ+μϑ)+β1ϑN(μϑ(ɛAϑ−1)(d1ϑ+θ1ϑ+r1ϑ)+θ1ϑɛAϑ(d1ϑ+r1ϑ)+μ2ϑ(ɛAϑ−1))+βϑN(−(Pϑ−1))(ɛAϑ−1)1N(β1ϑ−β2ϑ)(θ1ϑ+μϑ))+μϑ(β1ϑNδ2ϑNΛϑ(ɛAϑ−1)ɛAϑ+γ1ϑ(δ2ϑNɛAϑ(δ3ϑN+θ1ϑ+θ2ϑ)−β2ϑNθ1ϑ(ɛAϑ−1))+θ1ϑ(β1ϑNɛAϑ(d1ϑ+r1ϑ)+β1ϑN(−(d1ϑ+r1ϑ))+δ2ϑNδ3ϑNɛAϑ)+β2ϑδ2ϑN (Pϑ−1)2ɛAϑ−βϑN(Pϑ−1)(θ1ϑ(ɛAϑ−1)(β1ϑN−β2ϑN)+γ1ϑδ2ϑNɛAϑ+δ2ϑNɛAϑ(δ3ϑN+2θ1ϑ+θ2ϑ)))+μ2ϑ(βϑβ1ϑN2ɛAϑ−βϑβ1ϑN2−βϑβ2ϑN2ɛAϑ+βϑβ2ϑN2+2βϑδ2ϑN2ɛAϑ+θ1ϑ(β1ϑN(ɛAϑ−1)+β2ϑN)−β2ϑNγ1ϑɛAϑ+β2ϑNγ1ϑ+γ1ϑδ2ϑNɛAϑ+β1ϑNd1ϑ(ɛAϑ−1)+δ2ϑNδ3ϑNɛAϑ−βϑNβ1ϑNPϑɛAϑ+βϑNβ1ϑNPϑ+βϑNβ2ϑNPϑɛAϑ−βϑNβ2ϑNPϑ−2βϑNδ2ϑNPϑɛAϑ+β1ϑNr1ϑ(ɛAϑ−1))+r2ϑ(−β2ϑN(ɛAϑ−1)(γ1ϑ+μϑ)(θ1ϑ+μϑ)+β1ϑN(μϑ(ɛAϑ−1)(d1ϑ+θ1ϑ+r1ϑ)+θ1ϑɛAϑ(d1ϑ+r1ϑ)+μ2ϑ(ɛAϑ−1))−βϑN((Pϑ−1))(ɛAϑ−1)(β1ϑN−β2ϑN)(θ1ϑ+μϑ))+δ2ϑNθ1ϑɛAϑ(β1ϑNΛϑ(ɛϑ−1)+(βϑN(Pϑ−1)−γ1ϑ)(βϑN(Pϑ−1)−δ3ϑN))), e3=β1ϑNbBϑKBϑ(β2ϑδ2ϑθ1ϑɛAϑ+2βϑNγ1ϑδ2ϑθ1ϑɛAϑ+2βϑNδ2ϑδ3ϑNθ1ϑɛAϑ+3β1ϑNδ2ϑθ1ϑΛϑɛA2ϑ−3β1ϑNδ2ϑθ1ϑΛϑɛAϑ+3γ1ϑδ2ϑδ3ϑNθ1ϑɛAϑ+3β1ϑNd1ϑ(μϑ(ɛAϑ−1)(d2ϑ+θ1ϑ+r2ϑ)+θ1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ)−μ2ϑ)+d2ϑ(ɛAϑ−1)(3β1ϑNθ1ϑμϑ−3β2ϑNμϑ(γ1ϑ+θ1ϑ)−3β2ϑNγ1ϑθ1ϑ−2βϑN(Pϑ−1)(β1ϑN−β2ϑN)(θ1ϑ+μϑ)+3β1ϑNr1ϑ(θ1ϑ+μϑ))+β2ϑδ2ϑθ1ϑP2ϑɛAϑ+μϑ(β2ϑδ2ϑɛAϑ−2βϑβ1ϑN2θ1ϑ+2βϑβ1ϑN2θ1ϑɛAϑ+2βϑNβ2ϑNθ1ϑ−2βϑNβ2ϑNθ1ϑɛAϑ+2βϑNγ1ϑδ2ϑɛAϑ+2βϑNδ2ϑδ3ϑNɛAϑ+4βϑNδ2ϑθ1ϑɛAϑ+2βϑNδ2ϑθ2ϑɛAϑ+3β1ϑNδ2ϑΛϑ(ɛAϑ−1)ɛAϑ+3β2ϑNγ1ϑθ1ϑ−3β2ϑNγ1ϑθ1ϑɛAϑ+3γ1ϑδ2ϑδ3ϑNɛAϑ+3γ1ϑδ2ϑθ1ϑɛAϑ+3γ1ϑδ2ϑθ2ϑɛAϑ+3δ2ϑδ3ϑNθ1ϑɛAϑ+β2ϑδ2ϑP2ϑɛAϑ−2β2ϑδ2ϑPϑɛAϑ+2βϑNβ1ϑNθ1ϑPϑ−2βϑNβ1ϑNθ1ϑPϑɛAϑ−2βϑNβ2ϑNθ1ϑPϑ+2βϑNβ2ϑNθ1ϑPϑɛAϑ−2βϑNγ1ϑδ2ϑPϑɛAϑ−2βϑNδ2ϑδ3ϑNPϑɛAϑ−4βϑNδ2ϑθ1ϑPϑɛAϑ−2βϑNδ2ϑθ2ϑPϑɛAϑ−r2ϑ(ɛAϑ−1)(−3β1ϑNθ1ϑ+3β2ϑN(γ1ϑ+θ1ϑ)+2βϑN(Pϑ−1)(β1ϑN−β2ϑN))+3β1ϑNr1ϑ(ɛAϑ−1)(θ1ϑ+r2ϑ))−2β2ϑδ2ϑθ1ϑPϑɛAϑ−2βϑNγ1ϑδ2ϑθ1ϑPϑɛAϑ−2βϑNδ2ϑδ3ϑNθ1ϑPϑɛAϑ+2βϑNβ1ϑNθ1ϑPϑϑ−2βϑNβ1ϑNθ1ϑPϑϑɛAϑ−2βϑNβ2ϑNθ1ϑPϑϑ+2βϑNβ2ϑNθ1ϑPϑϑɛAϑ−3β1ϑNθ1ϑr1ϑr2ϑ+3β1ϑNθ1ϑr1ϑr2ϑɛAϑ−2βϑNβ1ϑNθ1ϑr2ϑ+2βϑNβ1ϑNθ1ϑr2ϑɛAϑ+2βϑNβ2ϑNθ1ϑr2ϑ−2βϑNβ2ϑNθ1ϑr2ϑɛAϑ+3β2ϑNγ1ϑθ1ϑr2ϑ−3β2ϑNγ1ϑθ1ϑr2ϑɛAϑ)+βϑN(Pϑ−1)ɛϑ(μϑ(θ1ϑ(γ1ϑ(β1ϑN−2β2ϑN)+β1ϑNd1ϑ−2βϑN(Pϑ−1)(β1ϑN−β2ϑN)+β1ϑNr1ϑ)+(βϑN(Pϑ−1)−γ1ϑ)(β2ϑNγ1ϑ−β1ϑNd1ϑ+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ))+θ1ϑ(βϑN(Pϑ−1)−γ1ϑ)(β2ϑNγ1ϑ−β1ϑNd1ϑ+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ)), e4=β1ϑNKBϑ(3(−β1ϑNδ2ϑΛϑμϑτϑɛA2ϑ+β1ϑNδ2ϑΛϑμϑτϑɛAϑ+β1ϑNθ1ϑμ2ϑτϑ+β1ϑNμ3ϑτϑ−β1ϑNθ1ϑμ2ϑτϑɛAϑ−β1ϑNμ3ϑτϑɛAϑ+β1ϑNd1ϑ(bBϑψ1ϑ(ɛAϑ−1)(d2ϑ+μϑ+r2ϑ)+μϑτϑ(μϑ−ɛAϑ(θ1ϑ+μϑ)))+bBϑψ1ϑ(δ2ϑɛAϑ(β1ϑNΛϑ(ɛAϑ−1)+γ1ϑδ3ϑN)+β1ϑNd2ϑ(ɛAϑ−1)(μϑ+r1ϑ)+β1ϑNr1ϑ(ɛAϑ−1)(μϑ+r2ϑ)+β1ϑNμϑr2ϑ(ɛAϑ−1))+β2ϑNbBϑψ1ϑ(μϑ(γ1ϑ−ɛAϑ(d2ϑ+r2ϑ)+d2ϑ+r2ϑ)−γ1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ))−γ1ϑδ2ϑθ1ϑμϑτϑɛAϑ−γ1ϑδ2ϑθ2ϑμϑτϑɛAϑ−γ1ϑδ2ϑμ2ϑτϑɛAϑ+β1ϑNd2ϑμ2ϑτϑ−β1ϑNd2ϑθ1ϑμϑτϑɛAϑ−β1ϑNd2ϑμ2ϑ τϑɛAϑ−δ2ϑδ3ϑNθ1ϑμϑτϑɛAϑ−δ2ϑδ3ϑNμ2ϑτϑɛAϑ+β2ϑNμϑτϑ(−γ1ϑθ1ϑ+μϑ(ɛAϑ−1)(γ1ϑ+d2ϑ+θ1ϑ+r2ϑ)+θ1ϑɛAϑ(γ1ϑ+d2ϑ+r2ϑ)+μ2ϑ(ɛAϑ−1))−δ2ϑθ1ϑμ2ϑτϑɛAϑ−δ2ϑθ2ϑμ2ϑτϑɛAϑ−δ2ϑμ3ϑτϑɛAϑ+β1ϑNμ2ϑr1ϑτϑ−β1ϑNθ1ϑμϑr1ϑτϑɛAϑ−β1ϑNμ2ϑr1ϑτϑɛAϑ+β1ϑNμ2ϑr2ϑτϑ−β1ϑNθ1ϑμϑr2ϑτϑɛAϑ−β1ϑNμ2ϑr2ϑτϑɛAϑ)+2βϑN(Pϑ−1)(β1ϑNμϑτϑ(ɛAϑ(θ1ϑ+μϑ)−μϑ)+δ2ϑɛAϑ(μϑτϑ(2θ1ϑ+θ2ϑ+2μϑ)−bBϑψ1ϑ(γ1ϑ+δ3ϑN))−β1ϑNbBϑψ1ϑ(ɛAϑ−1)(d2ϑ+μϑ+r2ϑ))−2βϑNβ2ϑN((Pϑ−1)(ɛAϑ−1)(μ2ϑτϑ−bBϑψ1ϑ(d2ϑ+μϑ+r2ϑ))+θ1ϑμϑτϑ(Pϑ(ɛAϑ−1)−ɛAϑ−1))+β2ϑbBϑδ2ϑ(Pϑ−1)2ψ1ϑɛAϑ)+βϑN(Pϑ−1)ψ1ϑɛϑ(βϑN(Pϑ−1)−γ1ϑ)(β2ϑNγ1ϑ−β1ϑN(d1ϑ)+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ), e5=−2(β1ϑNbBϑKBϑ(β2ϑδ2ϑθ1ϑɛAϑ+2βϑNγ1ϑδ2ϑθ1ϑɛAϑ+2βϑNδ2ϑδ3ϑNθ1ϑɛAϑ+3β1ϑNδ2ϑθ1ϑΛϑɛA2ϑ−3β1ϑNδ2ϑθ1ϑΛϑɛAϑ+3γ1ϑδ2ϑδ3ϑNθ1ϑɛAϑ+3β1ϑNd1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ)(θ1ϑ+μϑ)+d2ϑ(ɛAϑ−1)(θ1ϑ+μϑ)(−3β2ϑNγ1ϑ−2βϑN(Pϑ−1)(β1ϑN−β2ϑN)+3β1ϑNr1ϑ)+β2ϑδ2ϑNθ1ϑP2ϑɛAϑ−2β2ϑδ2ϑNθ1ϑPϑɛAϑ−2βϑNγ1ϑδ2ϑNθ1ϑPϑɛAϑ−21N2βϑδ2ϑδ3ϑNθ1ϑPϑɛAϑ+μϑɛAϑ(δ2ϑNδ3ϑN(2βϑN+3γ1ϑ−2βϑP)+βϑδ2ϑ(Pϑ−1)(βϑ(Pϑ−1)−2γ1ϑ)−2βϑ(Pϑ−1)r2ϑ(β1ϑN−β2ϑN)+3β1ϑNr1ϑr2ϑ−3β2ϑNγ1ϑr2ϑ)+μϑr2ϑ(3β2ϑNγ1ϑ+2βϑN(Pϑ−1)(β1ϑN−β2ϑN)−3β1ϑNr1ϑ)+2βϑβ1ϑNθ1ϑPϑϑ−2βϑNβ1ϑNθ1ϑPϑϑɛAϑ−2βϑNβ2ϑNθ1ϑPϑϑ+2βϑNβ2ϑNθ1ϑPϑϑɛAϑ−3β1ϑNθ1ϑr1ϑr2ϑ+3β1ϑNθ1ϑr1ϑr2ϑɛAϑ−2βNϑβ1ϑNθ1ϑr2ϑ+2βϑNβ1ϑNθ1ϑr2ϑɛAϑ+2βϑNβ2ϑNθ1ϑr2ϑ−2βϑNβ2ϑNθ1ϑr2ϑɛAϑ+3β2ϑNγ1ϑθ1ϑr2ϑ−3β2ϑNγ1ϑθ1ϑr2ϑɛAϑ)+βϑN(Pϑ−1)ɛϑ(θ1ϑ(βϑN(Pϑ−1)−γ1ϑ)(β2ϑNγ1ϑ−β1ϑN(d1ϑ)+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ)+μϑ(γ1ϑ(β1ϑN(βϑN+d1ϑ−βϑNPϑ+r1ϑ)+2βϑNβ2ϑN(Pϑ−1))−βϑN(Pϑ−1)(β1ϑN(βϑN+d1ϑ−βϑNPϑ+r1ϑ)+βϑNβ2ϑN(Pϑ−1))))), e6=23β1ϑNbBϑθ1ϑ(β2ϑδ2ϑɛAϑ+βϑNγ1ϑδ2ϑɛAϑ+βϑNδ2ϑδ3ϑNɛAϑ+β1ϑNδ2ϑΛϑ(ɛAϑ−1)ɛAϑ+γ1ϑδ2ϑδ3ϑNɛAϑ+β1ϑNd1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ)+d2ϑ(ɛAϑ−1)(−β2ϑNγ1ϑ−βϑN(Pϑ−1)(β1ϑN−β2ϑ)+β1ϑNr1ϑ)+β2ϑδ2ϑP2ϑɛAϑ−2β2ϑδ2ϑPϑɛAϑ−βϑNγ1ϑδ2ϑPϑɛAϑ−βϑNδ2ϑδ3ϑNPϑɛAϑ−βϑNβ1ϑNPϑɛAϑ+βϑNβ1ϑNPϑ+βϑNβ2ϑNPϑɛAϑ−βϑNβ2ϑNPϑ+β1ϑNr1ϑr2ϑɛAϑ−β1ϑNr1ϑr2ϑ+βϑNβ1ϑNr2ϑɛAϑ−βϑNβ1ϑNr2ϑ−βϑNβ2ϑNr2ϑɛAϑ+βϑNβ2ϑNr2ϑ−β2ϑNγ1ϑr2ϑɛAϑ+β2ϑNγ1ϑr2ϑ), e7=(18β1ϑNKBϑ(bBϑ(δ2ϑμϑψ2ϑɛAϑ(2βϑN+3γ1ϑ)−3β1ϑNd1ϑθ1ϑ(−d2ϑɛAϑ+d2ϑ+r2ϑ)+d2ϑθ1ϑ(3β2ϑNγ1ϑ+2βϑN(Pϑ−1)(β1ϑN−β2ϑN)−3β1ϑNr1ϑ)+3δ2ϑNμ2ϑψ2ϑɛAϑ,−2βϑNPϑ(δ2ϑNμϑψ2ϑɛAϑ+β1ϑNθ1ϑ−β2ϑNθ1ϑ)−3β1ϑNθ1ϑr1ϑr2ϑ−2βϑNβ1ϑNθ1ϑr2ϑ+2βϑNβ2ϑNθ1ϑr2ϑ+3β2ϑNγ1ϑθ1ϑr2ϑ)−μϑτϑ(3β1ϑNμϑψ1ϑ(ɛAϑ−1)−3β2ϑNμϑψ1ϑ(ɛAϑ−1)+δ2ϑNɛAϑ(3γ1ϑψ2ϑ+3μϑ(ψ1ϑ+ψ2ϑ)−2βϑN(Pϑ−1)ψ2ϑ)))3+27β2ϑβ13ϑγ12ϑKB2ϑ(bBϑKBϑ(γ1ϑδ2ϑNɛAϑ(δ3ϑNθ1ϑ+μϑψ2ϑ)+β1ϑNd1ϑθ1ϑ((d2ϑ+r2ϑ)+d2ϑθ1ϑ)(ɛAϑ−1)(β1ϑNr1ϑ−β2ϑNγ1ϑ)+δ2ϑNμ2ϑψ2ϑɛAϑ−β1ϑNθ1ϑr1ϑr2ϑ+β2ϑNγ1ϑθ1ϑr2ϑ(1−ɛAϑ))+βϑNγ1ϑd1ϑθ1ϑ(Pϑ−1)ɛϑ+β2ϑNd2ϑKBϑμϑτϑψ1ϑɛAϑ+β1ϑNKBϑμ2ϑτϑψ1ϑ−β1ϑNKBϑμ2ϑτϑψ1ϑɛAϑ+β2ϑNγ1ϑKBϑμϑτϑψ1ϑɛAϑ−β2ϑNKBϑμ2ϑτϑψ1ϑ+β2ϑNKBϑμ2ϑτϑψ1ϑɛAϑ−γ1ϑδ2ϑNKBϑμϑτϑψ1ϑɛAϑ−(γ1ϑψ2ϑ+δ3ϑNψ1ϑ)δ2ϑNKBϑμϑτϑɛAϑ−δ2ϑNKBϑμ2ϑτϑɛAϑ(ψ1ϑ+ψ2ϑ)+β2ϑNKBϑμϑr2ϑτϑψ1ϑɛAϑ−βϑNγ1ϑθ1ϑr1ϑɛϑ)−9)2, e8=β1ϑN(bBϑ(β2ϑδ2ϑN)θ1ϑɛAϑ(1+δ3ϑNγ1ϑ)+μ3ϑ(ɛAϑ(β1ϑN−β2ϑN+δ2ϑN)−β1ϑN+β2ϑN)+β1ϑNδ2ϑNθ1ϑΛϑɛAϑ(ɛAϑ−1)+γ1ϑδ2ϑNδ3ϑNθ1ϑɛAϑ+β1ϑNd1ϑ(ɛAϑ−1)(θ1ϑ+μϑ)(d2ϑ+μϑ+r2ϑ)+d2ϑ(ɛAϑ−1)(θ1ϑ+μϑ)(β1ϑNμϑ−β2ϑN(γ1ϑ+μϑ)−βϑN(Pϑ−1)(β1ϑN−β2ϑN)+β1ϑNr1ϑ)+β2ϑδ2ϑNθ1ϑP2ϑɛAϑ+μϑ(β2ϑδ2ϑNɛAϑ−βϑNθ1ϑ(1−ɛAϑ)(β2ϑN−β1ϑN)+βϑNδ2ϑNɛAϑ(γ1ϑ+δ3ϑN+2θ1ϑ+θ2ϑ)+β1ϑNδ2ϑNΛϑ(ɛAϑ−1)+β2ϑNγ1ϑθ1ϑ(1−ɛAϑ)+γ1ϑδ2ϑNδ3ϑNɛAϑ+γ1ϑδ2ϑNɛAϑ(θ1ϑ+θ2ϑ)+δ2ϑNδ3ϑNθ1ϑɛAϑ+β2ϑδ2ϑNP2ϑɛAϑ−2β2ϑδ2ϑNPϑɛAϑ+βϑNβ1ϑNθ1ϑPϑ−βϑNβ1ϑNθ1ϑPϑɛAϑ−βϑNβ2ϑNθ1ϑPϑ+βϑNβ2ϑNθ1ϑPϑɛAϑ−βϑNγ1ϑδ2ϑNPϑɛAϑ−βϑNδ2ϑNδ3ϑNPϑɛAϑ−2βϑNδ2ϑNθ1ϑPϑɛAϑ−βϑNδ2ϑNθ2ϑPϑɛAϑ−r2ϑ(ɛAϑ−1)(−β1ϑNθ1ϑ+β2ϑN(γ1ϑ+θ1ϑ)+βϑN(Pϑ−1)(β1ϑN−β2ϑN))+β1ϑNr1ϑ(ɛAϑ−1)(θ1ϑ+r2ϑ))−2β2ϑδ2ϑNθ1ϑPϑɛAϑ−βϑNγ1ϑδ2ϑNθ1ϑPϑɛAϑ−βϑNδ2ϑNδ3ϑNθ1ϑPϑɛAϑ+μ2ϑ(βϑNβ1ϑNɛAϑ−βϑNβ1ϑN−βϑNβ2ϑNɛAϑ+βϑNβ2ϑN+2βϑNδ2ϑNɛAϑ−β1ϑNθ1ϑ+β1ϑNθ1ϑɛAϑ−β2ϑNγ1ϑɛAϑ+β2ϑNγ1ϑ+β2ϑNθ1ϑ−β2ϑNθ1ϑɛAϑ+γ1ϑδ2ϑNɛAϑ+δ2ϑNδ3ϑNɛAϑ+δ2ϑNθ1ϑɛAϑ+δ2ϑNθ2ϑɛAϑ−βϑNPϑ(β2ϑN−β1ϑN)(ɛAϑ−1)+β1ϑNr1ϑ(ɛAϑ−1)+r2ϑ(ɛAϑ−1)(β1ϑN−β2ϑN))+βϑNβ1ϑNθ1ϑPϑ−βϑNβ1ϑNθ1ϑPϑɛAϑ−βϑNβ2ϑNθ1ϑPϑ+βϑNβ2ϑNθ1ϑPϑɛAϑ−β1ϑNθ1ϑr1ϑr2ϑ(1−ɛAϑ)−βϑNβ1ϑNθ1ϑr2ϑ+βϑNβ1ϑNθ1ϑr2ϑɛAϑ+βϑNβ2ϑNθ1ϑr2ϑ−βϑNβ2ϑNθ1ϑr2ϑɛAϑ+β2ϑNγ1ϑθ1ϑr2ϑ−β2ϑNγ1ϑθ1ϑr2ϑɛAϑ)+τϑ(−β2ϑδ2ϑNθ1ϑɛAϑ−βϑNγ1ϑδ2ϑNɛAϑ(θ1ϑ+δ3ϑN)+β1ϑNδ2ϑNθ1ϑΛϑ(1−ɛAϑ)−γ1ϑδ2ϑNδ3ϑNθ1ϑɛAϑ−β1ϑNd1ϑ(d2ϑ+r2ϑ)(θ1ϑ(ɛAϑ−1)−μϑ)+d2ϑθ1ϑ(ɛAϑ−1)(β2ϑNγ1ϑ+βϑN(Pϑ−1)(β1ϑN−β2ϑN)−β1ϑNr1ϑ)+β1ϑNd2ϑμϑ(βϑN(1−Pϑ)+r1ϑ)+βϑNγ1ϑδ2ϑNθ1ϑPϑ(βϑN+2ɛAϑ)−βϑNθ1ϑPϑ(β1ϑN−β1ϑNɛAϑ−β2ϑN)+β1ϑNr1ϑr2ϑ(θ1ϑ+μϑ)−β1ϑNθ1ϑr2ϑ(−r1ϑɛAϑ+βϑN−βϑNɛAϑ)+θ1ϑr2ϑ(βϑNβ2ϑN+β2ϑNγ1ϑ)(ɛAϑ−1)), e9=β1ϑNKB2ϑ(bBϑ(θ1ϑ(3β1ϑNd1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ)−(d2ϑ+r2ϑ)(−3β2ϑNγ1ϑ−β(Pϑ−1)(β1ϑN−β2ϑN)+3β1ϑNr1ϑ)+3β1ϑδ3ϑN2d2ϑɛAϑ)+δ2ϑNμϑψ2ϑɛAϑ(βϑN+3(γ1ϑ+μϑ)−βϑN−Pϑ))+μϑτϑ(−δ2ϑNψ2ϑɛAϑ(βϑN+3(γ1ϑ+μϑ))−3ψ1ϑ(μϑ(ɛAϑ−1)(β1ϑ−β2ϑN)+γ1ϑδ2ϑNɛAϑ+δ2ϑNɛAϑ(δ3ϑN+μϑ))+βϑδ2ϑN2PɛAϑ(2ψ1ϑ+ψ2ϑ))), e10=β1ϑNKBϑ2(bBϑ(θ1ϑ(3β1ϑNd1ϑ(ɛAϑ−1)(d2ϑ+r2ϑ)−(d2ϑ+r2ϑ)(−3β2ϑNγ1ϑ−βϑN(Pϑ−1)(β1ϑN−β2ϑN)+3β1ϑNr1ϑ)+3β1ϑNd2ϑr1ϑɛAϑ)+δ2ϑNμϑψ2ϑɛAϑ(βϑN+3(γ1ϑ+μϑ)+βϑN−Pϑ))+μϑτϑ(−δ2ϑNψ2ϑɛAϑ(βϑN+3(γ1ϑ+μϑ))−3ψ1ϑ(μϑ(ɛAϑ−1)(β1ϑN−β2ϑN)+γ1ϑδ2ϑNɛAϑ+δ2ϑNɛAϑ(δ3ϑN+μϑ))+βϑNδ2ϑNPϑɛAϑ(2ψ1ϑ+ψ2ϑ))), b1=−B∗d2ϑ(bBϑ−τϑ)ɛϑ−B∗r2ϑ(bBϑ−τϑ)ɛϑ−B∗μϑ(bBϑ−τϑ)ɛϑ+βϑNB∗(1−P)(βϑNB∗+B∗γ1ϑ+B∗μϑ−βϑNB∗P+γ1ϑKBϑ+KBϑμϑ)β1ϑN(ɛAϑ−1)(B∗+KBϑ)2, b2=B∗βϑ(1−Pϑ)+N(B∗+KBϑ)(μϑ+γ1ϑ)β1ϑ(ɛAϑ−1)(B∗+KBϑ), 5.1 Positivity and boundedness We will examine the positivity and boundedness of the solution of model (5) in this subsection. Let R+8=ζ(t)∈R8:ζ(t)≥0 and ζ(t)=[S(t),A(t),I(t),C(t),D(t),V(t),R(t),B(t)]T. With next lemma we will prove the non-negativity of the solution of system(5). Lemma 1 Generalized Mean Value Theorem Suppose that y(t)∈C[a1,b1] and 0CDtϑy(t)∈C(a1,b1] for 0<ϑ≤1 , then y(t)=y(a1)+1Γ(ϑ)0CDtϑy(σ)(t−a1)ϑ , where 0≤σ≤t,∀t∈(a1,b1] . Remark 1 If y∈C[0,b1] and 0CDtϑ(y(t))≥0,∀t∈(0,b1], then ∀ t∈[0,b1] the function y(t) is non-increasing. Theorem 3 The solution of model (5) including ICs has a bounded solution in R+8 . Proof We note that the non-negative region R+8, is positively invariant. Using system (5), we get 0CDtϑS\|S=0=Λϑ+(θ1ϑ+ψ1ϑ)V≥0, 0CDtϑA\|A=0=0≥0, 0CDtϑI\|I=0=β1ϑNɛAϑSA≥0, (11) 0CDtϑC\|C=0=βϑN(1−Pϑ)BB+KBϑS≥0, 0CDtϑD\|D=0=βϑ(1−Pϑ)BN(B+KBϑ)(A+I)+δ2ϑNCI+δ3ϑNSIC≥0, 0CDtϑV\|V=0=(θ2ϑ+ψ2ϑ)S≥0, 0CDtϑR\|R=0=r1ϑI+r2ϑC+r3ϑD+γ1ϑD≥0, 0CDtϑB\|B=0=ɛϑC≥0, If (S(0),A(0),I(0),C(0),D(0),V(0),R(0),B(0))∈R+8, then from system (11) and Remark 1, The solution of the model (5) can only be of high hyperplanes S=0,A=0,I=0,C=0,D=0,V=0,R=0 and B=0. Thus R+8 is positively invariant. Theorem 4 The region Q={(S(t),A(t),I(t),C(t),D(t),V(t),R(t),B(t))∈R+8,0<S(t)+A(t)+I(t)+C(t)+D(t)+V(t)+R(t)+B(t)≤Λϑμϑ} is a positive invariant set for the system (5) . Proof (5) yields the following: 0CDtϑN(t)=Λϑ−μϑ(S(t)+A(t)+I(t)+C(t)+D(t)+V(t)+R(t))−(−bϑ+τϑ)B−d1ϑI(t)−d2ϑC(t)−d3ϑD(t)+ɛϑC. This gives 0CDtϑN(t)≤Λϑ−μϑN(t)+μϑB+ɛϑC. LT is applied the previous equation, and the result is: sϑω(N)−sϑ−1N(0)≤Λϑs−μϑω(N)+μϑω(B)+ɛϑω(C), which further gives ω(N)≤s−1Λsϑ+μϑ+sϑ−1N(0)+μϑω(B)+ɛϑω(C)sϑ+μϑ. From Definition 3, Definition 4, we get that if (S0,A0,I0,C0,D0,V0,R0,B0)∈R+8, then N(t)≤ΛϑtϑEϑ,ϑ+1(−μϑtϑ)+Eϑ,1(−μϑtϑ)N(0)≤Λϑμϑ(tϑμϑEϑ,ϑ+1(−μϑtϑ))+Eϑ,1(−μϑtϑ)≤Λϑμϑ1Γ(1)=Λϑμϑ. Thus N(t) (the total population) is bounded and S(t),A(t),I(t),C(t),D(t),V(t),R(t),B(t) are bounded. 5.2 Stability of the equilibria Here, we will discuss the necessary conditions for the equilibrium points to be a stable. Theorem 5 If R0<1 then DFE¯ is locally asymptotically stable (LAS), if R0>1 then DFE¯ is unstable. Proof According to the DFE¯, the Jacobian matrix for system (5) as follows: J(DFE¯)=−μϑ−θ2ϑ−ψ2ϑ−S¯β1ϑN−S¯β2ϑN00θ1ϑ+ψ1ϑ0−βϑN(1−Pϑ)KBϑS¯0R01(γ1ϑ+μϑ)0000000β1ϑNɛAϑS¯a1(R02−1)00000000−a2000βϑN(1−Pϑ)KBϑS¯0000−a3000θ2ϑ+ψ2ϑ0000−μϑ−θ1ϑ−ψ1ϑ000γ1ϑr1ϑr2ϑr3ϑ0−μϑ0000ɛϑ000bϑ−τϑ, where S¯=Λϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(θ1ϑ+μϑ+ψ1ϑ+θ2ϑ+ψ2ϑ),a1=(d1ϑ+r1ϑ+μϑ),a2=(d2ϑ+r2ϑ+μϑ),a3=(d3ϑ+r3ϑ+μϑ). Therefore, the disease-free equilibrium DFE¯ is LAS if all the eigenvalues λi,i=1,2,…,8 of the matrix J(DFE¯) satisfy the condition (12) \|argeigJ(DFE¯)\|=\|arg(λi)\|>ϑπ2,i=1,2,…,8. We find the eigenvalues by solving the characteristic equation given by: (13) \|JDFE¯−λIˆ\|=0, where Iˆ is the identity matrix and λ is the eigenvalue. Thus : P(λ)=λ+μϑ(θ1ϑ+ψ1ϑ)(θ2ϑ+ψ2ϑ)(d3ϑ+r3ϑ+μϑ−λ)((d1ϑ+r1ϑ+μϑ)(1−R02)−λ)(R01(γ1ϑ+μϑ)−λ)=0. Hence, when R0=maxR01,R02<1 and all the eigenvalues of P(λ)=0 are negative. Thus, the DFE¯ point is LAS. In addition, when R0>1, then at least one of the eigenvalues of P(λ)=0 is positive. Therefore, if R0>1 the DFE¯ is unstable. Theorem 6 The endemic equilibrium EE^ of model (5) is LAS if R0>1 and unstable otherwise. Proof The Jacobian matrix JEE1^ is given by JEE^=a1−S∗β1ϑN−S∗(β2ϑN+δ3ϑNC∗)−S∗δ3ϑNI∗0θ1ϑ+ψ1ϑ0−βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2S∗a9a200000−βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2A∗a100a30000−βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2I∗βϑN(1−Pϑ)B∗(B∗+KBϑ)0−δ2ϑNC∗a4000βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2S∗δ3ϑNI∗C∗βϑN(1−Pϑ)B∗(B∗+KBϑ)a7a8a500βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2(A∗+I∗)θ2ϑ+ψ2ϑ0000a6000γ1ϑr1ϑr2ϑr3ϑ0−μϑ0000ɛϑ000bBϑ−τϑ, where a1=−(μϑ+β1ϑNA∗+β2ϑNI∗+θ2ϑ+ψ2ϑ+δ3ϑNI∗C∗)−βϑN(1−Pϑ)B∗(B∗+KBϑ), a2=β1ϑN(1−ɛAϑ)S∗−βϑN(1−Pϑ)B∗(B∗+KBϑ)−μϑ−γ1ϑ, a3=β2ϑNS∗−βϑN(1−Pϑ)B∗(B∗+KBϑ)−(d1ϑ+r1ϑ+μϑ),a4=−(d2ϑ+r2ϑ+μϑ+δ2ϑNI∗),a5=−(d3ϑ+r3ϑ+μϑ),a6=−(θ1ϑ+ψ1ϑ+μϑ), a7=βϑN(1−Pϑ)B∗(B∗+KBϑ)+δ2ϑNC∗+δ3ϑNS∗C∗,a8=δ2ϑNI∗+δ3ϑNS∗I∗,a9=β1ϑN(1−ɛAϑ)A∗,a10=β1ϑNɛAϑA∗+β2ϑNI∗. From the characteristic equation \|JEE^−λIˆ\|=0, we get P(λ)=(−λ−μϑ)(a5−λ)[B1+B2λ+B3λ2+B4λ3+B5λ4], where m1=βϑN(1−Pϑ)KBϑ(B∗+KBϑ)2,m2=βϑN(1−Pϑ)B∗(B∗+KBϑ), B1=−A∗a10β1ϑC∗δ2ϑm12S∗ɛϑ+A∗β1ϑm12m2S∗ɛϑ+a1a3a2m12S∗ɛϑ+a4(bBϑ−τϑ)+a10a2a4a6 bBϑβ2ϑC∗2δ2ϑ+a10a2a4a6bBϑC∗2δ2ϑδ3ϑ−a10a2a4a6β2ϑC∗2δ2ϑτϑ−a10a2a4a6C∗2δ2ϑδ3ϑτϑ+a10a2a6bBϑC∗δ2ϑδ3ϑ I∗S∗−a10a2a6C∗δ2ϑδ3ϑI∗S∗τϑ+a10a2δ2ϑm1ɛϑ+a10a2m1ɛϑ+a3(a2m12m2S∗ɛϑ+a2m1ψ1ϑψ2ϑɛϑ+a2m1ψ1ϑθ2ϑɛϑ+a2m1ψ2ϑθ1ϑɛϑ+a2m1θ1ϑθ2ϑɛϑ+a4a6a9β1ϑ(bBϑ−τϑ)+a6a9m1ɛϑ+bBϑδ3ϑI∗m2S∗−δ3ϑI∗m2S∗τϑ)+a6a9β1ϑI∗m1ɛϑ+C∗δ3ϑI∗m1ψ1ϑψ2ϑɛϑ+C∗δ3ϑI∗m1ψ1ϑθ2ϑɛϑ+C∗δ3ϑI∗m1ψ2ϑθ1ϑɛϑ+C∗δ3ϑI∗m1θ1ϑθ2ϑɛϑ, B2=a1a3a4−a1a3bBϑ−a4a6a9bBϑβ1ϑ−a10a2a4a6β2ϑC∗2δ2ϑ−a10a2a4bBϑβ2ϑC∗2δ2ϑ−a10a2a6bBϑβ2ϑC∗2δ2ϑ−a10a4a6bBϑβ2ϑC∗2δ2ϑ−a10a2a4a6C∗2δ2ϑδ3ϑ−a10a2a4bBϑC∗2δ2ϑδ3ϑ−a10a2a6bBϑC∗2δ2ϑδ3ϑ−a10a4a6bBϑC∗2δ2ϑδ3ϑ−a10a2a6C∗δ2ϑδ3ϑI∗S∗−a10a2bBϑC∗δ2ϑδ3ϑI∗S∗−a10a6bBϑC∗δ2ϑδ3ϑI∗S∗−a3δ3ϑI∗m2S∗−bBϑδ3ϑI∗m2S∗+a1a3τϑ+a4a6a9β1ϑτϑ+a10a2a4β2ϑC∗2δ2ϑτϑ+a10a2a6β2ϑC∗2δ2ϑτϑ+a10a4a6β2ϑC∗2δ2ϑτϑ+a10a2a4C∗2δ2ϑδ3ϑτϑ+a10a2a6C∗2δ2ϑδ3ϑτϑ+a10a4a6C∗2δ2ϑδ3ϑτϑ+a10a2C∗δ2ϑδ3ϑI∗S∗τϑ+a10a6C∗δ2ϑδ3ϑI∗S∗τϑ+δ3ϑI∗m2S∗τϑ+a1a4(−bBϑ+τϑ)+a3a6a9β1ϑ(−bBϑ+τϑ)−a3a4(bBϑ+a6a9β1ϑ+a9bBϑβ1ϑ−(1+a9β1ϑ)τϑ)−m1(a10(1+δ2ϑ)+a9(a6+β1ϑI∗)+a2m1(a1+m2)S∗+a2(ψ1ϑ+θ1ϑ)(ψ2ϑ+θ2ϑ)+a3(a9+m1(a1+a2+m2)S∗+(ψ1ϑ+θ1ϑ)(ψ2ϑ+θ2ϑ)))ɛϑ, B3=a4bBϑ+a4a6a9β1ϑ+a4a9bBϑβ1ϑ+a6a9bBϑβ1ϑ+a10a2a4β2ϑC∗2δ2ϑ+a10a2a6β2ϑC∗2δ2ϑ+a10a4a6β2ϑC∗2δ2ϑ+a10a2bBϑβ2ϑC∗2δ2ϑ+a10a4bBϑβ2ϑC∗2δ2ϑ+a10a6bBϑβ2ϑC∗2δ2ϑ+a10a2a4C∗2δ2ϑδ3ϑ+a10a2a6C∗2δ2ϑδ3ϑ+a10a4a6C∗2δ2ϑδ3ϑ+a10a2bBϑC∗2δ2ϑδ3ϑ+a10a4bBϑC∗2δ2ϑδ3ϑ+a10a6bBϑC∗2δ2ϑδ3ϑ+a10a2C∗δ2ϑδ3ϑI∗S∗+a10a6C∗δ2ϑδ3ϑI∗S∗+a10bBϑC∗δ2ϑδ3ϑI∗S∗+δ3ϑI∗m2S∗−a4τϑ−a4a9β1ϑτϑ−a6a9β1ϑτϑ−a10a2β2ϑC∗2δ2ϑτϑ−a10a4β2ϑC∗2δ2ϑτϑ−a10a6β2ϑC∗2δ2ϑτϑ−a10a2C∗2δ2ϑδ3ϑτϑ−a10a4C∗2δ2ϑδ3ϑτϑ−a10a6C∗2δ2ϑδ3ϑτϑ−a10C∗δ2ϑδ3ϑI∗S∗τϑ+m1(a9+m1(a2+m2)S∗+(ψ1ϑ+θ1ϑ)(ψ2ϑ+θ2ϑ))ɛϑ+a1(a3+a4+bBϑ−τϑ+m12S∗ɛϑ)+a3(a4+bBϑ+a4a9β1ϑ+a9β1ϑ(a6+bBϑ−τϑ)−τϑ+m12S∗ɛϑ), B4=(−a1−a3−a4−bB−a3a9β1ϑ−a4a9β1ϑ−a6a9β1ϑ−a9bBϑβ1ϑ−a10a2β2ϑC∗2δ2ϑ−a10a4β2ϑC∗2δ2ϑ−a10a6β2ϑC∗2δ2ϑ−a10bBϑβ2ϑC∗2δ2ϑ−a10a2C∗2δ2ϑδ3ϑ−a10a4C∗2δ2ϑδ3ϑ−a10a6C∗2δ2ϑδ3ϑ−a10bBϑC∗2δ2ϑδ3ϑ−a10C∗δ2ϑδ3ϑI∗S∗+τϑ+a9β1ϑτϑ+a10β2ϑC∗2δ2ϑτϑ+a10C∗2δ2ϑδ3ϑτϑ−m12S∗ɛϑ),B5=(1+a9β1ϑ+a10β2ϑC∗2δ2ϑ+a10C∗2δ2ϑδ3ϑ). By the criteria of Routh–Hurwitz, if B1>0,B4>0,B5>0 and B3B4>B2B5, B2B3B4>B22B5+B1B42, EE^ is locally asympotically stable. 5.3 Basic reproduction number The Next Generation Matrix Method (NGMM) [26], [27] is used to determine the basic reproduction number (“R0”) for the local stability of the disease-free equilibrium. The disease transmission coefficient “R0”, represents the number of people who can contract the virus and represents the extent of virus propagation. Biologically, when R0<1 the disease disappear, when R0>1, infection stays in the community. In order to calculate R0, which is thought to be the spectral radius of the following-generation matrix FV−1, we write the model as F−V, where F is the transmission part, which includes new infection, and V is the transition part representing the change in state. Therefore, F=0β1ϑNASβ2ϑNISβϑ(1−Pϑ)BN(B+KBϑ)Sδ3ϑNSIC000, and V=−Λϑ+(μϑ+β1ϑNA+β2ϑNI+θ2ϑ+ψ2ϑ+δ3ϑNIC)S+βϑ(1−Pϑ)BN(B+KBϑ)S−(θ1ϑ+ψ1ϑ)Vβ1ϑNɛAϑAS+βϑ(1−Pϑ)BN(B+KBϑ)A+μϑA+γ1ϑA−β1ϑNɛAϑA+βϑ(1−Pϑ)BN(B+KBϑ)I+(d1ϑ+r1ϑ+μϑ)I(d2ϑ+r2ϑ+δ2ϑNI+μϑ)C−βϑ(1−Pϑ)BN(B+KBϑ)(A+I)−δ2ϑNCI+(r3ϑ+d3ϑ+μϑ)D(θ1ϑ+ψ1ϑ+μϑ)V−(θ2ϑ+ψ2ϑ)S−r1ϑI−r2ϑC−r3ϑD−γ1ϑA+μϑR−ɛϑC−(bBϑ−τϑ)B. By using the NGMM [26], [27], the matrices F and V at DFE¯ are obtained by F=∂Fx(DFE¯)∂ty and V=∂Vx(DFE¯)∂ty, 1≤x,y≤5. Where L=FV−1, and Lij represents expected number of secondary cases in compartment i by an individual in compartment j. We take the equations of ‘infected’ compartments only (A,I,C,D,B), and defined F and V as a jacobian matrices by taking the derivative of these ‘infected’ compartments equations respect to these compartments (A,I,C,D,B) and then FV−1 has been calculated at DFE¯. This implies, F=β1ϑNS00000β2ϑNS0000000−βϑ(1−Pϑ)KBϑSN(B+KBϑ)20δ3ϑNSCδ3ϑNSI0000000, V=β1ϑNɛAϑS+βϑ(1−Pϑ)BN(B+KBϑ)+μϑ+γ1ϑ000−βϑ(1−Pϑ)KBϑAN(B+KBϑ)2−β1ϑNɛAϑSβϑ(1−Pϑ)BN(B+KBϑ)+d1ϑ+r1ϑ+μϑ00−βϑ(1−Pϑ)KBϑIN(B+KBϑ)20δ2ϑNCd2ϑ+r2ϑ+δ2ϑNI+μϑ00−βϑ(1−Pϑ)BN(B+KBϑ)−βϑ(1−Pϑ)BN(B+KBϑ)−δ2ϑNC−δ2ϑNIr3ϑ+d3ϑ+μϑβϑ(1−Pϑ)KBϑ(A+I)N(B+KBϑ)200−ɛϑ0−bBϑ+τϑ The basic reproduction number (R0 of the disease is calculated using the spectral radius of the matrix FV−1 at the equilibrium point DFE¯, which is given by two cases namely R01 and R02 : R01=β1ϑNΛϑ(1−ɛAϑ)(θ1ϑ+μϑ+ψ1ϑ)μϑ(γ1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ) R02=β2ϑNΛϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(d1ϑ+r1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ), in which (14) R0=maxR01,R02. 6 Sensitivity analysis In this section, we have analyzed the sensitivity of R01 and R02 as a function of parameters affecting the reproduction number. The sensitivity measure displays the relative changes in the variables that are affected by the variation of a certain parameter. We used the same method as in [28]. Given the importance of the reproduction number in calculating disease prevalence, it is necessary to assess the sensitivities of the parameters with respect to R0. The fractional derivative for R01 and R02 is given by ∂R01∂Λϑ=β1ϑN(1−ɛAϑ)(θ1ϑ+μϑ+ψ1ϑ)μϑ(γ1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)>0, ∂R02∂Λϑ=β2ϑN(θ1ϑ+μϑ+ψ1ϑ)μϑ(d1ϑ+μϑ+r1ϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)>0, ∂R01∂β1ϑ=Λϑ(1−ɛAϑ)(θ1ϑ+μϑ+ψ1ϑ)Nμϑ(γ1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)>0, ∂R02∂β2ϑ=Λϑ(θ1ϑ+μϑ+ψ1ϑ)Nμϑ(d1ϑ+μϑ+r1ϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)>0, ∂R01∂θ1ϑ=∂R01∂ψ1ϑ=β1ϑNΛϑ(θ2ϑ+ψ2ϑ)μϑ(γ1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)2>0, ∂R02∂θ1ϑ=∂R02∂ψ1ϑ=β2ϑNΛϑ(θ2ϑ+ψ2ϑ)μϑ(d1ϑ+μϑ+r1ϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1+ψ2ϑ)2>0, ∂R01∂θ2ϑ=∂R01∂ψ2ϑ=−β1ϑNΛϑ(1−ɛAϑ)(θ1ϑ+μϑ+ψ1ϑ)μϑ(γ1ϑ+μϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)2<0, ∂R02∂θ2ϑ=∂R02∂ψ2ϑ=−β2ϑNΛϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(d1ϑ+μϑ+r1ϑ)(θ1ϑ+θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)2<0, ∂R02∂d1ϑ=∂R02∂r1ϑ=−β2ϑNΛϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(d1ϑ+μϑ+r1ϑ)2(θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)<0, ∂R01∂γ1ϑ=−β1ϑNΛϑ(1−ɛAϑ)(θ1ϑ+μϑ+ψ1ϑ)μϑ(γ1ϑ+μϑ)2(θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)<0, ∂R01∂ɛAϑ=−β1ϑNΛϑ(θ1ϑ+μϑ+ψ1ϑ)μϑ(γ1ϑ+μϑ)2(θ2ϑ+μϑ+ψ1ϑ+ψ2ϑ)<0. It can be seen that R01 increases with parameters Λϑ,β1ϑ,θ1ϑ,ψ1ϑ and decreases with γ1ϑ,θ2ϑ,ψ2ϑ,μϑ,ɛAϑ. R02 increase with parameters Λϑ,β2ϑ,θ1ϑ,ψ1ϑ and decrease with θ2ϑ,ψ2ϑ,μϑ. By calculating the derivatives of R01 and R02, it is clear that the parameters with negative partial derivative values must be maximized in the population in order to limit the outbreak. 7 Parameter Estimation (PE) (PE) is an effective technique for approximating real data, and it has been addressed in many recent studies. It finds a suitable approximation for the real data by getting the best curve that fits the real data and reducing the absolute error between the real and approximate values to the lowest possible value. The PE method is used to calculate parameter values with the best approximation of the real values of the generated mathematical model. The best approximate values obtained by PE for the real data have been clarified in Table 2. The absolute error Ei which defined as Ei=\|Xi−Yi\| has been calculated for all real values Yi and corresponding approximated values Xi. The eleventh week in the table’s error value is discovered to be the highest E11=0.12990e+03, while the errors for the other values are smaller. This method increases the value of our study in terms of calculating the best approximation of the parameter values for the model. The working principle of this method can be expressed by the following algorithm: • Find coefficients c that solve the problem minc‖X(c,cdata)−ydata‖22=minc∑iX(c,cdatai)−ydatai2, given input data cdata, and the observed output ydata, where cdata and ydata are matrices or vectors, and X(c,cdata) is a matrix-valued or vector-valued function of the same size as ydata. • In the case where there are boundaries, lower and upper bounds lb, and ub can be determined, respectively. The arguments c, lb, and ub can be vectors or matrices. • In order to fit the real data we use MATLAB code lsqcurvefit where the user defines a function to calculate the function with the following vector value X(c,cdata)=X(c,cdata(1))X(c,cdata(2))X(c,cdata(3))⋮X(c,cdata(k)). Our model has a total of 24 parameter values. Using actual Cholera data from the Congo (found at https://www.reliefweb.int), we have fitted a total of 22 parameter values using the aforementioned algorithm. We then have estimated the recruitment rate (Λ) and natural death rate (μ) using the current life expectancy for the country of Congo (60.68 years) and the total population of the Congo, 94,816,562 (https://www.worldbank.org/en/home). We have covered the weekly cases from January 01 to July 31, http://www.plateformecholera.info. Congo’s total population and the initial asymptomatic and Cholera infected individuals are considered as A(0)=20, C(0)=72 since N(0)=S(0)+A(0)+I(0)+C(0)+D(0)+V(0)+R(0)+B(0), S(0)=94816490, I(0)=0, D(0)=0, the populations of recovered individuals, vaccinated individuals and Vibro cholera bacteria are calculated as R(0)=0, V(0)=0, and B(0)=0. Real Cholera cases are shown in Fig. 1. Table 1 contains the parameter values used in the modeling along with the best-fitted values determined by the least squares curve fitting approach (LCM). In addition, the second infection rate R01=6.7442389e−10, R02=4.5699168e−10, has been calculated using real data from Congo, from January 01 to July 31, 2021. In Fig. 1, the blue solid line is the model’s best-fit curve, and the red solid circles show real instances of Cholera. Since the execution of the algorithm relies on reducing the square of the error in each step, if the difference between the approximate value and the real value in the current step is taken as Ei=‖Xi−Yi‖, and the difference between the approximate value and the real value in the next step is taken as Ei+1=‖Xi+1−Yi‖, from the strategy of LCM working it can be seen that Ei+1<Eir, and limi→∞Ei+1Eir<M, where M>0,r>1, and r is the rate of convergence, r=log(Ei+1)log(Ei). Although it is obvious that the rate of convergence of the LCM approach is not linear, it is higher, it must be emphasized that the change in this rate relies on the change in our system’s appearance and parameters, as shown by various earlier research that can be found in [29], [30], [31]. Using real data from CONGO and its estimated data, it has been determined that the rate of convergence of the LCM approach for the model described in our study is 1.005≤r≤2.05. Fig. 1 Real cases of Cholera in Congo from January 1 to July 31, 2021 and the best-fit curve from the proposed model. 8 Numerical scheme for the provided Cholera-COVID-19 model in the Caputo derivative sense We examine the dynamics of the model (5) in this section. We used Adams-type estimator-corrector for the numerical simulation of model (5), the method [32], [33], [34], [35] is used. The following Cauchy type ODE is considered with respect to the Caputo operator of order ϑ: (15) 0CDtϑΦt=ϕt,Φt,Φb0=Φ0b,0<ϑ≤1,0<t≤τ, where b=0,1,…,n−1, and n=ϑ. Eq. (15) can be turned to the Volterra equation: (16) Φt=∑b=0n−1Φ0btbb!+1Γϑ∫0tt−sϑ−1Φs,Φsds. By considering this proposed predictor–corrector scheme associated with the Adam-Bashforth-Moulton algorithm [33] to have the numerical solutions of the proposed model, we can take h=τ/N,tz=zh, and z=0,1,…,N∈Z+, by letting Φz≈Φtz, it can be discretized as follows, i.e., the corresponding corrector formula [36] Sq+1=∑z=0q−1S0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(Λϑ−(μϑ+β1ϑNAz+β2ϑNIz+θ2ϑ+ψ2ϑ+δ3ϑNIzCz)Sz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Sz+(θ1ϑ+ψ1ϑ)Vz),+hϑΓϑ+2∑z=0qpq+1,q+1(Λϑ−(μϑ+β1ϑNAq+1PF+β2ϑNIq+1PF+θ2ϑ+ψ2ϑ+δ3ϑNIq+1PFCq+1PF)Sq+1PF−βϑ(1−Pϑ)Bq+1PFN(Bq+1PF+KBϑ)Sq+1PF+(θ1ϑ+ψ1ϑ)Vq+1PF), Aq+1=∑z=0q−1A0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(β1ϑ(1−ɛAϑ)AzSz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Az−μϑA−γ1ϑAz)+hϑΓϑ+2∑z=0qpq+1,q+1(β1ϑN(1−ɛAϑ)Aq+1PFSq+1PF−βϑ(1−Pϑ)Bq+1PFN(Bq+1PF+KBϑ)Aq+1PF−μϑAq+1PF−γ1ϑAq+1PF), Iq+1=∑z=0q−1I0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(β1ϑNɛAϑAz+β2ϑNIz)Sz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Iz−(d1ϑ+r1ϑ+μϑ)Iz+hϑΓϑ+2∑z=0qpq+1,q+1(β1ϑNɛAϑAq+1PF+β2ϑNIq+1PF)Sq+1PF−βϑ(1−Pϑ)Bq+1PFN(Bq+1PF+KBϑ)Iq+1PF−(d1ϑ+r1ϑ+μϑ)Iq+1PF, Cq+1=∑z=0q−1C0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(βϑ(1−Pϑ)BzN(Bz+KBϑ)Sz−(d2ϑ+r2ϑ+δ2ϑNIz+μϑ)Cz)+hϑΓϑ+2∑z=0qpq+1,q+1(βϑ(1−Pϑ)Bq+1PFN(Bq+1PF+KBϑ)Sq+1PF−(d2ϑ+r2ϑ+δ2ϑNIq+1PF+μϑ)Cq+1PF), Dq+1=∑z=0q−1D0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(βϑ(1−Pϑ)BzN(Bz+KBϑ)(Az+Iz)+δ2ϑNCzIz+δ3ϑNSzIzCz−(r3ϑ+d3ϑ+μϑ)Dz)+hϑΓϑ+2∑z=0qpq+1,q+1(βϑ(1−Pϑ)Bq+1PFN(Bq+1PF+KBϑ)(Aq+1PF+Iq+1PF)+δ2ϑNCq+1PFIq+1PF+δ3ϑNSq+1PFIq+1PFCq+1PF−(r3ϑ+d3ϑ+μϑ)Dq+1PF), Vq+1=∑z=0q−1V0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(−(θ1ϑ+ψ1ϑ+μϑ)Vz+(θ2ϑ+ψ2ϑ)Sz)+hϑΓϑ+2∑z=0qpq+1,q+1(−(θ1ϑ+ψ1ϑ+μϑ)Vq+1PF+(θ2ϑ+ψ2ϑ)Sq+1PF), Rq+1=∑z=0q−1R0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(r1ϑIz+r2ϑCz+r3ϑDz+γ1ϑAz−μϑRz)+hϑΓϑ+2∑z=0qpq+1,q+1(r1ϑIq+1PF+r2ϑCq+1PF+r3ϑDq+1PF+γ1ϑAq+1PF−μϑRq+1PF), Bq+1=∑z=0q−1B0ztq+1zz!+hϑΓϑ+2∑z=0qpz,q+1(ɛϑCz+(bBϑ−τϑ)Bz)+hϑΓϑ+2∑z=0qpq+1,q+1(ɛϑCq+1PF+(bBϑ−τϑ)Bq+1PF), where Sq+1PF=∑z=0q−1S0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(Λϑ−(μϑ+β1ϑNAz+β2ϑNIz+θ2ϑ+ψ2ϑ+δ3ϑNIzCz)Sz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Sz+(θ1ϑ+ψ1ϑ)Vz), Aq+1PF=∑z=0q−1A0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(β1ϑ(1−ɛAϑ)AzSz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Az−μϑA−γ1ϑAz), Iq+1PF=∑z=0q−1I0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(β1ϑNɛAϑAz+β2ϑIz)Sz−βϑ(1−Pϑ)BzN(Bz+KBϑ)Iz−(d1ϑ+r1ϑ+μϑ)Iz, Cq+1PF=∑z=0q−1C0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(βϑ(1−Pϑ)BzN(Bz+KBϑ)Sz−(d2ϑ+r2ϑ+δ2ϑNIz+μϑ)Cz), Dq+1PF=∑z=0q−1D0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(βϑ(1−Pϑ)BzN(Bz+KBϑ)(Az+Iz)+δ2ϑNCzIz+δ3ϑNSzIzCz−(r3ϑ+d3ϑ+μϑ)Dz), Vq+1PF=∑z=0q−1V0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(−(θ1ϑ+ψ1ϑ+μϑ)Vz+(θ2ϑ+ψ2ϑ)Sz), Rq+1PF=∑z=0q−1R0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(r1ϑIz+r2ϑCz+r3ϑDz+γ1ϑAz−μϑRz), Bq+1PF=∑z=0q−1B0ztq+1zz!+hϑΓϑ+1∑z=0qjz,q+1(ɛϑCz+(bBϑ−τϑ)Bz), and (17) pz,q+1=qϑ+1−q+1ϑq−ϑ,ifz=0,q−zϑ+1+q−z+2ϑ+1−2q−z+1ϑ+1,if1≤z≤q,1,ifz=q+1. where jz,q+1=q+1−zϑ−q−zϑ. 9 Numerical simulations and discussion For the values in Table 1, the numerical solution of model (5) has been discovered in this section using the Adams–Bashforth-Moulton Predictor-Corrector method. Parameters have a very important role in controlling the spread of disease. Using parameters in Table 1, the variation of each sub-population has been simulated over time for different values of ϑ by using the values given in Additionally, taking into account parameters that significantly change the behavior of system dynamics, graphics have been obtained for various values of these parameters. The dynamical behaviour of each state of the proposed Cholera and COVID-19 model is shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11, Fig. 12, Fig. 13, Fig. 14 for different values of ϑ and various parameter values. In Fig. 2, one can see that the susceptible individuals show a decreasing behavior until about day 10 and then reach a stable behaviour, with increasing values of ϑ. In other words, the susceptible population class achieves the stability state at the fractional order earlier than the integer order. The asymptomatic people in Fig. 3 undergo change throughout time. Up until the day when the behavior of susceptible individuals switches from decreasing to stability, the asymptomatic persons for varying fractional-order derivative exhibit an increase; beyond that day, the asymptomatic individuals exhibit a decrease in behavior. Also, as the value of ϑ decreases from 1, asymptomatic individuals need more time to stabilize in non-integer cases. According to Fig. 4, the number of I class grows up until the fifth day, around, at which point it starts to decline and eventually stabilizes. This statistic suggests that there will be no sick people in the population on the 100th day. In Fig. 5, the infection decreases after a certain time and reaches the stability as in Fig. 4, but the individuals infected with Cholera need more time to reach stability and equilibrium point compared to class I. Fig. 6 shows the change over time of people who have both Cholera and COVID-19. It can be seen that they initially show increasing behaviour and then become stable over time. Fig. 7 clearly shows that the number of vaccinated individuals increases over time and after 5th day the number of vaccinated individuals reach to stability. Fig. 8 shows that the recovered individuals increased over time and then stabilized. A rapid increase in recovered individuals for ϑ=1, whereas a slower increase in these individuals for fractional order values of ϑ=0.91 is remarkable. In Fig. 9, it can be seen that Vibrio cholera bacteria in the population did not grow before the day of 95. Here the slower increase in the number of bacteria for fractional values of ϑ is due to the memory effect of fractional systems. The figures show us that the observed changes in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9 are consistent with the expected phenomena related to true COVID-19 and Cholera disease. This is due to the memory effect in fractional derivatives, therefore the solution to equations of fractional order rely on every previous case. In Fig. 10, we have changed β1,β2 (the rate of disease transmission by contact with A class and I class, respectively) and kept the other parameters as in Table 1. In Fig. 10, it can be seen that the number of susceptible individuals decreases with increasing β1 over time and then reach stability, while the number of susceptible individuals decreases with increasing β2, but with a very small difference with respect to the change in β2. In Fig. 11, as β1 increases and when β1 value is higher than baseline (β1=0.252005), the asymptomatic cases reach stability more slowly, in order to get to the stability case faster, it is recommended to lower the rate of transmission by coming into touch with the asymptomatic group below the baseline. In Fig. 12, Fig. 13, as β1 and β2 increases, the number of Infected individuals with Cholera and COVID-19 increases over time and reach stability at higher values of β1 and β2 after longer time. This enables us to reach the conclusion that limiting contact with infected persons, regardless of whether they exhibit symptoms or not, enables the disease to be controlled and reaches a level of stability more quickly. In Fig. 14, with the increase of β1 and β2, the number of recovered individuals reach to stability more slowly. In terms of biology, if interactions with persons in the classes (A,I) had ceased, there would be a significant decrease in the number of asymptomatic Individuals (A), COVID-19 infected Individuals (I), Cholera infected Individuals (C), and infected Individuals with both COVID-19 and Cholera (D). Therefore, it is possible to control the spread of the illness and achieve a stable state more quickly when contact with infected people is reduced, regardless of whether they have symptoms or not. Additionally, adhering to the practices that have clearly reduced COVID-19 infection, such as maintaining good personal hygiene, isolating oneself, and receiving vaccinations, results in a decrease in Cholera cases. Fig. 2 Change of Susceptible individuals over time for some instances of ϑ. Fig. 3 Change of Asymptomatic individuals over time for some instances of ϑ. Fig. 4 Change of Infected individuals with COVID-19 over time for some instances of ϑ. Fig. 5 Change of Infected individuals with Cholera over time for some instances of ϑ. Fig. 6 Change of individuals who have been infected by Cholera and COVID-19 over time for some instances of ϑ. Fig. 7 Change of Vaccinated individuals over time for some instances of ϑ. Fig. 8 Change of Recovered individuals over time for some instances of ϑ. Fig. 9 Change of Vibrio cholera Bacteria populations over time for the varying fractional-order derivative. Fig. 10 Change of Susceptible Individuals over time for the varying β1 and β2 values, ϑ=0.97. Fig. 11 Change of Asymptomatic individuals over time for the varying β1 value with ϑ=0.97. Fig. 12 Change of Infected individuals with COVID-19 over time for the varying β1 and β2 values, ϑ=0.94. Fig. 13 Change of Infected individuals with Cholera and COVID-19 over time for the varying β1 and β2 values, ϑ=0.94. Fig. 14 Change of Recovered individuals over time for the varying β1 and β2 values, ϑ=0.94. 10 Conclusions In this paper, we first have been briefly informed about COVID-19 and Cholera diseases. We have studied the spread of COVID-19 along with the Cholera epidemic. We have also studied the effects of COVID-19 on people with cholera bacteria. We have created a new, dimensionally consistent fractional-order Caputo model. Using the theorem of fixed point, the system has been demonstrated to have a unique solution and the equilibrium points of the system have been calculated. LAS of the equilibrium points, consisting of a disease-free equilibrium point and an epidemic equilibrium point, has been examined. Then, considering the real data of Cholera disease from Congo, the sum of squares of the difference between the numerical solution for Cholera infected people and the real data has been minimized by LCM and the best fitting curve has been obtained. In addition, the best values for the model parameters have been calculated using the method PE to better and more realistically observe the dynamics of the system. In this way, we aim to predict the change in the population over time using more accurately generated parameters. Using the Adams–Bashforth-Moulton method, numerical simulations of the COVID-19 - Cholera model have been visualized for different fractional orders and different parameter values. We have investigated the effects of parameters on the increase and decrease in both the spread and decline of the pandemic. Using real data is very helpful for figuring out how accurate a complex mathematical model is. However, the biggest problem is often where to find this data and/or how to find the best fit curve for the data obtained. To date, a great deal of research has been done on the mathematical model of cholera and COVID-19. Most of these studies are on COVID-19 or Cholera only, and no studies using actual data from Congo have been found. To our knowledge, to date, there is no cholera study using real data from Congo using the fractional equations system. According to our results, it appears that the probability of Cholera-infected individuals contracting COVID-19 is higher than the probability of infection with COVID-19 from susceptible individuals (δ2>β2), also the probability of infected individuals with both COVID-19 and Cholera is higher than the probability of infection with COVID-19 from susceptible individuals (δ3>β2). The significant outcome of this study is that despite the obvious differences between them, it has been seen that there is much in common between the COVID-19 and Cholera outbreaks. It turns out that when COVID-19 cases decrease, Cholera cases also decrease, which explains why Cholera cases decrease when safety measures are taken to reduce COVID-19 such as reduced travel and increased attention to personal hygiene and social distancing. It also explains why the Cholera epidemic is resurging at the same time as the lifting of restrictions on the spread of COVID-19. Estimates indicate in the future that the severity of the Cholera epidemic cases will decrease with the decrease in cases of COVID-19, through the implementation and follow-up of safety measures that have been taken to reduce COVID-19 cases, which contributed significantly to reducing the number of Cholera cases. In addition to the fact that the risk of contracting Cholera will decrease when taking vaccinations and taking care of the provision of safe drinking water and sanitation facilities. It is possible to stop the spread of COVID-19 and cholera if the number of contacts with infected people can be decreased through practices like self-care and self-isolation, or if more people can be tested, i.e., if routine tests can be carried out not only on symptomatic but also on asymptomatic persons. Vaccination campaigns are also very useful in containing and controlling the spread of the disease. If the results obtained are taken into account, these studies will provide specialists working in the field of diseases and epidemics with the knowledge that can help them in controlling the spread of epidemics in the future and develop ways to treat them. This work may in the future provide insight into several studies in this area. Data availability No data was used for the research described in the article. Acknowledgments This work was supported by Research Fund of the Erciyes University . Project Number: FDS-2021-11059. ==== Refs References 1 Özköse F. Yavuz M. Investigation of interactions between COVID-19 and diabetes with hereditary traits using real data: A case study in Turkey Comput. Biol. Med. 2021 105044 2 Evirgen F. Uçar S. Özdemir N. Hammouch Z. System response of an alcoholism model under the effect of immigration via non-singular kernel derivative Discrete Contin. Dyn. Syst. S 14 7 2021 2199 3 Naik P.A. Zu J. Owolabi K.M. 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Appl. 58 8 2009 1573 1588	36471673	PMC9714208	NO-CC CODE	2022-12-10 23:15:29	no	J Comput Appl Math. 2023 May 15; 423:114969	utf-8	J Comput Appl Math	2,022	10.1016/j.cam.2022.114969	oa_other
==== Front Ain Shams Engineering Journal 2090-4479 2090-4479 THE AUTHORS. Published by Elsevier BV on behalf of Faculty of Engineering, Ain Shams University. S2090-4479(22)00369-0 10.1016/j.asej.2022.102058 102058 Article Effect of waste COVID-19 face masks on self-compacting high-strength mortars exposed to elevated temperature Durmuş Gökhan a Nur Çelik Damla ab Kılıç Demircan Rüya c Kaplan Gökhan d⁎ a Gazi University, Faculty of Technology, Department of Civil Engineering, Ankara, 06500, Turkey b Oregon State University, Department of Civil Engineering, Corvallis, Oregon, 97331, USA c Sinop University, Boyabat Vocational School, Department of Construction, Sinop, 57200, Turkey d Atatürk University, Faculty of Engineering, Department of Civil Engineering, Erzurum, 25030, Turkey ⁎ Corresponding author. 1 12 2022 1 12 2022 10205810 9 2022 27 9 2022 15 11 2022 © 2022 THE AUTHORS. Published by Elsevier BV on behalf of Faculty of Engineering, Ain Shams University. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. During the pandemic, it becomes customary to wear a disposable surgical (face) mask (SM) to guard against coronavirus illness 19 (COVID-19). However, because existing disposal procedures (i.e., incineration and reclamation) emit hazardous substances, vast generations of contaminated surgical masks pose an environmental risk. Therefore, many studies are currently being carried out worldwide to dispose of SM. The easiest and cheapest of these methods is the disposal of SMs in cement-based composites. This study cut waste SMs to macro size and used them in cement-based composites such as polypropylene fiber. The elevated temperature resistance of cement-based composites decreases as their compressive strength rises. Low-melting materials like polypropylene fiber are utilized to improve the high-temperature resistance of cement-based composites. Therefore, SM with a low melting temperature was used in the design of the mixtures. SM was added to the mix at rates of 0.3, 0.5, 0.8, and 1 by weight of cement. As the SM ratio increased, the workability of the mixtures decreased. Water absorption and apparent porosity increased as SM reduced the workability of composites. The mixes' 28-day compressive strength ranges from 36.6 to 79.4 MPa. It was observed that flexural strength raised in some mixtures when SM was used. In the mixes using 0.5% SM, about 40 MPa compressive strength was obtained after 800 oC. Additionally, SEM images showed that SM changed into microfibre during mixing. As a result, it has been determined that SM can be used at low rates to increase the elevated temperature resistance of cement-based composites. Keywords Waste surgical mask COVID-19 Self-compacted mortar High-temperature resistance Porosity ==== Body pmc1 Introduction A new coronavirus disease 19 (COVID-19) that is extremely infectious was identified in 2019 [1], [2]. COVID-19 was declared a pandemic by the World Health Organization (WHO) in March 2020. COVID-19 has started infecting millions of people on all continents [3]. Under these circumstances, most countries require non-pharmaceutical interventions to physically block COVID-19 transmission by providing personal protective equipment (PPE) (mask, glove, protective gown, etc.) [4], [5], [6]. As a result, the demand for PPE has grown, creating plastic waste [7]. Unfortunately, due to various factors, a viable PPE disposal platform has yet to be established [8]. Plastic pollution has increased as a result of the COVID-19 epidemic. The use of plastic is growing due to its heavy reliance on takeout and hygiene concerns, leading to the use of PPE. However, initiatives to reduce plastic use, such as a ban on single-use plastic bags, have been delayed, shelved, or outright banned [9]. Single-use face masks are the most popular personal protective equipment to stop the pandemic from spreading. Because many national and local governments have required single-use face masks, their use has grown commonplace. Face masks had a global market size of around 0.8 billion dollars in 2019, but by the end of 2020, it had grown to over 166 billion dollars [10]. Facemask waste has increased dramatically due to the increasing demand for face masks. Due to their complicated compositions and the potential for COVID-19 infection, they are difficult to recycle. Used masks are frequently disposed of inappropriately, mostly because waste management cannot keep up with the expanding amount of waste [11]. Many incorrectly disposed face masks are in the water, polluting marine habitats. According to OceansAsia, about 1.6 billion masks will reach the ocean in 2020, resulting in 4700-6200 tons of plastic pollution [12]. Therefore, scientists and local governments are looking for alternative solutions for the disposal of waste face masks. Using waste face masks in cement-based composites such as mortar and concrete may be an alternative solution. Concrete is widespread because it is the world's most widely utilized construction material [13]. Concrete is a non-flammable substance with a slower heat transmission rate (conductivity). As a result, concrete structures function admirably in the event of a fire in the majority of circumstances. However, high-strength concrete structures are sensitive to thermal spalling due to the dense microstructure of the concrete matrix. Spalling is the breaking off of pieces of concrete from the concrete member's surface. This results in the loss of the concrete section and, in some situations, the direct exposure of the reinforcement to the fire, which affects the concrete member's loading capacity and fire resistance. As a result, fire spalling is one of the most serious risks to concrete structures, especially those constructed of high-strength concrete [14], [15]. Concrete spalling is often explained by one of two mechanisms: (a) Pore pressure-induced spalling is mostly related to the thermal hydration process inside the heated concrete. As a result of heat transfer and moisture migration, pore pressure eventually builds up in the micropores. Spalling occurs when the subsequent tensile stress generated by the pore pressure exceeds the tensile strength of the concrete. (b) Delamination mechanism due to thermal stress. This is related to the generation of temperature gradients at the heated site and the resulting forced thermal expansion Pore pressure and thermal stress can act separately or in concert [15]. One of the thermo-chemo-mechanical challenges is fire damage to concrete structures. The disintegration of C-S-H gels, a plate-shaped crystal of Ca(OH)2, CaCO3, Al2O3–Fe2O3–tri (AFt), and AFm are all thermos-chemo-physical phenomena that should be included in computational modeling of cementitious composites at higher temperatures, as shown in Figure 1 . The thermos-chemo-physical reactions that occur during elevated temperatures are explained by the Japanese Concrete Institute [16]. Therefore, various methods have been developed to reduce the damage in high-strength concrete during fire. The most common of these methods is the use of polymer fibers with a low melting temperature. Adding polymer fibers to prevent spalling is a well established approach. To prevent HPC from spalling, 0.3 and 0.6 vol percent polymer fibers, such as nylon fibers, polypropylene (PP) fibers, and polyethylene (PE) fibers, are commonly required [17], [18].Fig. 1 The chemical change in hydration products [16] The cooling conditions of cement-based composites directly affect the mechanical properties. The firefighter usually extinguishes fires in the field with water or foam. Studies show the change in the mechanical properties of concretes exposed to elevated temperatures according to cooling conditions in the literature. Zhai et al. investigated the mechanical properties of concrete exposed to elevated temperatures after cooling with water. They observed an increase in strength in cooling with water. The content of Ca(OH)2 in the concrete reduces while the content of CaCO3 increases when heated to 200°C. When the water cools, it produces hydrated calcium silicate, and some of the free water fills the voids in the concrete simultaneously. The elevated temperature and water enhance the hydration reaction of the cement with the water in the concrete, and the concrete's peak stress is improved [19]. Air-entraining admixtures (AEAs) have been a major technological development in the construction sector over the last century. The development of air, gel and capillary pores is advantageous for AEA [20]. To increase workability and uniformity, stop bleeding and deterioration, and increase durability, AEA is frequently added to freshly mixed concrete [20], [21], [22]. Other advantage of AEA is that the increased air volume reduces thermal conductivity and raises resistance to heat flow. [23], [24]. The downside of AEA is that it reduces overall density by increasing porosity, which affects compressive strength. However, water-reducing admixtures can make up for the strength loss. Additionally, research has demonstrated that using this chemical to produce more air bubbles considerably impacts frost resistance [25]. This study examined cement-based composites with compressive strength (28 days) of more than 50 MPa for their elevated temperature endurance. As stated in the literature, polypropylene fibers improve the high-temperature resistance of high-strength or high-performance composites. This study used waste surgical masks instead of polypropylene fiber to produce the mixtures. Because in the research, it has been seen that surgical masks, which are protective equipment for COVID-19, will cause significant pollution in the future. One of the easiest ways to dispose of these masks is to use them as macro fiber in cement or concrete production. This way, the wastes released during the COVID-19 process will be disposed of safely. In addition, considering that surgical masks will not melt in a short time in the mixtures, an air-entraining admixture is used to reduce the internal vapor pressure that will form within the concrete. In this way, it is aimed to produce high-strength cement-based composites that are both environmentally friendly and resistant to elevated temperatures. 2 Materials and Analytical Methods Cement, fine aggregate, and water were used in this study as in conventional mortars. In addition, two chemical admixtures, superplasticizer and air-entraining (AEA), were used. Waste surgical masks (SM) were used instead of polypropylene fiber. 2.1 Raw Materials CEM I cement, which meets the requirements of EN 197-1[26], was used in this study. Cement mainly consists of 63.5% CaO and 17.1% SiO2. The ratios of Al2O3, Fe2O3, SO3, and MgO, which are minor cement components, are 3.7%, 2.9%, 3.5%, and 1.2%, respectively. The specific gravity of CEM I cement is 3.14. In preparing the mixtures, silica aggregate was used to conform to ASTM C 33 [26] and was well-graded. Table 1 shows the physical characteristics of silica aggregate.Table 1 Physical characteristics of silica aggregate Properties Standard Values Fineness modulus ASTM C136 2.72 Water absorption ASTM C128 0.75% Specific gravity ASTM C128 2.61 Dry Bulk density ASTM C29 1654 kg/m3 A polycarboxylate ether-based superplasticizer (PCE) was used to improve the workability of the mixtures. In addition, a lignin-based air-entraining admixture (AEA) was used to create micro air voids in the samples. The characteristics of the chemical admixtures used are given in Table 2 .Table 2 Physical characteristics of chemical admixtures PCE AEA Appearance Light brown Dark brown Specific gravity 1.06 1.14 Solid content 32% 18.4 Cl- content 0.1% <%0.1 Chemical structure Polycarboxylate ether-based Lignosulfonate based pH 6.3 10.2 The waste surgical masks (SM) used in the mixtures were cut and brought to the macro fiber size. The masks used against the COVID-19 epidemic are 3-ply. The general view and layer structure of these masks are given in Figure 2 [27]. SMs were prepared by cutting into 2 cm lengths. SMs were kept in an oven at 80 oC for 2 days to ensure decontamination. In addition, it was exposed to 400 oC temperature (1 hour) to observe the change of SMs in the mortar. The change in SM exposed to 400 oC temperature is given in Figure 3 . As seen in the figure, SM melted at 400 oC. However, a fibrous structure was observed after melting. Also, the TGA curve [27] of SM is presented in Figure 2. Yousef et al. analyzed the thermal behavior of COVID-19 masks analytically (TGA/DTG method). The results revealed that the face mask contains many volatile materials and can collapse in three phases at 360–500°C (with a mass loss of 67–96%) [27].Fig. 2 SM overview and layers [27] Fig. 3 General view of SM and TGA curve [27] 2.2 Mix Proportion Design In this study, different ratios of SM and AEA were used, and 15 mixtures were prepared. SM was used at ratios of 0%, 0.3%, 0.5%, 0.8% and 1% by weight of cement. AEA was used at the ratios of 0%, 0.35%, and 0.70% by weight of cement. The cement dosage was chosen as 800 kg/m3 to reduce the strength losses that AEA and SM may cause. The w/c ratio of all mixtures is 0.30. PCE was used at a ratio of 0.5% by the weight of cement in all mixtures. PCE ratio was determined in pre-experiments. The material amounts and ratios of the mixtures are given in Table 3 . Mortars with SM and AEA are prepared based on ASTM C305 [28] standard. First, cement and silica aggregate was mixed as a dry mixture for 1 minute. Afterward, 2/3 of PC and 75% water were added to the mixture and mixed for 4 minutes. After the wet mixture rested for 1 minute, the remaining water, PCE, and AEA were added. After the mixture was stirred for another 2 minutes, SM was added. Mixtures with SM were mixed for an additional 3 minutes. After the prepared mixtures were placed in their molds, they were kept under laboratory conditions for 24 hours.Table 3 Mixture properties Mix ID Mixing ratios (%) Quantities of materials (kg/m3) AEA SM Cement Aggregate Water PCE AEA SM M1 0 0 800 1285 240 4 - - M2 0.3 2.4 M3 0.5 4.0 M4 0.8 6.4 M5 1.0 1.0 M6 0.35 0 2.8 - M7 0.3 2.4 M8 0.5 4.0 M9 0.8 6.4 M10 1.0 1.0 M11 0.70 0 5.6 - M12 0.3 2.4 M13 0.5 4.0 M14 0.8 6.4 M15 1.0 1.0 2.3 Heating Procudere The mixtures were exposed to elevated temperatures with a special electric furnace (time-adjusted). The heating rate of the electric furnace is set at 5 min/oC. The samples were dried at 105 oC for 24 hours before being placed in the electric furnace. After the mixtures reached the target temperature, they were subjected to elevated temperatures for 2 hours. Afterward, two different cooling conditions, air and water, were applied to the mixtures. Air cooling; The samples were taken out of the electric furnace were kept in laboratory conditions until they cooled down. Water cooling; The samples were taken out of the electric furnace, put into the water tank, and cooled. The samples, cooled in water, were then dried again at 105 oC and their weight losses were determined. Additionally, the mixes exposed to elevated temperatures measured their compressive and flexural strengths. 40x40x160 mm prism samples were used to determine the elevated temperature resistance. Figure 4 shows the samples exposed to 800 oC and the heating process used in the experimental study.Fig. 4 Electric furnace and elevated temperature application process 2.4 Experimental methods The workability of the mixtures was compared according to their flow diameters. The method specified by EFNARC was used to determine the workability [29]. The fresh characteristics of the mixtures and the flow diameters of some mixtures are shown in Figure 5 . The high SM ratio induced segregation in some mixes, as can be seen in the image. In addition, bleeding was observed in some mixtures, but this situation was reduced with AEA.Fig. 5 Fresh properties of some mixtures The compressive and flexural strengths of the mixtures were used to determine their mechanical characteristics. Standard ASTM C349 [30] for compressive strength and ASTM C348 standard [31] for flexural strength were used. A 40 × 40 × 160 mm sample was utilized to determine the mechanical properties. On days 7 and 28, the mixtures' compressive and flexural strengths were also assessed. The mixtures' dry bulk density, apparent porosity, and water absorption were evaluated using the ASTM C642 standard. [32]. For the physical properties of the mixtures, cube samples of 50x50x50 mm were produced. Physical properties were measured on 28-day mixtures. All experiments were carried out on three samples (only flexural test with six samples). In addition, micro and macro studies were carried out on mixtures exposed to 800 oC. A scanning electron microscope (SEM) was used for micro-examinations. An optical microscope was used for macro examinations. Macro and micro research were carried out at Gazi University, Metallurgy and Engineering Laboratory. 3 Results and Discussion 3.1 Fresh Characteristics The effects of SM and AEA on fresh mortar properties are shown in Figure 6 . Flow diameters of mixtures without air-entraining admixtures vary between 28 and 41 cm. As 1% SM was added to non-AEA mixes, the flow diameter was decreased by 22.2 percent compared to the reference mix. It was found that the flow diameters showed similar properties if the air-entraining additive was 0.35. In the case of using 0.7% AEA, the flow diameters vary between 31-40 cm. The use of 1% SM in the mixtures reduces the spreading diameters. However, if air-entraining admixtures are used in these mixtures, flow diameters have been improved. In particular, if 0.35% of AEA was used, the flow diameter increased by 32.1%. If the air-entraining admixture ratio of 070% is used in mixtures with SM, the flow diameters are relatively reduced. The highest flow diameters were generally observed in mixes with 0.35% AEA. As the ratio of SM used in the mixtures increases, the flow time increases. The flow time was found to be 20.3 seconds when 1% SM was used in mixes without air entrainment admixture. However, when 0.35% AEA was used, the flow time was 17.7 seconds. If the AEA ratio was 0.70, the flow time decreased approximately four times. The flow times of the mixtures with 1% SM are relatively longer than the other mixtures. The rise in the AEA rate was more effective in the reference mixture. The use of air-entraining admixtures in self-consolidating mixtures generally increases viscosity. Therefore, the flow diameters were decreased when 0.70% AEA was used. Similarly, the increase in flow times occurred because of this. The workability of the mixtures decreased due to the water retention property of SM. In addition, the fact that SMs acted like fibers (Polypropylene fiber) and increased internal friction was also effective in this process.Fig. 6 Flow diameter and time of mixtures Many studies in the literature show the effect of air-entraining additives on workability. It can be explained that air bubbles entrained in concrete only last a short period if the mixture is not constantly mixed. A portion of the “ball bearing” effect is lost when these bubbles explode, sharply reducing the slump [33]. Similar findings were seen in the Dils et al. experiment. As the air-entraining additive ratio increased, the workability of the mixtures decreased. This effect is explained by increased yield stress and plastic viscosity of air-entraining admixtures [34]. The air bubble bridges provide a likely explanation for this phenomenon. Cement particles become positively charged when they hydrate. The cement particles, typically smaller than air bubbles, will be bonded to the anionic surfactants. The bubbles are coated with cement particles, which increases cohesiveness. These bridges cause increased flow resistance [35], [36]. In this study, the waste surgical masks acted like polypropylene fiber. Hassanpouret et al. stated that the workability of concrete decreases as the polypropylene fiber content increases [37], [38]. The friction between fibers and cement pastes and the usage of admixtures play a significant role in the workability loss of polypropylene fiber-reinforced concrete and mortars. Because polypropylene fiber increases granular structure and decreases maximum compactness, friction between fibers and cement particles increases, blocking granulate movement during the test. The slump value decreases, and the mixture's viscosity increases due to this behavior [39], [40]. While the AEA was 0.35%, the flow diameter was relatively increased, and the flow diameter was decreased again by 0.70% (Fig. 7 a). In this study, the optimum AEA ratio was found to be 0.35. When the optimum ratio of AEA is exceeded, the mixture's plastic viscosity increases and its workability decreases. Increasing the AEA ratio also increases the flow times. The rise in air bubbles decreased the workability of the mixes. The mixes' flow widths get smaller as the SM ratio rises, as seen in Figure 7b. Additionally, when SM increases, the flow time of the mixtures gets shorter. The fact that waste surgical masks have water absorption properties has reduced the workability of the mixes. In addition, the irregular shape of the SMs also increased the internal friction, and as a result, the properties of the fresh mortar were adversely affected. If the SM ratio was 0.3%, a slight increase in the flow diameter was observed. Since the paste volume is high in the composites, the flow diameter was relatively increased with the use of low SM. However, workability can be improved as air-entraining admixtures increase the paste volume. In this way, workability has been enhanced in low AEA use. The workability losses caused by SM are reduced with air-entraining admixtures.Fig. 7 Effect of AEA and SM on fresh mortar properties 3.2 Physical Characteristics As shown in Figure 8 a, the apparent porosity of the mixtures increases as the AEA volume rises. The porosity increased by 10.4% for the reference mixture when the AEA content was 0.35% and 107.2% when the AEA content was 0.70. The porosity values of the mixtures with an AEA content of 0.70 vary between .19-7.48%. AEA admixture entrains air bubbles into the paste. These entrained air bubbles increase the porosity of the mixtures. If SM is used in some mixes, the porosity decreases compared to the reference mixture. For example, when the SM content of the AEA-free mixture was 1%, the porosity was reduced by 15.8% compared to the reference mixture. Similar behavior was observed in mixtures with an AEA content of 0.7%. If the SM content was 0.3%, the porosity decreased by 20.8% compared to the reference mixture. It is thought that SM closes some of the voids between aggregates, as in polypropylene fibers. SM can act as micro aggregates like polypropylene fibers. Therefore, in some cases, SM has been observed to reduce the apparent porosity. It is seen in Figure 8b that the water absorption values are parallel to the apparent porosity. The mixes' water absorption values increase as AEA content rises. Even though the combinations without AEA have water absorption values below 2%, when the AEA level is 0.70, the values range from 2.63 to 4.45%.The highest water absorption value was obtained in the mixture with an AEA content of 0.70 and an SM content of 1%. The number of air bubbles in the paste rises with increasing AEA content. This increases the water absorption value of the mixture. In the case of SM of 0.8% or 1% in mixtures without AEA, the water absorption value decreased by about 12% compared to the reference mixture. Similar behavior was determined in mixtures with an AEA content of 0.70%. SM reduced the water absorption values in some mixtures (For example, Mixture without AEA and 0.8-1% SM) by separating the connections between the capillary voids. But because SM often makes materials less workable, mixes tend to absorb more water and become more porous.Fig. 8 Physical properties of mixtures The mixes' dry bulk densities range from 2198 to 2424 kg/m3, as shown in Figure 8c. The mixtures' dry bulk densities fall as AEA content rises. Since SM has a low specific gravity compared to the combinations' dry bulk densities, the change in SM concentration has little impact on those densities. Studies have shown that AEA increases the porosity of concrete while decreasing its density [41], [42]. Pundiene et al. determined that AEA reduced the density of concretes by approximately 17%. They also observed that mixtures with AEA increased the porosity value from 17.1% to 31% [43]. Zeng et al. stated that as the AEA content increases, the mixture's porosity increases and the water absorption increases [44]. Wu et al. determined that the oven-dried densities of the mixtures relatively decreased as the polypropylene fiber content increased [45]. Because more air spaces are held in the fresh mix by the high polypropylene fiber content, the porosity increases and the mechanical properties are negatively impacted [46]. However, it has also been reported that fibers sometimes reduce water absorption and porosity. Previous studies discovered that low fiber content reduces water absorption and porosity because the fiber blocks the pores [47], [48]. At high fiber content, the porosity of the mixtures increases due to the loss of workability. The formation of air voids causes moisture diffusion due to the high fiber content [48]. 3.3 Mechanical Properties As seen in Figure 9 a, the 7-day flexural strengths of the mixtures without air-entraining additives vary between 11.9-15.8 MPa. If the SM ratio was 0.3% and 1%, the flexural strengths increased compared to the reference mixture. This result shows that SM behaves like polypropylene fiber. The lowest flexural strength was obtained in the mix with 0.8% SM, with a value of 11.9 MPa. The flexural strengths decreased when using the air-entraining additive ratio of %0.35. As the air bubbles entrained into the paste reduce the porosity, their flexural strength has reduced. The flexural strength of the mıxture wıth 1% SM and 0.35% AEA decreased 40% compared to the reference mıxture. When the air-entraining admixture ratio was 0.70%, the low use of SM increased the flexural strengths relatively. If the SM is 0.3 or 0.5%, the strength loss caused by the air-entraining admixture is eliminated. In particular, flexural strengths of over 10 MPa were obtained for mixtures with 0.3 and 0.5 SM. As seen in Figure 9b, increased cure time increased the flexural strength of the mix. It is seen that the 28-day flexural strengths are parallel to the 7-day flexural strengths. Using SM in mixtures without air-entraining admixtures increased the flexural strengths relatively. This effect shows that the curing time increases the adherence between SM and paste. If the SM ratio was 0.3%, the 28-day flexural strength increased by 26%. If the AEA ratio is 0.35%, the flexural strength varies between 12.1-13.3MPa. The rise in the SM rate decreased the flexural strengths. Flexural strength has increased relatively in the use of 0.70% AEA. The flexural strength of only 1% SM mixtures decreased compared to 0.35% AEA mixes. Compared to the reference mix, the mix with 0.5% SM had a 7% lower flexural strength. Despite the AEA ratio rise, the flexural strength of 0.5% SM mixtures increases. When these mixes are compared, it is shown that the fresh state properties are very close to each other. The flow time of the mix with 0.5% SM, where the AEA was 0.70, decreased by 12.3% compared to the mix with 0.35 AEA. The uniformity of the mixtures has grown as the air-entraining additive ratio increases, improving the mixtures' workability. Thanks to the increased uniformity, the flexural strength of the mixtures with 0.5% SM increased.Fig. 9 Flexural strength of mixtures The mixtures' 7-day compressive strengths range from 30.2 to 63.9 MPa, as shown in Figure 10 a. In mixes lacking an air entrainment admixture, the SM ratio increases but the 7-day compressive strength drops. When 0.8% SM was used, the compressive strength decreased by 14.6%. If the air-entraining admixture ratio is 0.35%, the compressive strength declines below 60 MPa. In this group, the rise in the SM rate reduced the 7-day compressive strength. 0.3 or 0.5% SM in mixtures with 0.70% AEA improved the compressive strength. However, compared to the reference mixture, the compressive strength of the mixes containing 1% SM dropped by about two times. In Figure 10b is shown that the 28-day compressive strengths vary between 36.6-79.4 MPa. Using SM in mixtures without air-entraining additives slightly raised the compressive strengths. The flexural strength's adherence between the SM and the paste also raised the compressive strength. The 28-day compressive strength of the mixes using 1% SM increased by approximately 7%. Utilizing SM reduces the compressive strength of mixes containing 0.35% AEA. The mixtures in this group have compressive strengths beyond 50 MPa, nevertheless. In the case of AEA of 0.70%, the low proportion of SM improved the compressive strength. Especially if the SM was 0.30%, the 28-day compressive strength showed similar properties to the reference mixture. The fresh state properties of these two mixtures are also very close to each other. The lowest compressive strength with a value of 36.6 MPa was observed in the mixture with 0.70% AEA and 1% SM. The use of high ratios of AEA and SM in this mixture considerably reduced the compressive strength.Fig. 10 Compressive strength of mixtures The AEA used in the mixtures increased the porosity and decreased the compressive ve flexural strength. However, strength losses could be eliminated by SM in some cases. This effect indicates that SM behaves like a polypropylene fiber. SM reduces the strength losses by slowing down the crack development in the microstructure. In addition, it has been observed that the curing process is effective in mixtures with SM. It is also thought that SM retains some of the water during mixing, contributing to internal curing. In this way, the 28-day compressive strength of the mixtures with SM increased more. It has also been observed that very high compressive strengths, such as 70 MPa can be obtained in SM mixtures. In the study conducted by Özcan and Koç, the compressive strength of concretes decreased as the AEA ratio increased. Increasing porosity explains the decrease in concretes' compressive strength [49]. Compressive strength and concrete porosity are inversely connected, with strength increasing as porosity decreases [50]. Because the bubbles have taken the place of the concrete, this is the case. The bubbles cannot endure the pressure, which increases in severity over time [51]. According to research, the air-void structure's properties significantly impact the compressive strength of concrete [52]. Many studies in the literature show that the compressive and flexural strengths of concretes are increased by using polypropylene fiber [53], [54], [55]. Polypropylene fiber has a low elastic modulus, which can delay the creation and propagation of microcracks during the early stages of hardening, reducing the number of crack sources [56]. In this study, since the elasticity modules of the SMs used were lower, they prevented crack development at an early age. Crack development, which was prevented at an early age, improved the mechanical properties at later ages. At 28 and 90 days, using 0.20 percent, polypropylene fiber resulted in a slight increase in compressive strength [57]. It has been stated that polypropylene fibers' chemical and physical properties also develop strength [58]. The great fineness and varied length of fibers in staple PP fibers, which form a network that functions as a bridge and prevents the microcrack from propagating further, may explain this behavior of fiber-reinforced concrete. However, because of decreased workability and improper mixing, when the PP fiber concentration is higher, the fiber is placed non-uniformly in concrete. As a result, these fiber masses are collected to form relatively weaker places that behave as voids, making them more vulnerable to cracking and decreasing compressive strength [59]. In this study, it was determined that the optimum SM rate was 0.3%. The use of 0.3% SM in mixtures can reduce crack development. However, when the SM ratio was 0.8% and 1%, the compressive strengths decreased because weak points increased. Figure 11a shows that an R2 value of 0.83 was determined between the 7-day compressive and flexural strengths. On the 28th day, a value of 0.80 was noted. The mixtures' compressive and flexural strengths were found to be highly correlated. The flexural strength of the mixtures grows along with their compressive strength. Figure 11b illustrates the strong association between porosity and compressive strength (R2=0.79). It appears that mixtures with good compressive strength have little apparent porosity. Dry bulk density and compressive strength were found to have an R2 coefficient of 0.70. The compressive strength of the mixes grows along with their dry bulk density. Particularly, combinations with compressive strengths of 75 MPa and above have dry bulk densities of approximately 2400 kg/m3.Fig. 11 The relationship between flexural-compressive strength As seen in Figure 12 , a reduction in porosity results in an increase in compressive strength. If the apparent porosity is below 3.5%, compressive strengths of 70 MPa and above were obtained in the mixtures. However, in some mixtures, it is seen that the compressive strength decreases as the flow diameters increase. The reason for this is the use of AEA admixture in the mixes. On the other hand, as AEA increases the mixes' flow diameter, their compressive strength diminishes. As a result of AEA, the matrix becomes more porous and produces more air bubbles.Fig. 12 Relationship between compressive strength and physical properties 3.4 Transport Properties The water penetration depths of the mixtures without AEA vary between 2.8-3.6 mm (Figure 13 a). If the SM is 1% in the mixtures without AEA, the depth of water penetration decreased by about 1.3 times compared to the reference mix. Since the SM seals the connections between the capillary gaps, the depth of water penetration is decreased. However, in the combinations where the SM ratio was 0.8%, the water penetration depth was found to be 3.6 mm. The water penetration depths decreased by 3.5% compared to the reference combination when using a low SM fraction (0.3% or 0.5%). Figure 13b shows that the water penetration depths increase when the AEA ratio is 0.35%. The reference mixture with 3.5 mm had the lowest water penetration depth. Compared to the reference mixture, the mixture incorporating 0.8% SM had a 32.5% deeper water penetration depth. The water penetration depth closest to the reference mix was observed in the mixes with 0.3% SM content. In the mixtures using 0.35% AEA, the depth of water penetration generally increases as the SM volume increases. The converting of SM to microfibre structure during mixing and non-homogeneous mixing are crucial aspects of this process.Fig. 13 Water penetration depths of mixtures As seen in Figure 13c, when the AEA content is 0.70%, the depth of water penetration varies between 3.9-6.6 mm. In the case of using 0.3% SM, the water penetration depth decreased negligibly. However, if the SM was 1%, the water penetration depth increased 1.6 times than the reference mixture. This ratio was obtained 1.2 times in mixtures with 0.5% SM. The SM ratio is 0.8% or 1% in the mixtures using AEA increases the water penetration depths considerably. Since AEA affects the viscosity of the mixes, the self-compacting property may decrease. As a result, the depth of water penetration is increased by combinations with high SM concentration and AEA. Creating a blocking feature when SM material is used sparingly can lower the depth of water penetration. The relationship between water penetration depth and porosity was found to have an R2 value of 0.79, as shown in Figure 14 . The expected decrease in water penetration depths occurs when the mixes' porosity diminishes. The combinations with an apparent porosity value of less than 4%, in particular, have a water penetration depth of 3.5 mm or less. The correlation between compressive strength and water penetration depth was found to have a substantially lower R2 value (0.66). However, water penetration depths decrease as the mixes' compressive strengths rise. Mixtures with a 28-day compressive strength of more than 75 MPa have a water penetration depth of less than 3.5 mm. The findings showed an inverse link between strength and sorptivity, as Kanellopoulos et al. noted [60].Fig. 14 Correlations of water penetration depth Capillarity is accepted as a chemical resistance index in concrete technology. Because it is directly related to the resistance of materials to the penetration of aggressive chemicals [61]. The porosity and absorbency coefficient of cement-based composite materials raise with the increase in the pore volume [62]. In the investigation done by Elango et al., it was found that sorptivity rose as the mixtures' porosity rose [63]. Foam concretes with various foam contents were created by Bayraktar et al. The depths of the combinations' water penetration rise as foam concentration in foam concrete increases [64]. Usman Rashid showed that concretes' sorptivity decreased using polypropylene fiber and steel fiber [65]. Similar findings were also determined in the study by Akid et al. [66]. Studies in the literature show that fibers reduce sorptivity to a certain ratio [67]. The inclusion of fibers up to a specific volume percentage decreased the sorptivity, according to Ramezanianpour et al., but above that range, the sorptivity coefficient value raised again [67]. In this study, SM generally reduces the depth of water penetration at low ratios (0.3% or 0.5%). The water penetration depth rises when used at a 1% ratio, though. 3.5 Elevated Temperature Resistance 3.5.1 Compressive strength In Figure 15 a, the compressive strengths of the mixtures are exposed to temperatures up to 800 oC after cooling in the air. The compressive strength of the mixes diminishes as the air-entraining additive ratio rises, as seen in the figure. After 600 oC, the strength loss in mixtures utilizing air-entraining admixtures primarily accelerated. Particularly, after 800 oC, the compressive strength of the 0.70% AEA mixture declined around five times less than that of the reference mixture. This mixture was more susceptible to the effects of elevated temperatures since its 28-day compressive strength was likewise low. The mixes chilled in the air at 800 oC have compressive strengths ranging from 17.8 to 45.2 MPa. Since the air-entraining admixtures reduce the 28-day compressive strength, relatively lower strength values were determined after elevated temperatures (600 and 800 oC).Fig. 15 Effect of AEA on compressive strength after elevated temperature In Figure 15b, the compressive strengths of mixtures exposed to different temperatures are given after water cooling. The compressive strengths of the mixtures cooled with water vary between 13.6-88.3 MPa. Water cooling at low temperatures such as 100-150 oC did not damage the mixtures much. In the mixtures cooled in water, strength losses occurred from 400 oC. After 400 oC in air-entraining mixtures, water cooling reduced their compressive strength below 50 MPa. After 800 oC, the mixture containing 0.70 % AEA had an 85% lower compressive strength than the reference mixture. The rise in the air-entraining admixture ratio in the water-cooled mixtures reduced the compressive strength. However, using AEA in the cooling water application at 150-200 oC reduced the strength loss. Water cooling applied to the mixtures decreased their compressive strength more than air cooling—thermal shocks resulting from water cooling cause more damage to the ITZ between paste and paste-aggregate. As a result, the compressive strength decreases more in water-cooling. The compressive strength of the mixes reduces after elevated temperatures, as seen in Figure 16 a, as the SM ratio rises. The mixtures containing 1% SM produced compressive strengths of 50 MPa and higher up to 200 oC and 30 MPa and up to 600 oC. When the mixture with 1% SM was cooled in the air after 800 oC, the compressive strength decreased approximately three times compared to the reference mix. This ratio was found to be 1.5 times in mixtures without SM. Compressive strength increased after the 150 oC effect in mixtures without SM. This effect can be explained by the elevated temperature activating the non-hydrated cement particles. In this process, although 100 oC was ineffective, 150 oC helped to increase the strength. Since the slow cooling process did not damage the microstructure, the strength increased. In Figure 16b, strength increases up to 150 oC were observed in water-cooled mixtures. However, the compressive strength of 400 oC and later decreased considerably with the effect of water cooling. The rise in the SM rate reduced the compressive strength of the water-cooled mixtures. This effect shows that SM effectively reduces thermal shocks after 400 oC. When compared to the reference combination, the compressive strength of the mix with 1% SM after 800 oC fell roughly four times. Due to its low elastic modulus, the SM is unable to withstand the stresses caused by the heat effect.Fig. 16 Effect of SM on compressive strength after elevated temperature 3.5.2 Flexural strength The flexural strengths of the combinations cooled by air after elevated temperatures range between 1.1 and 9.1 MPa, as shown in Figure 17 a. The flexural strengths typically decrease as the AEA ratio rises. The flexural strength of the mixture with 0.7% AEA decreased approximately nine times after 800 oC. If the AEA ratio was 0.35%, the flexural strength values increased relatively after 150 and 200 oC temperatures. The vapor pressure formed in the microstructure at low temperatures is thought to contribute to internal curing. Since the rise in the AEA rate increased the porosity, the flexural strengths decreased. In addition, the flexural strength decreases as more micro-cracks occur with the thermal effect at temperatures 400 oC and beyond. As shown in Figure 17b, the flexural strengths of the water-cooled mixtures decreased below 9MPa. Relatively higher flexural strength was obtained in water-cooled mixtures at 150 and 200 oC temperatures. Since the internal vapor pressure formed at 150 and 200 oC temperatures is not very high, it increases the compressive strength. However, increasing steam pressure from 400 oC increased the microcracks in the microstructure and considerably reduced the flexural strengths. Since the water cooling process creates more thermal shock after 400 oC, the flexural strengths are decreased more than air cooling.Fig. 17 Effect of AEA on flexural strength after elevated temperature Flexural strengths can rise by up to 200 oC with an increase in the SM ratio, as shown in Figure 18 a. When cooled in the air after 100 oC, it has been found that the mixture containing 1% SM has characteristics similar to those of the reference mixture. After 800 oC, the flexural strength of the mix containing 1% SM declined almost 4.5 times less than that of the reference mixture. The flexural strength of the mixtures exposed to 800 oC fell below 3 MPa. Although the mixtures cooled slowly, most damage occurred at 600 and 800 °C. Figure 18b shows the effect of SM on water-cooled mixtures. The flexural strength is raised by using SM in water-cooled mixtures at temperatures of 100, 150, and 200 oC. In particular, the flexural strength of more than 5 MPa was obtained up to 200 oC in mixtures with 1% SM. In mixtures, the use of SM was not effective in compressive strength but more effective in flexural strength. However, this positive effect also disappears at temperatures of 400 oC and beyond. Since SM behaves like a polypropylene fiber, it increases flexural strength rather than compressive strength. SM melted relatively at elevated temperatures and increased its flexural strength by filling the air bubbles caused by AEA. In addition, the transformation of SM into a fibrous structure when melted could increase the flexural strengths after elevated temperatures. SM has determined that thermal shocks after elevated temperature are more effective in flexural strength. Although SM filled the gaps caused by AEA, it could not positively affect the compressive strength as it was soft and had low strength. However, after melting, the fibrous structure of SM improves its flexural strength.Fig. 18 Effect of AEA on flexural strength after elevated temperature Not many studies in the literature show the effect of AEA on mortars and concrete exposed to elevated temperatures. Akca and Özyurt Zihnioglu investigated the effect of AEA in high-performance concrete. The addition of AEA to the specimens reduced the loss in residual strength, although the results were unstable after 300°C for thick specimens [68]. In a study conducted by Seçer, AEA was utilized to entrain air into concrete in volume ratios of 4%, 6%, and 8%, with the concretes being exposed to temperatures of 300°C, 500°C, and 700°C. According to the findings, the loss in the strengths of concretes exposed to elevated temperatures decreased as the air content of the concrete increased [68]. Pliya et al. investigated the behavior of high-strength concrete at elevated temperatures (150, 300, 450, and 600°C) using polypropylene and steel fibers. The amount of polypropylene fibers with a 6 mm length was estimated to be between 1 and 2 kg/m3. The concrete samples were subjected to four heating and cooling cycles. The results showed that samples containing 1 kg/m3 polypropylene fiber had higher residual compressive and splitting tensile strength results when heated to 300°C. However, polypropylene fibers did not affect the remaining compressive strength after reaching 600°C [69]. Using a 15 mm long polypropylene fiber, Ding et al. examined the ultimate load, residual compressive strength, flexural toughness, fracture mechanism, and energy of self-compacting high-performance concrete maintained at 300, 600, and 900°C for 3 hours. The results demonstrated that while PP fibers had no significant effect on the material's mechanical properties, they significantly reduced the number of surface cracks [70]. Yermak et al. investigated the behavior of steel and polypropylene fiber-reinforced concrete at elevated temperatures. Polypropylene fibers can now shield steel fiber from visual fire damage by shield [71]. The thermal behavior of polypropylene fiber-reinforced concrete at elevated temperatures was the subject of Rudnik and Drzymaa's research. Temperatures of 100, 200, 300, 400, 500, and 600°C were applied to the samples. The researchers found that polypropylene fibers had no impact on the thermal stability of concrete samples. After being analyzed, the compressive strength values showed behavior similar to that of the samples before elevated temperature. This circumstance is consistent with the conclusions of the prior investigations. Compressive strength is unaffected by polypropylene fibers [72]. When polypropylene fibers melt at 170°C, they generate channels, which allow gases to escape; the pore pressure falls, and the degree of concrete damage decreases [73]. The melting of fibers allows water to be drained from the first centimeters of concrete, resulting in a significant rise in the permeability of the exposed concrete [74]. The cement matrix absorbs melted polypropylene as it approaches the melting temperature of fibers [74]. On the other hand, Fibers expand by 10% when they melt, resulting in the formation of cracks and an increase in concrete permeability [75]. In this study, SM realized these behaviours exhibited by polypropylene fiber. However, the fact that SM has a lower modulus of elasticity compared to polypropylene fiber has increased the strength losses. In addition, although SM closes the gaps belonging to the AEA when it melts, the strength losses increased because the gaps formed when it melted were larger. In the literature, increases in compressive strength have also been observed in concretes exposed to elevated temperatures. Abid et al. studied the high-temperature resistance of RPCs up to 900 oC. By 120 °C, compressive strength began to decline, but at 300 °C, all types of RPC showed a partial recovery [76]. The initial decline in compressive strength is caused by the interaction of stress and internal vapor pressure build-up [77]. The expansion of water between the C-S-H gel layers also reduced the adhesive forces [78]. The rise in Van der Walls forces (surface forces) due to the removal of free water was primarily responsible for the strength recovery at 300°C [79]. The porosity of something like concrete directly influences strength when free water is decreased [80]. When subjected to elevated temperatures, cementitious materials lose substantial mechanical strength [81]. It has been observed that concrete loses compressive strength mostly between the temperatures of 400°C and 800°C, with significant strength loss occurring between 600°C and 800°C [82]. When hydration products like Ca(OH)2 and C-S-H decay at elevated temperatures, the average pore size of cement paste grows, which causes a loss in compressive strength [83], [84]. According to Qi Zhang's research, the structure of C-S-H remains steady as the temperature rises from 105 to 400 oC. When the temperature rises above 500 oC, certain gels change into crystalline particles, and the content of capillary voids expands dramatically [85], [86]. In this study, some increases were observed in the strength of cement-based composites exposed to temperatures of 100, 150 and 200 oC. Since the permeability of high-strength cementitious composites is quite low, the efficiency of water curing application decreases. Therefore, internal curing method is recommended for high-strength cementitious composites. Hydration cannot be completed due to low permeability. However, the particles that cannot be hydrated by the effect of elevated temperature participate in hydration and increase strength. 3.5.3 Mass Loss Figure 19a shows that as the temperature applied to the mixtures and the AEA content rise, so make the mass losses. As a result of the applied temperature up to 200 oC, the weight losses in the mixtures are negligible. However, after 800 oC temperature application, the mass losses of the mixtures vary between 8.01-16.31%. At elevated temperatures, H2O in hydration products such as CSH and CH decomposes and evaporates. As shown in Figure 19b, the mass losses increased comparatively after the mixes were heated to an elevated temperature. Particularly, the range of mass losses for mixes heated to 800 oC is 13.01-21.53%. It was observed that the mass losses mainly increased at 600 and 800 oC. The mass losses increase with the increasing AEA rate in the water-cooling mix. The reason for the high mass loss in water-cooled mixtures is the breakage of parts due to thermal shock. Mass losses were significant since the compressive strength of the mixes frequently decreased as the AEA rate raised.Fig. 19 Effect of AEA on mass loss after elevated temperature As seen in Figure 20 a, similar properties were obtained up to 200 °C in air-cooled samples. Similar results were observed in mixtures exposed to 400 oC when the SM ratio was 0.8%. However, using 1% SM increased mass losses at 400 oC and above temperatures. While the mass losses of the mixtures exposed to 100 oC vary between 0.59-2.09%, the weight losses of the mixtures exposed to 800 oC vary between 11.09-14.35%. More weight losses occurred in the mixtures with 1% SM. As seen in Figure 20b, water cooling applied to the mixtures increased the mass losses. The rise in SM rate up to 200 oC did not affect the mass loss much. However, if the SM ratio was 1% in the mixtures exposed to 800 oC, the mass loss increased compared to the reference mix. In water cooling in mixtures exposed to 600 oC, the mass loss exceeds 10%. In mixtures with SM, the higher temperature than the usage rate affects the weight loss. As the temperature increases, the SM suffers more damage. In addition, SM could not greatly reduce the thermal shock effect of water cooling. Because in mixtures with SM, more mass loss occurred with the impact of water cooling. The reason for the mass loss is the breaking of parts due to the effect of thermal shock. SM polypropylene fiber has a low modulus of elasticity, so it has not prevented part breakage.Fig. 20 Effect of SM on mass loss after elevated temperature 3.5.4 The Variance of Compressive strength Changes in compressive strength were determined by comparison with 28-day compressive strength. The compressive strengths of the mixtures without air-entraining admixture increased up to 200 oC (Fig. 21 a). This effect was also observed in mixtures with 0.35% and 0.70% AEA. The compressive strength of the mixtures with 0.35% AEA increased by 16.8%-22.8%, depending on the temperature. Losses occurred in the compressive strength of the mixtures at 400 oC and above temperatures. As the AEA rate rises, the losses in the compressive strength of the mixes increase. As seen in Figure 21b, it was observed that the compressive strength raised (up to 200 oC) in the mixtures cooled with water. The fact that the ratio of AEA was 0.35% in the mixtures cooled with water increased the compressive strength more. The temperature effect of up to 200 oC increases the compressive strength of the mixtures. While there is no damage to the microstructure up to this temperature, unhydrated cement particles can react. However, at 400 oC and above temperatures, the crack network formed in the microstructure reduces the compressive strength.Fig. 21 Effect of AEA on compressive strength change after elevated temperature As seen in Figure 22 , the compressive strength reduces as the SM rate rises in both air and water cooling. The compressive strength of the mixture with 1% SM cooled in the air decreased by 54.4% (Fig. 22a). This ratio was observed as 63.2% in the mixtures cooled with water (Fig. 22b). When SM melts, it can fill some of the voids caused by AEA, but this is not enough. Therefore, as the SM rate rises, the compressive strength reduces. When SM melts, it creates larger voids and irregular voids than AEA, reducing its compressive strength.Fig. 22 Effect of SM on compressive strength change after elevated temperature 3.5.5 Visual Analysis The damage in the mixtures was more visible at 600 and 800 oC. Figure 23 a shows no significant damage occurred when the mixtures exposed to 600 oC were cooled in air. It was observed that only small parts were broken off from the corners. In Figure 23b, it was observed that the mixtures were damaged in water cooling. Especially in mixtures with low 28-day compressive strength, more damage occurred.Fig. 23 General view of mixtures exposed to 600 oC temperature As seen in Figure 24 a, spills occurred only at the corners of the mixtures cooled in air. In the samples cooled in water, both spills at the corners and map cracks appeared on the surface—the thermal shock created by water cooling after elevated temperature increases visual damage (Fig. 24b).Fig. 24 General view of mixtures exposed to 800 oC temperature As seen in Figure 24c, map cracks were observed in some mixtures. It was determined that the map cracks were mostly formed in the casting direction. In addition, it is seen that map cracks are more apparent in mixtures with AEA. Map cracks are relatively less in mixtures with high compressive strength. 3.5.6 Macrostructure after elevated temperature Figure 25 shows the macroscopic view of mixtures with AEA and SM exposed to 150 oC. The figure shows the regional filamentous structure of the SM. It has been determined that SM is mostly in the form of fiber clusters in the paste. After melting, the remaining threads of SM made bridging behavior like fibers. However, the bridging behavior was very weak due to the SM's very low modulus of elasticity.Fig. 25 Stereo Microscopy image of AEA:0.7%-SM:1% sample (at 150 oC-Air Cooling) Figure 26 shows the macro structures of the mixtures exposed to 800 oC. It is seen that SM forms square and elliptical structures on the paste surface after melting. Numerous cracks were observed on the surface where the SM came into contact with the paste. These figures show that the SM fills the ventilation slots in the mask after it melts.Fig. 26 Stereo Microscopy image of AEA:0.7%-SM:1% sample (at 800 oC-Air Cooling) 3.5.7 Microstructure after elevated temperature Figure 27 shows that the reference mixture (AEA:0%-SM:0%) has a dense microstructure at 25 oC. Needle CSH gels and hexagonal CH plates were detected in SEM images. Since no pozzolan was used in the mixtures, CH plates were frequently observed in the hydration products.Fig. 27 SEM image of AEA:0%-SM:0% sample (at 25 oC) SEM images of the mixture with an AEA content of 0.7% and SM ratio of 1% are given in Figure 28 . In SEM images, fiber-like threads are seen in some regions. These threads were formed on the cutting surfaces during the sizing of the SM. Furthermore, some washable masks release many microfibers during the washing process, which are classed as polluting elements [87], [88]. In this study, contact of SMs with mixing water and exposure to a basic environment transformed them into microfiber. In addition, the rapid mixing process in the mixer has transformed most of the SMs into microfiber. SM threads seen in SEM images are defined as microfibers. If SM was used at a low ratio (%0.3), these threads acted like fibers, increasing the flexural strength of the blends relatively. In addition, spherical air bubbles were observed with the effect of the air-entraining additive. SEM images show dense needle CSH gels. Hexagonal CH plates are also seen between the CSH gels.Fig. 28 SEM image of AEA:0.7%-SM:1% sample (at 25 oC) Figure 29 shows 1500 and 2500 magnification SEM images of the reference mixture exposed to 150 oC temperature. As seen in the figure, it was found that a dense tobermorite structure (CSH) was formed within the paste. In addition, hexagonal CH plates were also seen. It was observed that the CSH gels preserved their existence despite the temperature of 150 oC. A denser CSH structure was formed as the non-hydrated cement particles hydrated, thanks to the 150 oC temperature. This effect resulted in increased strength in mixtures exposed to 150 oC temperature.Fig. 29 SEM image of AEA:0%-SM:0% sample (at 150 oC-Air Cooling) SEM images of mixtures with SM and AEA at 150 oC are given in Figure 30 . The filamentous form of SM was determined in SEM pictures. It was also observed that SM had good adherence to paste. It has been determined that CSH gels and CH plates are frequently formed from hydration products. Since the water/cement rate of the mixtures was low, capillary voids were not observed much. It is known that the voids formed are mostly spherical, and they are formed due to the air-entraining admixture.Fig. 30 SEM image of AEA:0.7%-SM:1% sample (at 150 oC-Water Cooling) SEM image of the reference mixture cooled with water after 800 oC temperature is given in Figure 31 . It is observed that the water cooling effect damages the hydration products in the microstructure. It has been determined that the microstructure generally resembles a spongy structure with the effect of elevated temperature and shock cooling. In addition, many crack formations in the microstructure were detected.Fig. 31 SEM image of AEA:0%-SM:0% sample (at 800 oC-Water Cooling) Figure 32 shows that SMs melt under the influence of elevated temperatures. The thread form of SM melted to form air ducts. The width of the formed air ducts is about 10 microns. In addition, many air bubbles were observed with the effect of the air-entraining additive. The air ducts formed by the SM balance the internal hydrostatic pressure caused by the elevated temperature effect. However, if SM is used at a very high rate, high strength losses occur because the porosity increases too much. Crack development in air-cooled mixtures is relatively less than in water-cooled mixtures. Square-shaped structures were observed in SEM images as in stereo microscope images. These symmetrical shapes show the molten form of SM. The size of the square-shaped molten SMs ranges from 600-700 microns. It indicates that SM behaves like micro aggregate after melting. However, the low strength and modulus of elasticity of SM negatively affected the mechanical properties. Such patterns were obtained on the paste surface because the ventilation slots on the mask are square or elliptical.Fig. 32 SEM image of AEA:0.7%-SM:1% sample (at 800 oC-Air Cooling) 4 Conclusion If the SM content is more than 0.5%, the workability of the mixtures decreases. However, the air-entraining additive improved the workability in the mixtures using 1% SM. As the air-entraining additive content increased, the workability of the reference mixture increased. The flow times of the mixtures generally increase as the SM content increases. As SM increases internal friction, like polypropylene fiber, workability decreases. This is especially observed at high SM content (0.8% or 1%). For workability, optimum AEA content of 0.35% and SM content of 0.3% is appropriate. The combinations' apparent porosity and water absorption both rise when AEA level does. The porosity values increased due to AEA increasing the quantity of air bubbles in the mixture. Water absorption and porosity ratings typically rise as SM content does. The mix's air content rises as SMs often make it less workable. Low SM (0.3% or 0.5%) and high AEA (0.7%) content both reduced porosity and water absorption. By limiting the connection between the voids in mixtures with adequate workability, SMs also decreased porosity. The mixtures' dry bulk densities dropped as AEA and SM concentrations rose. The increase in SM content in the mixes without AEA raised the 28-day flexural and compressive strengths. At high AEA content (0.7%), the use of SM generally increases the 28-day flexural strengths. The microfiber behavior can explain the increase in flexural strength of SM. Similar results were observed for compressive strength. Although the modulus of elasticity of SM is low, it can still slow the propagation of cracks. This effect was more pronounced in mixtures with high porosity. Increasing the SM ratio in mixtures without AEA reduces the depth of water penetration; however, as the SM content rises in the mixtures using AEA, the water penetration depth of the mixes increases. Since AEA affects the viscosity of the mixtures, the self-compacting property decreases. As a result, SMs did not distribute homogeneously, increasing the water penetration depth of the mixtures. SM should be used in low proportions in mixtures with AEA or low workability. After 400 oC, the mixtures' mechanical qualities primarily started to deteriorate. After being exposed to elevated temperatures, the compressive strength of the combinations reduced as the AEA level rose. The compressive strength of the mixes exposed to elevated temperatures also reduced as the SM content rose. Water cooling after the elevated temperature in SM mixtures negatively affected the flexural strengths. Such an effect was observed as the shock cooling reduced the adherence between the SM and the paste. While the increase in SM content is not very effective in mass loss, the mass loss rises as the AEA content rises. It was observed that SM melted and filled some voids in the mixture. However, as the SM and AEA volume rises, the compressive strength reduces considerably. More than 50% strength loss has occurred in mixtures with 1% SM depending on water and air cooling conditions. SEM and stereomicroscope examinations revealed that the SM was partially converted to microfiber. It has also been determined that microfibers have bridging properties. It was seen in the SEM images that SM melted and filled some voids in the paste. In addition, CSH and CH structures were observed in the mixtures due to the high cement content. CSH gels (tobermorite) were also determined in the mixtures exposed to 150 oC temperature. A spongy microstructure and a network of cracks were observed in the mixtures exposed to 800 oC. As a result, it can be substituted for SM polypropylene fiber at low rates to create high-strength cement-based composites. It can be used in the AEA at modest rates to further lessen the detrimental effects of elevated temperatures. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Gökhan Kaplan: The author completed his undergraduate education at Süleyman Demirel University. He completed his doctorate education in Manisa Celala Bayar University in 2012. He conducts research on foam concrete, geopolymer and recycling agegas. Rüya Kılıç Demircan: The author completed his undergraduate, graduate and doctoral studies at Gazi University. She conducts research on the materials used in the repair of historical buildings. Damla Nur Kılıç: The author completed his undergraduate education at Gaziantep University. She is still continuing his doctorate education at Hacettepe University. She conducts research on durability issues in cement-based composites. Gökhan Durmuş: The author completed his undergraduate, graduate and doctoral studies at Gazi University. 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==== Front Lancet Reg Health Am Lancet Reg Health Am Lancet Regional Health. Americas 2667-193X The Author(s). Published by Elsevier Ltd. S2667-193X(22)00222-8 10.1016/j.lana.2022.100405 100405 Articles Severe COVID-19 outcomes in pediatrics: An observational cohort analysis comparing Alpha, Delta, and Omicron variants Bahl Amit a∗ Mielke Nicholas b Johnson Steven a Desai Ankita c Qu Lihua d a Department of Emergency Medicine, Beaumont Hospital, Royal Oak, MI, USA b Oakland University William Beaumont School of Medicine, Rochester, MI, USA c Department of Pediatric Infectious Diseases, University Hospitals Rainbow Babies and Children's Hospital, Cleveland, OH, USA d Department of Outcomes Research, Beaumont Health Research Institute, Royal Oak, MI, USA ∗ Corresponding author. Attending Physician, Department of Emergency Medicine, Beaumont Hospital, Royal Oak, 3601 13 Mile Rd, Royal Oak, MI 48073. 1 12 2022 2 2023 1 12 2022 18 100405100405 23 8 2022 3 10 2022 11 11 2022 © 2022 The Author(s) 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective COVID-19 can rarely lead to severe illness in pediatric patients. The aim of this study was to determine if severe outcomes in pediatric COVID-19 have changed over the course of the pandemic. Methods This was a multicenter, observational cohort analysis from a large regional healthcare system in metro Detroit using electronic health record data to evaluate emergency visits, hospitalization, and severe COVID-19 disease in pediatric patients. Consecutive pediatric patients presenting to the emergency department with a primary diagnosis of COVID-19 were included. Outcomes data was gathered from three distinct time intervals that coincided with Alpha, Delta, and Omicron variant predominance (Time interval 1 (T1) 1/1/2021–6/30/2021: Alpha, T2 7/1/2021–12/31/2021: Delta, T3 1/1/2022–6/16/2022): Omicron. The primary outcome was severe disease inclusive of composite intensive care unit admission, mechanical ventilation, multisystem inflammatory syndrome in children (MIS-C), myocarditis, or death. Secondary outcomes included severe outcomes considering viral coinfection and vaccination status. Results Between 1/1/2021 and 6/16/2022, there were 4517 emergency COVID-19 visits, of which 12.5% (566) of children were hospitalized. 24.4% (138), 31.6% (179), and 44.0% (249) of admissions occurred during T1, T2 and T3 respectively. Most patients were male (55.1%) and 59.9% identified as Caucasian. The median age was 5.0 (interquartile range 1.0, 13.0) with infants comprising 22.8% (129), toddlers 25.1% (142), children 23.0% (130), and teenagers 29.2% (165). Over the course of the pandemic, the proportion of infants in hospitalization increased from 16.7% in T1 to 19.6% in T2 to 28.5% in T3 (p < 0.01) while the proportion of teenagers in hospitalization decreased from 39.1% in T1 to 31.3% in T2 to 22.1% in T3 (p < 0.001). Oxygen therapy was required in a minority (29.9%) of cases with supplemental oxygen utilized the least in T3 (16.5%) and most in T2 (30.2%). Composite severe disease decreased throughout the pandemic occurring in 36.2% in T1, 27.4% in T2, and 18.9% in T3. A multivariable logistic regression analysis revealed the odds of composite severe disease was significantly lower in T3 compared to T1 (adjusted odds ratio [aOR] 0.35, 95% Confidence Interval 0.21–0.60, p < 0.001). Fully vaccinated or fully vaccinated and boosted admission rates remained low throughout all periods with 4.4% in T1, 4.5% in T2 and 8.4% in T3. Viral coinfection was most common during T2 (16.8%) followed by T3 (12.5%) and least common in T1 (5.1%) (p = 0.006). Coinfection occurred more commonly in younger children with a median age of 1.2 (0.0, 4.5) compared to those with mono-infection with a median age of 6 (1.0, 14.0) (p < 0.001). Severe outcomes occurred in 45.6% of coinfection cases compared to 22.1% without coinfection (p < 0.001). Conclusions While Omicron cases had the highest admission frequency, severe illness was lower than Delta and Alpha variants. Coinfection with respiratory viruses increased the risk of severe outcomes and impacted infants more than older children. Funding None. Keywords SARS-CoV-2 COVID-19 Omicron Delta Alpha Viral co-infection Pediatrics Children ==== Body pmc Research in context Evidence before this study On June 16, 2022, we utilized PubMed to review data regarding severe outcomes in pediatric patients infected with COVID-19. Given the relatively low hospitalization rate in this population compared to the adult population, limited data were available, especially during the recent Omicron-predominant period. We utilized the search terms (COVID-19 OR novel coronavirus OR SARS-CoV-2) AND (emergency visit OR hospitalization) AND (pediatric OR children OR infants). No time or language restrictions were used. Several large trials from early in the COVID-19 pandemic evaluated pediatric outcomes, however, there was limited data available from more recent periods of increased hospitalization among the pediatric population. Furthermore, data regarding vaccination effectiveness and viral co-infection was limited. Added value of this study Our observational cohort analysis of a large, eight-hospital healthcare system in metro Detroit, Michigan, United States, evaluated the emergency visits and hospitalizations of pediatric patients infected with COVID-19 and assessed for severe outcomes, including intensive care unit admission at any time during hospitalization, need for mechanical ventilation, multisystem inflammatory syndrome in children (MIS-C), myocarditis, or in-hospital death. We found that the odds of composite severe disease were significantly lower during the Omicron-predominant time period compared to the alpha-predominant time period (adjusted odds ratio 0.35, 95% Confidence Interval 0.21–0.60, p < 0.001). Additionally, viral co-infection was associated with a higher frequency of severe outcomes (p < 0.001). Implications of all the available evidence As the COVID-19 pandemic continues and mutations lead to new variants, it is important to continuously assess severe outcomes among the pediatric population. Introduction Since the beginning of the pandemic, over 14 million children have been infected with SARS-CoV-2 infection representing 18.6% of all cases in the United States.1 While hospitalization of children has been less frequent than their adult counterparts (21.37 admissions per 100,000 for adults age 70 and older),2 the rate of hospitalization for children has peaked recently at 1.25 admissions per 100,000 during the Omicron predominant time period.2 These hospitalization rates may be an overestimate, however, given the number of unconfirmed cases that occur.3 When hospitalization is required, children commonly require supplemental oxygen therapy but, in some cases, can deteriorate and require vasopressors or mechanical ventilation. Several larger trials have evaluated pediatric outcomes from earlier phases of the pandemic.4, 5, 6, 7 However, as the virus has mutated over time, it is unclear if the risk of severe outcomes in pediatrics has shifted, particularly including the Omicron predominant time frame. Although vaccination efforts have had more time to filter through the pediatric population, the rates of transmission and hospitalization have peaked in the past 6 months.8 Given this trend, more real-world data is needed to examine hospitalization and severe outcomes in children. Therefore, we aimed to examine and compare pediatric COVID-19 outcomes in three-time intervals that correspond with a predominant variant strain. Further, we explored the impact of vaccination and viral co-infection on severe outcomes. Methods Study design, setting, and participants This multicenter, observational cohort analysis utilized electronic health records (EHR; Epic Systems, Verona, WI, USA) to evaluate outcomes in pediatric patients over three distinct time periods during the pandemic. The study was conducted at Beaumont Health, an eight-hospital acute care regional health system caring for 2.2 million people across the communities within the Metro Detroit area. The hospitals range from a large tertiary care academic center to intermediate-sized and smaller community hospitals. Beaumont Children's sees over 200,000 children annually with pediatric and neonatal units at three sites. Consecutive patients less than 18 years old who presented to one of Beaumont Health's emergency departments (EDs) between January 1, 2021, and June 16, 2022, who had a principal diagnosis of COVID-19 (U07.1) were included. Patients were excluded if they had COVID-19 (U07.1) as a secondary diagnosis. Further, patients were excluded if transferred out of the health system and investigators did not have access to the transfer records. The Beaumont Institutional Review Board approved this investigation. Written informed consent was waived due to the retrospective nature of this study. Severe disease was defined as a composite outcome of intensive care unit (ICU) admission at any time during hospitalization, need for mechanical ventilation, multisystem inflammatory syndrome in children (MIS-C), myocarditis, or in-hospital death. Three distinct time segments, each approximating six months, were assessed during this study. Time interval 1 (T1) occurred 1/1/2021–6/30/2021 and coincided with Alpha variant predominance. T2 occurred 7/1/2021–12/31/2021 and coincided with Delta variant predominance. T3 occurred 1/1/2022–6/16/2022 and coincided with Omicron variant predominance. Demographic, clinical, and outcomes data were obtained from the EHR. Demographics included age, race, and sex. Clinical data included body mass index (BMI), in hospital therapies such as supplemental oxygen, high flow oxygen, mechanical ventilation, and intensive care admission. Outcomes data included severe disease, MIS-C, myocarditis, viral co-infection, length of stay, disposition, and death. Comorbid conditions were grouped via ICD-10-CM code classifications from the Pediatric Complex Chronic Conditions Classification System Version 2.9 Patients were classified as immunocompromised if their clinical record contained any historical International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes consistent with the immunocompromised state as defined by the Agency of Healthcare Research and Quality (AHRQ) at the time of their ED presentation.10 EHR data was used to confirm SARS-CoV-2 vaccination status. This data was available through our EHR and linked to the Michigan Care Improvement Registry (MCIR) and therefore captured patients who had been vaccinated outside of the Beaumont Health System.11 MCIR contains all SARS-CoV-2 immunization data for patients who received their vaccine within the state of Michigan. This data included vaccine type as well as the date of administration. Viral co-infection was determined by query of commonly performed laboratory testing for pediatric patients with a presumed viral illness. The query consisted of a respiratory virus panel, obtained by nasopharyngeal swab and tested via multiplex nucleic acid amplification (NAA), which detects Influenza A (including subtypes H1, H3, and H1N1), Influenza B, respiratory syncytial virus (RSV) (A/B), Metapneumovirus, Rhinovirus/Enterovirus, Adenovirus, Parainfluenza virus 1–4, Coronavirus (SARS-CoV-2, 229E, NL63, HKU1, and OC43), and Bocavirus. The query also consisted of an Influenza A/B and RSV NAA panel obtained by nasopharyngeal swab and an ED-specific panel for COVID-19, Influenza A/B, RSV by NAA. All tests were performed at Beaumont Laboratories. Outcomes and measurements The primary outcome of this study was severe disease and included composite ICU admission during hospitalization, mechanical ventilation, MIS-C, myocarditis, or death. Secondary outcomes included severe disease in patients with viral co-infection and with vaccination. Statistical analysis Descriptive analysis was used to summarize patient characteristics. Numerical variables were reported as means with standard deviations or medians with interquartile ranges. Categorical variables were expressed as counts and frequencies (percentages). The Chi-square or Fisher's exact test was used for categorical variables and the Kruskal–Wallis (exact) test was used for numerical variables. Logistic regression was used to assess the effect of the predominant variant at the time of COVID-19 infection on the composite severe outcome for hospitalized patients. All tests performed in this analysis were two-sided tests. Analysis was performed using R-4.1.2 (R Foundation for Statistical Computing) and Excel (Microsoft). Ethics committee approval This study was approved by the Beaumont Health Institutional Review Board. Role of the funding source This research received no specific grant from any funding agency in public, commercial, or not-for-profit sectors. Results Between 1/1/2021 and 6/16/2022, there were 4517 pediatric emergency room encounters for COVID-19. 17.6% (795) of encounters occurred during T1 – Alpha, 38.0% (1714) of encounters during T2 – Delta, and 44.5% (2008) occurred during T3 – Omicron. In total, 3951 (87.5%) were discharged while 566 (12.5%) were admitted. Of the 53 transferred patients, 26 (49%) were included within the hospitalized cohort and 27 (50.9%) were excluded due to a lack of access to outside hospital records. In our cohort, the median age was 5.2 (interquartile range (IQR) 1.0, 13.0) years old with infants (<1 year old) comprising 17.8% (806), toddlers (age 1–4) 28.2% (1274), children (age 5–11) 25.3% (1142), and teenagers (age 12–17) 28.7% (1295). Nearly half (48.4%) of the cohort was female, and a small minority were fully vaccinated or fully vaccinated and boosted (4.7%), with 95.3% being partially vaccinated or unvaccinated (Table 1 ).Table 1 Demographics, in-hospital therapies, and outcomes among emergency room encounters with COVID-19 during Alpha, Delta, and Omicron predominant time periods. Variablesa Time period p valued T1 (Alpha) T2 (Delta) T3 (Omicron) n 4517 795 (17.60%) 1714 (37.95%) 2008 (44.45%) Demographics Age, years Mean 6.88 (5.97) 8.34 (6.25) 7.45 (6.03) 5.81 (5.61) <0.001b Median 5.20 (1.00, 13.00) 9.00 (2.00, 15.00) 6.15 (1.00, 13.00) 4.00 (1.00, 10.00) Age, category Infant (age 0) 806 (17.84%) 100 (12.58%) 276 (16.10%) 430 (21.41%) <0.001c Toddler (age 1–4) 1274 (28.20%) 200 (25.16%) 444 (25.90%) 630 (31.37%) Child (age 5–11) 1142 (25.28%) 174 (21.89%) 445 (25.96%) 523 (26.05%) Teen (age 12–17) 1295 (28.67%) 321 (40.38%) 549 (32.03%) 425 (21.17%) Sex Female 2187 (48.42%) 396 (49.81%) 854 (49.82%) 937 (46.66%) 0.108c Male 2330 (51.58%) 399 (50.19%) 860 (50.18%) 1071 (53.34%) Race Black or African American 1974 (43.70%) 386 (48.55%) 817 (47.67%) 771 (38.40%) <0.001c White or Caucasian 2107 (46.65%) 332 (41.76%) 745 (43.47%) 1030 (51.29%) Other 436 (9.65%) 77 (9.69%) 152 (8.87%) 207 (10.31%) BMI, kg/m2 (n = 2745/519/1013/1213) Mean 20.39 (7.47) 21.99 (7.88) 21.21 (8.03) 19.01 (6.51) <0.001b Median 18.31 (15.53, 22.86) 19.58 (16.55, 25.42) 18.75 (15.80, 24.33) 17.48 (15.09, 21.01) Vaccination status Boosted 17 (0.38%) 0 (0.00%) 0 (0.00%) 17 (0.85%) <0.001c Fully vaccinated 195 (4.32%) 6 (0.75%) 41 (2.39%) 148 (7.37%) Partially vaccinated 50 (1.11%) 7 (0.88%) 17 (0.99%) 26 (1.29%) Unvaccinated 4255 (94.20%) 782 (98.36%) 1656 (96.62%) 1817 (90.49%) Discharge location Home 3951 (87.47%) 657 (82.64%) 1535 (89.56%) 1759 (87.60%) <0.001c Admit 566 (12.53%) 138 (17.36%) 179 (10.44%) 249 (12.40%) Comorbidities (n = 4440) Neurologic and neuromuscular 37 (0.83%) 8 (1.05%) 8 (0.47%) 21 (1.06%) 0.118c Cardiovascular 29 (0.65%) 9 (1.18%) 6 (0.36%) 14 (0.70%) 0.056c Respiratory 9 (0.20%) 1 (0.13%) 3 (0.18%) 5 (0.25%) 0.909c Renal and urologic 11 (0.25%) 2 (0.26%) 4 (0.24%) 5 (0.25%) 1.000c Gastrointestinal 32 (0.72%) 5 (0.66%) 11 (0.65%) 16 (0.81%) 0.836c Hematologic or immunologic 43 (0.97%) 8 (1.05%) 15 (0.89%) 20 (1.01%) 0.907c Metabolic 60 (1.35%) 10 (1.31%) 18 (1.07%) 32 (1.61%) 0.359c Other congenital or genetic defect 23 (0.52%) 6 (0.79%) 9 (0.53%) 8 (0.40%) 0.411c Malignancy 5 (0.11%) 0 (0.00%) 0 (0.00%) 5 (0.25%) 0.081c Premature and neonatal 13 (0.29%) 5 (0.66%) 1 (0.06%) 7 (0.35%) 0.021c Immunocompromise 93 (2.09%) 19 (2.49%) 29 (1.72%) 45 (2.26%) 0.360c Abbreviations: BMI = body mass index; T1 = time period 1 (1/1/21–6/30/21); T2 = time period 2 (7/1/21–12/31/21); T3 = time period 3 (1/1/22–6/16/22). a For continuous variables, medians (interquartile ranges, IQRs) and means (standard deviation, SD) were presented. For categorical variables, frequencies (percentage) were presented. b Kruskal–Wallis test. c Chi-squared or Fisher's exact test. d See Supplementary Table S1 for p-values from multiple comparisons test; post hoc Holm-Bonferroni procedure and the Tukey–Kramer method was used for categorical and numerical variables, respectively. Among the 566 hospitalized patients, 24.4% (138), 31.6% (179), and 44.0% (249) occurred during T1, T2, and T3, respectively. The median age was 5.0 (IQR 1.0, 13.0), with infants comprising 22.8% (129), toddlers 25.1% (142), children 23.0% (130), and teenagers 29.2% (165). Over the course of the pandemic, the proportion of infants in hospitalization increased from 16.7% in T1 to 19.6% in T2 and 28.5% in T3 (p < 0.01), while the proportion of teenagers in hospitalization decreased from 39.1% in T1 to 31.3% in T2 and 22.1% in T3 (p < 0.001). Most patients were male (55.1%) and 59.9% identified as White or Caucasian. Fully vaccinated or fully vaccinated and boosted admission rates remained low throughout all periods, with 4.4% in T1, 4.5% in T2 and 11.6% in T3. Oxygen therapy was required in a minority (29.9%) of cases, with supplemental oxygen utilized the least in T3 (24.5%) and the most in T2 (38.0%). Composite severe disease decreased throughout the pandemic, occurring in 36.2% in T1, 27.4% in T2, and 18.8% in T3 (Table 2 ). A multivariable logistic regression analysis revealed the odds of composite disease was significantly lower in T3 compared to T1 (adjusted odds ratio [aOR] 0.35, 95% Confidence Interval 0.21–0.60, p < 0.001) (Table 3 ). There was a significant difference in the number of new MIS-C cases among hospitalized patients, with 17.4% in T1, 10.1% in T2, and 6.4% in T3 (p < 0.001). There were three total deaths.Table 2 Demographics, in-hospital therapies, and outcomes among hospitalized patients with COVID-19 during Alpha, Delta, and Omicron predominant time periods. Variablesa All Time Period p valued T1 (Alpha) T2 (Delta) T3 (Omicron) n 566 138 (24.38%) 179 (31.63%) 249 (43.99%) Demographics Age, years Mean 6.68 (6.19) 8.05 (6.34) 7.16 (6.30) 5.57 (5.85) <0.001b Median 5.00 (1.00, 13.00) 7.55 (1.05, 14.35) 5.90 (1.00, 13.80) 3.00 (0.00, 10.00) Age, category Infant (age 0) 129 (22.79%) 23 (16.67%) 35 (19.55%) 71 (28.51%) 0.011c Toddler (age 1–4) 142 (25.09%) 31 (22.46%) 45 (25.14%) 66 (26.51%) Child (age 5–11) 130 (22.97%) 30 (21.74%) 43 (24.02%) 57 (22.89%) Teen (age 12–17) 165 (29.15%) 54 (39.13%) 56 (31.28%) 55 (22.09%) Sex Female 254 (44.88%) 61 (44.20%) 78 (43.58%) 115 (46.18%) 0.852c Male 312 (55.12%) 77 (55.80%) 101 (56.42%) 134 (53.82%) Race Black or African American 160 (28.27%) 49 (35.51%) 45 (25.14%) 66 (26.51%) 0.190c White or Caucasian 339 (59.89%) 73 (52.90%) 109 (60.89%) 157 (63.05%) Other 67 (11.84%) 16 (11.59%) 25 (13.97%) 26 (10.44%) BMI, kg/m2 (n = 547/133/171/243) Mean 20.13 (7.74) 21.07 (7.96) 21.10 (9.16) 18.93 (6.25) 0.018b Median 17.94 (15.57, 21.73) 18.28 (15.77, 22.93) 17.94 (15.96, 22.31) 17.66 (15.27, 20.31) Vaccination status Boosted 8 (1.41%) 0 (0.00%) 0 (0.00%) 8 (3.21%) 0.005c Fully vaccinated 35 (6.18%) 6 (4.35%) 8 (4.47%) 21 (8.43%) Partially vaccinated 15 (2.65%) 2 (1.45%) 3 (1.68%) 10 (4.02%) Unvaccinated 508 (89.75%) 130 (94.20%) 168 (93.85%) 210 (84.34%) In-hospital therapies O2 therapy 169 (29.86%) 40 (28.99%) 68 (37.99%) 61 (24.50%) 0.010c Nasal cannula/non-rebreather 125 (22.08%) 30 (21.74%) 54 (30.17%) 41 (16.47%) 0.003c High flow O2 39 (6.89%) 9 (6.52%) 19 (10.61%) 11 (4.42%) 0.043c Primary outcomes Composite severe disease 146 (25.80%) 50 (36.23%) 49 (27.37%) 47 (18.88%) <0.001c ICU-level care 119 (21.02%) 40 (28.99%) 41 (22.91%) 38 (15.26%) 0.005c Mechanical ventilation 12 (2.12%) 1 (0.72%) 3 (1.68%) 8 (3.21%) 0.249c MIS-C 58 (10.25%) 24 (17.40%) 18 (10.06%) 16 (6.43%) 0.003c Myocarditis 6 (1.06%) 5 (3.62%%) 0 (0.00%) 1 (0.40%) 0.005c Death 3 (0.53%) 2 (1.45%) 0 (0.00%) 1 (0.40%) – Secondary outcomes Viral co-infection 68 (12.01%) 7 (5.07%) 30 (16.76%) 31 (12.45%) 0.006c Length of stay, hours Mean 69.43 (165.55) 61.37 (57.46) 62.46 (64.89) 78.90 (239.65) 0.323b Median 42.00 (24.00, 71.00) 41.11 (24.04, 72.60) 46.00 (25.00, 72.00) 38.00 (22.25, 68.00) Abbreviations: BMI = body mass index; ICU = intensive care unit; MIS-C = multisystem inflammatory syndrome in children; T1 = time period 1 (1/1/21–6/30/21); T2 = time period 2 (7/1/21–12/31/21); T3 = time period 3 (1/1/22–6/16/22). a For continuous variables, medians (interquartile ranges, IQRs) and means (standard deviation, SD) were presented. For categorical variables, frequencies (percentage) were presented. b Kruskal–Wallis test. c Chi-squared or Fisher's exact test. d See Supplementary Table S2A for p-values from multiple comparisons test; post hoc Holm-Bonferroni procedure and the Tukey–Kramer method was used for categorical and numerical variables, respectively. Table 3 Association between predominant variant at time of COVID-19 infection and the composite severe disease. Effect Model 1a Model 2b ORa (95% CI) aORb (95% CI) Predominant variant at time T1 (Alpha) Reference Reference T2 (Delta) 0.66 (0.41–1.07) p = 0.092 0.68 (0.41–1.12) p = 0.129 T3 (Omicron) 0.41 (0.26–0.66) p < 0.001 0.35 (0.21–0.60) p < 0.001 Abbreviations: aOR = adjusted odds ratio; CI = confidence interval; OR = odds ratio; T1 = time period 1 (1/1/21–6/30/21); T2 = time period 2 (7/1/21–12/31/21); T3 = time period 3 (1/1/22–6/16/22). a Unadjusted multivariable logistic regression analysis in the hospitalized cohort. b Adjusted multivariable logistic regression analysis in the hospitalized cohort, adjusting for age group, gender, race, body mass index, and vaccination status. The presence of viral coinfection amongst hospitalized patients was most common during T2 (16.8%), followed by T3 (12.5%) and least common in T1 (5.1%) (p = 0.006). Coinfection occurred more commonly in younger children, with a median age of 1.2 (0.0, 4.5) years old compared to those without coinfection who had a median age of 6.0 (1.0, 14.0) years old (p < 0.001). Hospitalized patients with viral coinfection were more likely to require supplemental oxygen therapy (58.8% vs 25.9%; p < 0.001). High-flow oxygen therapy was utilized in 27.9% of coinfection cases compared to 4.0% of cases without coinfection (p < 0.001). Compared to those without coinfection, pediatric patients with viral coinfection were significantly more likely to have a severe outcome (45.6% vs 22.1%; p < 0.001) (Table 4 ).Table 4 Demographics, in-hospital therapies, and outcomes among hospitalized COVID-19 patients with and without viral coinfection. Variablesa All Viral coinfection p value Time period p valued Yes No T1 (Alpha) T2 (Delta) T3 (Omicron) n 566 68 (12.01%) 498 (87.99%) 7 (10.29%) 30 (44.12%) 31 (45.59%) Demographics Age, years Mean 6.68 (6.19) 3.10 (4.17) 7.17 (6.26) <0.001b 1.59 (2.39) 3.35 (5.00) 3.20 (3.61) 0.526b Median 5.00 (1.00, 13.00) 1.20 (0.00, 4.47) 6.00 (1.00, 14.00) 1.00 (0.00, 1.70) 1.00 (0.00, 3.97) 2.00 (0.00, 6.00) Age, category Infant (age 0) 129 (22.79%) 22 (32.35%) 107 (21.49%) <0.001c 3 (42.86%) 9 (30.00%) 10 (32.26%) 0.820c Toddler (age 1–4) 142 (25.09%) 29 (42.65%) 113 (22.69%) 3 (42.86%) 14 (46.67%) 12 (38.71%) Child (age 5–11) 130 (22.97%) 13 (19.12%) 117 (23.49%) 1 (14.29%) 4 (13.33%) 8 (25.81%) Teen (age 12–17) 165 (29.15%) 4 (5.88%) 161 (32.33%) 0 (0.00%) 3 (10.00%) 1 (3.23%) Sex Female 254 (44.88%) 26 (38.24%) 228 (45.78%) 0.297c 1 (14.29%) 13 (43.33%) 12 (38.71%) 0.397c Male 312 (55.12%) 42 (61.76%) 270 (54.22%) 6 (85.71%) 17 (56.67%) 19 (61.29%) Race Black or African American 160 (28.27%) 16 (23.53%) 144 (28.92%) 0.371c 2 (28.57%) 4 (13.33%) 10 (32.26%) 0.054c White or Caucasian 339 (59.89%) 46 (67.65%) 293 (58.84%) 4 (57.14%) 21 (70.00%) 21 (67.74%) Other 67 (11.84%) 6 (8.82%) 61 (12.25%) 1 (14.29%) 5 (16.67%) 0 (0.00%) BMI, kg/m2 (n = 547/66/481/7/29/30) Mean 20.13 (7.74) 16.93 (2.73) 20.57 (8.10) <0.001b 18.02 (3.39) 17.18 (2.85) 16.44 (2.42) 0.558b Median 17.94 (15.57, 21.73) 16.61 (15.09, 18.52) 18.23 (15.62, 22.33) 16.53 (15.26, 21.24) 16.64 (15.46, 18.55) 16.38 (14.98, 17.90) Vaccination status Boosted 8 (1.41%) 1 (1.47%) 7 (1.41%) 0.290c 0 (0.00%) 0 (0.00%) 1 (3.23%) 0.717c Fully vaccinated 35 (6.18%) 1 (1.47%) 34 (6.83%) 0 (0.00%) 0 (0.00%) 0 (0.00%) Partially vaccinated 15 (2.65%) 1 (1.47%) 14 (2.81%) 0 (0.00%) 0 (0.00%) 1 (3.23%) Unvaccinated 508 (89.75%) 65 (95.59%) 443 (88.96%) 7 (100.00%) 30 (100.00%) 28 (90.32%) In-hospital therapies O2 therapy 169 (29.86%) 40 (58.82%) 129 (25.90%) <0.001c 5 (71.43%) 17 (56.67%) 18 (58.06%) 0.878c NC/NRB 125 (22.08%) 22 (32.35%) 103 (20.68%) 0.043c 1 (14.29%) 13 (43.33%) 8 (25.81%) 0.232c High flow O2 39 (6.89%) 19 (27.94%) 20 (4.02%) <0.001c 4 (57.14%) 6 (20.00%) 9 (29.03%) 0.128c Primary outcomes Composite severe disease 141 (24.91%) 31 (45.59%) 110 (22.09%) <0.001c 6 (85.71%) 12 (40.00%) 13 (41.94%) 0.090c ICU-level care 119 (21.02%) 28 (41.18%) 91 (18.27%) <0.001c 5 (71.43%) 11 (36.67%) 12 (38.71%) 0.273c Mechanical ventilation 12 (2.12%) 2 (2.94%) 10 (2.01%) 0.645c 0 (0.00%) 1 (3.33%) 1 (3.23%) 1.000c MIS-C 58 (10.25%) 9 (13.24%) 49 (9.84%) 0.514c 2 (28.57%) 5 (16.67%) 3 (9.68%) 0.315c Myocarditis 6 (1.06%) 1 (1.47%) 5 (1.00%) 0.538c 0 (0.00%) 1 (3.33%) 0 (0.00%) 0.544c Death 3 (0.53%) 0 (0.00%) 3 (0.60%) – 0 (0.00%) 0 (0.00%) 0 (0.00%) – Secondary outcome Length of stay, hours Mean 69.43 (165.55) 57.64 (42.66) 71.04 (175.76) 0.228b 40.27 (16.42) 53.25 (44.50) 65.81 (44.02) 0.129b Median 42.00 (24.00, 71.00) 45.50 (28.00, 73.05) 42.00 (23.00, 71.00) 40.00 (28.50, 50.11) 44.11 (25.25, 69.75) 49.00 (38.00, 82.00) Abbreviations: BMI = body mass index; ICU = intensive care unit; MIS-C = multisystem inflammatory syndrome in children; NC = nasal cannula; NRB = non-rebreather; T1 = time period 1 (1/1/21–6/30/21); T2 = time period 2 (7/1/21–12/31/21); T3 = time period 3 (1/1/22–6/16/22). a For continuous variables, medians (interquartile ranges, IQRs) and means (standard deviation, SD) were presented. For categorical variables, frequencies (percentage) were presented. b Kruskal–Wallis test. c Chi-squared or Fisher's exact test. d See Supplementary Table S2B for p-values from multiple comparisons test; post hoc Holm-Bonferroni procedure and the Tukey–Kramer method was used for categorical and numerical variables, respectively. There were 286 admitted patients who were eligible to receive the COVID-19 vaccine during the study period, of which 43 (15.0%) were fully vaccinated or fully vaccinated and boosted. The median age of vaccinated patients was 15.0 (IQR 13.0, 16.0) compared to 12.0 (IQR 8.0, 15.0) for unvaccinated (p < 0.001). Compared to 35.4% of unvaccinated encounters, only 11.6% of vaccinated patients required supplemental oxygen therapy (p = 0.004). There was no difference in composite severe outcomes comparing unvaccinated (25.6%) and vaccinated (25.9%) (p = 1.000). Likewise, the need for ICU-level care was similar among pediatric patients regardless of vaccination status (25.6% vs 25.9%; p = 1.000). MIS-C occurred in 9.3% of vaccinated patients and in 15.2% of unvaccinated patients (p = 0.432). Of the four immunized patients who developed MIS-C, three cases occurred during T3 while one occurred during T2. There was no difference in the occurrence of myocarditis among vaccinated patients (2.3%) compared to unvaccinated patients (2.1%) (p = 1.000) (Table 5 ).Table 5 Demographics, in-hospital therapies, and outcomes among hospitalized COVID-19 patients based on vaccination status. Variablesa All Vaccination status p value Time period p valued ≥2 immunizations <2 immunizations T1 (Alpha) T2 (Delta) T3 (Omicron) n 286 43 (15.03%) 243 (84.97%) 6 (13.95%) 8 (18.60%) 29 (67.44%) Demographics Age, years Mean 12.01 (3.94) 14.00 (3.01) 11.66 (3.99) <0.001b 15.20 (1.19) 14.96 (1.54) 13.48 (3.45) 0.595b Median 13.00 (8.22, 15.70) 15.00 (13.00, 16.00) 12.00 (8.00, 15.00) 15.25 (14.38, 15.60) 15.50 (13.47, 16.00) 14.60 (12.00, 16.00) Age, category Child (age 5–11) 123 (43.01%) 7 (16.28%) 116 (47.74%) <0.001c 0 (0.00%) 0 (0.00%) 7 (24.14%) 0.246c Teen (age 12–17) 163 (56.99%) 36 (83.72%) 127 (52.26%) 6 (100.00%) 8 (100.00%) 22 (75.86%) Sex Female 132 (46.15%) 22 (51.16%) 110 (45.27%) 0.583c 1 (16.67%) 4 (50.00%) 17 (58.62%) 0.182c Male 154 (53.85%) 21 (48.84%) 133 (54.73%) 5 (83.33%) 4 (50.00%) 12 (41.38%) Race Black or African American 86 (30.07%) 9 (20.93%) 77 (31.69%) 0.287c 0 (0.00%) 2 (25.00%) 7 (24.14%) 0.579c White or Caucasian 169 (59.09%) 30 (69.77%) 139 (57.20%) 5 (83.33%) 5 (62.50%) 20 (68.97%) Other 31 (10.84%) 4 (9.30%) 27 (11.11%) 1 (16.67%) 1 (12.50%) 2 (6.90%) BMI, kg/m2 (n = 275/42/233/6/7/29) Mean 23.55 (9.25) 23.80 (7.51) 23.50 (9.54) 0.229b 26.09 (5.07) 25.54 (12.29) 22.91 (6.56) 0.200b Median 20.79 (17.27, 25.60) 21.73 (19.57, 25.40) 20.76 (17.06, 25.65) 24.06 (22.09, 30.41) 22.29 (18.44, 26.28) 20.24 (19.25, 25.01) In-hospital therapies O2 therapy 91 (31.82%) 5 (11.63%) 86 (35.39%) 0.004c 1 (16.67%) 1 (12.50%) 3 (10.34%) 0.803c NC/NRB 71 (24.83%) 5 (11.63%) 66 (27.16%) 0.048c 1 (16.67%) 1 (12.50%) 3 (10.34%) 0.803c High flow O2 17 (5.94%) 1 (2.33%) 16 (6.58%) 0.484c 0 (0.00%) 1 (12.50%) 0 (0.00%) 0.326c Primary outcomes Composite event 74 (25.87%) 11 (25.58%) 63 (25.93%) 1.000c 4 (66.67%) 3 (37.50%) 4 (13.79%) 0.013c ICU-level care 73 (25.52%) 11 (25.58%) 62 (25.51%) 1.000c 4 (66.67%) 3 (37.50%) 4 (13.79%) 0.013c Mechanical ventilation 7 (2.45%) 0 (0.00%) 7 (2.88%) 0.599c 0 (0.00%) 0 (0.00%) 0 (0.00%) – MIS-C 44 (15.38%) 4 (9.30%) 40 (16.46%) 0.007c 0 (0.00%) 1 (12.50%) 3 (10.34%) 1.000c Myocarditis 6 (2.10%) 1 (2.33%) 5 (2.06%) 1.000c 0 (0.00%) 1 (12.50%) 0 (0.00%) 1.000c Death 2 (0.70%) 0 (0.00%) 2 (0.82%) – 0 (0.00%) 0 (0.00%) 0 (0.00%) – Secondary outcomes Viral co-infection 17 (5.94%) 2 (4.65%) 15 (6.17%) 1.000c 0 (0.00%) 0 (0.00%) 2 (6.90%) 1.000c Length of stay, hours Mean 92.07 (228.14) 100.80 (164.62) 90.53 (237.86) 1.000b 60.50 (20.53) 102.96 (78.52) 108.55 (196.52) 0.540b Median 48.00 (26.00, 92.59) 48.63 (25.09, 100.72) 48.00 (26.00, 92.39) 68.82 (53.42, 73.04) 86.50 (37.95, 169.33) 34.00 (24.00, 76.00) Abbreviations: BMI = body mass index; ICU = intensive care unit; MIS-C = multisystem inflammatory syndrome in children; NC = nasal cannula; NRB = non-rebreather; T1 = time period 1 (1/1/21–6/30/21); T2 = time period 2 (7/1/21–12/31/21); T3 = time period 3 (1/1/22–6/16/22). a For continuous variables, medians (interquartile ranges, IQRs) and means (standard deviation, SD) were presented. For categorical variables, frequencies (percentage) were presented. b Kruskal–Wallis test. c Chi-squared or Fisher's exact test. d See Supplementary Table S2C for p-values from multiple comparisons test; post hoc Holm-Bonferroni procedure and the Tukey–Kramer method was used for categorical and numerical variables, respectively. Discussion This study highlighted trends over the course of the pandemic regarding severe COVID-19 outcomes in pediatric patients. While the overwhelming majority of cases in the community are self-limited, this study focused on encounters requiring the most resources and medical attention.12 , 13 It is notable that the three distinct time intervals represent roughly equal periods of Alpha, Delta, and Omicron variant dominance, therefore providing a balanced comparison. However, it is noteworthy that the variant data was not patient-specific and represents a good estimate rather than an exact comparison of variant type. Importantly, nearly half of all ED encounters and hospitalizations in the cohort were from the most recent Omicron period. There are several explanations for this observation. It has been well described that Omicron is more transmissible than other variants.14 One study found that the Omicron variant replicates 70 times faster than the Delta variant in airways.15 Further, as mask mandates and social distancing practices have diminished in various communities, the infectious potential of Omicron has likely increased.16 Based on this study and other existing evidence, it is likely that the high Omicron hospitalization rate relative to other variants reflects a substantially higher incidence of COVID-19 in the general community rather than a more virulent variant. In fact, in this study, severe outcomes declined over time with a peak of 36.2% during the Alpha period and 18.9% during the Omicron period. Other relevant clinical outcomes such as the need for supplemental oxygen or the development of MIS-C were also the least in the Omicron period. This phenomenon of less severe outcomes in hospitalized COVID-19 with Omicron has been noted in other reports with one study highlighting that Omicron has difficulty multiplying in the lungs compared to the Delta variant which may explain reduced respiratory impairment with Omicron.15 This study also has implications for vaccination in pediatrics. In the United States, COVID-19 vaccinations first received emergency use authorization (EUA) by the U.S. Food and Drug Administration for ages 16 or greater on December 11, 2020, ages 12–15 on May 10, 2021, ages 5–11 on October 29, 2021.17, 18, 19 Ages 6 months through 5 years old received EUA on June 17, 2022, which was outside of the study period.20 Overall, the vaccinated cohort represented only 7.6% of all hospitalized pediatric patients. Interestingly the proportion of hospitalized cases in teens, the group with the earliest access to vaccination in pediatrics, has declined substantially over time and may reflect a benefit from vaccination. While vaccination efforts in children have been rolled out at different time intervals during the pandemic, with infants still ineligible, the percentage of vaccinated children now approaches 50%, and the implication of this data is that vaccination may be reducing hospitalization and severe outcomes compared to unvaccinated children. Importantly, even when hospitalized, severe outcomes trended lower in the vaccinated group from T1 to T3. However, it is unclear if the lower rates of severe disease are due to vaccination efforts, a less virulent Omicron strain, or a combination. As more children continue to become vaccinated and the youngest become eligible for vaccination, the precise impact of vaccination may be assessed better. Previous literature on children has demonstrated mixed outcomes in viral coinfections, a subpopulation of interest. As one virus may synergistically or antagonistically alter the replication and proliferation of another pathogen, both positive and negative impacts on morbidity and mortality have been described.21 There are several animal and human studies demonstrating severe outcomes in the setting of other respiratory viral coinfections.22 , 23 In a study evaluating coinfections of RSV and Influenza in mice, researchers found increased airway resistance and reduced thoracic compliance in coinfected groups.24 In the pediatric literature, children with co-infections demonstrated greater lower respiratory tract infections and moderate to severe illness.25 In contrast, other evidence purports either neutral or opposite findings in viral coinfections. Scotta et al.26 performed a systematic review including 17,000 children and found no increase in severe outcomes in children with viral coinfection. In another systematic review on children less than five years of age, Lim et al.27 even identified a subgroup without comorbidities that had more severe outcomes with single virus infection. In our study, children with respiratory coinfections experienced more severe outcomes. This finding is consistent with some existing COVID-19 evidence. In a study of adults, viral coinfection with SARS-CoV-2 occurred in 8.4% of cases and coinfection with influenza increased the odds of mechanical ventilation. Further, coinfection with influenza and adenoviruses also increased the odds of death, which reinforces the importance of annual influenza vaccination in the pediatric population as well.28 While our findings highlight viral coinfection as a risk factor for more severe disease, the concept of viral coinfection in the setting of COVID-19 needs further exploration in children. Specifically, focusing on age, comorbidities, and precise viral–viral combinations may help identify children at the highest risk of severe outcomes. Limitations There were some limitations to this study. First, variant data was not patient-level data but rather represented the predominant variant strain during the time period. Thus, the precise impact of a particular variant strain upon severe outcomes cannot be extracted from this study. Second, this analysis does not account for infection in the community thus we cannot determine if increased encounters during a variant period was a function of severity of the disease or increased frequency of disease. Third, while a large number of pediatric patients that presented to emergency rooms across metro Detroit were included in this analysis, a relatively smaller cohort experienced hospitalization and even smaller additional severe outcomes. Further, the study included ages 0–17 and generalized findings for the entire cohort may not be evident for subgroups, specifically extremes of age. Fourth, outcomes data for some transferred patients were not available and these cases were excluded. It is possible that these patients had severe outcomes that were not included in our analysis. However, this group was small and included only 27 patients. Further, we performed a death instance query on 7/31/2022 on transferred patients and confirmed 18 were alive in this group with unknown status for only nine patients. Finally, data was retrieved from the EHR which may be incomplete or inaccurate at times. Specifically, while only patients with a principal diagnosis of COVID-19 based on EHR query were included in the analysis, it is possible that some patients with a secondary diagnosis of COVID-19 may have been inadvertently included. However, the number is likely small as this study used the same methodology that was employed in three previous large-scale (>40,000 encounters) COVID-19 investigations at the institution with <1% of cohort found to have secondary COVID-19.29, 30, 31 Conclusions Over the course of the pandemic, children have required hospitalization for COVID-19 and some have experienced severe outcomes. While Omicron represents a high proportion of hospitalized cases, clinical outcomes are less severe for this variant. A small percentage of all inpatients were vaccinated with a trend of less severe outcomes. Further larger research investigations are needed to determine the impact of vaccination on pediatrics. Contributors A.B., N.M., S.J., A.D., and L.Q. designed the study, had full access to the data, and take responsibility for the integrity and accuracy of the data analysis. A.B. and N.M. contributed to data and statistical analysis. All authors contributed to the writing and editing of the manuscript. All authors contributed to data acquisition, analysis and interpretation, and all reviewed and approved the final version of the manuscript. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Data sharing statement The data that support the findings of this study are available via a data access agreement. Please contact the corresponding author (AB) for this request. Declaration of interests All authors declare no relevant conflicts of interest relevant to this work. Appendix A Supplementary data Supplemental Table S1 Supplemental Table S2 Acknowledgements Special thanks to Dr. Nai-Wei Chen for his assistance and review of the statistical analysis. Also, thank you to Martha Garcia Hernandez and Srini Nimmagadda from Information Technology for data testing, authentication, and transfer from the electronic medical record. Funding: This research received no specific grant from any funding agency in public, commercial, or not-for-profit sectors. 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CNS Clinical Trials 34.5, P783 CNS drugs P448 CNV P534 Co-Abuse P621 Co-Activation Pattern Analysis P256 Cocaine 53.4, P619, P674, P688, P689, P720, P727, P730, P602 Cocaine Addiction 10.4, 25.5, 34.3, P635, P667, P703 Cocaine And Opioid Use Disorders 34.2, P621, P682 Cocaine Craving P603 Cocaine Seeking P613 Cocaine Self-Administration P603, P692, P725, P756 Cocaine Self-Administration And Reinstatement P761 Cocaine Sensitivity P669, P721 Cocaine Sex Differences P174, P727 Cocaine Use Disorder P656, P638, P734, P638, P734 Cocaine-Seeking Behavior P692 Co-Expression Network Analysis P540 Cognition 6, 30, 39.4, 40.2, 59.4, P12, P508, P13, P20, P131, P160, P235, P270, P408, P437, P484, P490, P494, P500, P506, P533, P534, P543, P553, P555, P786 Cognitive Behavioral Therapy 7.5, P44 Cognitive Control 40, P194, P649, P711, P751 Cognitive Decline P8, P17, P400, P397 Cognitive Effort P261 Cognitive Enhancement P45 Cognitive Flexibility 21.4 Cognitive Impairment 39.2, P486, P495, 32.3, P14, P247, P435 Cognitive Impairment Associated With Schizophrenia 9.4, 32.2, P505, P542, P560, P581 Cognitive Inflexibility P454 Cognitive Neuroscience 39.4, P262, P638 Cognitive Outcome Prediction P509 Cognitive Performance P801 Communication P123 Community Detection P123 Comorbid Insomnia Obstructive And Sleep Apnea (COMISA) P588 Comorbidity 22 Comorbidity depression and CVD 22.3 Complement Component 3 P312 Complement Pathway P318 Compulsive Behavior P752 Compulsive Drug Intake P599 Computational Cognitive Neuroscience P51, P126 Computational Genomics P531 Computational Modeling 39.2, 39.3, P54, P53 Computational Models Of Decision-Making 51.3, P280, P652, P653 Computational Neuroscience P641 Computational Psychiatry 39, 39.5, 49, P52, P126, P183, P518 Computational Reinforcement Learning Model 51, 51.1, 51.2, P789 Computerized Cognitive Training 3.4 COMT Inhibitor P711 Concussion P124 Conditioned Place Preference 4.3, P727 Conflict Monitoring P37 Connectivity P233, P255 Content Validity Of A Scale P337 Contextual Fear P96, P413 Continuous Performance Task P273 Convolutional Neural Network (CNN) P442 Copy Number Variants P205, P421, P524, 21.1, P537 Copy Number Variation 21, P151 Cortical Columns P808 Cortical Development P141 Cortical Morphology 32.5 Cortical Thickness P141 Cortical Volume 57.2 Corticosterone P25 Corticosterone Response To Stress 11.5 Corticostriatal Circuit 20, P168 Cortico-Striatal-Thalamo-Cortical Circuits P763 Corticotropin Releasing Hormone 11.3 Corticotropin-Releasing Factor (CRF) P104, P777, P747 Corticotropin-Releasing Hormone P92, P747 cortisol P684, P38, P648 Cortisol Response To Stress 17.3, P628 COVID-19 14, 14.5, P36, P38, P76, P264, P454, P798 COX-1 and COX-2 P24 Craving P635 C-Reactive Protein P68, P264, P627 CREB P4 CRH P119, P464 CRH+ neurons 11, 11.2 CRISPR/Cas9 P483 Critical Periods 42.4 Cross-Species Translation 7, 21.1, 29, P585 CSF P798 CSF sTREM2 P7 CSF1R P404 Cue Reactivity P622, P642 Cue Reinstatement P608 Cue-Induced Craving 15.3 Cue-Reactivity P626, P669 Cytochrome P450s 27.1 cytokines 17.1, P25, P114, P246, P313, P552, P553, P744, P797, P798, 46 Cytoskeleton P489 D1 agonist, D1-PAM P575 D1 Dopamine Receptors P575, P730 D2 Dopamine Antagonists P351 D2 Dopamine Receptor 43.3, P195, P202, P483, P565 D2 Medium Spiny Neuron P568 Data Science 12, 32.3, 47.2, 52 Database P466 DBS P230 d-cycloserine 3.3 Decision Making 56, P46, P249, P259, P473, P519, P641, P790, P791 Declarative Memory P19 Deep Brain Stimulation P322, P369 Deep Phenotyping P274 Default Mode Network (DMN) P49, P277, P557, P257 Defense Mechanism P794 Delay Discounting P195, P413, P793 Delirium P14 delta9-tetrahydrocannabinol 1.3, P115, P717 deltaFosB P206 Delusions P519 Dendrites 4.1, P111, P476 Dendritic Arborization P108, P358 Dendritic Spines P214, P688, P333, P437 Dentate Gyrus 45.4, P2, P113, P379, P383 Depression 3.4, 11.3, 14.3, 22, 27.3, 39.2, 39.4, 48.2, 48.3, 48.4, 50, P535, P208, P301, P7, P34, P69, P173, P218, P229, P231, P234, P239, P251, P257, P260, P268, P273, P281, P284, P292, P294, P302, P305, P315, P325, P327, P331, P334, P340, P345, P348, P351, P352, P353, P357, P367, P382, P386, P455 Depression And Anxiety P79, P176, P215, P264, P371 Depression And/Or Anxiety P276 Depression Inflammation Cytokine 14.2, P317 Depression Subtypes P389 Depression Treatment Response P286 Depression, Synaptic Aberations 18.4 Depressive Disorders P336 Depressive Symptoms P319 Depressive-Like Behavior P207 Depth Perception P808 Development 36.5 Developmental Neuroscience 25 Developmental Psychopathology 7.5, 12, 31.1, P56, P125, P800 Developmental Sensitive Period 42 Developmental Tools, Techniques, And Strategies 38 Developmental Trajectory 15.1, 31, 42.1, 42.3, P169 Dexmedetomidine, Agitation, Locus Coeruleus, Alpha2-Adrenergic P567 Diagnostic Specificity 7.5 Diet P81 Diffusion MRI P124, P138 Diffusion Tensor Imaging (DTI) 7.4, 21.4, 47.3, P9, P47, P230, P267, P743, P687 Diffusion Weighted Imaging 21.1, P146, P499 Digital Phenotyping 52, 52.2, P332, P518 Disease Modeling P221 Disorders Of Glutamate P546 Disparities 5.3 Diverse Populations 24, 24.4 Diversity And Inclusion 24.2 DLPFC P233, P477 DNA Methylation P60, P136, P296 DNA, Whole-Genome, Sequencing, Schizophrenia P530 DNMT P578 Dopamine 33.3, 37.3, 43, 43.2, 43.3, 44.5, 58.3, P342, P91, P101, P132, P157, P159, P185, P220, P253, P288, P359, P373, P393, P409, P463, P562, P597, P600, P601, P634, P644, P656, P659, P675, P721, P733, P738, P763, P771, P775, P807 Dopamine (D2, D3) Receptors P601 Dopamine Circuitry 50.2 Dopamine Circuits P34 Dopamine D4 Receptor P629 Dopamine Neuron P603 Dopamine Receptor P223 Dopamine Release P701 Dopamine Transporter P409, P730 Dopamine-Glutamate Co-Transmission 37.4, 37, 37.2 Dopaminergic System 33.1, P541 Dorsal Attention Network 51.1 Dorsal Hippocampus P97, P412 Dorsal Raphe P311, P357 Dorsal Striatum 4.4, 20.3, 20.4, P253, P357, P428, P767, P204 dorsolateral prefrontal cortex (DLPFC) P205, P535, P548, P655, 58.4, P540, P271 Dorsolateral Striatum P760 Dorsomedial Striatum P757 DREADDs P615, 15.2 Drinking P697 Drinking Disorders P653 Drug Addiction 10.3, 53, 53.3 Drug Development P804 Drug Discovery P722 Drug Discovery - New Approaches P128 Drug Discovery/Development P568 Drug Liking 35.3 Drug Metabolism P672 Drug Relapse P598 Drug Seeking P610, P772 Drug Self-Administration P671, P743 Drug Use Disorders 6, P405 Drug-Drug Interaction P728 Drugs 10 DSM-5 P287 Dual Orexin Receptor Antagonist (DORA) P588, P593, P720 Dynamic Functional Connectivity P273 Dynorphin P597, P765, P767 Dysphoria P71 Early Career Scientist Workshop 38 Early Intervention Services P528 Early Life Adversity 15, 15.1, 31.2, 42, 45.2, 45.5, P379 Early Life Stress (ELS) 5, 7.3, 11.3, 15.3, 28.4, 29, 31.1, P81, P278, P479, P601, P74, P119, P132, P134, P379 Early Life Trauma 15.4 Early Psychosis 18.2, 52.3, P504, P520, P528 Early Trauma P89 Early-Life Adversity 42.1, P92, P109 Eating Behavior P456 Eating Disorders P182, P183, P186, P189, P191, P192 Ecological Momentary Assessment 51.2, P164, P183, P268, P329, P496 EEG P118, P142, P262, P395, P439, P470, P494, P500, P573 EEG biomarkers 40.5, P77, P334, P469, P567, P573, P653, P751 EEG connectivity P154 EEG electrophysiology P503, P661, P778 EEG/ERP electrophysiology 39.4, P561, P584 Effective Connectivity P423 Effort P376 Effort Based Decision Making Task P554 Effort-Cost Benefit Task P809 E-I balance 15.2 Electrical Field Modeling P331 Electroconvulsive Therapy P283, P383 Electroencephalography P320, P507, P595 Electron Microscopy 21.4 Electron Transport P339 Electronic Cigarette (E-Cigarette) 1.5, P735 Electronic Health Record (EHR) P529, P558, P676 Electronic Medical Record 28.3, P284 Electrophysiological Approaches P34 Electrophysiology 44.2, 46.5, P175, P630 Elevated Plus Maze P77 Emotion Circuitry 25.4 Emotion Processing P271 Emotion Recognition 52.2 Emotion Regulation 28.2, P33, P268, P788 Emotional Arousal P72, P299, P648 Emotional Dysregulation P329 Emotional Memory 36.4 Emotional Regulation P48, P277, P298, P386 Emotional Stress P264 Empathy P473 Endocannabinoid System P63, P201 Endocannabinoids 35, P22, P61, P566, P600, P635, P719, P761, P773, P805 Endophenotypes P120 Endothelial Function P579 Energy Metabolism P190 Engram 54.4 Entorhinal Cortex 59.4, P394 Entourage Effect P425 Environment 9, 9.3, P769 Environmental Risk Factors P32 EPA P237 Epidemiology 39.5 Epigenetic Age Acceleration P683 Epigenetic Biomarkers P60, P306 Epigenetic Clock P15 Epigenetics 47.4, P107, P207, P219, P225, P297, P399, P448, P459, P598, P672, P680 Epigenome Wide Association Studies P296 Epigenomics P667 Epilepsy P410, P70 Episodic Memory 36.5, 39.3, P648 Episodic Memory Rodents P772 eQTL 57.2 ERP 26.1, P494 Escitalopram P317 Esketamine P354, P724 Estradiol P231, P305, P306, P591 Estrogen Receptor P84, P305 Estrous Cycle P736 Ethanol P745 Event Related Potentials P467 EWAS P60 Excitation-Inhibition Balance P411 Excitatory / Inhibitory Balance P398 Excitatory Synapses 4.1, 18.4, P111, P411, P570, P111, P411, P570, P611 Executive Function P138, P499, P671, P713 Exercise P5 Expansion Microscopy P548 Expectancy P802 Experience Dependent Plasticity 7.2 Experimental Methods 39.5, P31 Explainable-AI P125 Exploration 51, 51.1, 51.2 Explore-Exploit Dilemma P280, P790 Exposome 28, 28.3 Exposure-Response Model P29 Expression Quantitative Trait Loci (eQTLs) 24, 24.4, 44 Exteroception 49 Extinction 36.2, P35, P602, P632 Extinction and Reinstatement P753 Extinction learning P130 Extracellular vesicles P1, P297, P618 Extrapyramidal symptoms P558, P243 Eye Movements 8.2 Eye-Tracking P503, 31.3, P197 F-18 PET Imaging P796 FAAH P63 Face Emotion Processing P503 Face Expression 8 Face Expression Synchrony 8 Facial Affect 8.4 Facial Dysmorphology P517 Facial Expression 25.4 Familial Risk 28.5 Familial Risk for MDD P256 Familial Risk of Bipolar Disorder P304 Family Study P424 Fast Scan Cyclic Voltammetry P734 Fast-Acting Antidepressant 18.4, P254 Fatty Acid Amide Hydrolase 35.2 Fatty Acid Amide Hydrolase, PET, Cannabis P201 Fear P374, P87 Fear Conditioning 36.3, P22, P97, P102, P375 Fear Conditioning And Extinction 36, P33, P36, P50, P68, P82, P756 Fear Extinction P84 Fear Generalization 36.3, P26, P96 Fear Regulation P94 Fear-Potentiated Startle P82, P92 Feeding 56.2 Feeding Behavior 4.2, 56, P192, P193 Fentanyl P615, P729, P745, P758 Fetal Alcohol Spectrum Disorder P441 Fetal Brain P429 Fetal Brain Development 27.3 Fetal Neurobehavior P136 FGF-21 P364 Fiber Photometry P758, P62, P385, P458, P463, P634, P752, P763 First Episode Psychosis P493, P506, P551, P552 First Episode Schizophrenia P576 FKBP5 P479, P682 Flame Retardant P127 Fluorescence In Situ Hybridization P196 Fluornitrazene P391 Fluoxetine 27.2 fMRI 10.3, 26, P43, P46, P105, P166, P467, P474, P622, P643, P644, P646, P712, P768, P780, P782, P299, P326 fMRI Functional Connectivity 25.3 fMRI Negative Affective Stimuli P45 fMRI, Anxiety, Threat P39 FMRP P150 fNIRS P152, P197 Focused Ultrasound 53.2 Food Cues P456 Food Insecurity P144 Food Intake P190, P191 Food-Reinforced Instrumental Task 20.4 Fragile X Syndrome P118, P150, P152 Free Fatty Acids P485 Freesurfer P516 Frequency Optimization 22.4, P380 Frmi 31.4 Frontal Pole (Brodmann’S Area 10) P481 Frontolimbic Network P169 Frontoparietal Network 40.2, P49, P126 Frontopolar Cortex P655 Frustration P164 Functional And Structural MRI P39, P413 Functional Brain Network P321 Functional Capacity P560 Functional Characterization P391 Functional Connectivity 25, P155, P418, P462, P682, P155, P418, P462, P682, P368 Functional Genomics 45, 57, 57.4 Functional Impairment P685 Functional Magnetic Resonance Spectroscopy P64, P298 Functional Mri (fMRI) 28.2, P37, P41, P45, P56, P130, P147, P259, P262, P268, P470, P520, P543, P627, P638, P656, P706, P784, P788, P338, P512, P547, P699 Functional Near-Infrared Spectroscopy P685 Functional Network Connectivity 32.4, 45.2 Functional Neuroimaging P505 Functional Outcomes P505 Functional Segregation P547 Functioning 12 GABA P119, P221, P223, P302, P430, P511 GABA Neuron P432 GABA Transmission P765 GABA-A Receptors P110, P482, P667, P734 GABA-A, Positive Allosteric Modulators P31, P75, P77 GABA-B Receptors P734 GABAergic Interneurons P350 Gabaergic System 15.2, P80 Gabapentin P728 Galanin P607 Gamma Synchronisation 59.2 Gaze Dynamics 8 Gaze-Following 8.2 Gender Differences P433, P493, P543, P657 Gene Discovery P151 Gene Expression P110, P420, P429, P668, P670 Gene Priming P598 Gene Therapy 53, 53.2, 53.4 Generalizability P50 Generalized Anxiety Disorder P30, P42, P46, P75, P717 Genes P295 Genetic Ancestry 24.3, P429 Genetic Association Study P678 Genetic Disorder 21 Genetic Mapping P673 Genetic Mouse Models 21 Genetic Risk Factor 28.5 Genetic Variation P339 Genetically Encoded Sensor 59, 59.5 Genetics P562, P148, P421, P422, P475, P674 Genetics Of Depression 22.3 Genome-Wide Association Studies 24, P669, P795 Genomics 24.2, 28, 48, 48.3, 48.4, P58, P530, P536, P674 Geriatric 47, 47.1, 47.2, P13 Ghrelin P431, P690, P742 Ghrelin Receptor P742 Gliosis P186 Globus Pallidus P472 Glucocorticoid Antagonists P628 Glucocorticoid Receptor P213, P457 Glucocorticoids P66 Glutamate P64, P194, P366, P382 Glutamate GABA P599 Glutamate GABA Co-Release P777 Glutamate Receptors P564 Glutamate Transporter P414 glutamate transporter (EAAT3) P621 Glutamatergic P377 Glutamatergic Synapses 15.2 Glutamatergic Transmission 37.3, P459, P621 Glutamine P302 Glycosylation P475 Goal-Directed P636 Goal-Directed Behaviors 20.3, P767 Goal-Directed Control 20.4 GoNoGo P298 GPCRs 4, 4.2, P393, P27, P218 GPe P636 GRAB-5HT P204 Graph Theory P509 Graph-based Analysis P56 Gray Matter Volumes 9.4, P39 Grey Matter Morphometry P129 GrimAge acceleration 47.4, P365 Growth Factors P571 Gut Microbiome 5.5, 17, 17.1, 48.2, P69, P313, P314 Gut Microbiota 11.5 Gutamate P302 GWAS 24.5, 44.4, P59, P200, P403, P535, P676 Habenula 58.4 Habit Formation 20.5 Habits 20.3, 20.4, 20, P182, P636 Hallucinations P583 Head Twitch Response P349, P425 Headache 60 Health Disparities P665, P666 Health Disparity 41 Health Services P665 Healthy Individuals P157, P646 Heart Rate P86 Heart Rate Variability 17, 17.1, 17.3, 22.1, P86 Heartbeat Evoked Potential P30 Hepatitis C P181 Heritability P421 Heritability Of Depression P793 Heritability Of Substance Use Disorder P671 Heroin 35, P641 Heroin Seeking P766 Heroin Self-Administration P601, P611, P617, P724, P753, P774 Heterogeneous Stock Rats P612 High Fat Diet P733 High Potency Thc P620 High Resolution Fmri P808 High Risk P155 High Throughput Screening P128 High-Throughput P333 Hippocampal Function 7.3, P762 Hippocampal Subfields P523 Hippocampus 3.2, 36.4, 36.5, 45, 45.2, 54.4, P2, P5, P21, P26, P40, P66, P96, P113, P260, P308, P346, P355, P435, P586, P3, P87 Hipscs 44.2, P221 Histone Variants P217 HIV P84, P181 HIV and Inflammation 14 HIV-Associated Neurocognitive Disorder P405 Hopelessness P269 Hormones P643 Hot Flashes P591 Housing P260 HPA axis P213 Human And Non-Human Primates 8 Human Brain Imaging P472, P686 Human Connectome P282 Human Connectome Project (HCP) P442 Human Epigenetics/Microrna P472 Human Genetics 24.3, 57.4, P177, P539, P677 Human Immunodeficiency Virus P686 Human Laboratory P626 Human Microglia P214 Human Neuroimaging 12, 23, 25.3, 31.2, 45.3, 47, 48.2, P48, P153, P185, P308, P367, P523 Human Pluripotent Stem Cells 58 Human Post-Mortem Brain P537, P214 Human Postmortem Brain Tissue 44.3, P205, P532 Hyperactivity P192, P586 Hyperalgesia P446, P449 Hyperarousal P103 Hypertension 22.4 Hypofrontality P483 Hypomania P238 Hypothalamic Development P171 Hypothalamus 11.3, P192 Ibudilast P627 iDISCO mapping P474 IGF-1 P617 IL-6 P220 Imaging 28, P541 Imaging-Genetics 32.2, P366, P420 Immune Biomarkers P556 Immune Markers P114 Immune Modulation 46.3, P291 Immune Responses 15.4 Immune System 17, P372 Immunity & Neurodevelopment By Sex P158 Immunometabolism P158, P291 Immunotherapy 16.5, 35 Implicit Association Test P275 Impulsive Behavior P195, P675 Impulsivity P145, P200, P280, P669, P699, P711, P793 In Vivo Calcium Imaging 20.4, P21, P22, P104, P378, P417, P755 In Vivo Electrophysiology P168, P586, P600 In Vivo Fiber Photometry P34 In Vivo Imaging 59.3, P91, P373 In Vivo Microscopy 20.2 Incentive Motivation P775 Incentive Salience P203, P641, P713 Income P452 Incubation Of Cocaine Craving P174, P606 Incubation Of Sucrose Craving P606 Ind-Enabling Glp Studies P361 Independent Component Analysis P54 Individual Differences P203 Individualized Head Models P655 Induced Pluripotent Stem Cells (iPSCs) 44, 58.5, P216, P220, P406 Infancy 5.4 Infant Behavior P136, P147 Infant Brain P146 Infant Emotionality P146 Infection P66 Inflammation 14.3, 16.4, P161, P220, P237, P264, P346, P388, P435, P488, P554, P555, P627, P797, P798 Inflammatory Disturbances 9.2 Inflammatory Markers P1, P348 Infralimbic Cortex P35, P191, P749, P760, P774 Ingroup Bias 33.2, P196 Inhibitor 35.2 Inhibitory Control P643, P779, P800, P780 Inhibitory Synaptic Transmission P411, P478 Innate Immunity 17.2 Innovative Methods 47.2 Insomnia P592 Insomnia Disorder P589, P593, P596 Insula Connectivity P49 Insular Cortex P193, P354, P468 Insulin 43.3, P372 Insulin Resistance P17 Intellectual Disability P406 Interferon P312 Intergenerational Depression 45.4 Intergenerational Transmission 45 Intergenerational Transmission Of Stress P55 Intergenerational Transmission Of Trauma 31, P85 Interleukin 1 Receptor P311 Interleukin 1Beta P311 Interleukin-33 46.5 Internalizing Disorders 26.2, P258 International 47 Interneurons P581 Interoception 49, P30 Intracranial EEG P631 Intravenous Drug Self-Administration 1.3 Intrinsic Metabolic Risk P551 Intrinsic Motivation P809 Intrinsic Neural Timescale P131 In-Vivo Sirna Treatment P353 Ion Channels 44.2, P406, P571 iPSC P223 IQ P534 Irritability P336, P792 Irritability/Aggression P507 Isoform Co-Expression 57.1 Isoform Transcriptome 57.1 IV Alcohol P690 IV- Ketamine 3.4, P227, P341 Juvenile 42.3 Juvenile Social Isolation 42.2, P658 JZL184 P566 Kappa Opioid Receptor 50, 50.2, 50.4, P363, P409, P471, P741, P767 Kappa Opioid Receptor Antagonist 50.3, 50.5, P363, P739, P741 Keratinocyte 16.3 Ketamine 3, 3.5, P209, P218, P250, P275, P293, P318, P341, P372, P415, P416, P353 Ketogenic Diet P19, P733 Ketones P708 Kinome P487 Kynurenic Acid P522 Kynurenine Pathway P397 Lactate-To-Pyruvate Ratio P210 Language 10.1 Language Delay P123, P152 Late-Life Depression 47.1, P1, P316, P344, P13, P387 Latent Factor Analysis P279 Latent States 25.4 Lateral Habenula 42.1, P633, P758 Lateral Hypothalamus P203 Lateral Septum P432, P474, P609 Latin America 9 Learning 54.4 Lemborexant P588, P593 Lesion P65, P282 LFP P580 LGBTQIA+ needs 26.1, 26.3 Lifetime Stress P683 Lipopolysaccharide P335 Liver Brain Axis P181 Local Field Potentials P168, P770 Locomotor Activity 4.3, P757 Locus Coeruelus P630 Locus coeruleus (LC) 56.2, 56.3, P378 Long Term Potentiation P5 Long-Acting Injectable Antipsychotics P490 Longitudinal Analysis P162 Longitudinal Imaging P258 Longitudinal MRI P105, P524 Longitudinal Study P274 Long-Term Treatment P491 lorcaserin P497 LPS P311 LSD P349, P358 Lumateperone P228, P232, P238, P243 Lurasidone P356 M1 and M4 Muscarinic Receptors P480, P573, P575 M1 Muscarinic Receptors P481 M4 Muscarinic Receptors P573 Macaques 8.2 Machine Learning 32.4, 48, 52, P67, P125, P225, P330, P334, P341, P520, P547, P666, P752, P281 Machine Learning Classification 48.4, P657 MADRS P345 Magnetic Resonance Imaging P731, P131, P300 Magnetic Resonance Spectroscopy P511, P549 Magnetoencephalography P275, P576 Major Depression 48.5, P199, P259, P316, P479 Major Depressive Disorder (MDD) 2, 3.3, 22.4, 50.5, 54, P338, P235, P237, P244, P278, P290, P295, P330, P335, P201, P210, P226, P240, P241, P263, P361, P362, P383, P778, P788, P795, P342, P338, P235, P237, P244, P278, P290, P295, P330, P335, P201, P210, P226, P240, P241, P263, P361, P362, P383, P778, P788, P795, P342, P242, P245, P262, P501 Mania 47.1, P238, P339, P238, P339, P271 Marijuana 35, 35.1, P620, P654 Mast Cells P309 Maternal Behavior P746 Maternal Brain P660 Maternal Immune Activation P127 Maternal Inflamation P139 Maternal Stress P55 Matrix Metalloproteinase-9 (MMP-9) P556 Matrix Metalloproteinases P78 MCP-1/CCL2 P319 MDD P58, P319, P785 MDD, PTSD P464 MDMA 10.2, P453, P803 MDPV P768 MDPV self-administration P726 Mechanism Of Action P804 Mechanotransduction 16.3 Medial Amygdala P170 Medial Orbitofrontal Cortex P417 Medial Prefrontal Cortex 42.2, P24, P27, P94, P95, P97, P253, P310, P315, P502, P606, P723, P752, P779 Medication Development P710 Medication-Naive First Episode Psychosis 9.2, P521 Meditation P470 Medium Spiny Neuron P570 MEG 51.1, P269, P546 Melanocortin P191 Memantine P398 Memory 4.4, 21.4, 36, 54.4, 59, 59.5, P5, P11, P791 Memory And Learning P581, P725 Memory Engram Cell P26 Mendelian Randomization 48.3, P514, P681 Menopause 22.2, P11, P12, P308, P591 Mental Health And Health Disparities 5.2, 14, 14.5 Mental Health Disorders P32 Mental Illness P224 Mentalizing 8.2 Mesolimbic Circuitry P463 Mesolimbic Reward Circuitry P110, P646 Meta-Analysis P116, P239, P549, P799 Metabolic Biomarker P492 Metabolic Function P11 Metabolic Profile P232 Metabolic Side-Effects P180, P178 Metabolic Syndrome P178 Metabolites P184 Metabolomics 48, 48.2, 48.3, 48.4, 48.5, P551 Metabolomics Signature P227 Metabotropic glutamate receptor 2 (mGluR2) P27 Metabotropic glutamate receptor 3 (mGluR3) P570 metabotropic glutamate receptor 5 (mGluR5) P338, P639 Metagenomics 48 Metformin P497, P727 Methamphetamine 10.2, P405, P439, P646, P733, P762, P803 Methamphetamine Self-Administration P744 Methamphetamine Use Disorder P639, P707, P772 Methylphenidate P701, P166 mGluR5 receptors P606 Mice P468, P473 Microarray P291 Microbiome 5, 55, P159, P691 Microbiota-Gut-Brain Axis 11.5, P158 Microdialysis P167, P434, P721 Microdosing With Psychedelics 10.1 Microglia 11.2, P159, P160, P179, P310, P315, P404, P605, P651, P692, P7, P209 Microglial Activation P405, P693 Microrna P297 Midazolam P416 Mindfulness P49 Miniscope P22 Mirna 57.2, P278 Mirnas P335 Mismatch Negativity P580 Mitochondria P364, 48.3, P225, P289, P339, P477 Mitochondrial Dna 5.3, P81, P289, P530 Mitochondrial Dna Copy Numbers P15 Mitochondrial Dysfunction P210 Mitochondrial Respiration P3 Mobile Application P800 Mobile Phones 52.4 Model Bias 25.2 Model Systems P352 Moderators P415 Modest Dose P687 Molecular Genetics 24, P177, P373 Molecular Imaging P404 Monetary Incentive Delay Task P554 Monetary Reward 26.3, P265 Monocytes P489 Monoiodoacetate Model Of Osteoarthritis P447 Mood P456 Mood And Addiction 25 Mood And Anxiety Disorders P314 Mood And Cognition P274 Mood Disorders P99, P255, P277, P329, P332, P450, P261, P781, P797 Mood Dysregulation P304 Mood Stabilizers P343 Mood, Childhood Stress P308 Morphine 46, 46.3, P689 Morphometry P472 Motivated Behaviors P109 Motivation 56.2, P363, P443, P572, P703, P737, P763, P787, P791 Motor Learning P393 Mouse Genetics P200 Mouse Model P583 Mouse Models 46.5, P24, P97, P694, P731 mPFC 36, P169, P377, P461 MPRA 44.4 MR spectroscopy P325, P550 MRI 32.3, P283, P12, P294, P301, P390 mTOR P372, P606 Multi-Ancestry 24.5, P151 Multimodal Neuroimaging 5.4, 9, 25.4, 47.3, P146, P153, P402 Multiple Sclerosis P284 Multivariate Analysis 31.1 Multivariate Approaches, Deep Learning P442 Multivariate Pattern Analysis P50, P54 Multivariate Statistics 32.5 Mu-Opioid Receptor Agonist P718 Mu-Opioid Receptors P391, P724 Myd88 P605 Myelin Imaging P402 Myelination 7, 7.2, 7.3, 7.4, 21.2, 21.3, P47, P143 N170 P395 N-Acetylaspartate P549 Naltrexone P707 Narcolepsy P594 Native Americans P467 Natural Language Processing (NLP) 10.4, 52, 52.3, P14, P518, P641, P702 Natural Setting 10.1, P323 Naturalistic Drug Cues P642 Near-Infrared Light P325 Negative Affect P511 Negative Emotionality P713, P782 Negative Reinforcement P417, P634 Negative Symptoms P579 Negative Urgency P271 Negative Valence System P633, P797 Neighborhood Crime P147, P788 Neighborhood Poverty P83 Neocortex 57.3 Neonatal P161 Neonatal Opioid Withdrawal P673 Neonates 27 Network-Analysis P433, P164, P768 Networks P368 Neural Activity P354 Neural Circuit And Animal Behavior 33.1, P99, P305 Neural Circuits 22.2, P373, P379, P384 Neural Decoding P99 Neural Networks 18.3 Neuroactive Steroid P242, P246 Neurocircuitry P336 Neurocognitive Assessment P415, P783 Neurodegeneration 30, 37.2, 37.3, 53, P1, P316, P740 Neurodegenerative Diseases 22.2 Neurodevelopment 5, 45.2, 55, P129, P148, P160, P506, P539, P149, P489, P523 Neurodevelopmental Disorders 21.2, 21.4, P127, P168, P441, P537 Neurodevelopmental Disorders, Cannabidiol, CBDV, Translational Models, Clinical Trials P117, P620 Neuroeconomics P249 Neuroendocrine P191 Neuroendocrine Responses P628 Neuroendocrinology P308 Neuroepigenetic Editing P459 Neurofibrillary Tangle P394 Neurogenesis 57.2, P488 Neuroimaging 31.4, 32, 32.4, P13 Neuroimmune Activation P695 Neuroimmune Mechanisms 46.5, P696 Neuroimmunology P8, P68 Neuroinflammation 5.5, P179, P312, P315, P316, P694, P696, P796, P224, P309, P404, P744 Neurological Disorders P367 Neuromelanin-Sensitive MRI 50.4, P157, P390 Neurometabolism P158 Neuromodulation 4.3, 18, 53, 53.3, 53.4, P325, P331, P698 Neuronal Ensembles P604 Neuronal Oscillations P108, P413, P584 Neuron-Glial Interactions 11, 58.5 Neurons P220, P224 Neuropathic Pain 16.2 Neuropathology P400 Neuropeptide S P630 Neuropeptide Y P212 Neuropeptides 56.3, P4, P88, P98 Neuropharmacology 50.2, P272 Neurophysiology 59, 59.5, P533, P614, P806 Neuroplasticity 3, 3.4, 18.2, P263, P333 Neuroprotection P272, P569 Neuropsychiatric Disorders 4, 4.4, 24.2, 30, 40.3, 57.3, 58.5 Neuropsychiatric Disorders [Schizophrenia, Parkinson’s Disease, Major Depressive Disorder] P393, P531 Neuropsychiatric symptoms (NPS) P403 Neuropsychology P783 Neurostimulation 55, P799 Neuroticism P38 Neurotransmission P355 Neurovascular P579 NHP 53.5, 58 Nicotine 46, 46.3 Nicotine Addiction P608, P640, P693, P702, P732, P765 Nicotine Dependence P557 Nicotine Exposure P735 Nicotine Metabolism P735 Nicotine Vapor P735, P737 Nicotine/Substance Use Disorder P738 Nicotinic Acetylcholine Receptors P738 Nitrous Oxide P368 NMDA 3.2, P368 NMDA Antagonists P215 NMDA Glutamate Receptors P110, P482 NMDA Modulators 3 NMDA Receptor P79, P353, P806 NMDA Receptor Antagonist P235 NMDAR P350 Nociceptin 16.2 Nociception P446 Nociceptor 16.3 Non Human Primate P739 Noncoding RNA P207 Nonhuman Primate P729, P777 Nonhuman Primate Models 7.4 Non-Invasive Brain Stimulation P498 Non-Invasive Neuromodulation 18.3, P45, P321, P698 Nonlinear Analysis P515 Non-Suicidal Self-injury (NSSI) P156, P165 Noonan Syndrome P149 Noradrenergic Signaling 60.4 Noradrenergic System P103 Norepinephrine 56, 56.2, 56.3, 56.4, P90, P378, P460, P461, P750 Normative Modelling P521, P516 Nos1 P193 Novel Endpoints 2 Novel Object Recognition P772 Novel Therapeutics P31 Novelty Seeking P772 Nucleus Accumbens 18.5, 33.4, 58.4, P23, P78, P119, P133, P195, P250, P443, P601, P604, P632, P634, P675, P734, P753, P755, P760, P703 Nucleus Accumbens (NAA) P675 Nucleus Accumbens Glutamatergic Afferents P377, P762 Nucleus Tractus Solitarii P103 Nutrition P624 Obesity 4.2, 43.2, P69, P136, P187, P456 Observational Learning P97 Obsessive-Compulsive And Related Disorders P420 Obsessive-Compulsive Disorder (OCD) 20.2, P417, P418, P424, P426, P415, P419, P421, P427, P331, P416, P422, P786 Obsessive-Compulsive Symptoms P419 Obstructive Sleep Apnea P588 Occipital Cortex P550 Odor Deficits P578 Olanzapine 43.4 Older Adults P10, P14, P589 Oligodendrocytes 7.2, 21.2, 21.3 Onset P255 Open Chromatin Regions P531 Open Label Trials P227 Open-Label Follow-Up P322 Operant Behavior P770 Opioid P691, P766, P731, P408, P607 Opioid Abuse 35.4, P609, P659, P728, P729, P764 Opioid Addiction 25.5, 53.3, P616, P661, P677, P750 Opioid Agonist Treatment P665, P718 Opioid Antagonist Treatment P624, P729 Opioid Dependence P741, P746, P750 Opioid Overdose P728, P745 Opioid Peptides 16.5 Opioid System 58.3, P80 Opioid Tolerance P597, P769 Opioid Use Disorder 35.3, P198, P401, P624, P660, P696 Opioid Withdrawal P758, P609, P614, P621 Optical Biosensors P62, P90, P771, P773 Optogenetics 20.2, 33.3, 54.4, P458 Oral Self-Administration P758 Orbitofrontal Cortex (OFC) P658, P675, P630, P688 Orexin P594, P597, P748 Orexin Receptor Antagonist P449 Orexin/Hypocretin P458 Orphan Receptors P568 Oscillations P561 Ovarian Hormones P246 Oxidative DNA Damage P11 Oxycodone P612, P616, P736 Oxytocin 59.2, P453 Oxytocin Receptor 33.4 P300 P546, P584 PACAP P194 Pain 16, 16.2, 16.3, 16.4, 16.5, 40.2, P649 Pain Analgesia P764 Pain Models P407 Pain Therapeutics P173, P448 Pain Therapeutics Profiling P407 Panic P31 Panic Attacks P51 Parabrachial P614 Parabrachial Nucleus P754 Paralaminar Nucleus P114 Paraventricular Nucleus Of The Hypothalamus 11.4 Paraventricular Nucleus Of The Thalamus 33.2, P100, P109, P196, P203, P279, P414, P443 Parent - Child Dyads P130 Parietal Cortex P808 Parkinson’s Disease 37, 37.2, P382 parkinson’s Disease Psychosis P569 Partial Agonist 35.4 Parvalbumin Fast-Spiking Gabaergic Interneurons 40.3 Parvalbumin Interneurons P160, P478, P770 Parvalbumin Interneurons/Perineuronal Nets P582 Parvalbumin Neurons P502, P776 Patient Reported Outcomes P337 Pavlovian Conditioning P203, P760, P771 PDE10A P575 Pediatric P329, P424, P559 Pediatric Clinical Trials P163 Pediatric Irritability P164, P336 Perceived Stress 28.4 Perceptual Decision-Making P468 Perceptual Distortion Of Appearance P423 Perceptual Inference 49 Perforant Pathway (PP) 7.3 Periaqueductal Grey (PAG) P102 Perinatal Anxiety 17, 17.1, 17.3 Perinatal Development P522 Perinatal Stress 5.3 Peripheral Blood Marker 27, P296, P347 Peripheral Nervous System 33.3 Persistent Avoidance P427 Personal Space P808 Personalized Medicine P183, P286 PET Imaging P63, P198, P434, P484, P684, P696, P739, P106, P16, P186 PET Imaging Study P471 PET Tracer P198 PF-04958242 P495 PFC P106, P167, P251, P617 Pharmacodynamics P713 Pharmacoepidemiology P343, P665 Pharmacogenetics P172, P292, P344 Pharmacogenomics P292 Pharmacokinetics 53.5, P344, P362, P446, P644 Pharmacology 42.4, P806, P361 Pharmacotherapy 35, P410, P741, P746, P750 Phase 1 P75 Phase 2 P115 Phase II Clinical Trial P28, P29 Phosphoproteomics 44.3 Phosphorylation P501 Photobiomodulation P325 Photopharmacology P27 Pimavanserin P569 Placebo P802 Placebo Response P239 Placenta P514 Plasma Lactate And Pyruvate P210 Plasticity P356 Platelet Serotnin 27.2 Polygenic Risk Scores 44.2, 45.4, P538, 44, P505, P529, P533, P534 Polygenic Scores 45.5, P294 Polysubstance Abuse P200, P613 Polyunsaturated Fatty Acids P184 Population Genetics 24, P177 Population Neuroscience 23 Population Pharmacokinetics P445 Population-Based P452 Positive Affect 54, 54.3 Positive Allosteric Modulators P242, P722 Positive Valence 54.2, P797 Positron Emission Tomography (PET) P201, P327, P338, P438, P512, P644, P796, P651, P708, P686, P542 Post Traumatic Stress Disorder P23, P41, P52, P70, P78, P82, P172 Postmortem Brain Tissue 44, P112, P411, P535 Postmortem Brain Tissue Gene Expression P482 Postmortem Human Brain P224, P226 Postmortem Human Brain Study P401 Postmortem Human Brain Tissue P290, P460, P477, P481, P677 Postoperative Cognitive Dysfunction P408 Postpartum 27.1 Postpartum Anxiety 17.2 Postpartum Depression P307, P310 Postpartum Period 5.2 Posttraumatic Headache 60.4 Posttraumatic Stress Disorder 31.2, P40, P72, P85, P73 Post-Traumatic Stress Symptoms P38 Potential Mechanism P801 Prazosin 60, 60.2, 60.3, 60.4, 60.5 Precision Health 48 Precision Medicine For Depression P291, P341 Precision Medicine For Mood And Anxiety Disorders P60 Precision Psychiatry P229, P346, P418 Preclinical Alzheimer’s Disease P596 Preclinical Pharmacology 21.2, P410, P447 Predatory Imminence P374 Prediction 25.2, P330 Predictive Models P387, P654 Predictor Of Treatment Response P521 Prefrntal Cortex P759 Prefrontal Circuit Maturation 18.2 Prefrontal Cortex 1.3, P134, P3, P102, P197, P478, P550, P631 Pregabalin P728 Pregnancy 5, 17.2, 27, 27.1, 27.3, P147, P371, P451, P455, P659, P746, P798 Prelimbic P22 Prelimbic Cortex 46.4, P385, P457, P749, P760 Premenstrual Dysphoric Disorder P306 Prenatal Alcohol Exposure 40.5 Prenatal Antidepressant Exposure P55 Prenatal Drug Exposure 1.4, P731 Prenatal Exposure 22.1, 45.3 Prenatal Programming P138 Prenatal Stress Model 5.5 Prepulse Inhibition P566 Prescriptions P400 Preterm Birth 5.3 Prevention of Alzheimer’s disease P8 Primates 58.2 Probabilistic Reversal Learning P789 Probabilistic Reward Learning P377 Problem-Level Use P654 Processing Speed P513 Prodrome P122, P266, P267, P140 Progesterone P231, P693 Pro-Inflammatory Cytokines 14.2, 17.2, 46.4 Prolactin P527 Protective Factor P467 Protein Aggregation P486 Protein Kinase P487 Protein Trafficking P394 Proteomics P6, P112, P401, P618, P681 Proton Magnetic Resonance Spectroscopy 9.2, P546 Prucalopride P79 Psilocin P362, P723 Psilocybin 34, 34.3, 34.4, P234, P244, P314, P346, P352, P425, P470, P723, P252, P426 Psilocybin Analog P361, P362 Psilocybin Therapy P236, P240, P241 Psychedelic Medicine 34, 34.5, P234, P254, P349 Psychedelic Therapy P802 Psychedelics 10.1, 18.4, 19, 34.2, 34.3, 34.4, P218, P236, P244, P252, P349, P352, P360, P361, P362, P425, P572, P723, P801, P426 Psychiatric Comorbidity P663 Psychiatric Disorders 32, P287, P452, P514 Psychiatric Disorders Of Aging 22.2 Psychiatric Disorders, Cognitive Function P217 Psychiatric Genetics 57 Psychiatric Hospitalization P328 Psychological Wellbeing P470 Psychometric Properties P783 Psychomotor Symptoms P513 Psychopathology P794 Psychopharmacology P440 Psychopharmacotherapy P116 Psychophysiology P85, P784, P803 Psychosis 18.2, 39, P113, P175, P494, P511, P532, P541, P550, P553, P555, P559, P586 Psychosis And Memory P542 Psychosis-Risk P500, P517, P544 Psychostimulant 4.3 Psychostimulants P122 Psychotherapy 19, 34.3, 54.1, P60, P244 Psychotic Disorder P507, P544, P547, P583, P526 Psychotomimetic Effects P350 Psychotropic Medications P501 PTSD 36, 36.3, 36.4, P29, P33, P37, P41, P43, P49, P57, P58, P59, P64, P65, P67, P83, P84, P86, P96, P105, P173, P176, P213, P680, P784, P785 PTSD Depression 31.4 PTSD, Prediction, Causal Modeling, Trajectory Modeling, Cortisol P67 Puberty 15.1, 26.2, P137 Public Health P454 Punishment P726 Pyramidal Neuron P536 Quantitative Electroencephalography (qEEG) P369, 3.5 Quantitative Relaxometry (QR) 7.4 Race Disparities 5, 28.3, P784 Racial Differences 14.2, 14.5 Racial Ethnic Minority 5.3 Racial Segregation 14 Racial/Ethnic Differences P440, P664 Racism 14.4, 55 Randomized Balaam Crossover Design P322 Randomized-Controlled Trial 60.5, P172, P240, P241, P241 Rapid-Acting Antidepressant P215, P235 Rare Genetic Variation P151, P421 Rare Variation P422 Rats P733, P18 Rbfox1 P478 RCT P44, P415 Real-Time fMRI Neurofeedback 54.3, P45 Recent Onset Psychosis P490 Recollection And Familiarity P412 Recordings P774 Recruitment P450 Reelin P216 Reformulation P29 Reinforcement Learning P90, P658, P757, P791, P807 Reinforcement Learning Modelling P519 Reinstatement P736 Relapse P319 Relapse And Hospitalization P490 Relapse Biomarkers P647 Relapse Prevention 60.3 Reliability 39, 39.3 Rem Sleep P595 Remission P330, P538 Remitted Depression P259 Repetitive Transcranial Magnetic Stimulation (rTMS) P153, P239, P321, P323, P386, P423, P557, P806, P73, P281 Reproductive Affective Disorder P231, P306 Reproductive Psychiatry P455 Research Domain Criteria (RDoC) P262, 40, P781 Resilience P211, P301 Respiratory Depression P745 Resting State P685 Resting State Brain Imaging P266 Resting State Functional Connectivity 48.5, P40, P52, P146, P279, P281, P300, P346, P387, P389, P402, P499, P584, P633, P660, P735 Resting State Networks P557, P698 Resting-State fMRI 43.2, P138, P256, P304, P336, P419, P682 Retrieval Induced Forgetting P779 Reversal Learning P658 Reward 26.2, 33.3, 37.3, 56, P249, P351, P376, P384, P701, P732, P755, P786, P791, P204, P636, P770 Reward And Aversion P749 Reward Anticipation P189, P640, P699 Reward Circuitry 43, P109, P276, P757 Reward Deficit P370 Reward Devaluation P632 Reward Expectancy P188, P326 Reward Learning P604 Reward Neural Circuitry 26 Reward Neural Network P619 Reward Prediction Error P656 Reward Processing P285 Reward Representation P99 Reward Sensitivity 54.1, P270, P652 Reward-Based Decision-Making 43.2 Ribotag P171 Right Temporo-Parietal Junction 8.3 Risk P656 Risk And Resilience P48, P107 Risky Behaviors P179 Risky Decision-Making P202 RNA-Seq 7.3, 15.3, 58, 58.3, P532, P617, P59, P134, P288, P483, P170, P171, P113, P205 Rodent Models 22.1, 53.3 Rodents P688 Rtms P9, P42 Rumination P257 S1P Receptor P410 Safety Learning P72, P94, P101 Salience P98 Salience Network P277, P645 Salvinorin A P80 Sample Size And Power 23 Sappα P7 Sappβ P7 Schizophrenia (SCZ) 2, 8.4, 9, 9.3, 21.1, 24.4,39.2, 44, 44.2, 44.3, 44.4, 44.5, 50, 57.4, P6, P114, P176, P475, P476, P478, P479, P480, P482, P488, P489, P501, P503, P509, P512, P514, P519, P523, P527, P529, P532, P533, P535, P538, P539, P540, P543, P545, P549, P550, P552, P554, P556, P557, P561, P565, P572, P577, P584, P786, P795, P535, P481, P495, P522, P526, P562, P785, P486, P487, P508, P564, P578 Schizophrenia And Bipolar Disorders 39.4 Schizophrenia And Cognition P595 Schizophrenia Negative Symptoms P496, P570 Schizophrenia Novel Treatment P479, P492, P570 Schizophrenia- Novel Treatment P180, P568 Schizophrenia Spectrum Disorders P518, P579 Schizophrenia Subtype P485 Schizophrenia Subtypes P481 Schizophrenia Therapeutics P585 Schizophrenia, Synaptic Aberrations P484 Schizophrenia; Technology P496 Schizotypal Personality Disorder 39.5 SCN2A P111 Scopolamine P208 Seizure P410 Selective Serotonin Reuptake Inhibitors (SSRIs) 27.2, P270 Self-Administration 1, 1.5, 35.1, P674, P766, P674, P766, P615, P736, P737, P764 Self-Harm P165 Self-Referential Processing 31.3 Senescence P340 Sensitive Period 42.2 Sensitization 16.4, P430 Sensorimotor 4.4, P120 Sensory Gating P544 Sensory Neurobiology 49 Septum P432 Serotonin 18.5, P62, P106, P167, P179, P204, P311, P357, P371, P379, P434, P789 Serotonin 1A Receptors 28.4 Serotonin 5-Ht2A Receptor 35.4, P352, P360 Serotonin And Norepinephrine Reuptake Inhibitor P347 Serotonin Reuptake Inhibitors 27 Serotonin Transporter P347 Sertraline 27.1 Serum Levels P445 Severe Major Depressive Disorder 54.3 Sex 15 Sex And Gender P57 Sex Differences 1.2, 11.2, 15.4, 22, 22.1, 22.3, 22.4, 37, 37.2, 42.3, 56.4, P663, P3, P11, P19, P21, P47, P102, P170, P212, P313, P387, P461, P543, P622, P650, P651, P693, P704, P714, P726, P738, P761, P762, P790, P247, P664, P132, P214, P430, P736, P308, P92, P202 Sex Hormones 33.2, P219 Sex Steroid Hormone Receptors P196 Sex-Specific P605 Sex-Specific Effects P136, P279, P456, P678 Sexual Orientation P587 Sharp-Wave Ripples 59.2 Short-Term Synaptic Depression P611 Sickle Cell Disease P449 Sign-Tracking P632 Simulated Driving P705, P706 Simvastatin P245 Single Cell Omics 11, P58 Single-Cell RNA Sequencing 33.4, 57.3, 58.3, 58.5, P609, P616, P681, P668 Single-Nucleus RNA Sequencing 58.2, 58.4, P24, P211, P432, P460, P615, P677 Single-Unit Electrophysiology In Vivo 53.5, 56.4, P473, P632 Sirtuins P295 Skin Conductance Response P72 SLC6A1/GAT1 P221 Sleep P86, P206, P263, P592 Sleep Deprivation 45.3, P595 Sleep Disorders P593 Sleep Disturbances 30, P10, P125, P590, P620, P697, P591 Sleep Oscillations P589, P596 Sleep Spindles P544 Sleep-Dependent Learning P596 Slice Electrophysiology 20.5, P355, P385, P597, P756, P776 Slow Wave Sleep P257 Slow-Wave Activity P263 Smoking P652, P666 Smoking Cessation P640 Smoking Onset Age P679 SNP P295 SNP Variation 33.4 Social 8.2 Social And Behavioral Deficits P74, P133, P459, P473 Social Anxiety P74 Social Anxiety Disorder P63, P788, P28 Social Behaviors P457, 10.2, 11, 11.5, 18.5, 26.2, 33, 33.2, 42, 42.1, 42.3, 42.4, 45.5, P222, P414, P650 Social Buffering 26 Social Cognition 8.3, 8.4, P41, P638 Social Defeat Stress 33.1, P206, P211, P248, P313, P315, P357 Social Deficits 33, P458, P474 Social Determinants Of Health 5.4 Social Determinants Of Health Inequity P709 Social Factors And Functioning P165 Social Functioning 54.2, P525, P576 Social Hierarchy 14.4 Social Impact Model 41 Social Interactions 11.4, 33, P453 Social Isolation P458, P759 Social Media Use P450 Social Perception 8 Social Play Behavior P170 Social Processing 42.2, P650 Social Recognition Memory P412 Social Rejection 26.1, P792 Social Reward P787 Social Stress Buffering P25 Social Touch 33.3 Somatostatin P385, P208 Sonic Hedgehog Signaling 4.2 Spatial Memory 37.4, P96, P135 Spatial Transcriptomics 58.4, P2, P460, P668 Spine Morphogenesis P358 Splice Site Mutations 57.1 Splice Variants P20 Splicing 57 SSRI P285 Startle P42 Statins P245 Statistical Methods 24.3, P162, P642 Stem Cells P293 Stigma 26.1, 41 Stimulant Use Disorder P707 Stimulants P451, P453, P725 Stratification P516 Stress P143, P256, P370, P582, P684, P20, P209, P247, P251, P310, P376, P680 Stress And Adversity P147 Stress And Anxiety Behavior P68, P378 Stress And Depression 6, 26.3 Stress And Trauma 36.5 Stress Coping P253, P358 Stress Hormones P688 Stress In Adolescence P110 Stress Models P171 Stress Resilience P253 Stress Resilience And Susceptibility P55, P248 Stress Response Circuitry 60.2 Striatal Dopamine Signaling P771, P809 Striatum 20.2, 43.3, P583, P699, P763 Structural Adversity 45.4 Structural And Functional Connectivity P647 Structural Connectivity P801 Structural Covariance Analyses P545 Structural MRI 9.3, P513 Structural Racism 5.2 Structural Stigma P587 Structural Stressors 45 Subchronic Ketamine Model P474 Subcortical Volume P521, P637 Subgenual Anterior Cingulate Cortex P233 Subgroups P488 Subjective Effects P240, P241, P439, P716 Substance Abuse 53.4, P683 Substance Abuse Disorders 34.4, P705, P629 Substance Use 14, P704 Substance Use Disorders P676, 34, 56.5, P644, P669, P699 Substantia Nigra P157 Sudden Cardiac Death P526 Suicidal Behavior 28.4, 51.2, P142, P590 Suicidal Ideation 28.2, P142, P246, P268, P590, P780, P300 Suicidality P799, P332 Suicide 28, P142, P231, P269, P280, P466, P467 Suicide Attempt 28.3, 48.4, P165, P296, P300, P365, P465, P471 Suicide Behavior Severity P165 Suicide Mechanisms P465 Suicide Risk Factors 28.5, P213, P261, P275, P464, P465, P466 Superior Orbitofrontal Cortex P188 Supplementary Motor Cortex 20.2 SV2A PET imaging P484 Symptom Dimensions P281 Synapse P3, P310 Synapse Loss P312 Synapses P114, P209, P382 Synaptic Connections P776 Synaptic Density P198 Synaptic Function P617 Synaptic Orgnization P548 Synaptic Plasticity 18, 19, 20.5, P250, P353, P355, P610 Synaptogenesis P327 Synaptosomes P401 Synthetic Cannabinoids 1.2 Synthetic Psychoactive Cathinones P744 Systemic Inflammation P272 Systems Neuroscience P108 Systems Pharmacology P670 TAAR 1 P574 TAAR1 P180, P492 TAAR1 Knockout Mice P574 Tail Suspension Test P425 Tardive Dyskinesia P491, P558 Target Engagement 54.1, P564 Targeted Cognitive Training P785 Task fMRI 51, 51.1, P44, P187, P512, P648 Task-Based Functional Connectivity 25.2, P299 Taste P468 Tau P394 TBI P26, P37 TCB-2 P360 Technology P592 Telemetry P206 Telomere Length P15 Telomeres P17 Temporal Pole P70 Test-Retest Reliability P515 Thalamo-Cortical Connectivity P524, P544 Thalamus P637, P757, P769 THC 1.5, P18, P618, P714, P743 the COVID-19 pandemic 14.3, 14.4, P276, P713 The Dimensional Anhedonia Rating Scale P337 Therapeutic Alliance P240 Therapeutic Approach 42 Therapeutic Drug Monitoring P563, P445 Therapeutic Interventions 22.2 Therapy 16 Theta Band Oscillatory Measures 59.2 Theta Burst Transcranial Magnetic Stimulation 3.3, P71, P320, P498 Theta-Burst Stimulation P324, P326 Threat Conditioning P169 Threat Faces 36.4 Threat Reactivity P70 Thyroid Hormone P265 Tianeptine P285 Time Estimation P712 Time Varying Functional Connectivity P768 Time-Frequency P778 Timing P600 TMS P65, P233, P331 TMS targeting P282 TMS-EEG P324, P366, P380 TNF-alpha P554 Tobacco 34.4, P676, P719 Tobacco Dependence P686 Toll-Like receptors (TLRs) P160 Tools For Building Your Successful Career 38 Touch 16.3 Touchscreen 40.4, 40.5, P751 Touchscreen Cognitive Testing P502 Tourette Syndrome P116 Trace Amines P574 Trajectories P400 Transcranial Current Stimulation P494 Transcranial Direct Current Stimulation (tDCS) P655 Transcranial Magnetic Stimulation P389 Transcript Isoforms 57.3, P536 Transcriptome 44.5, P290, P603, P689 Transcriptome Biology P612 Transcriptomics 37.3, 58, 58.2, P8, P293, P307, P356, P420, P602, P659, P667 Transcutaneous Auricular Vagus Nerve Stimulation (taVNS) 22.4 Transdiagnostic 7, 32.4, P782, P794 Transgender 26, 26.2 Transgenerational P756 Transition To Broad Psychosis Spectrum Psychopathology 12, 18.2, P546 Translational Animal Models 1.4, P314, P671 Translational Approaches To Drug Development 13 Translational Biomarker Development 40.5 Translational Cognitive Testing 40.3 Translational Neuroscience P469 Translational Research 13, 18, 29, 40, 59, 59.4, 59.5 Translational Studies 16 Translocator Protein (TSPO) P651 Transplantation P190 Trauma P43, P684 Trauma Exposure P493, P661 Traumatic Brain Injury 60.4, P413 Treatment P173 Treatment Mechanisms 7, 7.4 Treatment Outcome P624 Treatment Outcome Prediction P286, P520 Treatment Prediction P334 Treatment Resistant Depression P230, P300, P322, P366, P210, P216, P227, P234, P244, P245, P318, P321, P323, P324, P341, P372 Treatment-Resistance P317, P538, P562, P388 Treatment-Resistant Schizophrenia P563 Treatment-Response P545 TSPO P186 Tumor Necrosis Factor P25 TWAS 24.5, P514 Two-Photon Calcium Imaging P468, P580 Ubiquitination P486 Ulotaront P492 Ultra-High Field Mri P298 Ultrasonic Vocalization P102 Ultrasonic Vocalization (USV) Model P359 Unbiased Effect Size Estimation 23 Uncertainty P90, P428, P519, P53 Unconditioned Responses P82 Vaccine 35.3 Vaccine Hesitancy P454 Valbenazine P491 Valence P95, P381 Value-Based Decision-Making P428, P653 Vapor 1.4, P743 Vasopressin 1A Receptor Antagonist P469 Vasopressin 1B Antagonist P229 Venlafaxine P344 Ventral Hippocampus 45.4, P35, P55, P100, P377, P582, P776 Ventral Pallidum P381, P599 Ventral Striatum P98, P427 Ventral Tegmental Area (VTA) 50.2, 58.3, P288, P607, P775, P211, P748 Ventral Visual Stream P258 Ventrolateral Prefrontal Cortex P427 Ventromedial Prefrontal Cortex 25.4, 31.3, 36.2 Verbal Episodic Memory 9.4 Verbal Memory P639 Vesicular Glutamate Transporter (VGLUT) 37.2 veterans P173, P529, P780, P73, P261 Viloxazine P106, P167, P434 Violence P528 Viral Vectors 53, 58.2 Virtual Reality P71, P72 Virus Infection P397 Visual Cortex 7.2 Visual Perception P423 Visuospatial Working Memory P536, P502 vlPFC P326 VMAT2 P438 VTA P430 Wake-Promoting P594 Wheel Running P190 White Matter Development 21.1, 31.1, P144 White Matter Microstructure 7, 7.5, P47, P124 Whole-Body Plethysmography P745 Whole-Brain Rodent Imaging P249, P769 Williams Syndrome P402 Withdrawal 1.2, P598, P719, P741, P746, P750 Women P86, P643 Women’S Health P16, P308 Women’S Mental Health P84, P591, P665 Word Embedding Clustering 52 Working Memory 39, 39.3, P18, P64, P131, P509, P510, P512, P786 Working Memory Capacity 39.5 Working Memory Fmri P277, P442 X Chromosome P219 Xanomeline P480 Young Adults P276 Youth P289, P329 Zebrafish P128, P148 Zif268 P766 Zinc P730 Zuranolone P242 Α5 GABAA Positive Allosteric Modulator P437 ΔFosB P309	36456697	PMC9714386	NO-CC CODE	2022-12-06 23:15:03	no	Neuropsychopharmacology. 2022 Dec 1; 47(Suppl 1):555-569	utf-8	Neuropsychopharmacology	2,022	10.1038/s41386-022-01489-w	oa_other
==== Front Neuropsychopharmacology Neuropsychopharmacology Neuropsychopharmacology 0893-133X 1740-634X Springer International Publishing Cham 36456692 1483 10.1038/s41386-022-01483-2 Abstracts Collection ACNP 61st Annual Meeting: Panels, Mini-Panels and Study Groups 1 12 2022 12 2022 47 Suppl 1 162 © The Author(s), under exclusive licence to American College of Neuropsychopharmacology 2022 issue-copyright-statement© American College of Neuropsychopharmacology 2022 ==== Body pmc December 4-7, 2022 Phoenix, Arizona Sponsorship Statement: Publication of this supplement is sponsored by the ACNP. All content was reviewed and selected by the Program Committee, which held full responsibility for the abstract selections. Only disclosures for presenting authors are listed. Individual contributor disclosures may be found within the abstracts PANEL 1. Chasing the Green Dragon: Challenges and Progress in the Development of Rodent Models of Cannabis and Cannabinoid Self Administration 1.1 Spontaneous and Precipitated Cannabinoid Withdrawal in Male and Female Rats Following Self-Administered or Experimenter Administered WIN55, 212-2 Delivery Sade Spencer The University of Minnesota, Minneapolis, Minnesota, United States Background: Synthetic cannabinoids (SCBs) gained popularity as a designer drug alternative to cannabis in the 2000s. WIN55,212-2 (WIN) is an aminoalkylindole derivative, and drugs from this class are common in SCB products. SCBs produce significant adverse effects, and users of SCBs report more severe withdrawal compared to people using plant cannabis. This study assessed withdrawal in male and female rats following two methods of administration of the SCB WIN. Methods: Adult male and female Long Evans rats received jugular catheter implantation. In experimenter-administered drug studies, rats were given escalating doses of WIN (0.2, 0.4, 0.6, and 0.8 mg/kg) via twice daily iv infusions (vehicle injected animals as controls). Withdrawal was precipitated with the CB1R inverse agonist SR 141716A (10 mg/kg, ip) four hours after the final infusion (n = 8/group). Spontaneous withdrawal was observed at 6, 14, 24, 48, 72, and 96 hours (n = 7-8/group). In the self-administration studies (SA), rats were given access to WIN (6.25 ug/kg for females; 12.5 ug/kg for males) or vehicle under standard FR1 SA parameters. Withdrawal was precipitated with SR 141716A four hours after the final SA session (n = 3-4/group), and spontaneous withdrawal was scored at 14, 24, 48, 72, and 96 hours (n = 3-6/group). Somatic withdrawal signs were observed over 30 minutes to generate a global withdrawal score (GWS). Results: There was a main effect of WIN treatment on GWS (F(1,26) = 6.611, p = 0.0162) with precipitated withdrawal from experimenter administered WIN. Sidak’s multiple comparison testing revealed a significant WIN treatment effect in males only (p = 0.035). There was a main effect of WIN treatment (F(1,41) = 22.15, p < 0.0001) and a Time x Sex interaction (F(5,119) = 2.502, p = 0.0342) on GWS with spontaneous withdrawal from experimenter administered WIN injections. Tukey’s multiple comparison testing indicated a significant difference between WIN treated male and female rats at 48 hours with males showing a higher GWS (p = 0.0415). Equivalent analysis was performed after WIN SA. Conclusions: There is a robust spontaneous cannabinoid withdrawal syndrome observed following spontaneous withdrawal from experimenter-administered or self-administered SCB in male and female rats. Cannabinoid withdrawal was more robust in male rats in the non-contingent model, contrary to our expectations. Disclosure: Nothing to disclose. 1.2 Assessing the Long-Term Impact of Intravenous Cannabinoid Self-Administration in Adolescent Male and Female Rats Sierra Stringfield University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Cannabis use during adolescence is associated with cognitive deficits and risk of psychiatric disorders in adulthood. Cannabidiol (CBD) is a component of cannabis that may diminish the aversive properties of THC during self-administration or protect against cognitive impairments associated with drug. We investigated the impact of CBD on intravenous THC self-administration in adolescent male and female rats and establish if it protected against diminished performance on a cognitive task. We also investigated the protracted and acute effects of THC on prefrontal cortical activity during the cognitive task. Methods: Adolescent male and female rats self-administered escalating doses of THC (3, 10, 30, and 100 µg/kg/infusion) alone or combined with CBD (10:1 ratio). In adulthood, rats were trained on a delayed-match-to-sample working memory task which requires responding after increasingly difficult delays. A separate group of adolescent rats intravenously self-administered THC or a control vehicle solution throughout adolescence. Rats expressing CaMKII.GCaMP6f in the prelimbic prefrontal cortex performed the working memory task during single-photon calcium imaging sessions. These animals were given acute injections of THC (0.5 mg/kg, i.p.) or vehicle 30 min before testing. Results: Male and female adolescent rats acquired THC or THC + CBD self-administration with no effect of drug solution or sex. No significant difference in working memory performance emerged based on sex or adolescent self-administration condition. In rats that self-administered THC or vehicle during adolescence that received acute injections of THC, females significantly reduced responding on the working memory task compared to males regardless of prior drug exposure. Neuronal activity increased across all groups but the proportion of differentially modulated neurons corresponding to task accuracy was altered only in females without adolescent THC exposure. Conclusions: These results demonstrate that the presence of CBD did not promote THC self-administration in adolescence or impact performance on a cognitive task in adulthood. Additionally, acute THC enhanced cortical activity regardless of adolescent exposure yet primarily impacted behavior and the associated neuronal response in adult females that were not exposed to THC during adolescence. Disclosure: Nothing to disclose. 1.3 A Response-Contingent Cannabis Vapor Delivery Approach for Interrogating Protracted Effects of Maternal Cannabis Use in Developing Offspring Ryan McLaughlin Washington State University, Pullman, Washington, United States Background: Cannabis use during pregnancy is a growing public health concern and its impacts on developing offspring remain elusive. This is partly due to a lack of animal studies that use volitional drug delivery models in pregnant dams. To this end, we developed a model of maternal cannabis use that employs response-contingent delivery of vaporized cannabis extracts in rat dams and examined whether prenatal cannabis exposure alters emotionality, vapor self-administration, and corticostriatal inputs in adulthood. Methods: Sprague Dawley rat dams self-administered cannabis vapor (69.8% Δ9-THC; 150 mg/mL) (n = 11) or vehicle (4:1 PG:VG) (n = 13) vapor on a fixed-ratio-1 schedule twice daily in hour-long sessions throughout mating and gestation. Isolation-induced ultrasonic vocalizations (USVs) were measured in early life and behavior in the elevated plus maze and novelty-suppressed feeding tests were assessed in adulthood. Other offspring self-administered cannabis vapor or vehicle vapor according to an escalating schedule of reinforcement over 21 days during adulthood. Remaining offspring received injections of fluorescent retrobeads (200 nl/side) into the nucleus accumbens core (NAc) during adulthood and excitatory postsynaptic currents (sEPSCs) were measured in NAc-projecting medial prefrontal cortex (mPFC) neurons. Results: Cannabis dams showed greater discrimination for the cannabis-paired operandum (p = .003) and earned more vapor reinforcers than vehicle dams (p = .066). Cannabis-exposed pups weighed less than vehicle- (p < .001) and air-exposed pups (p = .039) on postnatal day (PND) 1 and emitted more USVs on PND6 (p < .05) but no differences in anxiety-like behavior were observed in adulthood. Female offspring self-administered significantly more cannabis vapor and had higher break points during a progressive ratio challenge irrespective of prenatal exposure condition (both p’s < .05). Conversely, male cannabis-exposed offspring showed reduced responding for all vapor in adulthood (p = .032). Cannabis-exposed offspring had higher sEPSC frequencies in NAc-projecting mPFC neurons, indicating altered excitability within the corticostriatal pathway. Conclusions: These results support the use of the cannabis vapor self-administration approach to investigate long-term effects of maternal cannabis use in developing offspring. Disclosure: Nothing to disclose. 1.4 Progress and Hurdles in Establishing Vapor Self-Administration in Rodents Jacques Nguyen Baylor University, Waco, Texas, United States Background: Cannabis is commonly used via inhalation, i.e., smoking and, more recently vaping, of the plant material and/or cannabis extracts. Due to a high base rate of exposure, there are more people who meet criteria for cannabis dependence than have used cocaine in the past month, or ever tried heroin. Pre-clinical models of the self-administration of cannabis, extracts or active constituents have been limited and there are only a few recent attempts at inhalation self-administration in rodents. Methods: Groups (N = 8) of male Wistar rats were permitted to respond on nose-poke manipulanda for deliveries of propylene glycol (PG) vapor adulterated with ∆9-tetrahydrocannabinol (THC; 50-100 mg/mL) delivered into sealed vapor exposure chambers through the use of controllers that trigger commercial e-cigarette tanks. The response requirement was incremented from Fixed Ratio 1 to 5 and dose substitution (12.5, 25, 100 mg/mL) was assessed in a counterbalanced order. In one group intake was limited to 5 vapor deliveries for 24 sessions before evaluating ad libitum responding. Results: Rats exposed themselves to 10-20 minutes of THC vapor inhalation, a duration which produces cannabinoid typical effects with non-contingent exposure. Mean vapor deliveries were not systematically changed across up to 50 sessions of training, even as the FR was incremented. Rats obtained more infusions of the 12.5 mg/mL concentration than when 100 mg/mL was available. Responses on the drug-associated manipulandum were below 80% of responses. Ad libitum intake was increased after release of the limitation to 5 vapor deliveries. Conclusions: Rats will make responses for deliveries of THC infused vapor in daily sessions, exposing themselves sufficiently for a physiologically significant effect. Dose-dependent changes in behavior can be observed as well as defense of exposure against increasing workload. Major limitations to the model include a lack of behavioral tuning on the drug-associated lever, shallow dose-effect functions and individual differences. Much validation work remains to result in a convincing model of self-administration. Disclosure: Nothing to disclose. STUDY GROUP 2. Developing Novel Clinical Outcome Assessments (COAs) for Psychiatric Illnesses Linda Brady, Sonya Eremenc, Richard Keefe, Madhukar Trivedi, Michael Sand, Kenneth Koblan, Elizabeth Stafford, Rachel Streiff, Sarah Lisanby National Institute of Mental Health, Rockville, Maryland, United States Study Group Summary: There are many challenges in the development of drugs for the treatment of psychiatric disorders. This panel will focus on the development of fit-for-purpose clinical outcome assessments (COAs) to assess treatment benefit in clinical trials. COAs are instruments that measure or reflect how patients feel, function, or survive. COA measures include patient-reported (PRO), clinician-reported (ClinRO), observer-reported (ObsRO), and performance (PerfO) outcome measures. Optimizing the match between trial population, instrument, and endpoint will increase the validity and power of clinical trials, especially for the development of new treatments. Ongoing and recent efforts as well as innovative approaches will be considered. Examples of new COAs for MDD to be discussed include the Symptoms of Major Depressive Disorder Scale (SMDDS), which is a PRO measure with a 7-day recall period that was developed by the Critical Path Institute (C-Path) PRO Consortium Depression Working Group (WG). The SMDDS was qualified by FDA as a PRO measure within a limited context of use (COU) in clinical trials to assess self-reported depression symptom severity in adults with MDD. The Depression WG2.0 is developing two new COAs for MDD with the ability to assess the effectiveness of rapid acting antidepressants: 24 hour-recall and momentary assessment measures. Letters of Intent (LOIs) for each measure were submitted to FDA’s COA Qualification Program (COAQP) and both measures were accepted into the qualification program. FDA operates the COAQP under the 21st Century Cures Act as part of its Drug Development Tool efforts. Qualification of a COA is a regulatory conclusion that the FDA finds the COA to be a well-defined and reliable assessment of patients’ symptoms, overall mental state, or functioning within a specific context of use. FDA can also review COAs under a sponsor’s drug development program. Another example of a new COA to be discussed is a PerfO measure for schizophrenia, the Virtual Reality Functional Capacity Assessment Tool (VRFCAT), a computerized tool developed as a measure of functional capacity with the potential to translate to real-world functional improvements associated with cognitive change. The VRFCAT LOI was accepted into the COAQP as a proposed co-primary outcome measure in schizophrenia treatment trials. It is currently being used as a secondary endpoint measure in sponsor drug trials in schizophrenia. The psychometric properties of VRFCAT and ongoing development efforts will be discussed. With these case examples in mind, study group participants will discuss the COAQP process and lessons learned in the development of fit-for-purpose COAs for use in clinical trials. Participants will also explore areas of unmet need for new COAs to enable innovation in treatment development. The participants include academic and pharmaceutical investigators and clinical trialists, C-Path, advocacy organizations, an individual with lived experience, NIMH, and FDA. Collaboration is key for successful development of a new COA. Qualitative research with patients, caretakers, and healthcare providers offers the foundational insight for instrument development and selection of the appropriate tool to fit the research need. This study group offers a venue for Q and A with multiple stakeholders to consider the challenges and opportunities for advancing the development of new COAs for psychiatric disorders. Disclosure: Nothing to disclose. PANEL 3. Novel Synergistic Treatment Combinations to Leverage Plasticity Induced by Drugs Acting at NMDA Receptors 3.1 A Tale of Two Receptors: Simultaneous, Prophylactic Targeting of NMDARs and 5-HT4Rs Exerts Additive Effects in Protecting Against Stress-Induced Anxiety-Like Behavior Christine Ann Denny Columbia University Irving Medical Center, New York, New York, United States Background: Serotonin (5-HT) receptors and N-methyl-D-aspartate receptors (NMDARs) have both been implicated in the pathophysiology of stress and depression. Here, we evaluated whether combined dosing of (R,S)-ketamine, an NMDAR antagonist, and prucalopride, a 5-HT type 4 receptor (5-HT4R) agonist, would have additive effects, resulting in improvements in stress-induced maladaptive behavior. Methods: A single injection of saline, (R,S)-ketamine, prucalopride, or a combined dose of (R,S)-ketamine + prucalopride was administered 1 week before a 3-shock contextual fear conditioning (CFC) paradigm in 129S6/SvEv mice of both sexes. Drug efficacy was assayed using a variety of behavioral tests, including the forced swim test (FST), elevated plus maze (EPM), open field (OF), marble burying (MB), and novelty-suppressed feeding (NSF). Neural activity (e.g., c-fos immunoreactivity) was quantified throughout the hippocampus. Results: Prophylactic (R,S)-ketamine + prucalopride administration attenuated learned fear in male mice (n = 6-15 mice per group; ANOVA, p = 0.0035) and decreased behavioral despair in both male and female mice (n = 6-15 mice per group; RMANOVAs, p = 0.0202 and p = 0.0133, respectively). Prophylactic (R,S)-ketamine + prucalopride administration had an additive effect of decreasing stress-induced hyponeophagia in male and female mice (n = 5-15 mice per group; Mantel-Cox, p < 0.0001 and p = 0.0007, respectively). Notably, prophylactic (R,S)-ketamine + prucalopride administration at lower doses was effective in the NSF, but not when administered separately at those doses. Combined (R,S)-ketamine + prucalopride administration, but not either drug alone, significantly increased c-fos expression throughout the hippocampus (n = 5-9 mice per group; ANOVAs, p’s < 0.0001). Conclusions: Together, these results indicate that combined (R,S)-ketamine + prucalopride prophylactic administration exerts additive neural and behavioral effects compared to administration of either drug alone. Our results suggest that combined administration of an NMDAR antagonist and 5-HT4R agonist has additive benefits for stress-induced pathophysiology, providing preliminary evidence that future clinical studies using this combined treatment may prove advantageous. Disclosures: Silo Pharma: Contracted Research (Self), Janssen, McLean: Honoraria (talk given) (Self), Columbia University (numerous patents and provisional patents): Patent (Self), NIH Study Section: Honoraria (Self), Transformative Award, NINDS R21, NIA R21, Columbia Trx Grant: Grant (Self), For the Love of Travis (donation to lab): Other Financial or Material Support (Self) 3.2 Efficacy of Adjunctive D-Cycloserine to Intermittent Theta Burst Stimulation for Major Depressive Disorder: A Randomized Clinical Trial Alexander McGirr Hotchkiss Brain Institute, University of Calgary, Calgary, Canada Background: Transcranial magnetic stimulation is a non-invasive treatment for major depressive disorder (MDD) that is believed to depend on synaptic plasticity in targeted circuits. Protocols such as theta-burst stimulation (TBS) are N-methyl-D-aspartate (NMDA) receptor dependent, suggesting that targeting the NMDA receptor could be an effective means of enhancing treatment outcomes. Here, we test whether low doses of the NMDA receptor partial-agonist, D-Cycloserine, can enhance intermittent TBS (iTBS) treatment outcomes in MDD. Methods: In this single site 4-week randomized double-blind placebo-controlled trial, fifty participants with treatment resistant MDD were randomized 1:1 to iTBS+Placebo or iTBS+D-Cycloserine (100mg). Participants were asked to take the adjunct or placebo at least 60 minutes prior to daily iTBS treatments for the first two weeks, and iTBS continued without an adjunct for weeks three and four. The primary outcome was change in depressive symptoms as measured by the Montgomery Asberg Depression Rating Scale (MADRS) at the conclusion of treatment. Secondary outcomes included clinical response, clinical remission, and clinical global impression (CGI). Results: The iTBS+D-Cycloserine group had greater improvements in MADRS scores compared to the iTBS+Placebo group (mean difference -6.15, 95%CI -2.43 to -9.88; Hedges’ g = 0.99, 95%CI 0.34-1.62). Rates of clinical response were higher in the iTBS-D-cycloserine than in the iTBS+Placebo group (73.9% vs 29.3%), as were rates of clinical remission (39.1% vs. 4.2%). This was reflected in lower CGI severity ratings, and greater CGI-improvement ratings. No serious adverse events occurred. Conclusions: Adjunctive D-Cycloserine is a promising strategy for enhancing transcranial magnetic stimulation treatment outcomes in MDD using iTBS. Disclosure: Provisional Patent: Patent (Self) 3.3 A Brief, Fully Automated Neurocognitive Training Intervention Extends the Antidepressant Effect of a Single Ketamine Infusion: Clinical and Neuroimaging Findings Jared Kopelman UC San Diego, San Diego, California, United States Background: Intravenous ketamine (ket) is posited to rapidly reverse depression by enhancing neuroplasticity. We posited that ketamine would provide a ‘window of opportunity’ in which to introduce automated neurocognitive training techniques to consolidate adaptive forms of cognitive processing while neuroplasticity remains high. Methods: 154 unipolar depressed adults who failed at least one antidepressant medication were randomized to one of three arms: (1) a single infusion of ket (0.5mg/kg over 40min) followed by 4 days (~2.5 hrs total) of active ‘automated self-association training’ (ASAT), targeting self-worth through ‘evaluative conditioning,’ (ket+ASAT;n = 53); (2) ket infusion followed by 4 days of sham training (ket+Sham;n = 50); or (3) saline (sal) infusion followed by 4 days of training (sal+ASAT;n = 51). The Montgomery-Asberg Depression Rating Scale quantified depression severity over a 30 day period. In a subset of patients (67 ket, 31 sal) whole-brain Diffusion Tensor Imaging (DTI) data were collected prior to and 24 hrs post-infusion. DTI mean diffusivity (DTI-MD), a putative marker of neuroplasticity, was calculated for 7 ROIs (left and right BA10, left and right amygdala, and left and right hippocampus, and vACC). Results: Ket rapidly reduced depression scores at 24 hrs post-infusion (β=-1.30;t150 = -4.29;p < .0001; 52% responders vs. 25% in sal arm). Over the 30-day acute phase, ket+ASAT produced a stable enduring decrease in depression relative to saline+ASAT (β=-0.61;t148 = -3.62;p = .0004). By contrast, depression scores following ket+Sham followed an increasing linear trajectory from 24 hrs to 30 days, as the post-ket effect waned and depression approached sal+ASAT levels (grouptime relative to saline+ASAT: β=0.015;t568 = 2.35;p = .019). In follow-up, the ket+ASAT arm showed continued benefits on self-reported depression 3 mo post-infusion (d = .57;t83 = 2.6;p = .01). Our neuroimaging results indicated that individual differences in DTI-MD in medial prefrontal and limbic ROIs were associated with improvement in depression scores (p’s < .05), primarily in the ket group. Conclusions: In the present study, ASAT extended the rapid antidepressant effect of a single dose of ket to at least 30 days. In addition, our DTI data indicate the acute effects of ket on depression may be mediated, at least in part, by acute changes in plasticity. Disclosure: Nothing to disclose. PANEL 4. Cilia GPCRs in Neuropsychiatric Disorders 4.1 Primary Cilia Signaling Shapes Excitatory Synaptic Connectivity Jiami Guo Hotchkiss Brain Institute, University of Calgary, Calgary, Canada Background: In the mouse cerebral cortex, pyramidal neurons, the main class of excitatory neurons in the cortex, elaborate a tree-like structure called a dendrite, which contains thousands of small protrusions called dendritic spines to receive synaptic inputs from other neurons. The appropriate size and shape of dendritic arbors and spines is a key determinant of how neurons receive, integrate, and encode circuit information. Knowing how dendrites and dendritic spines acquire their form during development is thus essential to understanding the emergence of functional neural circuits. It is clear that environmental signals, such as diffusible morphogen cues, have major influences on dendrite morphogenesis12. We recently found that primary cilia serve as centralized signaling hubs regulating dendrite morphogenesis. This discovery challenges the existing view that the extracellular environmental cues primarily act directly on the dendritic membrane to control dendritic shape. Instead, it identifies an additional, undefined, centralized signaling mechanism that stems from the primary cilia positioned at the neuronal soma. Methods: We generated mice with ciliary gene deletion specifically in glutamatergic excitatory neurons. Dendrite and spine morphology was analyzed by confocal microscopy. Synaptosome was analyzed using proteomics. Behavioral changes were analyzed by behavioral assays. AAV viruses were injected into the prefrontal cortex in the adulescent stage to induce deletion of ciliary genes and changes in social dominance were examined. Results: Dendritic growth is reduced, and spine morphology is changed in ciliary mutant neurons. Synaptosome shows changes in protein composition in ciliary mutants. Mutant mice display changes in motor and cognitive behaviors. Induced ciliary gene deletion in the adulescent stage leads to changes in spine dynamics and social dominance. Conclusions: Upon completion of this project, we will have gained transformative insights into how neuronal primary cilia function to regulate dendrite morphogenesis. These efforts will help uncover hitherto undefined cell biological mechanisms fundamental for neurodevelopment and functional wiring of the brain. Disclosure: Nothing to disclose. 4.3 Primary Neuronal Cilia Loss Impacts Behavioral Responses to Psychostimulants in Cell-Specific Manners Jeremy McIntyre University of Florida, College of Medicine, Gainesville, Florida, United States Background: In recent years, neuronal primary cilia have garnered much attention for their role in a variety of neurobehavioral contexts including drug responses, thus offering a new landscape to explore. Primary cilia are microtubule-based organelles that project from the surface of nearly all mammalian cells, including neurons. They function as signaling hubs and are enriched with a diverse array of GPCRs, including several known to be associated with motivation and drug-related behaviors; however, our understanding of how cilia regulate neuronal function and behavior is still limited. We previously showed that neuronal cilia loss alters locomotor responses to amphetamine in a cell-type specific manner. The objective of the current study was to investigate the contributions of primary cilia on specific neuronal populations to behavioral responses to cocaine. Methods: To test the consequences of cilia loss on cocaine-induced locomotion and reward-related behavior, we selectively ablated cilia from dopaminergic or GAD2-GABAergic neurons in male and female mice, using DAT:CRE or GAD2:Cre respectively. Locomotor effects were tested to both acute and repeated administration of cocaine. Conditioned-Place preference was used to analyze differences in the rewarding effects of cocaine in mice lacking cilia. Data were analyzed use two-factor ANOVAs. Results: Cilia ablation on either dopaminergic or GAD2-GABAergic neurons failed to significantly alter acute responses to cocaine at a range of doses (3, 10, and 30mg/kg). With repeated administration, mice lacking cilia on GAD2-GABAergic neurons exhibited enhanced locomotor sensitization to cocaine at 3mg/kg compared to wild-type littermates ((F(1,21) = 6.448, p = .019)), whereas mice lacking cilia on dopaminergic neurons exhibited reduced locomotor sensitization to cocaine at 10 and 30mg/kg (F(1,17) = 6.458, p = .021)). To test the rewarding effects of cocaine we used a conditioned place preference test. Mice lacking cilia on GAD2-GABAergic neurons show no difference in cocaine CPP. However, mice lacking cilia on dopaminergic neurons exhibit reduced CPP compared to wild-type littermates. Conclusions: Combined, our results show that behavioral effects of cilia ablation are cell- and drug type-specific, and that neuronal cilia regulate both the locomotor-inducing and rewarding properties of cocaine. Disclosure: Nothing to disclose. PANEL 5. Stress and Social Determinants Affect Pregnancy and Early Development: A Critical Window for Generational Studies 5.2 Unequal Beginnings: Maternal Life Stress and Health Inequities Affect the Next Generation Catherine Monk Columbia University Vagelos College of Physicians and Surgeons, New York, New York, United States Background: Preterm birth (PTB) affects 1 in 10 women, is the leading cause of neonatal mortality and morbidity, and is associated with future risk for poor mental (ADHD) health. In the U.S., racial and ethnic disparities in PTB exist independent of socio-economic status: overall, 11.37-9.38% for Hispanic versus 9.10% for non-Hispanic White women. Psychosocial stress and childhood trauma are associated with risk for PTB. PTB has an intergenerational impact: mothers born preterm are more likely to give birth preterm. Biomarkers to predict PTB have proven unsuccessful, and do not account for the recognition of intergenerational transmission via maternal heritage. Mitochondria, which contain their own genome, the mitochondria DNA, are inherited from the mother and represent a potential intersection point between psychosocial experiences and their biological embedding in PTB. Methods: In early 2nd trimester in n = 187, 27 data points are obtained from questionnaires, ambulatory diaries, and physical assessments to phenotype stress groups. In a subsample of women, placental RNA-seq data is currently available allowing for summary scores of cellular energetic pathways to determine whether maternal stress phenotypes differ in mitochondrial-related gene expression in the placenta (n = 37). Results: Three latent profiles of maternal stress were identified: 66.8% Healthy; 17.1% Psychologically Stressed, 16% Physically Stressed. Hispanic vs Non-Hispanic PTB was 16% vs 9%. For Hispanic women but not non-Hispanic White women, stress group was associated with risk of PTB (OR = 5, 95% CI 1.26-19.9; OR = 1.33, 95% CI: 0.13-14.2). Psychologically stressed mothers showed higher transcript levels for mitochondrial biogenesis and respiratory chain transcript accompanied by lower metabolic sensing and lower glycolysis transcript expression, suggesting they might rely more on placental mitochondrial energy production than healthy pregnant women (Fig. 1). Conclusions: These findings from RNA-seq show that psychologically stressed mothers have higher expression of a gene involved in making new mitochondria and mitochondrial respiratory chain proteins, suggesting that placenta from these mothers are making more ATP; we plan to look directly at the enzymatic activity of the respiratory chain and test that hypothesis. Disclosure: Curio: Advisory Board (Self) 5.3 The Impact of Social Determinants of Health on Infant Brain Development Cynthia Rogers Washington University School of Medicine, St. Louis, Missouri, United States Background: Social determinants of health like exposure to poverty, neighborhood crime, and racial discrimination are increasingly recognized as having deleterious effects on development. What is less well known is how exposure to these social determinants during the prenatal period may impact the developing brain. This presentation will review data from ongoing longitudinal studies of prenatal exposure to social determinants and neonatal and infant brain development. Methods: These studies include approximately 400 caregiver-infant dyads that were predominantly Black and enriched for exposure to adversity. Participants were recruited during pregnancy with assessments of income to needs, neighborhood poverty, neighborhood crime exposure, racial discrimination, depression, perceived stress, and stressful life events. Infants underwent MRI scans during the neonatal period. Image analyses included resting state functional connectivity, diffusion tractography of white matter tracts and structural analyses of volume and surface area with a focus on brain regions related to emotion regulation and emotion processing. Latent factors encompassing social disadvantage variables and psychosocial stress variables were created and related to neonatal brain measures. Results: Prenatal social disadvantage was significantly related to the functional connectivity of cortical networks (R2 = 0.29, p < .05) but particularly with frontoparietal, default-mode, and ventral attention networks. Similarly, prenatal social disadvantage was related to neonatal diffusion measures of the cingulum bundle (β = -.24, p < .001) the uncinate fasciculus (β = -.16, p < .01) and the fornix (β = -.11, p < .05). Social disadvantage was also related to hippocampal (R2 0.22, p = .009) and amygdala (R2 0.41, p = .009) volumes. Psychosocial stress was not significantly related these measures when controlling for social disadvantage. Conclusions: Prenatal exposure to social determinant of health factors particularly those that index social disadvantage and poverty were related to multiple measures of neonatal brain development including cortical networks, white matter tracts and subcortical regions that are related to emotion regulation and emotion processing. Future work will relate these findings to early childhood developmental outcomes. Disclosure: Nothing to disclose. 5.4 Translational Approaches to the Impact of Stress on Maternal Microbes and the Next Generation Tamar Gur Ohio State University College of Medicine, Columbus, Ohio, United States Background: Previous studies show that exposure to prenatal stress has long term consequences on offspring. Work from our lab pinpoints the maternal microbiome and the chemokine CCL2 as key mediators of long term neuroinflammation and reductions in social behavior in rodents. Here we address the central role of fetal CCL2 and extend our preclinical findings into a prospective cohort clinical study. Methods: Preclinical Study: Intra-amniotic injections of recombinant CCL2 or saline were performed on E16.5. Tissues were collected 24 hours after the procedure to measure concentration of CCL2 in maternal and fetal tissues by ELISA. A second cohort went through parturition and social behavior was measured in adulthood in the 3 chamber social approach paradigm. Clinical Study: A prospective longitudinal study (N = 38) was performed in the peripartum period with maternal rectal and vaginal swabs and cord blood collected. Assessment included measures of perceived stress (PSS), anxiety, depression (CESD). PacBio full-length 16S rRNA was used to identify microbial communities. Results: Preclinical: Following injection of CCL2 into the amniotic sac, CCL2 was increased in the fetal plasma, fetal liver, and fetal brain (n = 6-7; p < 0.001.) CCL2 concentrations were not impacted in the maternal plasma or placenta. Female offspring exposed to CCL2 demonstrated increased neuroinflammation in prefrontal cortex (CCL2, TNFα; n = 6-7; p < 0.05) and did not exhibit social preference (n = 6-7; p = 0.134), in contrast to the control group (n = 6-7; p = 0.00029). Clinical: Fecal diversity metrics in 2nd trimester were associated with increased stress (PSS) and depression (CESD) scores. In the 2nd Trimester, Fecal Prevotella was increased in mothers that reported higher PSS. PSS metrics at delivery were associated with decreased CCL2 levels in the cord blood. Conclusions: In a preclinical model we show amniotic CCL2 injection is sufficient to induce neuroinflammation and long-term reduction in social behavior. In a clinical study we found maternal perceived stress and depressive symptoms are associated with alterations in maternal microbiota and cord blood CCL2 levels. Leveraging preclinical and clinical research can expedite our mechanistic understanding of how prenatal stress is transmitted to the next generation. Disclosure: Nothing to disclose. STUDY GROUP 6. Navigating the Maze: How You and Your University Can Prepare and Protect Your Lab Against The Risks Associated With Animal Research Georgia Hodes, Margaux Kenwood, Andre Der-Avakian, Eric Nestler, William Carlezon, James Jentsch, Mar Sanchez, Zoe Donaldson, Edythe London, Jill Turner Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States Study Group Summary: Animal research has produced some of the most important scientific developments of the past century, including the recent development of a new class of mRNA vaccines that has saved countless human lives. Calls to protect research animals over the past 40 years have produced important regulations that aid in the welfare of animals and humans alike. However, there is a strong political voice calling for the end of all animal research. As a result, there is a growing danger in the form of threats and harassment to scientists, their laboratories, and institutions. The members of this study group will discuss their personal experiences with being targeted by animal rights activists. The participants have a wide range of experiences with different forms of animal research and will include information on how institutional support, or lack thereof, impacted their response to threats and harassment. We will use this as a starting point for a discussion with the audience about the following:Key measures to take before a lab is targeted, including discussions to have with your lab members and institution, so they are prepared and know how to respond. What to do if an anti-science group targets you, or your laboratory. How to talk to the public about animal research so they can understand and appreciate the importance of this form of science and the vast regulations already protecting research animals. Our study group discussions will help basic and clinical scientists to better understand the issues faced by animal researchers, along with the regulations and protections already instituted to protect research animals. By engaging in this discussion, we hope to help scientists safely navigate the growing complexity surrounding this area of science, which is critical to treating physical and mental health. Disclosure: Nothing to disclose. PANEL 7. Is the Modification of White Matter Microstructure a Viable Treatment Strategy for Psychopathology? 7.1 Myelination Regulates Experience-Dependent Neuronal Plasticity in the Mammalian Cortex Wendy Xin UCSF, San Francisco, California, United States Background: Developmental myelination is a protracted process in the mammalian brain, occurring postnatally in mice and continuing through the first three decades of life in humans. One theory for why oligodendrocytes mature so slowly posits that myelination may stabilize neuronal circuits and temper neuronal plasticity as animals age. We tested this hypothesis in the visual cortex, which has a well-defined critical period for experience-dependent neuronal plasticity. Methods: To prevent myelin progression, we conditionally deleted Myrf, a transcription factor necessary for oligodendrocyte maturation, from oligodendrocyte precursor cells (Myrf cKO) in adolescent mice. To induce plasticity, adult control and Myrf cKO mice were monocularly deprived by eyelid suture. Functional and structural neuronal plasticity in visual cortex were assessed by in vivo optical intrinsic imaging (n = 8 control, 9 cKO) and in vivo longitudinal two photon imaging of dendritic spines (n = 5 CTL, 4 cKO), respectively. Both male and female mice were included in histological and in vivo imaging experiments. Results: The density of mature oligodendrocytes and myelin sheaths progressively increased from P28 to P60 in control mice but plateaued by P28 in Myrf cKO mice. Following monocular deprivation, visual cortex activity in response to visual stimulation of the deprived eye remained stable in adult control mice, as was expected based on previous experiments in wildtype mice. By contrast, visual cortex responses to the deprived eye decreased significantly following monocular deprivation in Myrf cKO mice, reminiscent of the plasticity observed in adolescent mice (mean difference in visual cortex responses 4 days post-monocular deprivation: control -0.33% vs cKO -17.78%; p = 0.0003). Furthermore, the density of dendritic spines in visual cortex was reduced following monocular deprivation in Myrf cKO, but not control, mice. Conclusions: These results support the concept of myelin acting as a brake on neuronal plasticity during development and suggest that oligodendroglia and myelination play a critical role in shaping the maturation and stabilization of cortical circuits. Disclosure: Nothing to disclose. 7.2 Early Life Stress Impairs Perforant Pathway (PP) Development Arie Kaffman Yale University School of Medicine, New Haven, Connecticut, United States Background: Early life stress (ELS) impairs normal myelination across diverse mammalian species, but the mechanisms responsible for this developmental abnormality are yet to be clarified. Here we show that pups raised with limited bedding (LB), a mouse model of ELS, have significant myelination deficits in the developing perforant pathway (PP) in the hippocampus that are due to sparse axonal innervation from the entorhinal cortex. Methods: Balb/CByj mice were exposed to control condition (500 cc bedding, 5 X 5 nestlet) or LB (125cc, no nestlet) from postnatal day 0 (P0) to P25. RNA was harvested from the hippocampus at P17 and sequenced at a depth of 80MR/sample (n = 7-9 mice per rearing and sex group). Expressed transcripts (TPM > 3, total of 13,245 genes) were analyzed using DESeq2 script in R to identify differentially regulated genes with false discovery rate (FDR) < 0.05, and to map major pathways affected by LB using MetaCore™ (Clarivate Analytics). Confocal microscopy confirmed abnormal myelination and revealed reduced axonal innervation in the PP (n = 4-5, P17 pups per rearing and sex group). Additional tissue was collected after contextual fear conditioning at P33 for high resolution dMRI and retrograde tracing (n = 6-8 mice per rearing and sex group). Results: RNA-seq identified 747 differentially regulated genes (FDR < 0.05) with abnormal myelination as the most significant pathway impaired by LB (FDR = 3.83e-11). LB reduced the expression of genes necessary for oligodendrocyte differentiation but not oligodendrocyte progenitor cells proliferation. Follow-up Immunohistochemistry studies showed that the PP is one of the most myelinated structures in the developing hippocampus and confirmed reduced oligodendrocyte maturation in the PP of LB mice (F (1, 16) = 35.56 P < 0.0001, np2 = 0.69). Abnormal myelination was associated with reduced axonal markers (F (1, 16) = 9.88, P < 0.0063, np2 = 0.38) suggesting that LB impaired axonal pathfinding and connectivity between the entorhinal cortex and the dorsal hippocampus. This assertion was further confirmed using diffusion MRI tractography and retrograde tracing and was correlated with abnormal contextual freezing in P33 juvenile mice. Conclusions: Sparse PP connectivity between the entorhinal cortex and the dorsal hippocampus contributes to hippocampal-dependent deficits in juvenile LB mice. Disclosure: Nothing to disclose. 7.3 Assessing the Effects of Solifenacin, an Antimuscarinic Agent, on Anxious Behaviors and White Matter Microstructure in Non-Human Primates Nakul Aggarwal University of Wisconsin, Madison, Wisconsin, United States Background: Myelination subserves efficient neuronal communication, and alterations in white matter (WM) microstructure have been implicated in numerous psychopathologies, including pathological anxiety. Recent work in rodents suggests that muscarinic antagonists may enhance myelination with behavioral benefits. Here, we present data from a first-in-primate study exploring the effects of solifenacin on anxious behaviors and WM microstructure in non-human primates (NHP), as a potentially translatable therapeutic strategy for human pathological anxiety. Methods: 12 preadolescent rhesus macaques (6 control, 6 experimental; 8 F, 4 M) were included in a pre-test/post-test control group study design. The experimental group received 3 months of daily IM injections of solifenacin succinate (0.5 mg/kg); controls received vehicle. Subjects underwent pre- and post-assessments of: 1) anxious temperament (AT)-related behaviors in the potentially threatening no-eye-contact (NEC) paradigm (30-min); and 2) WM imaging metrics, including diffusion tensor imaging (DTI) and quantitative relaxometry (QR). Results: We found significant Time (pre vs. post) by Group (control vs. experimental) interactions with respect to both freezing and cooing during the NEC. Specifically, experimental subjects, compared to controls, exhibited effects consistent with a reduction in anxiety: reduced freezing (p = 0.01) and increased cooing (p = 0.004) post-treatment. Whole-brain voxelwise analyses of post-minus-pre differences in DTI and QR metrics suggested increases in WM specific to the experimental group (all p’s < 0.005, uncorrected), including: increased fractional anisotropy in left stria terminalis and superior cingulum; decreased radial diffusivity (low values~greater myelination) in left sagittal striatum and stria terminalis; increased axial diffusivity (high values~greater axonal integrity) in right uncinate fasciculus, right external capsule, left dorsal prefrontal WM, and left superior corona radiata; and increased longitudinal relaxation rates (high values~greater myelination) in bilateral inferior frontal gyrus. Conclusions: Our findings from this first-in-primate study support further investigation of the utility of antimuscarinic agents in targeting WM microstructure as a means to reduce levels of pathological anxiety. Disclosure: Nothing to disclose. 7.4 Specificity and Causality of White Matter Aberrancies in Youth Psychopathology Julia Linke National Institutes of Health, NIMH, Bethesda, Maryland, United States Background: Aberrant maturation of white matter (WM) tracts and changes in WM microstructure in reaction to specific experiences (e.g., early adversity, psychopathology) have been proposed as pathophysiological mechanisms across mental disorders. However, tracts implied in different disorders largely overlap. Further, causality within these WM-behavior relationships still needs to be established. Data presented here addresses questions regarding specificity and causality. Methods: We obtained diffusion tensor imaging (DTI) data in two independent samples. Specificity questions were addressed in 144 youth (mean age 12.9 years (8-18), 68 girls, with anxiety disorders, attention-deficit/hyperactivity disorder, disruptive mood dysregulation disorder, or without a diagnosis). Youth and parents provided anxiety, irritability, inattention, and hyperactivity ratings that were subjected to a bi-factor model, thereby parsing unique and shared aspects of these co-occurring symptoms. Preliminary analyses regarding causality were conducted in the context of an ongoing trial, where DTI data are acquired before and after an exposure-based cognitive behavioral therapy (CBT; 23 anxious youth: 12.3 years (8-15), 13 girls; 20 age and gender-matched controls). DTI data was processed with TBSS. Results: In our cross-sectional analyses the shared factor was associated with widespread alterations in interhemispheric and fronto-limbic connections (x = 12, y = -9, z = 31, k = 17688, pFWER = .007). The anxiety-specific factor was associated with alterations in the splenium of the corpus callosum (x = -17, y = -52, z = 23, k = 435, pFWER = .008), while the irritability-specific factor was linked to the corticospinal tract (x = -25, y = -19, z = 34, k = 401, pFWER = .009). There were no findings for the ADHD-specific factors. In the treatment study, lower fractional anisotropy in the clusters associated with the shared (ab path = .031) and anxiety-specific factor (ab path = .031) in the cross-sectional analysis, partially mediated reduction in anxiety symptoms. Conclusions: Findings implicate alterations in fronto-limbic WM circuitry as a transdiagnostic mechanism of youth psychopathology. Furthermore, they suggest changes in WM microstructure as a mediator of CBT-response. Disclosure: Nothing to disclose. MINI PANEL 8. Face Expression and Gaze Dynamics in Schizophrenia and Its Risk States 8.1 Gaze Following in Third-Party Observers of Simulated Social Conflict Shows Few Reflexive and Many Mentalizing Features Katalin Gothard The University of Arizona, Tucson, Arizona, United States Background: Most primates exchange gaze-mediated social signals in the form of gaze-following and joint-attention saccades. Gaze following saccades start from the eye/face of the social partner and follow their gaze direction. Joint-attention saccades land on the specific object that is explored by the social partner. Methods: We quantified the scan paths of 3 macaques who watched videos of simulated social conflict between conspecifics. Conflict was simulated by juxtaposing two videos depicting a threatening and an appeasing individual, oriented toward each other, with the timing of the facial and bodily displays adjusted to mimic an exchange of social signals. Motion and the presence of facial expressions were registered in ethograms. Gaze-following and joint-attention saccades (henceforth JAGF) were hand-scored based on pre-established criteria. Results: From the 3 viewers we have analyzed 3,743 trials and identified 14,686 saccades that meet the criteria for JAGF. In two viewers, JAGF saccades occurred during frames that had significantly less motion than average (chi-square p < 0.001, N = 306); the third viewer showed the opposite effect (chi-square p < 0.001, N = 86). All 3 viewers produced more JAGF saccades in response to facial expressions (chi-square p < 0.001, N = 14,185). Viewers followed preferentially the gaze of the threatened monkeys, who elicited more joint attention than gaze-following saccades. This way the viewer explored vicariously the source of threat perceived by the appeasing individual. The temporal distribution of JAGF saccades showed clusters aligned to specific video segments. Some clusters showed significant increases in JAGF saccades across all 3 viewers, others were viewer-specific. Clusters often contained sequences of JAGF saccades, with the attention of the viewer rapidly alternating between the eyes and face of the interacting individuals. Conclusions: The clustering of JAGF saccades and their enhancement by facial expressions argue for reflexive responses to visual cues. The low probability of JAGF saccades (>20%) however, argues against simple reflexivity. The biased attention to the threatened animal, joint attention with animals who point with their gaze to a source of danger, and the large individual differences, suggest that the scan paths of observers of conflict are informed by cognitive processes. Disclosure: Nothing to disclose. 8.2 Visual Scanning and Brain Dynamics of Naturalistic Social Perception in Schizophrenia Gaurav Patel Columbia University, New York, New York, United States Background: Visual scanning of naturalistic social scenes requires the dynamic interaction of cortical systems underlying visual processing, face-emotion recognition, attention, and theory-of-mind (ToM) operations. Recently we found that these systems converge in the temporoparietal junction/posterior superior temporal sulcus (TPJ-pSTS) and that TPJ-pSTS abnormalities affect social perception in schizophrenia participants (SzP). To expand upon this work, first we detail similarities in visual scanning deviations between SzP and those at clinical high-risk for schizophrenia (CHR). Second we examine differences in movie-evoked dynamic brain states between SzP and healthy controls (HC) and their relationship to social perception. Methods: In Experiment 1, 46 HC, 43 SzP, and 27 CHR performed The Awareness of Social Inference Test (TASIT) with eye-tracking. For each TASIT video frame, eye-positions were scored as a z-transformed distance from the mean HC eye-position (distance/1 SD distance, HCs compared to a leave-self-out mean) and compared between intervals where SzP were divergent (z-transformed distance>2) or not. In Experiment 2, 27 SzP and 21 HC watched a 15-minute video clip while BOLD-fMRI data was collected. K-means clustering was used to cluster the BOLD time-courses into co-activation pattern (CAP) states. Results: In Experiment 1, the CHR eye-positions deviated more during the SzP divergent vs. non-divergent periods (t(26) = 5.6, p < 10-5). In Experiment 2, 5 CAP states were isolated, including one where all TPJ-pSTS areas were activated. Fractional occupancy of this state was lower in SzP (t(46) = 3.1, p = 0.003), correlated with TASIT accuracy (r = 0.51, p = 0.008), and the degree of visual motion in the movie (p < 10-13). We also found a mode consisting of the TPJ-pSTS, ToM and frontoparietal CAP states that was evoked only when viewing social interactions. Conclusions: In Experiment 1, the shared gaze divergence in CHR and SzP suggests a common underlying mechanism. In Experiment 2, the involvement of the TPJ-pSTS state in viewing and understanding of social interactions highlights the importance of coordinated activation of underlying areas for processing of those scenes. The results demonstrate the utility of naturalistic stimuli in identifying moments of abnormalities, which may aid in refining the timing of neurostimulation delivery (e.g., TMS) to enhance treatment effects. Disclosure: Pfizer, Inc, Employee, Spouse 8.3 Correlation of Inter-Speaker Facial Emotion Estimates During Dyadic Interactions in People With Schizophrenia and its Risk States Stephen Heisig Icahn School of Medicine At Mount Sinai, New York, New York, United States Background: Abnormal face expression is a sign of schizophrenia typically assessed by clinical scales. We characterize individual facial expressions in 10 healthy controls (HC), 7 schizophrenia (SZ), and 20 clinical high risk (CHR) patients and their correlations with interviewer expressions in the context of open-ended interviews using software models. We hypothesize group differences in individual expression and dyadic correlation. Methods: Open-ended 30-minute interviews were recorded using Zoom. We used the OpenFace facial analysis toolkit 2.0 (Baltrušaitis et. al. IEEE 2018) to estimate face action units. 48 emotional intensity estimates of facial expression per video frame were generated using HUME AI (Cowan et.al. Nature 2020) software. We calculated the overall mean and percent present for each of the 18 action units captured by OpenFace, focusing on AU07, (orbicularis oculi), which is key in social signaling. From the time-locked interviewer and participant video frames, we generated the dyadic correlation of each emotion estimate throughout the interview. The action unit and emotional estimate model labels are treated as objective, mechanistic features to describe the faces. Results: We replicated prior findings in SZ of decreased overall AU means (p = 0.001) and in particular AU07, (orbicularis oculi) (p = 0.018) extending these findings to CHR patients. Using HUME AI features no group differences were found in emotion estimates. However, we did find significant differences in patient/interviewer dyadic correlations (as compared to healthy/interviewer dyads) for 20 of the 48 emotion estimates (Doubt, Joy, Confusion, Contempt, Realization, Amusement, Determination, Anxiety, Anger, Empathic Pain, Tiredness, Contemplation, Entrancement, Awkwardness, Distress, Envy, Concentration, Disappointment, Excitement, Boredom), (all p < 0.03.) Conclusions: Employing a novel computational technique, we found decreased and disordered facial synchrony during naturalistic conversation in patients with SZ and individuals at CHR for SZ. Decreased facial synchrony in patients may be related to decreased face motor activity, as well as deficits in visual and/or face emotion processing, or disturbance in gaze; this will be the focus of future study. Disclosure: Nothing to disclose. MINI PANEL 9. Highlighting Psychosis Neuroimaging Research in Latin America: Inflammation, Accelerated Aging, and Environment 9.1 Systemic Inflammation and Cortical Neurochemistry in Never-Medicated Individuals With Psychosis Camilo de la Fuente-Sandoval Instituto Nacional de Neurologia y Neurocirugia, Mexico City, Mexico Background: Studies on cellular and cytokines profiles have contributed to the inflammation hypothesis in schizophrenia; however, precise inflammatory dysfunction markers remain elusive. Proton magnetic resonance spectroscopy (1H-MRS) studies have shown higher levels of myo-inositol (mI) and choline-containing compounds (Cho) in patients with first-episode psychosis (FEP), suggesting neuroinflammation. Here, we present inflammatory profiles in antipsychotic-naive FEP patients and age-and-sex matched healthy controls (HC), as well as cortical mI and Cho levels using 1H-MRS. Methods: Inflammatory profiles were analyzed using cytokine spontaneous production of peripheral blood mononuclear cells (PBMCs), and upon mitogenic stimulation, in 48 FEP patients and 23 HC. 1H-MRS was performed on a 3T scanner, centered on the medial prefrontal cortex and analyzed with LCModel (FEP = 27, HC = 17). Inflammatory markers were compared between groups and correlations with 1H-MRS findings were also explored. Results: FEP patients (duration of untreated psychosis 232 (±415) weeks (range 1-1720) showed higher proportion of proinflammatory Th1/Th17 subset (median .265) compared with HC (median .11; two tailed Mann Whitney U = 325, p = .03). Also, FEP patients revealed an increased spontaneous production of IL-6 (median 2340, HC median 2.695; U = 193, p = .02), IL-2 (median 11.61, HC median 3.86; U = 196, p = .03), and IL-4 (median 3.785, HC median 2.05, U = 144 p < .01). Similarly, FEP subjects showed higher Cho levels (mean 1.69 ± 0.22, HC mean 1.37 ± 0.56, t = 2.72, df = 42, p = .01). No differences in mI levels or other neurometabolites were found. Lastly, Cho levels correlated with T regulatory cells (r26 = .44, p = .02), and with classic monocytes (r26 = -.53, p = .004). Conclusions: FEP subjects were characterized by immune dysregulation, affecting both the innate and adaptive immune response, with a predominantly Th2 signature. Furthermore, increased Cho levels were also found and correlated with T-regulatory cells. The intense Th2 signature and proinflammatory response found in subjects, along with the 1H-MRS findings, suggest changes that can be associated with both systemic and central inflammation processes in schizophrenia. Disclosure: Janssen: Contracted Research (Self) PANEL 10. Let’s Talk: Psychoactive Drugs Alter the Structure and Content of Social Speech 10.1 Dose-Dependent Effects of Psilocybin on Language Produced Under Natural Conditions Enzo Tagliazucchi Universidad Adolfo Ibañez, Buenos Aires, Argentina Background: While the effects of low and high doses of psilocybin have been investigated in laboratory settings using standardized tasks and questionnaires, the validity of this approach can be questioned due to the influence of contextual factors. We report the results of two studies conducted under natural conditions based on the analysis of free unconstrained speech: a study of language produced during the effects of a psilocybin microdose (study 1) and a study of verbal interactions between a group of participants who consumed psilocybin during a retreat (study 2). Methods: Study 1 followed a double-blind placebo-controlled design (N = 35, 0.5 g of dried psilocybin mushrooms or an inert placebo). Natural language was recorded during the acute effects, when participants answered a series of questions about different topics. Study 2 was a single blind randomized study (N = 100 participants divided into two matched groups, receiving 1 g or 3 g of dried psilocybin mushrooms, administered by a facilitator in the context of a retreat). Natural language was recorded during group interactions after the acute effects. Both studies included male and female participants. Language samples were analyzed in terms of verbosity, sentiment analysis, semantic coherence, and topic detection. Pairwise comparisons were conducted using Wilcoxon (Study 1) or Mann-Whitney (Study 2) non-parametric tests. Mushroom samples were analyzed using liquid chromatography-mass spectrometry. Results: In Study 1 we found that a low dose of psilocybin (close to 1 mg of psilocybin) resulted in enhanced positive sentiment and higher verbosity. In Study 2 we found that a higher dose of psilocybin (in the range of 10 mg) resulted in both positive sentiment and higher verbosity (with longer turns taken by participants in the conversation), and decreased semantic coherence, but with smaller effect sizes than those reported in a previous study of LSD. The prevalence of topics related to visual perception and ego dissolution was significantly increased in the high vs. low dose condition. Conclusions: We conclude that natural language is sensitive to the reported effects of low and high doses of psilocybin consumed in different contexts. Natural language analysis should be further explored as a potentially valuable tool to quantify the contents of unconstrained reports after psychedelic experiences. Disclosure: Nothing to disclose. 10.2 Effects of MDMA and Amphetamine on Speech and Social Connection During Dyadic Conversations Hanna Molla University of Chicago, Chicago, Illinois, United States Background: Although MDMA and amphetamine are drugs that increase social behavior, MDMA has been characterized as a unique ‘empathogenic’ drug that induces pro-social effects, which may be observed through quantitative linguistic analysis. Methods: We conducted a double-blind, placebo-controlled study in which male and female participants (N = 36) engaged in a dyadic conversation with partners of the same-sex after MDMA or amphetamine. Results: The study is still ongoing and the results we present will be new data. Conclusions: The study is still ongoing and the results we present will be new data. Disclosure: Nothing to disclose. 10.3 Natural Language Processing of Movie Recall and Drug Fluency, and Associated Brain Function, in Cocaine and Heroin Addiction Rita Goldstein Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Individuals with addiction attribute excessive salience to drug/drug-related cues, which could enhance performance on neuropsychological probes. For example, individuals with cocaine addiction outperform healthy controls (HC) when prompted to generate drug-related words. Here we tested this effect in individuals with heroin use disorder (iHUD), in addition testing for verbal recall following watching of a heroin-related movie, before and after 14-weeks of treatment. We aimed to test for group differences and the potential reduction in drug-related verbal fluency and recall biases following inpatient treatment. Methods: The drug fluency task was performed by 32 inpatients with iHUD (age=40.8 ± 9.2, 6 women) and 16 HC (age=43.8 ± 10.3, 6 women). Participants watched the first 17 min of the movie Trainspotting, containing drug and nondrug scenes. After watching the movie, participants verbally recalled it. Fourteen iHUD performed the tasks again following 14 weeks of treatment, when adherence was tracked by daily EMAs. Results: The iHUD named more drug-related words than HC on the verbal fluency task (18.4 ± 5.8 vs. 15.6 ± 3.3 p = .02). In the recall task, iHUD proportionally recalled more drug>nondrug scenes (recall frequency: .7 ± .2 vs. .6 ± .2, p = .045) with a similar pattern for recall duration (p = .024). While fluency, recall frequency or duration did not change with treatment (ps > .60), there was a trend for less decrease in drug over nondrug recall frequency between the sessions to be associated with longer lifetime heroin use (r = .5, p = .051). Also, longer drug over nondrug recall duration at the post-treatment session was associated with worse treatment adherence in the iHUD (r = .6, p = .018). Conclusions: Here we identify simple and naturalistic, ecologically-valid, speech-based neuropsychological measures that tap language, memory/retrieval and higher order executive functions, revealing a drug bias in drug addiction. Associations with addiction severity and treatment adherence suggest these contextually-biased measures may be biomarkers of clinical endpoints. Current efforts are geared towards the use of machine learning (e.g., natural language processing) for the exploration of these speech-based biomarkers vis-à-vis brain function. Disclosure: Nothing to disclose. 10.4 Artificial Intelligence Approaches to Study Speech Production Related to Drug Use Guillermo Cecchi IBM Research, Yorktown Heights, New York, United States Background: The assessment of psychological states prior, during and after psychoactive drug use traditionally relies on a combination of subjective reports and clinicians’ evaluation. The development of automated language analytics allows for the application of a limited but consistent metric across arbitrarily large drug-related speech datasets, including samples produced in different media and in naturalistic settings. Methods: We studied 60 regular users of cocaine engaged in an abstinence trail, analyzing baseline open-ended speech samples in which they describe the positive and negative consequences of withdrawal. Applying Natural Language Processing (NLP) techniques, we measured the semantic similarity of the samples to concepts related to drug use and quality of life, in order to predict outcomes at 12 months after baseline. The predictions were validated using 10-fold subject-based cross-validation. Results: Several of the outcome variables at 12 months, including Cocaine Selective Severity Assessment and Cravings, were predicted from baseline speech samples with significant accuracy, whereas baseline clinical and demographic variables including age, gender, CSSA and years of use did not provide any predictive value. Conclusions: These preliminary results suggest that psychological processes present at baseline and expressed in open-ended speech can be captured by NLP and used to significantly constrain the expected range long-term outcomes of abstinence treatment. Disclosure: IBM Corporation: Employee (Self) PANEL 11. New Tricks for Old Cells: Plasticity and Novel Roles of Hypothalamic CRH Neurons in Mental Health and Disease 11.3 An Unexpected Role for CRHPVN Neurons in the Social Buffering of Stress Jaideep Bains Hotchkiss Brain Institute, University of Calgary, Calgary, Canada Background: The presence of others, either during or soon after a stressful experience can mitigate the harmful effects of stress. This phenomenon, known as social buffering, has been described extensively, but the mechanistic underpinnings of social buffering remain elusive. The CRH neurons of the paraventricular nucleus of the hypothalamus (CRHPVN) are necessary for the social transmission of stress, but their potential role in social buffering of stress is not known. Methods: Here, in mice, we use in vivo imaging, optogenetics, behavioral evaluations and ex vivo electrophysiology to evaluate the role of CRH neurons in the paraventricular nucleus in social buffering after acute stress. Results: We used an established novel object recognition (NOR) task to test for the immediate effects of acute stress on memory. After one day of training in the NOR chamber, we footshocked mice and re-exposed them to the chamber. We calculated a discrimination index (DI) to evaluate memory (DI = (Timenovel − Timefamiliar)/ (Tnovel + Tfamiliar). Acute stress (footshock) decreased the discrimination index in male and female mice, consistent with a memory impairment. In female mice, the presence of a naive conspecific in the homecage after stress prevented stress-induced impairments in memory. By contrast, in males, the presence of a conspecific had no effect on stress-induced memory impairments. In females, social interactions after stress decrease synaptic metaplasticity that relies on increased CRHPVN activity (Sterley et al, 2018). To determine whether CRHPVN neurons contribute to memory impairments after stress, we optogenetically silenced these cells in single FS females immediately after stress. This was sufficient to restore novel object discrimination. Conclusions: Social buffering, which occurs when a stressed individual interacts with an unstressed conspecific, helps relieve negative effects of stress specifically in females. This includes a decrease in stress-induced memory impairments specifically. This effect of social interaction on memory is mimicked in single-housed stressed mice by photoinhibition of CRHPVN neurons. This direct inhibition also results in a reduction in stress-induced short-term potentiation of glutamate synapses on these cells, implicating CRHPVN neurons as key mediators of the stress alleviating effects of social contact. Disclosure: Enveric Biosciences: Consultant (Self) STUDY GROUP 12. Developmental Cohort Studies Past, Present, and Future: Lessons Learned and Design for Clinical Impact Aristotle Voineskos, Deanna Barch, Damien Fair, Theodore Satterthwaite, Michael Milham, Stephanie Ameis, Judith Ford University of Toronto, Toronto, Ontario, Canada Study Group Summary: The present study group application will highlight efforts to build developmental cohorts to better understand risk trajectories of mental illness, in the context of biological, psychological, and social factors. There is growing appreciation for the developmental onset of mental illness during childhood or adolescence. Nearly 75% of cases of mental illness onset before the age of 25, and mental illnesses such as depression and schizophrenia are the top causes of disability in youth. There is even evidence showing that disorders that onset early in the lifespan, such as autism or ADHD, can themselves be risk factors for disorders that onset in later adolescence such as psychotic disorders. Large cohort studies across the developmental phase of life offer the opportunity to understand different trajectories of illness and brain development that children and youth experience. Through data sharing and re-use, hundreds if not thousands of investigators around the world are now asking new questions accelerating the pace of scientific discovery. Cohorts are often population, or catchment-area based, but more recently new studies are recruiting help-seeking young people (and families). Studies have also evolved from cross-sectional to longitudinal. The advent of digital tools and technologies mean that additional real-world data can be collected that might index physiological functions and offer new insights regarding symptoms that can be difficult to characterize, such as insomnia. Finally, there is growing awareness of structural racism and other social disadvantages that minority populations face. Not only are such assessments now included, but active equity-based approaches to ensure inclusion and retention of such populations is now expected. The title of the study group, reflecting past, present, and future, aims to take lessons learned, successes, and challenges of recently completed, and in progress cohort studies, and help the field in optimizing design of newly initiated, or to-be initiated studies. The Philadelphia Neurodevelopmental Cohort, a study of ~ 1,600 children and youth from the greater Philadelphia area (population-based) will serve as an exemplar of a cohort study recently completed. The Adolescent Brain and Cognitive Development (ABCD) study that is now underway having completed its first wave on ~10,000 9 and 10 year olds will serve to provide early lessons learned, as well as possibilities for adjustments in real time. Similarly the Human Brain Network (HBN) study, studying children 5-21 years of age (total sample anticipated n~10,000) based on ascertainment in the greater New York area via parents concerned about potential symptoms or distress in their children will provide an example of how to pivot following a pilot phase analysis. Finally, the newly launched Toronto Adolescent and Youth (TAY) Cohort Study, aims to recruit ~3,000 children and youth from 11-24 years of age, who are referred to an academic mental health hospital (in Toronto) to child and youth psychiatric services. The objectives (for this study group) relate to discussing challenges and opportunities in the following categories:Population-based vs. clinical cohorts Data Sharing and Reuse Engagement and Recruitment of marginalized populations Data collection strategies (focus on tech and wearables) Clinical Impact/Practice Change that could emerge from results Disclosure: Nothing to disclose. STUDY GROUP 13. Fostering Translational Research and Academic/Industry Collaboration to Accelerate Development of New Mental Health Treatments Kimberly Vanover, Alik Widge, Andre Fenton, Danielle Graham, Bita Moghaddam, Kerry Ressler, Mark Schmidt, Sade Spencer, Stephen Strakowski, Jared Young Engrail Therapeutics, Inc., San Diego, California, United States Study Group Summary: The current biomedical basic research model and current organization of funding opportunities are not designed to facilitate the translation of basic research to clinical practice in Psychiatry. Cost and complexity are among the top challenges for translational research projects. Furthermore, academic / industry collaborations remain infrequent or are not incentivized for discovery and translation. However, the focus on translational research is accelerating and impacts academic as well as industry research and is the focus of many governmental initiatives. This study group will emphasize the importance of T1 translational research, bringing findings from basic research into early clinical testing in humans, as well as T2 translational research, establishing effectiveness in humans and establishing clinical guidelines. We will discuss what needs to change to facilitate more translational research to accelerate the development of new treatments for disorders of the central nervous system as well as what is at risk to be lost with a change in focus and funding. Specifically, better tools and broader funding opportunities are needed to support translational models for the translational researcher. This Study Group brings together representatives from academia and industry, across different stages of career, and inclusive of diversity. Our objective is to bring together leaders to discuss translation of basic research to clinical practice and the opportunities and challenges for academic / industry collaboration. Questions for discussion will include, but are not limited to:What are the major gaps facing translational neuroscience in psychiatry? How can academic/industry collaborations help fill the gaps? How do we better de-risk target pipelines at the preclinical level to achieve better prediction of translation in humans? How do we enhance discovery pipelines for translationally robust biomarkers for precision medicine in Psychiatry? Are standards and rigor in animal research sufficient? How do we keep up with the focus on reproducibility and larger sample sizes now required in human work? Do the current funding structures favor basic research and/or should more funding be invested in translational work and appropriately powered clinical trials? Do many journals favor basic research or clinical studies, but not translational findings? Is there a gap in expertise, such that preclinical data may not be well understood by clinical reviewers and vice versa, diminishing competitiveness of papers with both animal and human data? Disclosure: Engrail Therapeutics: Employee (Self), Intra-Cellular Therapies: Stock / Equity (Self), Evolution Research Group: Consultant (Self) PANEL 14. Racial Disparities in Mental Health During the COVID-19 Pandemic 14.1 Race, Cytokines, and Depression: The Role of the Exposome Olusola Ajilore University of Illinois at Chicago, Chicago, Illinois, United States Background: Previous studies have demonstrated that racial discrimination and the perceived stress of racism have been associated with elevations in pro-inflammatory cytokines. Given the well-documented relationship between pro-inflammatory cytokines and major depression, the goal of the present study was to examine whether there were racial differences in the relationship between inflammation and major depression using a secondary analysis of a ethnically diverse dataset. Methods: 121 subjects were recruited as part of a larger program of research investigating major depressive disorder (MDD) at the University of Illinois at Chicago (UIC). 46 were Black (21 healthy comparison subjects, 25 with MDD) and 75 were White (34 healthy comparison subjects, 41 with MDD). The final inclusion criterion for depressed subjects was a score of ≥15 on the Hamilton Rating Scale for Depression. Severity of depression was also assessed using the Center for Epidemiological Studies – Depression scale. In addition to C-reactive protein (CRP), levels of pro-inflammatory cytokines were determined in plasma/serum aliquots by enzyme-linked immunosorbent assay (ELISA) using commercially available Quantakine® kits (R and D Systems, Inc., Minneapolis, MN) for human IL-1β, IL-6, and TNF-α. Socioeconomic factors were obtained from the Chicago Health Atlas and linked to each participant by the neighborhood where they resided at the time of the study. Results: In the non-depressed sample, Black participants had significantly higher levels of CRP and IL-6 than White participants. In the context of major depression, there was no difference between groups. After controlling for BMI, CRP was no longer significant in the non-depressed group, but IL-6 remained significantly different between groups. After controlling for socioeconomic factors, IL-6 levels were no longer significant between the two groups. Additionally, IL-6 was not correlated with depression severity in Black participants but was in White participants. Conclusions: The well-known relationship between pro-inflammatory cytokines and major depression may be moderated by race due to high levels of cytokines at baseline in Black participants. These elevations may be related to the stressful life events due to discriminatory experiences as indexed by socioeconomic factors and environmental stressors. Disclosures: KeyWise AI: Founder (Self), Embodied Labs, Blueprint, Milken Institute, Sage Therapeutics: Advisory Board (Self) 14.2 Racial Disparities in COVID-19 Stress: The Role of Inflammation April Thames University of California, Los Angeles, California, United States Background: The COVID-19 pandemic has disrupted the lives of billions, raising concerns about overall mental health. Further, data from the Centers for Disease Control demonstrate significant racial disparities in COVID-19 outcomes for African Americans. The current study examined the predictive association between baseline symptoms of depression and inflammation on COVID-19 related stress outcomes (as measured by the Pandemic Stress Index [PSI]; Harkness, 2020). African American (n = 66) and non-Hispanic White participants (n = 50) aged 30-60 (Mage = 45.4; SD = 9.0) were recruited from a larger study on racial disparities in cognitive outcomes. Methods: African American (n = 66) and non-Hispanic White participants (n = 50) aged 30-60 (Mage = 45.4; SD = 9.0) were recruited from a larger study on racial disparities in cognitive outcomes. For circulating (plasma) levels of inflammation-related biomarkers, we will assess both pro- and anti-inflammatory cytokines using a multiplex assay (IL-6, IL-10, TNF-a) (R and D Systems Luminex Performance Human High Sensitivity Cytokine Panel). Additional biomarkers (sCD14, CRP, MCP-1/CCL2) will be determined by ELISA (R and D Systems Human Quantikine ELISAs), as they cannot be multiplexed with the cytokines or with each other due to differences in circulating concentrations and assay requirements. Results: Higher PSI scores were reported among African Americans in comparison to non-Hispanic Whites. African Americans demonstrated higher levels of CRP in comparison to non-Hispanic Whites, F (2, 113) = 7.66, p = .02. Linear regression was used to examine predictive associations baseline depression, race, CRP as well as interactions on PSI scores. As expected, the overall model predicted PSI scores, (r2 = .18, p < .0001). Independent associations were found between depression (b = .57, p < .001), CRP (b = .32, p < .02), race (b = -.27, p = .03) and the CRP X depression interaction (b = .22, p = .04) on PSI outcomes. The association between depressive symptoms and CRP on PSI scores were in the expected direction. The relationship between depression and PSI scores were strongest among those with high levels of CRP. Conclusions: Study results provide preliminary evidence for the role of inflammation in the relationship between depression and COVID-19 pandemic stress scores. Disclosure: Nothing to disclose. 14.3 Associations Between Segregation, Coronavirus Racial Bias, and Covid-19 Pandemic Distress Rodman Turpin Department of Global and Community Health, Fairfax, Virginia, United States Background: Racial/ethnic minorities are disproportionately impacted by the COVID-19 pandemic, as they are more likely to experience structural and interpersonal racial discrimination, and thus social marginalization. Based on this, we tested for associations between pandemic distress outcomes and four exposures: racial segregation, coronavirus-related racial bias, social status, and social support. Methods: We collected data as part of a larger longitudinal national study on mental health during the pandemic (n = 1,301). We tested if county-level segregation, individual-level social status, social support, and coronavirus racial bias were associated with pandemic distress using cumulative ordinal regression models, both unadjusted and adjusted for covariates (gender, age, education, and income). Results: Both our segregation index (PR = 1.19; 95% CI 1.03, 1.36) and coronavirus racial bias scale (PR = 1.17; 95% CI 1.06, 1.29) were significantly associated with pandemic distress. Estimates were similar after adjustment for both segregation (aPR = 1.15; 95% CI 1.01, 1.31) and coronavirus racial bias (PR = 1.12; 95% CI 1.02, 1.24). Social status (aPR = 0.74; 95% CI 0.64, 0.86) and social support (aPR=0.81; 95% CI 0.73, 0.90) were associated with lower pandemic distress after adjustment. Conclusions: Segregation and coronavirus racial bias are relevant pandemic stressors, and thus have implications for minority health. Future research exploring potential mechanisms of this relationship, including specific forms of racial discrimination related to pandemic distress and implications for social justice efforts, are recommended. Disclosure: Nothing to disclose. 14.4 Stressors During COVID: Racial Bias and Drug Use Brenda Curtis National Institute of Health/NIDA, Baltimore, Maryland, United States Background: Black, Asian, and Latinx people are disproportionately impacted by the COVID-19 pandemic and they are more likely to experience coronavirus-related racial discrimination. Prior to the COVID-19 pandemic, racial and ethnic minority adults reported similar or lower levels of alcohol and other drug use than their white counterparts. The pandemic appears to have begun to reverse these trends with racial and ethnic minorities reporting larger increases in substance use during the pandemic. This study examined the association among coronavirus-related victimization distress, perceptions of pandemic associated increase in societal racial biases, and substance use risk among Asian, Black, Latinx, and non-Hispanic White adults. Methods: Participants were part of a larger longitudinal survey study on mental health and substance use during COVID-19. Adults (N = 1336) who self-identified as Asian (8.53%), Black (10.55%), Latinx (10.93%), and non-Hispanic White (69.99%) completed measures that included demographic and COVID-19 related stressors, the coronavirus victimization distress scale (CVD), the coronavirus racial bias scale (CRB), and drug use consumption (alcohol, tobacco, cannabis, and other drugs). Results: Across race/ethnicity, binary logistic regression analyses controlling for demographic variables indicated coronavirus victimization distress was associated with higher odds of tobacco use risk (AOR = 1.36, 95% CI [1.01, 1.81]) and coronavirus racial bias beliefs were associated with higher odds of drug use including cocaine, hallucinogens, inhalants, and methamphetamine (AOR = 1.31, 95% CI [1.00, 1.71]). Logistic regressions for each racial/ethnic group found different patterns of relationships between CVD, CRB and substance use disorder (SUD). Conclusions: Results highlight the significance of examining how the current pandemic has exacerbated racial/ethnic systemic inequalities through COVID-19 related victimization. The data also suggest that across all racial/ethnic groups perceptions of pandemic instigated increases in societal racial bias is a risk factor for SUD. The study calls for further empirical research on substance use treatment sensitive to specific needs of diverse populations during the current and future health crises. Disclosure: Nothing to disclose. PANEL 15. The Forces That Shape Us: Interactions of Early Experience, Sex, and Puberty on The Development of Circuitry and Behavior 15.1 Sex-Specific Influences of Early Experience and Pubertal Timing on Threat Responsiveness in Rats Heather Brenhouse Northeastern University, Boston, Massachusetts, United States Background: Individuals who endured traumatic environments in childhood commonly struggle with responding appropriately to threats throughout life. Genetic sex contributes to the nature and timing of these effects, with evidence that risk is unmasked in females following periods of hormonal activation, including puberty. Pubertal timing itself appears to be affected by early life adversity, evidenced by earlier puberty initiation in females. I will present data from an animal model illustrating sex-specific changes to threat responses in rats exposed to maternal separation (MS), using a novel behavioral paradigm. I will also discuss potential mechanisms underlying precocial puberty after MS, as well as preliminary findings suggesting that heightened estrogen signaling can cause enhanced threat responsiveness in females. Methods: Males and females (n = 6-12) underwent control rearing or MS postnatal days (P)2-20. One cohort was tested for baseline acoustic startle response (ASR) on P25, P35, or P55 on Day 1, and on Day 2 ASR was preceded with presentation of a social threat cue [playback of a 22 Hz ultrasonic vocalization (USV)] in the ASR chamber. Another cohort was sacrificed at P10, P15, or P15 and hypothalamus was collected for qPCR analyses of puberty-related gene expression (GnRH, RFRP, Kisspeptin). A third cohort received AAV-shRNA against estrogen receptor (ER)alpha into the basolateral amygdala (BLA) at P14, then were tested for baseline and USV-potentiated ASR at P55. Results: MS enhanced baseline ASR at P35 in females (p < 0.001), which was correlated with earlier age of puberty initiation. MS females had heightened age-related increases of Kisspeptin mRNA in the anteroventral periventricular nucleus of the hypothalamus compared to controls. While USV presentation potentiated ASR in P55 controls, MS blunted this potentiation, and females overall showed lower USV-potentiation compared to males (p < 0.05). Conclusions: These data suggest that kisspeptin driven puberty acceleration is associated with enhanced ASR in MS females. We are currently testing the extent to which ERalpha signaling in the BLA drives this heightened response. I will also discuss a new translational model demonstrating a blunted ASR potentiation to social threat after early adversity, with potential mechanisms of risk, resilience, and adaptation in males and females. Disclosure: Nothing to disclose. 15.2 Early Life Adversity Arrests Prefrontal Cortical Maturation During Adolescence Kuei Tseng University of Illinois, Chicago, Illinois, United States Background: Early life experiences have been known to impact the development and maturation of corticolimbic connectivity and its regulation of cognitive and affective behaviors. For instance, individuals with a history of early life trauma are often at higher risk for anxiety-like disorders in adolescence, which are thought to result from a dysregulation of affective and cognitive processes, and the maturation of the prefrontal cortex (PFC) and associated neural circuits. It is therefore conceivable that early life events contribute to shape the developmental trajectory of PFC maturation during adolescence. Methods: To test this idea, we implemented an early-life adversity paradigm (i.e., maternal separation, MS) and assessed to what extent synaptic activity in the PFC and its maturation from postnatal day (P) 30 to adulthood becomes disrupted in both male and female rats. Both excitatory and inhibitory synaptic activity were recorded and compared in PFC pyramidal neurons using a combination of ex-vivo whole-cell patch-clamp and DREADD techniques. Results: Data revealed that the normal developmental gain of PFC GABAergic synaptic activity was not observed in animals exposed to MS, while AMPA-mediated transmission was not affected. As a result of the selective GABAergic disruption in MS animals, the excitatory-inhibitory (E-I) ratio that normally becomes balanced by P50 remains unbalanced (e.g., >1.0) in adulthood, resembling the level of E-I synaptic activity typically found in the PFC of P30-40 rats. Moreover, transient chemogenetic inhibition of PFC GABA interneurons during this early adolescent period was sufficient to elicit similar E-I imbalance that endures through adulthood. Conclusions: Collectively, our findings indicate that PFC inhibitory synapses are preferentially susceptible to early life adversity, likely through a disruption of activity dependent mechanisms that are needed to enable the GABAergic maturation during adolescence. In turn, a disinhibited PFC state could increase the risk for psychiatric disorders during adolescence with abnormal affective and cognitive responses. Disclosure: Nothing to disclose. 15.3 Early Scarcity Alters the Basolateral Amygdala Transcriptome and Addiction-Related Behaviors Amelia Cuarenta Temple University, Philadelphia, Pennsylvania, United States Background: Adversity is a risk factor for psychiatric disorders, however, stress that is not overwhelming can promote resilience. We use limited bedding and nesting (LBN) to model mild adversity. Our prior work shows LBN induces neurobiological alterations that reduce some addiction-related behaviors including reducing morphine taking. These behaviors rely on cues as a driver of behavior and performance. Exposure to cues previously paired with drug taking can induce craving and drug-seeking behavior following periods of abstinence. We are investigating whether LBN alters incubation of morphine craving, a cue-driven behavior; whether LBN alters cocaine self-administration; the molecular changes induced by LBN in the basolateral amygdala (BLA), a region important for responses to stress and for the integration of cues. Methods: Rats were reared in LBN or control housing from postnatal days 2 - 9. In LBN, dams and pups were given a single paper towel for nesting material; a metal grate prevented access to bedding. Control animals were reared in standard housing conditions.Rats were placed in operant boxes and permitted to lever press on a fixed ratio 1 (FR1) schedule for morphine infusions. Presses on the active lever resulted in one infusion accompanied by a 5 s light cue and then a 20 s timeout period during which the house light was off; lever presses were recorded but drug was unavailable. Rats were tested for behavioral signs of drug seeking on day 1 and day 30 of abstinence utilizing a within-subjects design. Rats had 6 hr cocaine self-administration access on an FR1 schedule for 10 days. RNA sequencing was conducted to delineate the effect LBN had on the transcriptional profile of the BLA in adult rats. Results: All groups showed the incubation effect, pressing more after 30 days of abstinence than 1 day. There was no difference in lever pressing between conditions in males or females. Preliminary results suggests that LBN male rats self-administer lower levels of cocaine than controls. LBN-induced sex-specific changes in transcription. RRHO analysis revealed distinct genes upregulated and downregulated in males and females due to LBN. Conclusions: LBN reduces morphine and cocaine drug taking in male rats but does not affect cue-driven incubation of morphine craving. LBN also induces sex-specific patterns of gene transcription within the BLA. Disclosure: Nothing to disclose. 15.4 The Forces That Shape Us: Interactions of Early Experience, Sex, and Puberty on the Development of Circuitry and Behavior Anne Murphy Georgia State University - Neuroscience Institute, Atlanta, Georgia, United States Background: Infants born prematurely are more likely to be admitted to the Neonatal Intensive Care Unit (NICU) where they experience upwards of 10-18 painful procedures each day, often with no anesthesia or analgesia. Both pre-clinical and clinical studies have shown that early exposure to pain disrupts normal CNS development resulting in lifelong changes in response to stress and pain. Here we present novel data on the effects of neonatal injury on the response to an immune challenge in adulthood. Methods: Male and female rats were exposed to a short-term inflammatory insult induced by intraplantar administration of 1% carrageenan (CGN) on the day of birth (P0). In adulthood (P60-P90), rats were implanted with Thermicron iButtons to monitor core body temperature; 14 days later, rats were injected with lipopolysaccharide (LPS) to elicit an immune response. Rats were sacrificed after 24 hours or at their peak fever point and brain tissue collected for analysis of VGat, VGlut2, Fos and prostaglandin receptor 3 within the median preoptic area (MnPO), a key site for pyrexia. Peripheral and central cytokine/chemokine levels were also determined. Results: LPS induced a febrile response in all rats beginning ~3-5 hours post injection that lasted ~5-7 hours. Early life pain (ELP) exposed males (n = 11) and females (n = 7) showed a significantly exaggerated febrile response to LPS in comparison to handled rats (males, n = 9; females n = 11). Center of gravity analysis revealed a significant rightward shift in the ELP females (t11 = 3.163; p = 0.009). Peak fever was also significantly higher in ELP females (F1,16 = 4.619, p < 0.0473). Analysis of EP3R, VGlut2, and VGat at peak fever revealed no differences between groups for EP3R (F(1,18) = 0.0102; p = 0.92) and VGlut2 (F(1,18) = 0.51; p = 0.48). Two-way ANOVA of VGat signal intensity showed a sex difference with females exhibiting higher expression compared to males (F(1, 18) = 5.777; p = 0.0272) with no effect of treatment (F1,18 = 0.036, p = 0.852). Conclusions: Our results indicate that unresolved ELP alters neural circuitry underlying immune-based fever response that persists into adulthood and is potentiated in females. Together, these studies may elucidate a mechanism through which children experiencing unresolved pain during the perinatal period show an increased severity of sickness behavior and attenuated immune signaling. Disclosure: Nothing to disclose. PANEL 16. Human Nervous System Based Translational Models Towards Discovery of Novel Pain Therapeutics 16.1 Pain Target Identification in Human Dorsal Root Ganglion (DRG) and Spinal Cord Studies Theodore Price University of Texas at Dallas, Richardson, Texas, United States Background: Pain therapeutic development has been plagued by clinical failures. Many rationales for this have been given, but one that is often overlooked are species differences in transcriptomes and proteomes for neurons and other cells in pain circuits. The goal of this talk will be to impart detailed information about how human nociceptors and spinal cord putative projection neurons differ between rodents and humans and how this information can be used to improve success in analgesic development. Methods: We have recovered dorsal root ganglion (DRG) and spinal cords from organ donors and DRGs from people having thoracic vertebrectomy and C1/C2 fusion surgeries. Tissues have been subjected to bulk, single cell and spatial transcriptomic assays to understand cellular transcriptomes and how these transcriptomes change in chronic pain states in people. Male and female samples have been used for all studies, and sex differences will be presented. Results: We have identified 12 subtypes of human sensory neurons and investigated how these subtypes differ between humans and mice. Similar comparisons have been made with spinal cord neurons between mice and humans, with a focus on differences in gene expression in neurons in the dorsal horn that are likely projection neurons. We find important species differences that have implications for therapeutic development. In the DRG, the most important difference is that human nociceptors all have peptidergic qualities while mouse nociceptors fall into both peptidergic and non-peptidergic subsets. Major differences in GPCR and neuropeptide expression are found between mice and human, and many such receptors and peptides are exclusively found in humans. In projection neurons in the spinal cord, human neurons express Nav1.7 mRNA and protein, while mouse projection neurons do not. These findings may have important implications for Nav1.7 clinical development. Conclusions: There are profound differences between transcriptomes and proteomes for human versus mouse neurons of the pain pathway. We propose that this is a primary difference that causes clinical failures. Better understanding of the molecular architecture of human nociceptors and projection neurons can improve our ability to create effective analgesics. Disclosures: Acadia Pharma: Other Financial or Material Support (Self), 4E Therapeutics, Doloromics, NuvoNuro: Board Member (Self), Grunenthal, Merck, Abbvie, Hoba Therapeutics: Grant (Self) 16.2 Communication Between Human Skin Cells and Nociceptors as a Discovery Platform for Pain Targets Cheryl Stucky Medical College of Wisconsin, Milwaukee, Wisconsin, United States Background: Keratinocytes, which constitute >95% of the epidermis, are innately sensitive to mechanical force, are capable of releasing a wide array of neuroactive factors and form close “synapse-like” connections with intraepidermal nerve fibers. Keratinocyte activity is critical for normal sensory neuron and behavioral responses to mechanical and thermal stimuli. Skin becomes sensitized to external mechanical and thermal stimuli following many types of tissue injury, nerve injury and disease. The contribution of keratinocytes to this injury-associated sensitization is unknown. Methods: We used a variety of preclinical models of pain in both sexes of animals together with keratinocytes from skin of human female and male donors in order to determine what ion channels, receptors and signaling molecules mediate the responses of keratinocytes from normal and tissue injury conditions to tactile and thermal stimuli. We utilize behavioral assays, calcium imaging and patch clamp electrophysiology to define the roles of keratinocytes in vitro and in vivo. Appropriate samples sizes based on power analyses were used for all studies. Results: PIEZO1 is a bona fide mechanically-gated, non-selective cation channel that is highly expressed in skin. Keratinocytes isolated from mouse and human skin respond to the PIEZO1 agonist Yoda1, PIEZO1 expression is critical for keratinocyte mechanical sensitivity. Furthermore, we demonstrate that loss of epidermal PIEZO1 decreases the firing rate of sensory nerve fibers in response to mechanical stimulation of the skin and blunts behavioral responses to both innocuous and noxious mechanical stimuli in vivo. Furthermore, keratinocytes isolated from mice with chronic painful diabetic neuropathy, sickle cell disease, traumatic nerve injury and chemotherapy-induced neuropathy exhibit sensitization of PIEZO1 in the form of increased keratinocytes responding and increased magnitudes of responses. Moreover, treatment of human keratinocyte with compounds that induce chemotherapy neuropathy in patients acutely sensitize PIEZO1 function. Conclusions: These data demonstrate that epidermal PIEZO1 is critical for normal touch sensation and that keratinocyte PIEZO1 is sensitized in multiple models of neuropathic pain in preclinical models and in human skin. Disclosure: Nothing to disclose. 16.4 Human Cancer Biopsies Elucidate Cancer Pain Targets Nicole Scheff University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Head and Neck squamous cell carcinoma (HNSCC) causes severe pain, beyond what is reported in other cancer types. We hypothesized that cancer immunosurveillance is impaired by the immunosuppressive actions of exogenous opioids and immune cell-mediated endogenous opioid analgesia could be leveraged as a novel therapeutic tactic for cancer pain treatment. Methods: HNSCC patient-derived single cell RNA sequencing data were used to evaluate opioid receptor and opioid precursor gene expression in tumor infiltrating leukocytes (TIL) and peripheral blood. A syngeneic mouse oral cancer orthotopic transplant model was used to evaluate analgesic morphine on the tumor immune response as well as immune cell-mediated endogenous opioid expression. Mouse oral cancer cell line 1 or 2 (MOC1, MOC2) tumor-bearing male and female mice were assessed for all studies. Results: Single immune cell RNAseq analyses from of the 26 HNSCC patients (70% male, median age=60.5) and 6 healthy donors (50% male, median age=55.5) found that opioid receptor OPRM1 is expressed by T lymphocytes; there was a significant increase in OPRM1 expression in CD8+ T cells in tumor compared to blood (padj=0.03). HNSCC patients with low reported pain had significantly higher immune-derived PNOC expression, the precursor for opioid, nociceptin (padj = 0.002). These clinical data led to investigation of both morphine-impaired CD8+ T cell infiltration and immune-mediated endogenous analgesia in preclinical mouse models. Immune infiltration has been quantified in a carcinogen-induced oral cancer mouse models; tumor-infiltrating neutrophils express and release beta-endorphin suppressing nociceptive behavior. However, intratumor neutrophils can promote tumorigenesis. Exploration of opioid gene expression in alternative tumor-infiltrating immune cell subtypes was assessed using fluorescence-activated cell sorting and qPCR. We found that tumor infiltrating B cells express more PNOC compared to B cells isolated from sham mice. Conclusions: Together these data suggest that exogenous opioids given for pain management may directly impair tumor immunosurveillance and weaken immune checkpoint immunotherapy (ICI) therapeutic efficacy. Alternatively, ICI may be employed to stimulate the immune response for endogenous opioid-mediated cancer pain treatment. Disclosure: Nothing to disclose. MINI PANEL 17. The Conundrum of Perinatal Anxiety: Using Physiological Measurements to Refine Assessment and Treatment 17.1 Anxiety Through the Perspective of the Microbiota-Gut-Brain Axis Mary Kimmel University of North Carolina, Chapel Hill, North Carolina, United States Background: One in five meet criteria for an anxiety disorder during pregnancy or the postpartum period; Generalized Anxiety Disorder (GAD) the most common. Anxiety can serve a role in protecting parent and child, but anxiety is also associated with negative outcomes for parent and child. The microbiome and host cytokines hold promise to better understand anxiety and its impacts on child development. Heart Rate Variability (HRV) as measured by respiratory sinus arrhythmia (RSA) is thought to reflect vagal activity; the vagus a key communicator of the microbiota-gut-brain axis. The objective of this work is better understanding of the interactions of maternal microbes and cytokines in relation to maternal anxiety and infant vagal activity. Methods: Self-assessment of anxiety symptoms included the three questions of the Edinburgh Postnatal Depression Scale (EPDS) associated with anxiety (EPDS 3A) and the Generalized Anxiety Disorder-7 (GAD-7); and were collected alongside maternal fecal samples at two visits in pregnancy and one visit postpartum. Infant RSA assessed by Actiheart was measured over five minutes at rest (baseline). Maternal microbial composition was characterized by 16S rRNA sequencing and alpha-diversity was calculated by Faith’s PD, observed OTUs, and Shannon. The microbial compositions and cytokine levels in the third trimester and postpartum of matched perinatal individuals with higher and lower GAD-7 scores were compared in network analyses. Key microbes from these networks, prior work, and the literature (including commonly used probiotics) were chosen, and their relative abundance averaged across the visits for each parent. Pearson’s correlations compared infant RSA and maternal alpha diversity. Regression modeling assessed these microbes in relation to maternal anxiety and infant RSA. Results: In a subset of the larger cohort of 94 perinatal individuals, 40 postpartum parent-infant dyads had infant HRV data. Network analysis in the third trimester found IL-23 as a central hub that associated with microbes including Akkermansia muciniphila, Bifidobacterium, Faecalibacterium; which were associated with anxiety. Network analysis in the postpartum visit no longer had IL-23 as a central hub, although IL-23 did associate with Blautia and Bacteroides; microbes including Blautia, Akkermansia, and Bacteroides were important nodes in the network and associated with maternal anxiety. Bacteroides also associated with IL-10. When Bacteroidies uniformis, Enterococcus, and Blautia, were included in a linear regression model, all three were significantly associated with the EPDS 3A (p = 0.05, 0.03. 0.03). Lower maternal OTUs associated with lower infant baseline RSA (p = 0.03). Lack of maternal Bifidobacterium adolescentis was significantly associated with infant baseline RSA (p = 0.006). Conclusions: Network analysis shows how the immune system and the microbiota differ in the third trimester versus the postpartum period for individuals with higher and lower anxiety. Utilizing a hypothesis-driven analysis of a common screening tool, three microbes with functions such as tryptophan metabolism and in inducing inflammation were associated with maternal anxiety. Lower maternal alpha diversity associated with lower infant vagal tone; a microbe related to GABA production associated with infant vagal tone. Disclosure: Abbvie: Stock / Equity (Spouse) 17.2 Innate Immunity and Perinatal Anxiety Lauren Osborne Weill Cornell Medicine, New York, New York, United States Background: Immune dysregulation has been linked to both psychiatric illness and pregnancy morbidity, including perinatal depression, but little is known about the immune phenotype of perinatal anxiety. Here, we sought to identify the unique immune profile of antenatal anxiety. Methods: Pregnant women (n = 107) were followed prospectively at 2nd and 3rd trimesters (T2, T3) and 6 weeks postpartum (W6). Each visit included a blood draw and psychological evaluation, with clinical anxiety assessed using the Spielberg State-Trait Anxiety Scale. Multiplex assays and flow cytometry methodology were used to examine the association of anxiety symptoms with secreted immune markers and PBMC-derived immune cells. Results: K cluster means revealed three clusters of anxiety symptomatology; due to low numbers in the highest severity group, these were collapsed into two groups: Healthy and Anxiety. Women within the Anxiety group demonstrated a lower level of innate immune cytokines during pregnancy (β = 0.371, SE = 0.173, t = 2.14, p = 0.035), but experienced a rise in levels postpartum, whereas the Healthy group demonstrated a decline; this difference was at a trend level .(β = 0.381, SE = 0.226, t = 1.69, p = .096) Immune cell populations differed between our two groups, where women within the Anxiety group showed a decrease in the ratio of B cells to T cells from pregnancy to postpartum while Healthy women increased (β = 4.289, SE = 1.955, t = 2.19, p = 0.031). Women in the Anxiety group also demonstrated an increased ratio of cytotoxic to helper T cells throughout, a modest increase in the TH1:TH2 ratio across pregnancy, and a lower ratio of TH17:TREG cells in the postpartum when compared to the Healthy women (β = 0.537, SE = 0.243, t = 2.21, p = 0.029). Conclusions: These data suggest that the innate immune response throughout the antenatal period differs for women with trait anxiety symptoms compared to healthy women, suggestive of a unique immune phenotype of perinatal anxiety. Disclosure: Nothing to disclose. 17.3 Autonomic System Measures of Anxiety in Pregnancy Julia Riddle University of North Carolina, Chapel Hill, North Carolina, United States Background: We sought to explore the understand stress responsiveness in pregnancies with and without anxiety during psychological stressor task using HRV, cortisol, and alpha amylase. Methods: We followed pregnant women with and without clinical anxiety (n = 95) at four timepoints in pregnancy and postpartum, with baseline psychiatric diagnosis determined by SCID-5 and symptoms tracked by psychological scales at each visit. At the third trimester visit, a subset of participants (n = 54) completed a laboratory psychological stressor. We measured baseline, in-stressor, and recovery HRV. Linear mixed effects models were used to analyze HRV measures at three time points by scales and anxiety diagnoses. Results: The sample was predominantly white and high income. Mean age was 33 and mean pre-pregnancy BMI was 27.2. When controlling for age and BMI, the two groups (anxious and non-anxious) were significantly different in their HRV RMSSD responsiveness between baseline and recovery (p = 0.0253). In secondary analysis, lower HRV was associated with worse sleep (Pittsburgh Sleep Quality Index, p = 0.0092) and higher subjective stress (Perceived Stress Scale, p = 0.03896). There was a non-significant trend with high subjective worry (Penn State Worry Questionnaire, p = 0.0547) and high trait anxiety (Spielberg Trait Anxiety Inventory Scale, p = 0.05367) Conclusions: Our findings demonstrate that, in this small sample in late pregnancy, anxiety and subjective perceptions of stress and poor sleep were related to differences in autonomic functioning. Longitudinal studies with a larger population may help to determine clinical applications. Disclosure: Nothing to disclose. STUDY GROUP 19. Trips and Tryptamines: What Makes Psychedelics Work in Psychiatric Disorders, If They Do? Bernard Lerer, Harriet de Wit, Bryan Roth, Gabriella Gobbi, Michael Mithoefer, Celia Morgan Hadassah-Hebrew University Medical Center, Jerusalem, Israel Study Group Summary: After a hiatus of three decades, there is growing interest in the potential role of psychedelic drugs in treating psychiatric disorders. Although the current focus is on a small number of compounds, particularly psilocybin and LSD, there has been a burgeoning effort to identify novel drugs, both natural and chemically synthesized compounds, to optimize therapeutic benefits. Growing interest in psychedelic research is motivated by the limitations of current psychopharmacological treatments in psychiatry, and by frequent anecdotal reports of efficacy of psychedelic drugs. On this background, several highly influential controlled studies have yielded promising results in major depression and posttraumatic stress disorder (PTSD), and many other studies are in progress. These advances have fueled enthusiasm, but at the same time ignited debate including methodological concerns and uncertainty about mechanisms of action. Key issues for discussion include:What is the appropriate ‘placebo’ comparison, given the intense psychological effects of psychedelic drugs? Are the trip-inducing effects essential to their therapeutic action, and if so, what are the appropriate comparison conditions? Is it possible to develop new drugs, or administer doses of existing drugs, that induce minimal or no psychedelic effects but are still effective in treatment? What are the optimal doses and dosing regimens? Is psychotherapy crucial to the therapeutic effects of psychedelics These issues will be the focus of this Study Group. The discussion will be led by participants with expertise in areas critical to developing psychedelics for psychiatry. Bryan Roth (University of North Carolina) has contributed pivotally to our understanding of the pharmacology of classical and novel psychedelics and will introduce discussion on the feasibility of “non-hallucinogenic” psychedelics. Gabriella Gobbi (McGill University, Quebec) has conducted exciting new research on the mechanism of action of low doses of LSD in anxiety and social behavior, and the potential antidepressant effects of psychedelics in animal behavior models. She will introduce important brain-behavior mechanisms to the discussion Michael Mithoefer (MAPS Public Benefit Corporation) will discuss the challenges in designing clinical trials with MDMA and other psychedelic drugs, and ways to mitigate concern about effective blinding. He will also address the unknowns about the role of subjective experiences in therapeutic efficacy. Celia Morgan (University Exeter, UK) will consider the practical and ethical challenges of conducting clinical trials of psychoactive substances and testing the role of therapy in the context of a clinical trial of ketamine assisted psychological therapy in Alcohol Use Disorder. The Study Group is characterized by diversity in several key areas including clinical, preclinical and translational focus, geographical origin and gender, and includes a mix of ACNP Fellows and non-members. Drs. Lerer (Israel) and de Wit (USA) will chair the Study Group and moderate the discussion ensuring active audience participation and interaction with the Study Group members. Disclosures: Back of the Yards Algae Sciences, Parow Entheobiosciences, Contracted Research (Self), Negev Capital, Consultant (Self) PANEL 20. Striatal Mechanisms of Repetitive Behavior and Habit 20.1 The Role of Cortico-Striatal Interactions in Repetitive Sequenced Behaviors Susanne Ahmari University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Smoothly linking individual actions into sequences is critical for execution of complex behaviors, but we still have a limited understanding of how behavioral sequences are encoded. Accumulating evidence suggests striatal activity patterns are linked to performance of sequenced behaviors, but the role of cortical inputs in initiation and control is less clear. Our recent work showed that SAPAP3-KOs, which display repetitive sequenced grooming behavior, have ~6 fold increase in drive to central striatum (CS) from anterolateral motor area (ALM). This suggested repeated selection of motor programs could be caused by excessive striatal drive from ALM. Methods: Male and female C57Bl6 mice were injected with AAV-GCaMP6m, AAV-jRGECO1a, AAV-ChrimsonR, or control viruses and implanted with optogenetics/photometry fibers or GRIN lenses in CS or ALM to visualize Ca2+ activity during spontaneous behavior. Raw Ca2+ videos were spatially/temporally down sampled, motion-corrected, and processed (CNMFe). Raw fluorescence was Z-scored to average fluorescence and standard deviation of the trace. Behavior was quantified using automated postural tracking (SLEAP) and manual behavior annotation, and compared between conditions using support vector machine classifiers (SVMC). Results: Surprisingly, we found a selective increase in CS activity at initiation of grooming (and not other behaviors) using fiber photometry. Optogenetic stimulation of CS (to mimic this activity increase) evoked immediate-onset, short, grooming-related fragmented movements distinguishable from normal grooming behavior using a SVMC. Fiber photometry also showed selective activation of ALM during grooming. Unexpectedly, peak ALM activity correlated with grooming bout length. Stimulation of ALM-CS terminals also caused evoked grooming bouts resembling naturalistic grooming. Finally, dual-color, dual-region photometry revealed that CS activation precedes ALM activation at initiation of grooming bouts. Conclusions: These results suggest that CS selects or initiates naturalistic grooming behavior, while ALM plays a role in sustaining effective bouts. ALM-striatal interactions may thus be a key contributor to regulation of sequenced and repetitive behaviors. Ongoing work is testing the causal role of ALM in sustaining vs. terminating sequenced behavior. Disclosure: Nothing to disclose. 20.2 Opposing Amygdala-Striatal Pathways Enable Chronic Stress to Hasten Habit Formation Jacqueline Giovanniello University of California of Los Angeles, Santa Monica, California, United States Background: To make decisions, we consider our possible actions and their consequences. This goal-directed strategy allows us to adapt but is cognitively taxing. To counter this, we also use habits. Habits are executed without thought of their consequences and so, are more resource-efficient but inflexible. Balance between goal-directed actions and habits permits adaptive and efficient behavior. Overreliance on habit is an endophenotype of many psychiatric conditions such as obsessive-compulsive disorder. Stress, a major contributing factor to these conditions, can prematurely promote habit. Despite this, the brain mechanisms that allow stress to promote habit formation remain unclear. Amygdala input to the dorsomedial striatum (DMS) may mediate the influence of stress over habits. The DMS is indispensable for goal-directed learning. Inhibition of this region reduces goal-directed learning and promotes habit. The basolateral (BLA) and central (CeA) regions of the amygdala are known stress hubs in the brain. Excitatory BLA-DMS neurons are known to exist. Recent evidence revealed direct CeA input to the dorsal striatum and we identified these inhibitory projections target the DMS. Thus, BLA and CeA are well positioned to differentially regulate the ability of stress to influence behavioral control. Methods: We developed a task to model chronic stress-induced instrumental habits in male and female mice and coupled it with in vivo pathway-specific neural activity monitoring and manipulation techniques. Results: We found that stress promotes habits in males and females. DMS projecting-BLA and -CeA neurons show opposing activity during instrumental learning. CeA-DMS activity opposes goal-directed learning and is necessary for stress to promote habits. Conversely, BLA-DMS activity opposes this and is sufficient to rescue goal-directed control in stressed mice. Conclusions: These data reveal a function for the BLA-DMS and newly identified CeA-DMS pathways. While BLA-DMS activity promotes the learning that supports flexible, goal-directed actions and prevents stress from promoting habits, CeA-DMS activity enables stress to promote habits. These findings have important implications for psychiatric conditions influenced by stress and characterized by maladaptive habits, such as obsessive-compulsive disorder and substance use disorder. Disclosure: Nothing to disclose. 20.3 Cell Type Specific Contributions of Striatal Projection Neurons to Habit Learning Melissa Malvaez University of California, Los Angeles, Los Angeles, California, United States Background: Optimal behavior relies on a balance between two distinct strategies; one goal-directed in which the consequences of actions are considered, and one habitual, where tasks are conducted without forethought of their consequences. The balance between these systems allows adaptive and efficient behavior, however, dysfunction in this balance can lead to symptoms characteristic of several psychiatric diseases. Goal-directed and habit learning are known to rely on the anatomically distinct dorsomedial (DMS) and dorsolateral (DLS) striatum, respectively. However, the subregion-specific contribution of the two major subtypes of striatal output neurons, the direct (dSPNs) and indirect (iSPNs) striatal projections neurons, is unknown. Methods: We used subregion-specific chemogenetic approaches to modulate dSPNs and iSPNs in mice, as well as cellular resolution calcium imaging to monitor cell-specific activity, during instrumental habit learning. Results: In the pDMS, both dSPNs and iSPNs mediate goal-directed learning. Chemogenetic inactivation of dSPNs (WT, n = 7; D1-cre, n = 9; Two-way RM ANOVA with Bonferroni MCT: p = 0.009) or iSPNs (WT, n = 9; A2Acre, n = 7; Two-way RM ANOVA with Bonferroni MCT: p = 0.031) during instrumental training prevents goal-directed learning, while activation of dSPNs (WT, n = 7; D1-cre, n = 7; Two-way RM ANOVA with Bonferroni MCT: p = 0.030) or ISPNs (WT, n = 11; A2A-cre, n = 9; Two-way RM ANOVA with Bonferroni MCT: p = 0.050) promotes goal-directed behavior even after overtraining. Conversely, in the DLS, we found that dSPNs (WT, n = 10; D1-cre, n = 11; Two-way RM ANOVA with Bonferroni MCT: p = 0.046), but not iSPNs (WT, n = 12; A2A-cre, n = 11; Two-way RM ANOVA with Bonferroni MCT: p > 0.999), mediate the acquisition of habits. In the pDMS, both dSPNs (n = 466-538) and iSPNs (n = 411-499) are active during instrumental performance. dSPNs maintain representation of action initiation throughout training and the transition to habit, while iSPNs become recruited to the initiation of instrumental performance as habits form. Conclusions: dSPNs and iSPNs differentially contribute to behavioral strategy in a subregion-specific manner. In the pDMS the two major striatal outputs coordinate the transition to habits. These findings challenge the canonical views of striatal function and identify a neural substrate of adaptive strategy learning. Disclosure: Nothing to disclose. 20.4 Plasticity of Striatal Fast-Spiking Interneuron Networks in Habit Formation Nicole Calakos Duke University Medical Center, Durham, North Carolina, United States Background: Striatal fast-spiking interneurons (FSI) occupy a powerful position within the striatal microcircuitry to influence striatal output. The behavioral transition from goal-directed to habitual requires dorsomedial (DMS) and dorsolateral (DLS) striatal regions. DLS FSI activity is required for habitual performance of a learned task and FSI excitability positively correlates with habitual, as opposed to goal-directed behavior. Here we examine the cellular and circuit mechanisms contributing to FSI excitability in habitual mice. Methods: Adult parvalbumin (Pv) Cre mice were trained in a lever press task. Habitual behavior was quantified by sensitivity of the behavior to outcome devaluation. For electrophysiology, acute brain slices were prepared after devaluation test. For behavioral tests of FSI activity, adult Pv Cre mice received intracranial AAV for Cre-dependent expression of Halo-Tag. Drugs Acutely Restricted by Tethering (DART) methodology was used to concentrate a modified GABAR antagonist, gabazine at FSIs. The sufficiency of GABAR inhibition on FSIs to modify habitual behavior was tested by infusing drug on the day of devaluation testing. Results: Intrinsic membrane excitability differed between FSIs in goal-directed and habitual mice, with habitual mice FSIs being more excitable (p < 0.05). FSI excitability differences were similar between dorsomedial and dorsolateral striatal regions. Notably, chamber control mice showed FSI excitability similar to habitual mice. Preliminary findings suggest that selectively reducing GABAR transmission in FSIs may be a sufficient mechanism to correspondingly influence habitual behavior (p = 0.0009). Conclusions: Our results provocatively update working models for habit learning and expression. First, rather than a model of acquiring plasticity with continued training from the goal-directed to habitual periods, data from naïve control mice support a model in which plasticity that dampens FSI excitability occurs in the early stages of goal-directed learning, followed by a return to “baseline” levels of FSI excitability in habitual mice. Second, even though DLS and DMS circuits are generally associated with habitual and goal-directed behavior, respectively, our results indicate that at least at the level of FSI plasticity, it occurs similar across these two regions. Disclosure: Nothing to disclose. PANEL 21. Bad Wrap: Oligodendrocytes as a Common Cellular Substrate for Genetically-Identifiable Neurodevelopmental Disorders in Humans and Mouse Models 21.1 Developmental Disruption of White Matter Microstructure in 22q11.2 Copy Number Variants: A Cross-Species Investigation Carrie Bearden University of California, Los Angeles, California, United States Background: Chromosome 22q11.2 deletions(22qDel) are among the greatest known risk factors for schizophrenia (SCZ). In the largest multisite diffusion-weighted magnetic resonance imaging (dMRI) study of 22qDel to date, we recently found widespread white matter (WM) microstructure alterations, characterized by higher fractional anisotropy (FA) relative to healthy controls (HC) in callosal tracts, but lower FA in long association tracts, suggesting altered frequency of layer 2/3 projection neurons. We also found intriguing gene dosage effects, involving opposing patterns of WM disruption in the reciprocal 22q11.2 duplication, which is putatively protective against SCZ. Here, we investigated the neurodevelopmental trajectory of these disruptions in a cross-species investigation. Methods: Our human study included 594 participants: 334 with 22qDel (mean age 16.88 ± 6.43, 153 females) and 260 healthy controls (HC, mean age 16.55 ± 8.01, 123 females). Across species we used tract-based spatial statistics to interrogate WM. Given findings of nonlinear dMRI trajectories in healthy individuals, we fit Poisson nonlinear models for each group separately. In parallel, we conducted dMRI studies in the LgDel mouse model (Juvenile WT = 22, LgDel=13; Adult WT = 21; LgDel=18; both sexes), using ex-vivo overnight imaging at 7 Tesla in PFA perfused samples. Animals were injected with Gsk3β in early postnatal days, shown previously to rescue cognition in a 22q11.2 mouse model. Results: Human 22qDel carriers showed robustly increased FA in callosal regions of interest (Cohen’s d = 0.37-0.58; all p < 2.69e-5). Further, the mean age of peak FA was significantly older in 22qDel vs HC (t = -3.03; p < .007). Consistent with human findings, adult LgDel mice had significantly higher FA in callosal fibers relative to WT(t > 2.1, cluster-corrected at p = 0.01). This effect was also present in juvenile mice, although its magnitude was slightly smaller (2- way ANOVA, genotype, p < 0.02; F(1,70) = 20.79). Lastly, FA alterations were not rescued by Gsk3β antagonism (t(36) = 0.88, p = 0.39). Conclusions: Findings of developmental WM disruptions, particularly impacting callosal fiber tracts, are consistent across species. Delayed WM maturation in 22qDel may be secondary to altered axonal diameters, a precursor of a delayed myelination process in 22qDel. Disclosure: Nothing to disclose. 21.2 Enhancing Myelination as a Therapeutic Intervention for Autism Spectrum Disorder Brady Maher Lieber Institute for Brain Development, JHMI, Baltimore, Maryland, United States Background: Autism spectrum disorder (ASD) is a genetically heterogeneous disorder with convergent symptomatology that suggests the potential for identifying common dysregulated pathways that could be amenable to therapeutic intervention. Mutations in Transcription Factor 4 (TCF4) cause a syndromic ASD known as Pitt-Hopkins Syndrome (PTHS). We have previously shown that transcriptional and biological profiling of PTHS mouse models indicated a disruption in oligodendrocytes (OLs). Moreover, we demonstrated a convergence of differential expression related to OL biology was also observed in two additional ASD mouse models (Pten and Mecp2). Remarkably, the eigengene of these convergent genes was effective at separating postmortem ASD brain samples from controls. Taken together, these results indicate that defects in myelination are a common pathophysiology in ASD and opens the door to developing ASD therapies that targets myelination. Methods: Clemastine (CF) is an antihistamine that is effective at promoting myelination through its block of muscarinic receptors. We tested the effects of CF (10uM) on primary OL cultures derived from Tcf4+/tr and WT littermates (male and female), as well as in vivo using daily i.p. injections (10mg/kg) between postnatal day 28 and 42. Results: In vitro CF treatment of Tcf4+/tr OL cultures resulted in a significant increase in the OL density compared to vehicle treated cultures (F = 5.34, p = 0.03, n = 6-10 samples/genotype/treatment). In vivo administration of CF also significantly increased the OL population (F = 6.78, p = 0.014, n = 8-13 animals/genotype/treatment) compared to vehicle treated mice. Moreover, in Tcf4+/tr CF treatment increased the proportion of newly formed uncompacted myelin as measured by electron microscopy (t = 4.19, p < 0.0005, vehicle n = 79, CF n = 89) and rescued electrophysiological deficits in compound action potentials (t = -2.49, p = 0.03, vehicle n = 11, CF n = 14). Remarkably, CF treatment also rescued behavior in Tcf4+/tr mice (F = 25.03, p < 0.0001, n = 8 animals/genotype/treatment). Conclusions: Treatment with CF, an FDA approved compound, is effective at rescuing the OL population, electrophysiology and behavior in a PTHS mouse model. These findings provide novel preclinical data that suggests CF or other remyelinating agents may be beneficial to PTHS patients and potentially other related ASDs. Disclosure: Nothing to disclose. 21.3 Roles of Oligodendroglial Sodium Channels in Myelination and Auditory Processing Deficits Related to Autism Jun Hee Kim The University of Texas Health Science Center, San Antonio, Texas, United States Background: Auditory processing abnormalities are common features of neurodevelopmental disorders such as autism spectrum disorder (ASD). However, the causes and mechanisms have not been sufficiently explored. The gene, SCN2A, encoding the alpha subunit of the voltage-gated Na+ channel 1.2, is highly linked to ASD. This presentation will highlight novel data on how loss of oligodendroglial SCN2A impacts myelination and neural connectivity in the auditory system, leading to auditory processing disorders. Methods: We used a novel Scn2a conditional knockout mouse (Scn2a cKO) to specifically delete SCN2A in oligodendrocytes (OLs), by crossing tamoxifen -inducible Pdgfra-CreER with Scn2a floxed mice. All mice were in a C57BL/6 genetic background. For auditory brainstem responses (ABRs) test, control and Scn2a cKO mice (20 mice per group, both sexes) were anesthetized with 2% isoflurane during recording (1 l/min O2 flow rate). ABR recordings was done in a sound attenuation chamber. Results: To determine the role of SCN2A in OL development and axonal myelination, we evaluated the population of OLs and myelin properties in the auditory brainstem using transmission electron microscopy (TEM). We found that SCN2A deletion impairs OL differentiation. In TEM analysis, Scn2a cKO mice showed a higher g-ratio in the brainstem, compared with control (control:0.81 ± 0.065, n = 1154 axons vs cKO: 0.85 ± 0.002, n = 432 axons; 5 mice/genotype, t = 7.233, p < 0.0001), indicating that axons have a thinner myelin in cKO mice. Alterations in myelination impact neuronal activity and axonal conduction. The nerve terminals in Scn2a cKO mice displayed a number of action potential failures in response to axon fiber stimulation (at 200 Hz, control:25 ± 6.1%, n = 11 cells vs Scn2a cKO: 40 ± 6.8%, n = 16 cells in cKO). Finally, in vivo ABRs showed Scn2a cKO mice had a reduced threshold (control:35 ± 3.7 dB, n = 23 mice vs cKO: 23 ± 4.1 dB, n = 13 mice, t = , 1.943, p = 0.044) and an elevated amplitude of ABRs (control: 1.9 ± 0.22uV, n = 23 mice vs cKO: 3.0 ± 0.26 uV, n = 13 mice, t = 3.483, p < 0.01), suggesting that Scn2a cKO mice have auditory hypersensitivity. Conclusions: SCN2A is important for oligodendrocyte differentiation, myelination, and neuronal activities in the auditory nervous system. Loss of SCN2A in OLs during early development can cause auditory processing disorders related to ASD. Disclosure: Nothing to disclose. PANEL 22. SeXX, Brain, and Cardiometabolic Health: What Do They Have in Common and Why Should Psychiatry Care? 22.1 Sex-Specific Regulation of Autonomic Function – Impact of Gonadal Hormones and in Utero Glucocorticoid Exposure Taben Hale University of Arizona, Phoenix, Arizona, United States Background: Prenatal insults leading to increased fetal glucocorticoid exposure can sex-selectively impact future disease risk. We have shown that when pregnant rat dams are treated with the glucocorticoid, dexamethasone (DEX), for the last 4 days of gestation, female-specific changes in stress-responsive cardiovascular function, autonomic function, depression-, and anxiety-like behaviors are detected in their adult offspring. The present study evaluated the degree to which the sex-specific changes in autonomic function are due to the activational effects of gonadal steroid hormones. Methods: Pregnant rat dams were administered DEX (0.4mg/kg per day, s.c.) or vehicle (VEH) on gestation days 18-21. Rats underwent a gonadectomy (GDX) or sham surgery post-puberty; 1-week later radiotelemetric transmitters were implanted for direct recording of heart rate variability (HRV) from arterial pressure waveforms to assess autonomic function. HRV was measured 2-3 weeks post GDX, and response to acute stress was assessed using a restraint tube for 20 minutes. Testing was performed on diestrus in sham females. Frequency domain analysis of HRV was performed during baseline and stress periods, with high frequency (HF) power reflecting parasympathetic and low frequency (LF) power reflecting both sympathetic and parasympathetic outflow. Results: There was a main effect of GDX resulting in reduced HF power (F (1, 21) = 4.946, p = 0.037) and LF power (F (1, 21) = 4.581, p = 0.044) in females. In females there was a significant interaction for LF/HF ratio (F (1, 21) = 5.314, p = 0.031), whereby LF/HF was increased during stress period in VEH, but not DEX-exposed rats. LF/HF was increased following GDX in DEX, but not VEH exposed females (F (1, 21) = 4.130, p = 0.055). In males, there was no significant impact of GDX, prenatal exposure, or stress on HF and LF HRV. LF/HF was significantly increased during stress in males (F (1, 23) = 4.784, p = 0.039). Conclusions: Findings revealed sex-specific regulation of autonomic function, whereby activational effects of gonadal steroids impact autonomic control of cardiac function in females, but not males. In utero DEX exposure induced dysregulation of sympathovagal balance in female rats that was exacerbated by gonadal hormone loss. Findings may lead to future sex-selective therapeutic considerations particularly in post-menopausal women. Disclosure: Nothing to disclose. 22.2 Trajectories of Risk for and Resilience Against Neurodegenerative Diseases of Aging: Lessons Learned From Midlife Neuro-Transitions Roberta Brinton University of Arizona, College of Medicine, Center for Innovation in Brain Science, Tucson, Arizona, United States Background: Midlife aging transitions can be critical periods for neural circuit reorganization, metabolic reprogramming and function which can provide insights into windows of opportunities to promote resilient vs vulnerable brain aging. The menopause in women and the andropause in men can be a critical period for determining risk for age-associated neurodegenerative disease and initiating the prodromal phase of disease. Methods: RNA-Seq was conducted on rat hippocampal RNA at 100 bp paired-end on NovaSeq. Metabolomics from rat brain and plasma was performed utilizing Global Metabolomics and Complex Lipids mass spectrometry. Mmedical claims study included women with or without claim records of hormone therapy medications. Relative risk ratios and 95% confidence intervals for combined Alzheimer’s, Parkinson’s, Multiple Sclerosis and ALS were determined. Results: Transcriptomic analysis revealed chronological and endocrinological aging specific shifts in bioenergetic systems of biology that were paralleled by bioenergetic dysregulation in midlife aging female brain. Metabolomic and lipidomic analyses revealed dynamic adaptation of the aging female brain from glucose centric to utilization of auxiliary fuel sources that included amino acids, fatty acids, lipids, and ketone bodies. At the population level of analysis, in 379,352 women with or without claim records of hormone therapy, use of HT was associated with significantly reduced risk for combined neurodegenerative diseases of aging (RR 0.42, 95% CI0.40–0.43,P < 0.001).Greatest reduction in risk of AD, and dementia emerged in patients aged 65 years or older. Conclusions: Midlife aging transitions can be critical periods of neural circuit reorganization, metabolic reprogramming, brain function and therapeutic interventions to prevent neurological and psychiatric disorders of aging. Disclosure: Nothing to disclose. 22.3 Evaluation of Sex Differences in the Genetic Relationship Between Cardiovascular Disease, Depression, and Chronic Inflammation Lea Davis Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: Cardiovascular disease (CVD) and depression are highly comorbid. Prior research finds that polygenic scores (PGS) which increase the odds of depression, also increases the odds of cardiovascular disease, but do so to a greater extent in females than in males, after accounting for canonical CVD risk factors. In contrast, coronary artery disease (CAD) PGS does not associate with clinical depression (OR, 0.93 [95% CI, 0.87-1.00]; P = 0.07). These findings raise the hypothesis that canonical CVD risk factors (e.g., lipids, hypertension, etc.) may have differential importance in males and females and that sex-differentiated biology may illuminate new relationships between depression and CVD. Methods: Motivated by prior results, we employed a data-driven approach (laboratory measurement-wide association study, i.e., LabWAS) in a retrospective study design. We first tested the relationship between depression PGS and 315 routinely collected laboratory measurements available in the VUMC electronic health records of 70,704 individuals of primarily European ancestry and 12,384 individuals of primarily African ancestry. Using linear regression models, we corrected for the cubic splines of age at lab measurement, top 10 principal components from genetic data, and sex (in the sex-combined models). We then further adjusted for the presence of clinical depression or anxiety disorders. Finally, we stratified the data by sex and tested the models separately in males and females. Results: After multiple testing correction, the LabWAS of depression PGS implicated significant associations with multiple elevated immune markers. Upon stratification by sex, all markers except for WBC count showed sex differential association with depression PGS (see Statistics). Lastly, mediation analyses (N = 20,749) demonstrated that markers of inflammation mediate ~30% (p = 0.017) of the risk for CAD that is conferred by genetic risk for depression. Conclusions: We show that multiple markers of inflammation are associated with the genetic risk for depression, even in the absence of clinical depression. Furthermore, increased inflammation accounts for a substantial portion of the risk for CVD that is conferred by depression genetics. Finally, sex differences point towards inflammatory processes as important mediators of the comorbidity between depression and CVD. Disclosure: Nothing to disclose. 22.4 Optimization of Non-Invasive Vagal Nerve Stimulation for the Modulation of Mood and Cardiovascular Function in Major Depression and Hypertension Ronald Garcia Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, United States Background: Our recent studies have suggested that a novel, non-invasive neuromodulation technique called Respiratory-gated Auricular Vagal Afferent Nerve Stimulation (RAVANS) may effectively modulate the stress response circuitry and have beneficial effects on cardiovagal activity and mood regulation. However, optimal stimulation parameters for this technique have not been established. Here we present the results of studies aimed to identify frequency-dependent effects of RAVANS on the regulation of depressed mood and anxiety symptoms in female patients with major depression (MDD) and sex- and frequency-dependent effects on the regulation of blood pressure values in patients with hypertension. Methods: Study 1 included women with recurrent MDD in an active episode (n = 13, 30.5 ± 6.0 years). Subjects underwent five stimulation sessions, during which they received exhalatory-gated stimulation at frequencies of 2, 8, 30, and 100 Hz or sham stimulation, in a randomized order. Electrodes were placed over vagal-innervated auricular regions (cymba concha) in the left ear. Subjects completed a Beck’s Depression Inventory (BDI) and State-Trait Anxiety Inventory (STAI) at the beginning and end of each stimulation session. Study 2 included subjects with hypertension (n = 20, 54.5 ± 6.2 years) in which the effects of five different RAVANS stimulation frequencies (2, 10, 25, 100 Hz or sham) on blood pressure levels were evaluated. Results: In MDD subjects, RAVANS administration at a 100 Hz frequency resulted in a significant reduction in depressive (BDI score) (β = -3.45, p = 0.026) and anxiety symptoms (STAI score) (β = -6.13, p = 0.034). In hypertensive subjects, RAVANS at 2 and 100 Hz frequencies resulted in a significant reduction of diastolic blood pressure (β = -5.10, p = 0.004; β = -8.28, p = 0.003) and mean arterial pressure values (β = -5.89, p = 0.003; β = -7.45, p = 0.02), but only in black female participants. Conclusions: Our results demonstrate the optimized, frequency-dependent effects of RAVANS administration on the modulation of mood and anxiety symptoms in MDD, and blood pressure regulation in hypertensive subjects. Furthermore, these findings indicate a potential differential response to this stimulation based on sex and race with important implications for the use of this technique in the treatment of comorbid MDD and cardiovascular disease. Disclosure: Nothing to disclose. STUDY GROUP 23. Quo Vadis: The Implications of Small Effect Sizes in Population Neuroimaging Studies Martin Paulus, Wesley Thompson, Chun-Chieh Fan, Brenden Tervo-Clemmens, Hae Kyung Im, Monica Rosenberg, Cynthia Rogers, Roman Kotov, Alexander Shackman, Damien Fair Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Study Group Summary: Large scale, population-representative imaging studies are beginning to examine the utility of structural and functional neuroimaging to better understand the neural mechanisms underlying psychiatric disorders and to provide predictive biomarkers that could be pragmatically useful. These studies have yielded the basis for a much-needed correction to the problems of small samples, capable of providing more accurate estimates of brain-behavior association effect sizes. However, the tools and techniques that are applied to the large studies to evaluate the utility of these brain-behavior associations are just beginning to be developed. One crucial question is what measures should be used to assess the “practical” utility of imaging data when effects are too small to be of individual patient level clinical importance (e.g., for neuropsychiatric outcomes). Moreover, these tools and techniques need to provide answers to the questions whether brain-behavior effects are truly small in a mechanistic (causal) sense, or whether they are they only apparently small, e.g., due to random or systematic measurement error, or whether they are due to group heterogeneities that leads to small average effect sizes? In this Study Group, we will focus on four main challenges of how to formulate a taxonomy of analytic designs for neuroimaging-behavioral studies going forward: 1) how to think about power and sample size (e.g., when do you need very large datasets, and when can you get by with smaller studies?); 2) generalizability to populations of interest (external validity); 3) estimation of effect sizes potentially biased by confounding, measurement error, and selection; 4) application of results of neuroimaging studies to diverse/understudied groups. Our panel of experts will focus on these issues, with the aim of moving beyond the current realization that effect sizes are often (though not always) quite small in brain-behavior relationships. Emphasis will be placed on a path to move forward and for providing guidance to stakeholders (e.g., NIH, neuroscience researchers) on how to fund and conduct studies going forward that maximize efficiencies and opportunities for scientific and clinical utility. Disclosures: UpToDate: Royalties (Self), Spring Health: Board Member (Self), Engrail Therapeutics: Employee (Spouse) PANEL 24. Extending Psychiatric Genetics to Multiple Ancestries 24.1 The Latin American Genomics Consortium: Increasing the Representation and Engagement of Latinx/Hispanic Individuals in Psychiatric Genomics Studies Paola Giusti-Rodriguez University of Florida, College of Medicine, Gainesville, Florida, United States Background: Psychiatric genome-wide association studies (GWAS) are making clear progress in identifying variants linked to psychiatric disorders and related traits. However, nearly 80% of all published GWAS still contain only European ancestry individuals. Admixed individuals in particular are routinely excluded from genetic studies due to concerns over population structure, contributing to the concerning health disparities documented across ancestries. Methods: Incorporating non-European and mixed-ancestry populations in genetic analyses can help to identify genetic effects that generalize across populations, pinpoint causal variants, and ascertain population-specific genetic variants. Results: The Latin American Genomics Consortium (LAGC) was founded in 2019 to accelerate psychiatric genetics research in Latinx/Hispanic populations. The LAGC includes over 100 active members, representing 8 Latin American countries/territories, as well as the U.S. This initiative aims to not only address the enormous underrepresentation of Latinx/Hispanic individuals in psychiatric genomics studies, but to facilitate access to training and resources, and engage in collaborations globally. Conclusions: To address the complexity of GWAS analysis in Latinx populations and conduct well-powered genetic studies, we have assembled an international team of experts equipped with sophisticated tools that will allow us to adequately account for admixture. We have secured access to multiple relevant datasets from the US and Latin America and are conducting meta-analyses focused on psychiatric traits captured across datasets. We continue to work on procuring access to additional cohorts and bringing in members from other Caribbean and Latin American countries. Disclosure: Nothing to disclose. 24.2 The Power of Including All Ancestries: New Methods and Results Kai Yuan Massachusetts General Hospital, Cambridge, Massachusetts, United States Background: The scale of non-European genomic data has started to grow in recent years. Methods to leverage genomic data across multiple ancestries, however, have been lagging. Most studies used naïve methods for multi-ancestry studies, failing to realize the full potential of the data. Here we present two new methods, with applications to psychiatric genetics, that leverage data across ancestries for variant discovery (fine-mapping) and polygenic risk prediction (PRS). Methods: Statistical fine-mapping refines a GWAS locus to a set of likely causal variants (credible set). We developed a novel method for cross-ancestry fine-mapping integrating data from multiple ancestries through explicitly modeling population-specific linkage disequilibrium (LD) and accounting for multiple causal variants in a genomic region. PRS are less effective when ported across populations. We developed the first principled Bayesian PRS method, PRS-CSx, that jointly models GWAS summary statistics from multiple populations to improve cross-ancestry PRS. PRS-CSx couples genetic effects across populations via a shared continuous shrinkage prior, enabling more accurate effect size estimation. Results: We applied our new fine-mapping method to traits from the Taiwan Biobank and UK Biobank (UKBB). Compared with fine-mapping in UKBB alone, we significantly reduced the size of credible sets and increased the PIP of the most probable variant. We then applied our method to schizophrenia genetics data of East Asian (EAS) and European (EUR) ancestries. Compared with the published fine-mapping results we reduced the size of credible sets in 70% of the loci. Similarly, we showed PRS-CSx substantially improved the prediction accuracy even if only a small non-European GWAS was included. The median R2 increased by 76% for EAS individuals when the Biobank Japan samples were added to the UKBB EUR samples to train the PRS. By integrating schizophrenia genetics data from EAS and EUR, we more accurately predicted schizophrenia risk in EAS individuals, showing 52% and 97% improvement in the liability R2 relative to PRS constructed using EAS or EUR summary statistics only. Conclusions: We developed two new methods that model genomics data across multiple ancestries to realize the full potential of the emerging non-European resources. Disclosure: Ono Pharma: Consultant (Self), Xian Janssen Pharmaceutical Ltd.: Honoraria (Self), Biogen: Contracted Research (Self) 24.3 Enhanced Interpretation of Schizophrenia GWAS of Diverse Population With Brain Regulatory Architecture With African American and East Asian Descent Yu Chen The Broad Institute, Cambridge, Massachusetts, United States Background: Previous genetic studies of schizophrenia (SCZ) focused on disproportionate majority of European Population. GWAS of non-European populations highlighted the importance of generalizability of genetic studies. Yet, the scarcity of expression quantitative trait loci (eQTL) data of non-European population brains restricted our understanding of how population genetic diversity is involved in the risk of schizophrenia. Methods: We analyzed genotype and RNA-seq of African Americans (AA, n = 158) and Europeans (EUR, n = 408) from the PsychENCODE consortium and East Asians (EAS, n = 217) from the Chinese Human Brain Bank. We compared population differences of eQTLs from three analyses: differences in allele frequency, population-specific variants, and heterogeneity in eQTL effects. We next used the brain eQTLs to explain SCZ GWAS from diverse ancestries and observed that eQTLs better explained SCZ GWAS in the matched population. We also prioritized risk genes via transcriptome-wide association analysis and colocalization in the matched populations. Results: In total, 1,996,544 significant independent eQTL signals involving 11,622 genes were identified. 173,211 eQTLs (8% of all eQTLs) involving 1,386 genes are novel captured in the non-EUR populations. The trans-ancestry differences represented by the 110,855 (64% of all) novel eQTLs can be explained by allele frequency differences. Although the effect size of eQTLs showed a strong correlation across populations, we identified 913 (2% of all) eQTLs involving ten genes that have opposite eQTL effects across populations. Fourteen novel SCZ candidate genes were identified based on EAS population eQTL with EAS SCZ GWAS. Conclusions: This characterization of brain regulatory architecture across diverse populations highlighted the importance of studying brain eQTL from non-EUR populations and provides a comprehensive resource for novel insights into schizophrenia. Disclosure: Nothing to disclose. 24.4 Leveraging Local Ancestry to Enhance Transcriptome-Wide Association Studies in Admixed and Multiethnic Populations Eric Gamazon Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: We have previously shown that use of local ancestry in genome-wide association studies (GWAS) in multiethnic populations may improve characterization of the heritability of complex traits and lead to more accurate mapping of genetic associations. Methods: We develop a framework for transcriptome-wide association studies (TWAS) in admixed and multiethnic populations. We investigate the methodological implications of a local ancestry aware TWAS (LA-TWAS). TWAS is a two-stage procedure, which exploits information on the genetic regulation of gene expression to conduct gene-level association testing. In the first stage, TWAS trains predictive models of gene expression using local genetic variation; in the second stage, the association between the genetically determined expression generated from each gene model and the trait under study is evaluated. Results: We show that LA-TWAS leads to significantly higher transcriptome prediction performance than conventional TWAS approaches. LA-TWAS jointly estimates the heritability of gene expression and the ancestry specificity attributable to local genetic variation. In addition, in systematic application to BioVU, Vanderbilt’s DNA biobank linked to electronic health records (N > 100k), LA-TWAS leads to significantly increased power for discovery of gene-level associations across a range of genetic architectures and degree of admixture. We present an application to neuropsychiatric traits in African Americans. We present both cross-population and population-specific gene-level findings. Conclusions: Thus, integration of local ancestry enhances modeling of gene expression and identification of expression-mediated complex traits. Our study contributes to ongoing efforts to enhance portability and generalizability of genetic methodologies to traditionally underrepresented populations. Disclosure: Nothing to disclose. PANEL 25. Beyond Resting State: Connectivity-Based Analyses of Evoked Brain Responses as an Optimal Approach to Understanding Brain-Behavior Clinical Associations 25.1 Characterizing Brain-Phenotype Relationships Across Brain States in Health and Disease Abigail Greene Yale School of Medicine, New Haven, Connecticut, United States Background: Predictive modeling may reveal the macroscale neural bases of complex phenotypes and identify individualized targets for clinical intervention. This talk will demonstrate the impact of brain state and measure bias on the performance, interpretation, and utility of brain-phenotype models. Methods: First, we used task- and rest-based functional connectivity (FC) to predict intelligence measures from 2 datasets (sample 1): the Human Connectome Project (HCP; n = 515, 241 male) and the Philadelphia Neurodevelopmental Cohort (n = 571, 251 male). Next, we used psychophysiological interaction (PPI), predictive modeling, and novel inter-subject PPI analyses in the HCP (n = 703; sample 2) to explore how tasks increase predictive model accuracy. Finally, we used task- and rest-based FC from a new, demographically and clinically heterogeneous dataset (n = 129; sample 3) to predict performance on 16 neuropsychological measures and explored in whom models failed. Results: In sample 1, task-based models consistently outperformed rest-based models (P = 0.018 via Mann-Whitney U test), with the best task explaining 12.8% of the variance in fluid intelligence (P < 0.001) and the best rest explaining 3.9% (P = 0.05). In sample 2, FC significantly predicted intelligence (all P < 0.01) independent of task-evoked activation. Activation is useful for prediction only if the task is related to the predicted phenotype (r = 0.96, P < 0.05). Further, some tasks are more useful for prediction than others (e.g., 3.4-12.8% in HCP sample 1), with the best task varying by sex: emotion models outperformed working memory models in females, while the opposite was true in males (all P < 0.02). In sample 3, we explored such group-specific models, finding that model failure is reliable in a subset of participants, phenotype specific (r = 0.49, P < 0.0001), generalizable across datasets (all P < 0.0001), and related to sociodemographic variables such as education and race. Conclusions: Tasks amplify phenotype-relevant patterns of brain activity via distributed, task-general denoising. Care must be taken, however, in interpretation, as models often reflect not unitary cognitive constructs, but rather stereotypical profiles. Together, this work furthers optimization of brain-based predictive modeling to reveal the neural bases of cognitive processes relevant to health and disease. Disclosure: Nothing to disclose. 25.3 Triangulating Multimodal Representations of Affective Experiences During Naturalistic Movie Viewing Luke Chang Dartmouth College, Hanover, New Hampshire, United States Background: Emotions reflect coordinated, multi-system responses to events and situations relevant to survival and well-being. These responses emerge from appraisals of personal meaning that reference one’s goals, memories, internal body states, and beliefs about the world. Consequently, vmPFC activity involved in processing these subjective appraisals appears to be highly idiosyncratic across individuals. We developed a novel computational framework to characterize the spatiotemporal dynamics of the vmPFC in processing our ongoing experiences. Methods: Participants watched the 45-minute pilot episode Friday Night Lights while undergoing fMRI in Study 1 (n = 13) and Study 2 (n = 35) and while we recorded their their facial expressions (Study 3, n = 30) and 16 subjective emotional feelings (Study 4, 183). In study 5 (n = 14), we recorded local field potentials from stereotactic electrodes implanted in patients undergoing intervention for epilepsy. In study 6, we performed a meta-analysis across n = 38 previously published datasets to identify the consistency of inter-subject correlations across the brain across a variety of naturalistic stimuli. Results: Overall, we observed very little evidence of synchronization in brain activity in the vmPFC compared to sensory cortex. This finding replicated across Study 1, 2, 5, and 6. We used a hidden markov model (HMM) to identify discrete latent state changes across different measurement modalities and performed an inter-experiment factor analysis across measurement modalities to triangulate changes in affective states that manifested across Studies 1-5. We found that the vmPFC slowly transitions through a series of discretized states that broadly map onto affective experiences. Although these transitions typically occur at idiosyncratic times across people, participants exhibited a marked increase in state alignment during high affectively valenced events in the show. Conclusions: Our work suggests that the vmPFC ascribes affective meaning to our ongoing experiences and that affective states can be objectively inferred based on patterns of brain activity. Furthermore, we observed strong evidence that participants are rarely occupying the same affective state in response to observing the same stimuli, which poses new theoretical and methodological challenges for studying affective experiences. Disclosure: Nothing to disclose. 25.4 Clinical Relevance of Task-Evoked Patterns of Functional Connectivity Sarah Yip Yale School of Medicine, New Haven, Connecticut, United States Background: Emerging data from basic human neuroscience indicates that functional connectivity data acquired during task performance (vs. resting state) is optimal for accurate brain-behavior modeling of trait associations. This approach has only recently been introduced to clinical research. Methods: In Study 1, we tested the utility of different types of task-based fMRI data for predicting outcomes among poly-substance-using individuals using a connectome-based machine learning approach. In Study 2, we used a similar approach to test whether the accuracy of predictive models of future alcohol use also differed as a function of task type—hereafter referred to as brain state—in a longitudinal sample of youth (N~1,200). In Study 3, we present ongoing work using a dense sampling approach (i.e., weekly scanning over 2 months) to study changes in brain-based signatures of abstinence (such as those identified in Study 1), as assessed across multiple brain states among individuals in methadone treatment. Results: Study 1: Findings demonstrated that the accuracy of machine learning models of abstinence during treatment (% of drug-negative Utox samples over three months) differed as a function of brain state. Reward-related brain states were specific for predicting future cocaine use, whereas inhibitory-related brain states were specific for predicting future opioid use. Based on this striking distinction—that different brain states are specific for predicting different substance-use behaviors in the same individuals—we next tested the effect of brain state in predicting risky alcohol-use in youth. Study 2: Results indicated significant sex divergence in the accuracies of brain-behavior models of future alcohol-use severity, such that female-only models consistently outperformed male-only models. Specifically, female-only models successfully predicted future and current severity across both reward-related and inhibitory control states. In contrast, male-only models were successful in predicting current severity using connectivity data acquired during inhibitory control—but not reward—brain states, indicating brain-state specificity of predictive models of alcohol-risk in males only. Conclusions: Brain state manipulation via performance of in-scanner tasks is optimal for prediction of complex, real world, clinical outcomes. Disclosure: Nothing to disclose. MINI PANEL 26. Minority Stress and its Mechanisms: From Systemic and Proximal Social Context to Mental Health Disparities in Sexual and Gender Minority Youth 26.1 Adolescents’ Sexual Orientation and Behavioral and Neural Reactivity to Peer Acceptance and Rejection: The Moderating Role of Family Support Kirsty Clark Vanderbilt University, Nashville, Tennessee, United States Background: Adolescents who face peer rejection are more likely, compared to their accepted peers, to anticipate and react severely to peer rejection (Downey at el., 1998) and to display blunted neural reactivity to peer acceptance (Rappaport et al., 2019), which has been linked with higher depressive symptoms (Kujawa et al., 2019). Sexual minority adolescents frequently endure stigma-related peer rejection, yet scant research examines sexual orientation differences in behavioral and neural reactions to peer feedback. Further, while family support can buffer negative mental health impacts of peer rejection, no studies have examined the protective (i.e., moderating) role of family support in studies assessing sexual orientation and behavioral and neural reactivity to peer feedback. The present study examined associations between sexual orientation, behavioral and neural reactions to peer feedback, and the potentially protective role of family support. Methods: In a community sample of adolescents approximately 15 years old (47.2% female; n = 36 sexual minority, n = 310 heterosexual) participants completed the Island Getaway task while EEG data were recorded (Kujawa et al., 2014). The Island Getaway task is a computerized social interaction task where, across several rounds of game play, participants vote to reject or accept peer co-players (who are, in fact, pre-set computerized co-players) and receive rejection and acceptance feedback from these peer co-players. Voting data were used to calculate behavioral measures of ingratiation (i.e., total number of votes to ‘keep’ co-players who had voted the participant off in the previous round) and alliance-building (i.e., the total number of votes to ‘keep’ co-players who had voted to keep the participant in the previous round). The reward positivity, an event-related potential (ERP) thought to index reward processing, measured individual differences in neural reactivity to peer acceptance versus rejection (i.e., residual RewP). Family support was assessed through the Multidimensional Scale of Perceived Social Support (MSPSS; Zimet et al., 1988). Results: Sexual minority adolescents reported significantly lower family support (M = 20.44) than heterosexual adolescents (M = 22.98, p = 0.013). Linear regression models showed that sexual minority adolescents engaged in a significantly higher average number of ingratiation efforts than heterosexual adolescents (b = 1.31, p = 0.046). Main effects for alliance-building and residual RewP were not significant at p < 0.05. An interaction between sexual orientation and family support predicted residual RewP (p-value = 0.039). Decomposing family support by sexual orientation interaction into simple slopes demonstrated that the sexual orientation difference in residual RewP was significant at low levels of family support (t = -2.68, p = 0.007) but not at mean (t = -1.28, p = 0.202) or high levels of family support (t = 0.29 p = 0.770). Conclusions: Results found that sexual minority adolescents show distinct behavioral and neural reactions to peer feedback and suggest that sexual minority adolescents with low family support exhibit particularly blunted neural reactivity to peer acceptance, which has been implicated in the development of depressive symptoms. These results may have implications for developing preventive interventions, especially for sexual minority adolescents with low family support. Disclosure: Nothing to disclose. 26.3 Medial Prefrontal Cortex Activity to Reward Outcome Moderates the Association Between Victimization Due to Sexual Orientation on Depression in Youth Kristen Eckstrand University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Sexual minority youth and young adults (SMY) are 3x more likely to experience depression than heterosexual peers. Minority stress theory posits that this association is explained by sexual orientation victimization, which acts as a stressor to impact depression. Emerging evidence suggests that stress increases depression severity by altering activity in neural reward systems. For those vulnerable to the effects of stress, victimization may worsen depression by altering activity in neural reward systems. However, imaging studies among SMY have primarily focused on neural differences by sexual orientation identity alone and no studies have examined the influence of sexual orientation victimization on neural reward system function. The purpose of this study was to examine whether neural reward activity moderates the relationship between sexual orientation victimization and depression, and whether these patterns differed by sexual orientation. Methods: 81 participants ages 16-22 years old (41% SMY, 59% self-reported female sex, 52% racially marginalized) were enrolled into the study. Participants completed self-report measures of depression using the Center for Epidemiological Studies-Depression (CESD) scale; anhedonia using the Snaith Hamilton Pleasure Scale (SHAPS); and identity victimization experiences based on sexual orientation, race, and gender. Participants underwent functional magnetic resonance imaging during a standardized monetary reward task to measure neural response to reward. Neural reward activity was determined by significant neural response (pFWE <0.05) to reward>neutral outcome within the Neurosynth reward mask in SPM12. BOLD activity was extracted for subsequent analyses. Age was included as a covariate in all models. Results: Bilateral ventral striatum (VS), medial prefrontal cortex (mPFC), dorsal anterior cingulate cortex, and right orbitofrontal cortex were significantly activated during reward outcome. SMY experienced higher severity depression (β = 14.96, p = <0.001, 95CI = 9.68, 20.25) and anhedonia (β = 5.98, p = 0.017, 95CI = 0.79, 7.75), and more victimization events based on sexual orientation (t[1,75]=-5.69, p = <0.001). Higher depression severity was also associated with more victimization based on sexual orientation (β = 0.95, p = 0.047, 95CI = 0.14, 1.87). More sexual orientation victimization predicted higher mPFC response to reward (β = 0.18, p = 0.04, 95CI = 0.01, 0.36). In an interacting moderation model (R2 = 0.493, p < 0.001) mPFC reward activation moderated the relationship between sexual orientation victimization and depression (β = 4.79, p = 0.04, 95CI = 0.22, 9.37), with higher mPFC activation and higher sexual orientation victimization being associated with higher depression. Sexual orientation did not moderate this interaction. No other regions of significant neural reward activity moderated orientation victimization-depression relationships. Conclusions: Depression in SMY was related to sexual orientation victimization, but only in the context of higher mPFC activation, a pattern observed in depressed youth. These novel results provide evidence for neural reward sensitivity as a vulnerability factor for depression in SMY, suggesting mechanisms for disparities, and is a first step towards a clinical neuroscience of minority stress in SMY. Disclosure: Nothing to disclose. MINI PANEL 27. Serotonin Reuptake Inhibitors (SRIs) and Pregnancy: Serial Monthly Changes in Plasma Sertraline Concentrations, the Use of Platelet Serotonin to Assess SRI Bioeffect, and the Association of Maternal and Cord Blood Platelet Concentrations With Fetal-Neonatal Neurobehavior 27.1 Changes in Sertraline Plasma Concentrations Across Pregnancy and Postpartum and Relationship to Cytochrome P450 2C19, 2C9 and 2D6 Enzyme Activities Katherine Wisner Northwestern University School of Medicine, Chicago, Illinois, United States Background: Serotonin reuptake inhibitors are prescribed to 5-8% of pregnant persons. Sertraline (SERT) is the most frequently prescribed drug, although few data about its pharmacokinetics in pregnancy is available. The primary metabolic pathway is the conversion of SERT to its weakly active metabolite, desmethylsertraline (DMSERT). SERT metabolism is catalyzed by CYP3A4, CYP2C9, CYP2C19, CYP2B6 and CYP2D6. CYP3A4 comprises more than 30% of the Phase I metabolic capacity and is the primary driver of SERT metabolism. The goal of this study was to characterize plasma SERT concentration to dose (C/D) ratios across pregnancy and postpartum as well as evaluate the effect of pharmacogenetic variability on SERT elimination. Methods: This prospective observational cohort study was conducted in pregnant people receiving maintenance SERT therapy and who elected to continue during pregnancy. The study was conducted at 3 NICHD-funded Obstetrical-Fetal Pharmacology Research Center sites. Pregnant people with a singleton pregnancy ≤18 weeks gestation and with at least one previous episode of MDD were eligible. Blood samples were obtained 24-hours post-dose every 4 weeks across pregnancy and twice postpartum for measurement of plasma concentrations of SERT and DMSERT. We analyzed the changes in trough plasma SERT C/D ratios as well as plasma SERT to DMSERT (S/DS) ratios (a measure of metabolic capacity). Plasma SERT and DMSERT concentrations were measured by high performance liquid chromatography-tandem mass spectrometry. We explored the effect of genetic variability in enzymes responsible for SERT metabolism on the SERT C/D ratios. Participants were genotyped for variants in CYP2C9, CYP2C19, and CYP2D6, using commercial allelic discrimination assays with Taqman probes. Results: Pregnant people (N = 47) were enrolled. Overall mean SERT C/D ratios decreased early in pregnancy and remained low until after delivery. In the last 4 weeks of pregnancy, the mean SERT C/D ratio, 0.25 (95% CI, 0.19-0.3) was less than the postpartum ratio, 0.32 (95% CI, 0.27-0.37). The model-estimated mean S/DS ratios were highest after birth, demonstrating that SERT elimination in pregnancy was comparatively increased. C/D ratios in those with functional CYP2C19 activity did not significantly change, while ratios in those with poor or intermediate CYP2C19 activity decreased by 51%. This decrease likely reflects increased SERT clearance by CYP3A4. Although CYP2C19 activity decreases in pregnancy, those with poor/intermediate CYP2C19 phenotypes do not decrease activity to offset the increased activity of other CYPs. Conclusions: An increase in SERT metabolism and declining concentrations in pregnancy compared to postpartum occurred for all genetic phenotypes. Although some experts recommend CYP2C19 testing prior to SERT therapy in nonpregnant adults to avoid adverse effects from elevated concentrations in people with CYP2C19 poor and intermediate metabolizers, these same individuals, when pregnant, are at risk for undertreatment as SERT concentrations decline. No meaningful SERT concentration differences were associated with CYP2C9 and CYP2D6 polymorphisms. Disclosure: Nothing to disclose. 27.2 Assessment of Serotonin Reuptake Inhibitor Bioeffect by Measurement of Platelet Serotonin George Anderson Yale University School of Medicine, New Haven, Connecticut, United States Background: Serotonin reuptake inhibitors (SRIs) are widely used to treat depressed mood and anxiety. Limited neuroimaging studies indicate that typical clinical doses of the SRIs result in approximately 80% occupancy of the central serotonin (5-HT) transporter (SERT). Researchers have also attempted to assess the SRI inhibitory bioeffect by examining decreases in platelet 5-HT level. This more accessible approach depends upon the fact that all platelet 5-HT is taken up via SERT over the platelet’s 10-day lifespan. The platelet measure provides a time-averaged index of SRI bioeffect and has advantages compared to plasma drug level when assessing SRI exposure. We will overview the theoretical basis for the measure and its practical applications and present a systematic analysis of the reported apparent bioeffects of SRIs. Of special relevance to this panel is the use of platelet 5-HT in assessing maternal and infant SRI exposure when SRIs are used to treat maternal perinatal depression. Methods: A systematic literature search used search terms including platelet 5-HT, whole blood 5-HT, antidepressant, selective reuptake inhibitor, selective serotonin reuptake inhibitor, serotonin-norepinephrine reuptake inhibitor, as well as individual SRIs. Bibliographies of identified papers were also searched for relevant papers. Studies were included if mean pre-drug and on-drug 5-HT values were given, if standard clinical doses were used, and if treatment lasted for at least two weeks. The mean value reported for the on-drug 5-HT value as a percent of baseline was extracted and the mean, standard deviations (SDs) and median values of the study means were calculated for the combined SSRI group and for individual SRIs with 3 or more studies. Results: Mean (SD) and median on-drug 5-HT values as a % of baseline for the different groups are given in Table 1., along with number of studies included in each group. The on-drug 5-HT values in the clomipramine group were significantly lower than in the combined SSRI group: Kruskal-Wallis H statistic 6.13, p = 0.013 Table 1. SRI Bioeffect: On-Drug Platelet 5-HT Values as a Percent of BaselineGROUP n MEAN (SD) MEDIAN Combined SSRI 38 17.4 (11.3) 14.2 Clomipramine 15 12.3 (7.7) 8.0 Fluoxetine 10 14.6 (7.8) 14.6 Paroxetine 8 11.4 (10.7) 9.7 Fluvoxamine 4 13.8 (2.1) 14.1 Sertraline 4 19.6 (13.5) 14.5. Conclusions: This systematic review of the use of platelet serotonin to assess the bioeffect of clinical doses of SRIs indicated that: 1) studies have found mean and median on-drug 5-HT values to be 8-20% of baseline values; 2) clomipramine showed significantly greater reductions in platelet 5-HT compared to other SSRIs. The overall results are consistent with limited neuroimaging data and within vitro evidence. The clomipramine results are consistent with prior indications that typical clinical doses of clomipramine lead to relatively greater SERT inhibition. In general, the results support the use of platelet 5-HT to assess SRI bioeffect. Disclosure: Nothing to disclose. 27.3 Fetal Neurobehavior in the Context of Bioeffect From Gestational Selective Serotonin Reuptake Inhibitors Amy Salisbury Virginia Commonwealth University, Richmond, Virginia, United States Background: More than a third of women with depressive disorders in pregnancy are treated with selective serotonin reuptake inhibitor (SSRI) antidepressants during the gestational period. The benefits of treating depression in pregnancy with SSRIs has been shown to outweigh potential risks to the fetus. However, gestational SSRI exposure alters serotonin signaling and has been associated with adverse respiratory, gastrointestinal, and neurobehavioral signs in the newborn. Although these signs diminish over the first postnatal month, recent evidence suggests potential long-term changes in serotonin-related functions. However, there is no consensus on the mechanisms of these early or later alterations. Therefore, this study examined fetal neurobehavior at the time of SSRI exposure compared to no exposure, with and without concurrent maternal depression and associations between fetal measures, SSRI bioeffect, and later infant outcomes. Methods: Pregnant women (N = 243) were enrolled into a prospective, longitudinal observational study examining the impact of prenatal SSRI use on fetal and infant neurodevelopment. Women were enrolled in second and third trimesters and completed standardized assessments to determine depression diagnosis and gestational SSRI use, dosage, and timing. Standardized fetal assessments were conducted on one or two occasions between 26- and 36-weeks gestation using ultrasound-based video recordings. Women who were using SSRIs concurrent with the fetal assessments (N = 50) and who were not using SSRIs at any time in pregnancy (N = 133; Total N = 183) were included in these analyses. At the time of delivery, samples of maternal peripheral blood (N = 103) and fetal cord blood (N = 97) were collected in a subset of participants to measure maternal and fetal whole blood platelet serotonin levels and SSRI blood concentrations. Fetal bioeffect was previously confirmed with lowered platelet serotonin levels in women using SSRIs. Mixed models were used to examine group differences for fetal measures; correlation analyses were used to examine relationships between continuous variables. Results: Compared to fetuses of women with (N = 61) and without depression (N = 72) and no SSRI use in pregnancy, fetuses with SSRI exposure (N = 50) had lower fetal movement quality, higher jerky to smooth movement ratio, shorter quiescent periods, and fewer fetal breathing movements. There was a significant negative relationship between fetal movement quality and maternal platelet serotonin levels at birth. Gestational age at birth was positively correlated with cord blood platelet serotonin levels, but not maternal levels. Conclusions: These results suggest that the adverse signs observed in some infants after delivery may be present prior to birth and removal of the SSRI exposure; suggesting that withdrawal or adaptation may not be the primary mechanism. Fetal neurobehavioral patterns found here suggest immaturity of motor and respiratory function which may have implications for later development. We will discuss these findings in relation to previously reported findings in the newborn period. Disclosure: Nothing to disclose. PANEL 28. How the Exposome Influences Suicidal Behavior 28.1 Suicidal Ideation in Response to Negative Life Events: The Role of Emotion Regulation Maria Oquendo Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: A subset of suicidal individuals responds to negative events with increased suicidal ideation (SI), placing them at greater risk for suicidal behavior during those periods. Whether these SI increases relate to lack of coping strategies such as emotion regulation (ER) is unknown. We hypothesized that those responding to negative events with lower SI increases would engage in ER more during exposure to painful memories. Methods: Individuals with Major Depression (n = 27) participated in an fMRI task, recalling painful autobiographical memories, and engaging in ER when instructed. Ecological Momentary Assessment (EMA) at 3, 6, 12, 18 and 24 months after baseline provided 7 days of responses about stressors, affect and SI. A neural decoder trained to detect ER effects in fMRI data from 77 participants identified a network comprising inferior frontal gyrus, orbitofrontal cortex (OFC), Anterior Cingulate Cortex (ACC), frontal operculum, left caudate, and insula. A linear mixed effect regression tested whether the ER neural decoder output moderated the effect of stressors on epoch-to-epoch SI change, adjusted for time, lagged values for SI and affect, and for 2-way interactions between lagged affect and decoder, and lagged affect and stressors. The neural decoder output was residualized for bold activity. Results: ER decoder output moderated negative events’ impact on SI; those who appeared to use ER more robustly when recalling negative memories responded to life events with lower changes in SI. This was in contrast to findings in those with lower ER during memory recall (interaction term: value = -3.87; S.E. = 1.34; df=3126, t = -2.89; p = 0.004). The ER decoder’s moderation of SI change in response to stress remained significant after adjusting for depressive symptoms before the life event through main effect and interactions. Conclusions: These pilot findings suggest that those who respond to life events with lower changes in SI may be able to harness ER as a coping strategy by engaging OFC and ACC when confronted with painful stimuli. Interventions for suicidal individuals such as dialectical behavioral therapy, which teach emotion regulation are available. Studying whether individuals who respond to life events with greater SI can leverage these interventions to display less propensity towards increased SI in response to stress would be instructive. Disclosure: C-SSRS: Royalties (Self), Bristol Myers Squibb: Employee (Spouse) Otsuka, MindMed, Alkermes, Sage Therapeutics: Advisory Board (Self), St George’s University: Board Member (Self) 28.2 Contribution of Individual and Structural Psychosocial Exposome to Suicide Attempts in Diverse Youth Ran Barzilay Perelman School of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Rates of youth suicidal behavior are rising, most steeply among Black youth. Distal and proximal psychosocial stressors are key suicidality risk factors. The network of environment and social determinants of health is referred to as the “exposome”. We use exposome data to better understand youth suicide risk and its related racial disparities. Methods: We combine multi-layer data of individual exposome with geocoded neighborhood exposome in 3 settings: (i) Electronic health record (EHR) of Children’s Hospital of Philadelphia (CHOP), where we complied data from N = 19,879 youth (Mage=15, 55% Black) presenting to emergency department. (ii) Research and clinical integrated dataset of 1,514 youth from the Philadelphia Neurodevelopmental Cohort (PNC) study that have longitudinal data on suicide attempts from CHOP EHR. (iii) Research data from the Adolescent Brain Cognitive Development (ABCD) Study that follows ~12k diverse adolescents across 21 sites in the US. Results: In CHOP data, 10% of patients reported past suicide attempt, with higher rates in Black (10.9%) Vs. White (8.5%) youth (P < .001). Prevalence of stressors associated with suicide attempt were different between races, with Black youth experiencing 63% more physical bullying, 29% more sexual abuse, 2-fold more romantic partner violence and 3-fold more involvement in fights. Conversely, White youth experienced 27% more cyberbullying (all P’s < .001). In PNC, we found an interaction effect of exposure to assault with geocoded neighborhood measures in association with suicidal ideation (Wald = 8.19, p = .004). Longitudinal analyses of PNC youth with data on suicide attempts years later revealed that geocoded neighborhood measures (e.g., percent of vacant lots) are among the top ranked predictors in models predicting future suicide attempt. In ABCD, peer stressors like discrimination, bullying and cyber-victimization were associated with suicide attempts (controlling for demographics, odds ratios, 95%CI = 1.3, 1.2-1.4; 1.6, 1.5-1.7; 3.7, 2.8-4.8, respectively, P’s < .001). Conclusions: Results converge across three different data sources to suggest a critical role for exposome in youth suicide attempt risk. Data highlight the potential of integrating individual with structural exposome to study youth suicide attempt, its related racial disparities, and optimize its prediction. Disclosure: Taliaz Health: Advisory Board (Self), Taliaz Health: Stock / Equity (Self), Zynerba Pharmaceuticals: Advisory Board, Consultant (Self) 28.3 Childhood Adversity and Recent Life Stress Brain Relationships in Depression and Suicide J. John Mann Columbia University, New York, New York, United States Background: Childhood adversity and recent life stressors increase major depression and suicidal behavior risk in adulthood. Little is known of genetic and epigenetic mediators. The serotonergic system is abnormal in suicidal behavior. We therefore examined relationships between serotonin (5-HT) system components and childhood adversity (Study 1) and recent life stress (Study 2) in major depression and suicidal behavior. Methods: Study 1: N = 192 MDDs, and N = 88 healthy volunteers (HV), majority female, were assessed for childhood adversity and recent life changes and genotyped for rs6295 in the 5HT1AR gene. DNA methylation was assayed at three upstream promotor sites (-1017, -1007, -681). 5-HT1A receptor binding (BPF) was quantified by PET with [11C]WAY100635 in a subgroup (N = 119). Study 2: Recent life stress was measured by Ecological Momentary Assessment (EMA) over one week in 25 depressed MDD participants (12 suicide attempters, majority female) before undergoing serotonin transporter (5-HTT) and 5-HT1A receptor PET imaging. Results: Study 1: Recent stress correlated positively with methylation at the -681 CpG site promotor site, adjusted for diagnosis, sex and age, and had positive and region-specific correlations with 5-HT1A BP in MDD, but not HVs. In MDDs, but not in HVs, methylation at only the -1007 CpG site had positive and region-specific correlations with 5-HT1A BP Childhood adversity was not associated with DNA methylation or 5-HT1A BP. Study 2: In suicide attempters, but not in non-attempters, region-specific associations between both 5-HTT (p = 0.006) and 5-HT1A (p = 0.01) BPND and EMA stress emerged. 5-HT1A BPND correlated positively with EMA stress in attempters in 8/10 regions (p’s<0.05) and negatively with 5HTT binding in one region. Conclusions: Study 1 Recent stress increased 5-HT1A receptor BP via DNA methylation of inhibitory transcription factor binding sites, thereby impacting MDD psychopathology and the risk of suicidal behavior. Study 2: Spatially localized lower 5-HTT binding and widespread higher 5-HT1A binding indicate impaired serotonin signaling in suicide attempters and not in non-attempters. Epigenetic relationships suggest a pre-existing genetic or epigenetic effects of childhood adversity, but this was not detected. Disclosure: Research Foundation for Mental Hygiene: Royalties (Self) STUDY GROUP 29. Bridging the Gap Between Pre-Clinical and Clinical Studies: The Promise and Challenges of Forward and Reverse Translational Approaches Jennifer Blackford, Marisa Silveri, Danny Winder, Tallie Z. Baram, Michael Yassa, Mohammed Milad, Ned Kalin, Daniel Pine, Kerry Ressler, William Carlezon, Isabelle Rosso, Jennifer Blackford Munroe-Meyer Institute, University of Nebraska Medical Center, Omaha, Nebraska, United States Study Group Summary: There have been extensive advances in our knowledge of the neurobiology of mental health disorders over the past decade. The translation of findings from bench to bedside, however, remains slow, costly, and is often fraught with substantial limitations. There has been much discussion around how to improve components of the translational process—for example, by using different animal models, developing novel and innovative experimental tasks, or selecting appropriate research tools. Nevertheless, a major challenge in translational research has been the wide gap between the scientists conducting preclinical animal studies and those conducting clinical human studies. Most scientists focus exclusively on only animal or human models, leading to silo mentality. Among factors isolating camps include the use of different research approaches, methods, and terminology, as well as presenting data at conferences and publishing findings in journals most associated with one line of research. Funding mechanisms and study section assignments also contribute to separation of animal from human studies, and vice versa. Despite these longstanding challenges, new approaches are beginning to emerge. This study group will highlight work by scientists dedicated to bridging gaps between animal and human studies through several perspectives. The first perspective is from scientists who have cross-trained in both human and experimental model research. A second perspective arises from scientists collaborating tightly across research modalities. Combining these perspectives can help mitigate silos by developing transdisciplinary language, methods, and interpretations. Especially compelling are scientific partnerships that provide a foundation for rapid and iterative forward and reverse translational approaches. Forward translation has been a hallmark of the bench to beside approach. However, reverse translation from human to animal models is equally important, as identifying the power and limitations of experimental systems and matching human questions to appropriate models is crucial. An iterative approach, that supports real-time forward and reverse translation, shows great potential for moving the field forward. A combination of reverse and forward translation can enhance identification of causality and mechanisms in animal models that can then be tested in human disease, and can encourage the application of findings from human studies to develop novel approaches and hypotheses to be tested in preclinical studies. An iterative, transdisciplinary process is critical for advancing construct-validated investigations of disorder-relevant neural circuitry, with the ultimate goal to guide the identification of novel treatment targets, development of novel therapeutics, and enhancement of current therapies. This study group will include scientists who have cross-trained in animal models and human studies (Ressler, Milad, Silveri) and pairs of scientists who are actively collaborating to achieve iterative forward and reverse translations across their research programs (Winder-Blackford, Kalin-Pine, Baram-Yassa, Carlezon-Rosso). Disclosure: Nothing to disclose. STUDY GROUP 30. Mechanisms of Dysregulated Sleep and Circadian Rhythms across the Psychiatric Spectrum: Clinical Studies to Decipher the Transdiagnostic Role of Sleep in Mental and Cognitive Health Ellen Lee, Jennifer Goldschmied, Kathleen Merikangas, Brant Hasler, Bryce Mander, Bengi Baran, Michael Irwin, Rebecca Hendrickson, AJ Schwichtenberg, David Kupfer University of California, San Diego, La Jolla, California, United States Study Group Summary: Sleep and circadian rhythm disturbances are core features of most neuropsychiatric disorders, but can also be viewed as modifiable targets with important implications for the progression of psychiatric disorders as has been shown by the improvement in depressive symptoms following cognitive behavioral interventions for sleep. Sleep/circadian factors are intricately tied to the neural circuitry of emotion regulation, reward processing, cognitive control, and sensory processing, suggesting trans-diagnostic mechanistic roles in the development and maintenance of neuropsychiatric disorders. This study group will unite nine of the field’s most relevant researchers–balanced by gender and career stage–to discuss sleep/circadian rhythms impairment across depression, bipolar disorder, schizophrenia, post-traumatic stress disorder, neurodegeneration, autism, and substance use. The non-ACNP member participants have expertise in aspects of sleep and/or circadian rhythms in depression (JG), substance use (BH), neurodegeneration (BM), psychotic disorders (BB), autism (AS), and post-traumatic stress disorder (RH). We propose that the study of sleep and circadian rhythms is essential to the investigation of the pathophysiology, maintenance, and treatment of psychiatric disorders. We will address the following questions:A broad range of underlying biological mechanisms connect sleep/circadian rhythms to cognitive and psychiatric functioning – e.g., inflammation, amyloid and tau deposition, slow-wave activity-mediated neuroplasticity changes, alterations in neural circuitry underlying reward function and cognitive control, oxidative stress, and sympathetic dysregulation. What is the current evidence of how these sleep/circadian-related mechanisms overlap with the pathophysiology of psychiatric conditions? Accurate assessment of sleep, circadian rhythms, and associated mechanisms are a longstanding challenge for researchers. Gold standard approaches like In-lab polysomnography for sleep and in-lab melatonin or core body temperature assessments for circadian rhythms are expensive, and burdensome. Novel wearable and environmental sensors can offer complementary information about sleep and circadian parameters, including night-to-night variability of sleep, over time and under naturalistic conditions. What are the strengths and limitations of different sleep/circadian assessments? How acceptable and feasible are such approaches for psychiatric populations? Sleep/circadian rhythm disorders are often attributed to primary psychiatric disorder, and thus, underdiagnosed, and untreated. Psychiatric treatments can also adversely affect sleep architecture and quality. How can clinical studies systematically examine the impact of sleep/circadian treatments on psychiatric symptoms and of psychiatric treatments on sleep/circadian problems? Which treatment approaches improve both sleep/circadian function and psychiatric symptoms? Which treatment approaches that target sleep/circadian function show promise in preventing psychiatric disorders such as depression? Disclosure: Nothing to disclose. PANEL 31. Neurobiological Pathways From Early Adversity to Psychopathology 31.1 Developmental Timing and Key Dimensions of Early Adversity: Multivariate Associations With White Matter Microstructure Dylan Gee Yale University, New Haven, Connecticut, United States Background: Delineating links between early adversity, neurobiology, and psychopathology is critical to identifying trajectories of risk and resilience following adversity exposure. Microstructural remodeling of white matter pathways represents one potential mechanism linking childhood adversity exposure with psychopathology across development. Given vast heterogeneity in the nature of early adversity and outcomes, conceptual models of early adversity have increasingly focused on key dimensions of adversity exposure. The ways in which specific aspects of adversity—such as the developmental timing of an exposure—may uniquely shape developing white matter have yet to be elucidated. Methods: N = 107 adults (ages 18-30) underwent a diffusion-weighted imaging scan and completed a modified version of the UCLA PTSD Reaction Index designed to examine key dimensions of lifetime stress exposure. We examined multivariate associations between specific features of adversity exposure (developmental timing, threat, deprivation, predictability, controllability) and 43 white matter tracts using regularized canonical correlation analysis. We then examined associations between the canonical modes that were identified and symptoms of psychopathology. Results: Results identified 11 canonical modes that differed significantly from chance (Bonferroni corrected; p < .01). Several meaningful modes emerged. One mode predominantly indexed adversity characterized by deprivation that was experienced across childhood and adolescence and alterations in the optic radiation tracts. A second mode predominantly indexed adversity that was perceived as predictable and controllable and experienced in adolescence and alterations in the cingulum cingulate. Further, the canonical mode characterized by deprivation was associated with internalizing symptoms in adulthood (p = .01). Conclusions: Our findings suggest that sensory processing tracts may be most affected by experiences characterized by deprivation, whereas frontolimbic and frontostriatal tracts may be more sensitive to experiences characterized by controllability and predictability. These results highlight the utility of multivariate approaches for parsing heterogeneity related to early adversity and indicate that such techniques can yield clinically meaningful information. Disclosure: Nothing to disclose. 31.2 Neurostructural Alterations Following Traumatic Experiences During Child and Adulthood Maurizio Sicorello Central Institute of Mental Health, Mannheim, Germany Background: Animal studies have shown that adverse experiences, and their neuroendocrine mediators, can lead to volumetric changes in hippocampus and amygdala. Similar volumetric alterations have also been observed in humans suffering from posttraumatic stress disorder and, for the hippocampus, even healthy trauma-exposed individuals. Recent evidence suggests that alterations can be dependent on the neurodevelopmental timing of events in childhood and adolescence. Still, these studies have thus far not compared effects to traumatic experiences occurring in adulthood and the specificity to psychopathology vs trauma-exposure is often not tested. Methods: A total of 155 women were allocated into one of six age-matched groups according to timing of traumatization (childhood vs adulthood) and psychopathology (PTSD vs trauma-exposed healthy vs trauma-naïve healthy). Volumes of amygdala and hippocampus were compared between these groups. Six additional exploratory regions of interest (ROI) were included based on a recent meta-analysis. Statistical tests were controlled for the number of ROIs, with additional correction for pair-wise post-hoc comparisons. Results: There was a significant disordinal interaction between timing and group for amygdala volume (p < .001, η2 = .08): For childhood trauma, participants with PTSD had lower volumes than healthy trauma-naive controls in both hemispheres (left: p = 017, g = 0.81; right: p = .31, g = 0.75). For adulthood trauma, the PTSD group had larger bilateral volumes in comparison to both trauma-exposed controls (left: p = .002, g = 1.01; right: p = .013, g = 0.83) and trauma-naive controls (left: p = .002, g = 1.01; right: p = .002, g = 0.73). The interaction between time and group was not statistically significant for the other regions (all p > .08). Conclusions: Timing of traumatization was associated with amygdala volumes throughout the lifespan, with opposite effects dependent on age at trauma occurrence. These findings have to be viewed in light of fundamental confounds that distinguish trauma during child- and adulthood. Regardless of these confounds, the opposing effects for amygdala volume have important implications for the interpretation of trauma-contingent structural findings in this region. Disclosure: Nothing to disclose. 31.3 Looking Into Troubled Waters: Childhood Emotional Maltreatment and Neural Responses to Prolonged Gazing Into One’s Own and Others Eyes Bernet Elzinga Leiden University, Leiden, Netherlands Background: Eye contact is crucial for the formation and maintenance of relationships, not only with others, but also with oneself. Childhood emotional maltreatment can disrupt the development of positive views about self and others. One potent way the psychological consequences of emotional maltreatment may become apparent in daily life is when gazing into one’s own or other people’s eyes. Methods: To investigate how emotional maltreatment may affect people’s responses when looking into one’s own or others’ eyes adult participants (mean age=49.9 years; n = 79, n = 35 men and n = 44 women) viewed videos of direct versus averted gaze of themselves and an unfamiliar adult while neural activity was recorded using fMRI, as well as self-reported mood and time spent looking into one’s own and others’ eyes using an eye tracker. Mood and gaze responses were analyzed in R (R Core Team (42), version 3.6.1) with lme4 for mixed model analysis. Significance for analyses on mood and gaze responses was set at p < 0.05 (two-tailed) and Cohen’s d effect sizes were calculated for significant effects. To examine associations between CEM and participants’ neural responses to self and unfamiliar adult, two separate regression analyses were conducted in SPM including CEM as covariate of interest and neural responses to either self or unfamiliar other adult as outcome. All whole-brain results were corrected for multiple comparisons with Family-Wise Error (FWE) cluster correction at p < 0.05 (with a p < 0.001 cluster-forming threshold). Results: Participants who experienced more maltreatment reported lower mood after videos of direct, but not of averted gaze (of themselves and unfamiliar adults). Individuals who reported higher levels of maltreatment exhibited increased activity in ventromedial prefrontal cortex when looking at videos of themselves. Maltreatment did not correlate with gaze or neural responses during eye contact with an unfamiliar adult. Conclusions: These results suggest that childhood emotional maltreatment may have a long-lasting impact on social-affective processes during eye contact and on self-referential processes that may particularly surface when looking into one’s own eyes. These findings may be an interesting point of departure for clinical interventions to strengthen self-views. Disclosure: Nothing to disclose. PANEL 32. From Big Data to Personalized Prediction in Psychiatry 32.1 Personalized Vulnerability Indexes for Severe Mental Illnesses: Translating Big Data Finding to the Level of Individual Subject Peter Kochunov Maryland Psychiatric Research Center, University of Maryland School of Medicine, Ellicott City, Maryland, United States Background: Big Data research in major depressive (MDD), bipolar (BD), and schizophrenia spectrum (SSD) disorders led to stable and reproducible neuroimaging deficit patterns. We propose the Regional Vulnerability Index (RVI) to quantify an individual’s brain-wide similarity to the expected SMI patterns, analogous to polygenic risk scores (PRS) that measure an individual’s similarity to genome-wide risk patterns. We hypothesized that RVI is a brain intermediary between genome and symptoms and is sensitive to both genetic and environmental risks for mental disorders. We tested this hypothesis in clinical samples as well as using it to evaluate the impact of genetic and environmental factors on development of illness in children. Methods: Sensitivity and specificity analysis in UKBB sample (N = 37285) evaluated RVI in mental illnesses and compared it to PRS. RVI-SSD was evaluated as a predictor of treatment resistance in sample of N = 122 of SSD subjects. The RVI-MDD was tested as predictor of higher symptoms of depression in N = 8,089 UKBB subjects with clinical and sub-clinical MDD. Adolescent Brain and Cognitive Development (ABCD) (N = 8940, age=10 years) was used to evaluate if ancestral history (AH) for SSD, perinatal risk factors and negative early life experiences can lead to higher individual similarity with the adult SSD brain patterns in children. Results: RVI for mental illnesses were sensitive and specific, more so than PRS. Treatment-resistant patients showed significantly higher RVI-SSD than that in treatment-responsive patients (p = 0.01). Group-average increase in depressive symptoms was mirrored by increases in RVI-MDD (r = 0.85). In children, RVI-SSD had captured more variance from family history of SSD, prenatal and negative life events than any whole-brain or individual regional brain measurements. Higher RVI-SSD scores in children were already associated with worse performance on the overall cognition (p < 10-20), working memory (p < 10-10) and processing speed (p < 10-5). Conclusions: Big Data analyses provided stable and reproducible illness deficit patterns that can be used to derive individual indexes of similarity. RVI can be used translate these patterns into sensitive and specific biomarkers that can be used in clinical and epidemiological samples, as well as to evaluate and quantify risk factors for mental illness in ABCD cohort. Disclosure: Nothing to disclose. 32.2 Predicting Cognitive Impairment and Amyloid Positivity Using a Data-Driven Vulnerability Index Lauren Salminen Keck School of Medicine University of Southern California, Marina del Rey, California, United States Background: Composite neuroimaging measures have shown greater utility in identifying cognitively normal individuals at risk for Alzheimer’s disease (AD) when compared to region-of-interest (ROI) methods that focus on a single structure (e.g., hippocampal volume). Different “metaROI’s” have been identified in the literature, but the structures included in these measures vary across studies and are typically based on a priori assumptions of the temporal sequence of AD pathophysiology. Here we evaluated the utility of a data-driven measure of individual vulnerability to differentiate stable cognitively normal controls (N = 184) from unstable cognitively normal controls who later developed MCI or dementia (N = 128) in the ADNI and OASIS-3 datasets. Methods: We created two regional effect size templates (REST) using Cohen’s d effect sizes for group differences in 68 FreeSurfer-derived regional cortical thickness and surface area measures, and 7 subcortical volumes between clinically determined controls (n = 836) and dementia cases (n = 452), and separately between amyloid- controls (n = 168) and amyloid+ dementia cases (n = 403) in ADNI and OASIS-3. Two regional vulnerability index (RVI-Dx, RVI-DxAB) scores were then calculated as the total correlation between the 75 regional Z scores and REST in a test set of 312 stable and unstable controls from both datasets. Results: Random effects linear regression showed that unstable converters had significantly higher RVI-Dx (p = 0.018) and higher RVI-DxAB (p = 1.6e-10) than non-converters, adjusting for age, sex, ICV, and site as a random effect, indicating greater vulnerability to dementia. Both RVI measures predicted clinical conversion with 79% AUC using logistic regression, but RVI-DxAB better predicted amyloid positivity (78.1% AUC) compared to RVI-Dx (65.9% AUC). Conclusions: Collectively these results support the use of data-driven methods for quantifying individual vulnerability to dementia, as well as amyloid positivity. Disclosure: Nothing to disclose. 32.3 Combining Big and Clinical Neuroimaging data: Analytic Strategies for Studying Categorical and Dimensional Neuroimaging Markers of Mental Illness Vince Calhoun Georgia State University, Atlanta, Georgia, United States Background: The neuroimaging community has seen a number of large open data collection efforts which have vastly increased the amount of data available to the community. However, the use of such data for studying mental illness is not trivial and comes with major challenges including 1) balancing computational resources and analytic flexibility, 2) harmonization/standardization across studies, 3) integrating categorical versus dimensional approaches to (multimodal) data. In this session I will present specific examples of strategies to address each of these points, with a focus on leveraging big data without ‘losing the forest for the trees.’ Methods: We apply three computationally efficient data-driven approaches (spatially constrained independent component analysis (ICA), constrained joint ICA, and a pre-trained deep neural network) to capture information about resting fMRI, structural MRI, and polygenic risk score (PRS) data in the large UK Biobank and ABCD datasets. We evaluate results relative to several transdiagnostic multisite clinical datasets including schizophrenia, major depression, autism spectrum, schizoaffective disorder, and bipolar disorder. Results: PRS associated neuroimaging patterns derived from typical data were associated with robustly decreased function and volume in frontotemporal, hippocampus complex, thalamus and insula. These patterns predict schizophrenia from controls with high accuracy, and predict cognition and symptoms for schizophrenia across four independent cohorts. Functional network connectivity stability was highly predictive of the individual and predictive of cognitive and psychiatric scores. The similarity of the functional patterns to clinical/control references showed a strong negative skew towards the clinical reference which was also significantly associated with prodromal scores. Finally, the deep fusion results were highly predictive diagnosis, outperforming unimodal prediction. Conclusions: Strategies for working with large heterogeneous multimodal neuroimaging data are numerous and fraught with trade-offs. Here we show several ways to efficiently work with large typically developing or aging datasets in combination with clinical data. Results highlighted potentially important relationships between neuroimaging genomics data and the mental illness spectrum. Disclosure: Nothing to disclose. 32.4 Does Multivariate Modelling in Pediatric Big Datasets Yield Reproducible and Replicable Relationships Between Cortical Thickness and Psychopathology? Hajer Nakua University of Toronto, Mississauga, Canada Background: Identifying latent variables (LVs; linked dimensions between two data matrices) can delineate brain-psychopathology relationships across the general population. These relationships can help describe potential risk or resilience factors of mental health trajectories across adolescent development. Canonical correlation analysis (CCA) and partial least squares (PLS) are two approaches that identify LVs based on maximizing the correlation or covariance between them, respectively. Here, we compared the identified LVs as detected by CCA and PLS, and the reproducibility of each LV, to assess whether one approach produces more robust relationships compared to the other. Methods: T1-weighted imaging and psychopathology data were accessed from the Adolescent Brain Cognitive Development dataset (n = 8643; ages=9-11). The brain matrix consisted of cortical thickness estimates from the Desikan-Killiany Atlas. The psychopathology matrix consisted of 11 subscale scores from the parent-reported Child Behavioral Checklist. CCA and PLS models were separately applied to both matrices. Statistical significance of each LV was assessed using permutation testing. Reproducibility of each LV was assessed using split-half resampling. Results: The first LV from CCA and PLS models revealed distinct linked dimensions between cortical thickness and psychopathology domains (singular values:CCA = 0.12,PLS = 0.35, p < 0.001) with CCA identifying spatially distributed positive and negative relationships. Split-half resampling did not reveal reproducible relationships. Post-hoc analysis linking cognitive task performance measures to cortical thickness revealed that summary scores from the NIH toolbox had a reproducible first LV for CCA and PLS models (singular values:CCA = 0.2,PLS = 0.47,p < 0.001). Conclusions: CCA and PLS did not yield reproducible relationships between cortical thickness and psychopathology. The results of the post-hoc analysis may suggest that population-level variation of cortical morphology can be better delineated by variation in cognitive performance instead of psychopathology. However, delineating reproducible brain-psychopathology relationships may be optimized by exploring clinically relevant subtypes within large-scale general populations instead of examining relationships across the sample. Disclosure: Nothing to disclose. PANEL 33. Neurocircuitry of Social-Emotional Behavior 33.1 Behavioral and Dopaminergic Signatures of Resilience Annegret Falkner Princeton Neuroscience Institute, Princeton, New Jersey, United States Background: Chronic stress can have lasting adverse consequences in some individuals, yet others are resilient to the same stressor. While previous work has found differences in the intrinsic properties of mesolimbic dopamine (DA) neurons in susceptible and resilient individuals after stress was over, the causal links between DA activity during stress, dynamic stress-evoked behavior, and individual differences in susceptibility and resilience are not known. Methods: We subject mice to a 10-day chronic defeat stress and then perform post-hoc tests of affective state. We record high speed video during stress and perform supervised and unsupervised behavior analysis to detect behavioral signatures of resilience (~14m video frames, n = 32 males, n = 8 females). In addition, using fiber photometry, we record the activity of the VTA-NAc and the SN-TS projecting populations across defeat and during post-hoc testing (n = 19 males, n = 8 females). Lastly, we develop a novel system to perform fast behavior-triggered optogenetic perturbation. Results: We find that resilient (R) and susceptible (S) individuals employ different behavior strategies during defeat. In males, resilience was associated with fight-back. Using unsupervised analysis individuals could be classified into R or S groups. Female behavior during defeat revealed a single cluster that was significantly correlated with R. In addition, we find that NAc-DA activity (but not TS-DA activity) predicts R in proximity to the aggressor during defeat in both sexes. This correlation persists during the post-hoc test of social avoidance. We find that both closed-loop triggered stimulation timed to an R-associated behavior and open-loop stimulation during defeat similarly bias individuals towards resilience. Both of these regimes also similarly reorganized behavior towards R-associated patterns of behavior during defeat. Conclusions: Overall we find that resilient and susceptible individuals are differentiated by their behavior strategies during defeat and by their dopaminergic correlates. Activation of NAc-DA during defeat timed to an R-associated behavior or untimed bias individuals towards resilience and reorganize behaviors towards patterns of resilient behavior. This provides evidence for a causal link between DA during defeat and resilience. Disclosure: Nothing to disclose. 33.2 Optogenetic Stimulation of ESR1-Expressing Neurons in PVT Reveals a Putative Neural Substrate for Outgroup, but Not Ingroup, Aggression Brandy Briones University of Washington, Seattle, Washington, United States Background: The Paraventricular Thalamus (PVT) is a critical node for integrating sensory features and emotional state information within novel and aversive contexts. However, few studies have investigated integration of social information in the PVT in these contexts. Navigating novel social environments is complex, multisensory, and cognitively demanding, where mammals often rely on social heuristics to guide behavior. This can lead to increased perceived threat and aggression towards an unfamiliar outgroup member, a phenomenon known as ingroup bias. While sex hormone receptors have been shown to modulate appetitive aggressive behavior, the neural circuits, and mechanisms critical for ingroup versus outgroup social behavior remain to be determined. Methods: Using a multiplexed hybridization chain reaction assay, we characterized sex hormone receptor-related genes to identify distinct cell clusters in the PVT of male (n = 3) and female (n = 4) mice. We virally targeted sex hormone receptor-expressing neurons in the PVT of Esr1-Cre + /- mice to investigate the role they play in defensive and social behaviors. After testing a variety of behavior assays with optogenetics (n = 8), we uncovered a male-specific aggression phenotype and focused the remainder of our study on males (n = 9-10). Results: We analyzed ~10,000 cells across the anterior-posterior axis of the PVT. Estrogen and androgen receptor-related genes Esr1, Esrra, and Rora, were highly co-expressed (>50%) in central and posterior PVT neurons, corroborated by expression patterns of viral-labeled neurons in Esr1-Cre + /- mice. Optogenetic activation of Esr1+ PVT neurons elicited aggressive attacks time-locked to light stimulation towards outgroup (indicated by differences in coat color), but not ingroup, mice (p < 0.05), providing us with a putative neural circuit to dissect outgroup aggression further. Conclusions: From the data we have collected thus far, our results suggest that activation of central and posterior PVT neurons expressing estrogen and androgen receptor-related genes gate intergroup aggression. Future directions of our study will aim to investigate 1) in vivo Esr1+ PVT neural activity during freely moving social behavior in the presence or absence of circulating hormones and 2) whether estrogen and/or androgen receptors in the PVT are necessary for outgroup aggression. Disclosure: Nothing to disclose. 33.3 Identification of Touch Neurons Underlying Dopaminergic Pleasurable Touch and Sexual Receptivity Ishmail Abdus-Saboor Columbia University, New York, New York, United States Background: The pleasure of a partner’s caress or a child’s embrace begins with mechanical signals transduced by neurons in our skin. Despite the centrality of socially rewarding touch in our daily lives, the neurons in the skin that detect social touch and shape the valence of perception generated in the brain, remain unknown. This gap in knowledge is critical, especially when considering the nature of neurodevelopmental disorders like autism spectrum disorder, where gentle touch and socially rewarding behaviors are aversive. Methods: Transdermal optogenetic stimulation of sensory neurons was performed on 8-16 week-old male or female mice that were habituated to mesh platform under a plastic chamber for 1 hour on each of two days prior to behavioral testing. Experimenter was present with lights, camera, and laser running during habituation to mimic entire sensory experience of test day. Results: A population of sensory neurons labeled by the G-protein coupled receptor Mrgprb4 detect stroking touch in mice, however, these neurons have never been implicated in any natural social behaviors. Here, we study the social relevance of Mrgprb4-lineage neurons by genetically engineering mice to allow activation or ablation of this population and reveal that these neurons are required for sexual receptivity and sufficient to induce dopamine release in the brain. Even in social isolation, optogenetic stimulation of Mrgprb4-lineage neurons through the back skin is sufficient to induce a conditioned place preference and a striking dorsiflexion resembling the lordotic copulatory posture in females. In the absence of Mrgprb4-lineage neurons, female mice no longer find male mounts rewarding sexual receptivity is supplanted by aggression and a coincident decline in dopaminergic release in the mesolimbic reward pathway. Conclusions: In summary, the work described here has revealed the sufficiency of peripheral inputs to regulate social reward independent of context and other sensory cues. We believe this study draws new attention to the importance of elucidating skin-brain circuits, analogous to the importance of gut-brain circuits. Moreover, this work points towards the therapeutic potential of peripheral manipulations for enhancing intact or impaired social reward systems, including sexual receptivity, or simulating social reward during periods of isolation. Disclosure: Nothing to disclose. 33.4 Genetic Variation in Oxytocin Receptor (OXTR) Alters Expression of OXTR and Autism Spectrum Disorder (ASD) Risk Genes in Reward Circuitry in Prairie Voles Larry Young Emory University School of Medicine, Atlanta, Georgia, United States Background: Variation in oxytocin receptor (Oxtr) expression increases diversity in social behavior within and across species. SNPs in human OXTR are associated with autism spectrum disorder (ASD) phenotypes and functional connectivity of reward circuitry in ASD. In monogamous prairie voles, variation in Oxtr expression in nucleus accumbens (NAc) is associated with variation in pair bonding, alloparentingr and resilience to neonatal social neglect. A set of non-coding Oxtr SNPs largely explain individual variation in Oxtr expression (r2 > 0.7) in NAc. Here we explore links between Oxtr SNPs, Oxtr expression, and transcriptional profiles in NAc. Methods: To investigate links between Oxtr SNPs, altered NAc Oxtr expression, and transcriptomic profile, we used ATAC-seq, bulk and single nucleus (sn)RNA-seq (N = 6-8/group, both sexes). The combination of these techniques allowed us to compare differences in chromatin accessibility and transcriptomic profile between genotypes (low- and high-NAc Oxtr expression) and across brain regions that show variable (NAc), stable (insular cortex, INS) or no (superior colliculus, SC) Oxtr expression. Results: ATAC-seq revealed that SNP in the Oxtr intron are more likely to be near areas of open chromatin than 5’ flanking SNPs and are better candidates for directly affecting Oxtr expression. RNA-seq showed differential expression between low- and high-NAc Oxtr expressing genotypes, in both bulk RNA and snRNA-seq data, with Oxtr as a top hit (q < 1x10-7). The differentially expressed gene sets are highly enriched for ASD risk genes (SFARI 2.0 database) in both bulk and snRNA-seq data sets (p < 0.0001). Most genes that are differentially expressed between genotypes localize to glia, which show little if any Oxtr expression. Conclusions: SNP-dependent gene expression may be the consequence of direct effects of the SNPs on trans gene expression, of individual variation in OXTR signaling in the NAc, or a combination of both. We are using CRISPR/Cas9 to differentiate between these possibilities. Our results suggest that variation in OXTR signaling in the NAc alters circuit wide-transcriptional profiles, enriched for ASD risk genes, in diverse cell types. This study will be discussed in the context of our rsfMRI studies in ASD subject showing that intranasal oxytocin normalizes reward circuit connectivity in ASD. Disclosure: Levo Therapeutics, Inc: Consultant (Self) PANEL 34. Substance Use Disorder Treatment With Psychedelic Drugs 34.1 Breakthrough the Barriers: Psychedelics to Control the Cycle of Dysfunction Induced by Opioids and Cocaine Noelle Anastasio University of Texas Medical Branch, Galveston, Texas, United States Background: A record >107,000 fatalities due to drug overdoses was reported in the U.S. for the twelve months preceding April 2022. Opioids account for most deaths, but deaths involving cocaine also steadily increased. Despite availability of medications for opioid use disorder, this polysubstance epidemic has crystalized the need to identify novel therapeutics to improve the odds of successful recovery. Recently, there has been a resurgence of interest in serotonergic psychedelics for the treatment of psychiatric disorders. While a clinical trial of the psychedelic psilocybin for cocaine use disorder is ongoing (NCT02037126), the nature and directionality of serotonergic dynamics and adaptations in the 5-HT2A receptor (5-HT2AR) system engaged by opioids and cocaine are elusive. We tested the hypothesis R-(-)2,5-dimethoxy-4-iodoamphetamine [(-) DOI] will disrupt opioid and cocaine-evoked effects in preclinical rodent models. Methods: Two cohorts of Sprague-Dawley rats (Cohort 1, n = 13; Cohort 2, n = 16) were utilized. Cohort 1 was subjected to the drug discrimination paradigm to model the interoceptive effects of the opioid oxycodone to establish ligand potency and effectiveness in vivo. Cohort 2 was trained to self-administer cocaine (0.25 mg/kg/inf) on an FR1-FR5 schedule of reinforcement to model human cocaine-taking and drug reinforcement. Selectivity was ascertained in the presence of the 5-HT2AR antagonist M100907. Results: Substitution tests indicated that all (-)-DOI doses (0-0.2 mg/kg) resulted in percent oxycodone-lever responding significantly different from the training dose of oxycodone (p < 0.05). (-)-DOI (0.1, 0.2 mg/kg) reduced the percent oxycodone-lever responding vs vehicle (p < 0.05), which was blocked by M100907 (p < 0.05). (-)-DOI reduced cocaine infusions (p < 0.05) at all doses tested (0-0.3 mg/kg) without altering the number of inactive lever presses (n.s.). M100907 prevented the (-)-DOI-induced suppression of intake (p < 0.05). Conclusions: The psychedelic (-)-DOI via actions at the 5-HT2AR does not substitute for oxycodone, suppresses the stimulus effects of oxycodone as well as the reinforcing effects of cocaine. Taken together, these studies provide support for expanded interrogation of the efficacy of 5-HT2AR agonists in counteracting the behavioral effects of opioids and cocaine. Disclosure: Nothing to disclose. 34.2 Psilocybin in the Treatment of Cocaine Use Disorder Peter Hendricks University of Alabama, Birmingham, Alabama, United States Background: Cocaine use disorder (CUD) affects millions of people in North America as is associated with significant medical and psychiatric comorbidity. However, there are no approved pharmacotherapies for CUD. The 5HT2A receptor agonist psilocybin shows evidence of transdiagnostic efficacy for a range of mental health conditions and may represent a promising treatment for CUD. Methods: 40 participants with CUD received 4 sessions of “preparatory” psychotherapy consisting of client-centered counseling and cognitive-behavioral therapy for CUD before being randomized to receive either psilocybin (25 mg/70 kg) or diphenhydramine (100 mg). Following drug administration, participants received 4 sessions of “integration” psychotherapy focused on translating insights from their drug experience to adaptive changes in cognition and behavior. Assessments occurred 90 and 180 days after end-of-treatment. Results: Whereas the two conditions reported no differences in percentage of cocaine use days prior to drug administration, following drug administration those in the psilocybin condition reported a significantly greater percentage of abstinent days than those in the diphenhydramine condition. Conclusions: Psilocybin shows preliminary signals of efficacy in the treatment of CUD. Future study with larger samples is needed to further interrogate psilocybin in the treatment of CUD, with attention to putative mechanisms of action. Disclosure: Bright Minds Biosciences, Eleusis Benefit Corporation, Reset Pharmaceuticals: Advisory Board (Self) 34.3 Psilocybin in the Treatment of Tobacco Use Disorder Matthew Johnson Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Early research from the 1950s to 1970s investigated classic psychedelics in the treatment of substance use disorders. Over the last 20 years research has once again investigated psychedelics as potential medications. Methods: The presenter is conducting randomized, open-label, comparative efficacy study randomizing 100 treatment-seeking cigarette smokers to a single psilocybin session or a course of nicotine patch treatment (n = 50 per group), both combined with cognitive behavior therapy. Both sexes were included. Point-prevalence abstinence including at 6- and 12-month follow-up visits was biologically verified with urinary cotinine and breath carbon monoxide samples. These interim data were analyzed via chi-square (2-tail, alpha = .05). Results: Current results (n = 61) show better results for psilocybin (n = 31), with a 12-month biologically confirmed 7-day point-prevalence abstinence rate of 52%, compared to 27% for nicotine patch. Conclusions: Current results are promising and if confirmed would suggest a potential role for psilocybin treatment in tobacco smoking cessation. Disclosure: AJNA Labs LLC, AWAKN Life Sciences Inc, Beckley Psychedelic Ltd, Entheogen Biomedical Corp, Mind Medicine Inc, Silo Pharma: Advisory Board (Self), Field Trip Psychedelics Inc., Otsuka Pharmaceutical Development and Commercialization Inc: Consultant (Self) PANEL 35. Clinical Research on Novel Pharmacologic and Immunologic Approaches to Treating Cannabis Use Disorder and Opioid Use Disorder 35.1 Effect of AEF0117, a Signaling-Specific Inhibitor of the CB1 Receptor, on the Reinforcing and Subjective Effects of Smoked Cannabis Margaret Haney Columbia University, New York, New York, United States Background: Cannabis use disorder (CUD) is an escalating problem, and there is no FDA-approved medication to facilitate its treatment. AEF0117, a signaling-specific inhibitor of the cannabinoid type 1 (CB1) receptor (Aelis Farma), may show promise as a potential pharmacotherapy. AEF0117 selectively inhibits THC activation of one intracellular signaling pathway (MAPK) without affecting CB1 agonist binding or THC’s other signaling cascades. In healthy volunteers, AEF0117 was safe and well-tolerated. Here we describe a Phase 2a placebo-controlled, human laboratory study testing the effects of AEF0117 on cannabis’s positive subjective effects and its self-administration. Evidence of precipitated withdrawal was also evaluated by measuring sleep, food intake and mood. Methods: Nontreatment-seeking cannabis users completed two 5-day inpatient periods separated by a ≥ 14-day outpatient washout. Two doses of AEF0117 (0.06 and 1 mg/day) were tested in two distinct cohorts (n = 13/cohort). Participants received AEF0117 or placebo once/day in counterbalanced order for 5 days. Capsules were administered at 09h00 and participants smoked a controlled amount of cannabis 3.5 hours later (12h30). Ratings of subjective effects were assessed over 2 hours. Participants had four opportunities (from 14h30 to 20h30) to self-administer cannabis. Results: Daily cannabis smokers (1F,25M), averaging 3.3 + 1.7 grams/day, completed the study. In analysis of the crossover design, AEF0117 (1 mg/day) significantly reduced both the positive subjective effects of cannabis and its self-administration (p < 0.05), while the 0.06 mg dose did not. However, a significant sequence by treatment interaction (p < 0.01) showed that AEF0117 had effects after 14 days of washout. A parallel-group analysis looking at the first treatment period only confirmed a significant (p < .01) reduction in the subjective and reinforcing effects of cannabis by AEF0117. AEF0117 was well tolerated, with no evidence of precipitated withdrawal. Conclusions: AEF0117 safely and robustly attenuated the positive subjective and reinforcing effects of cannabis in participants with CUD. Favorable pharmacokinetic and safety profiles combined with the human laboratory data support an upcoming Phase 2b, placebo-controlled multi-site clinical trial testing AEF0117 in patients seeking treatment for CUD. Disclosure: Pleo Pharma: Advisory Board (Self), Pleo Pharma: Stock / Equity (Self) 35.2 Multisite Randomized, Double-Blind, Placebo-Controlled, Parallel Group Study of the Efficacy, Safety and Tolerability of the Fatty Acid Amide Hydrolase (FAAH) Inhibitor JZP150 (PF-04457845) in Adults With Cannabis Use Disorder (CUD) Deepak D’Souza Yale University School of Medicine, West Haven, Connecticut, United States Background: With the liberalization of cannabis laws, the rates of and demand for the treatment of cannabis use disorder (CUD) are anticipated to increase. However, there are no FDA-approved or clinically accepted treatments for CUD. One approach to treating CUD is by potentiating endocannabinoid (eCB) signaling. Fatty acid amide hydrolase (FAAH) inhibitors which increase levels of anandamide, a principal eCB, reduce cannabis withdrawal in THC-dependent animals. Compared to cannabis and THC, FAAH-inhibitors do not have psychoactive or rewarding effects, are not associated with tolerance and do not increase the abuse liability of other addictive drugs. PF-04457845 aka JZP-150 is an orally active, long-acting, potent and selective FAAH inhibitor. In a completed proof of concept (POC), single-site, double-blind, randomized, placebo-controlled study (n = 60), JZP150 reduced CWS, reduced cannabis consumption, and attenuated stage 3 sleep deficits in CUD participants. Methods: Building on the positive results of the completed POC study, the efficacy, safety and tolerability of JZP150 in reducing cannabis use was evaluated in a 4-site randomized, double-blind, placebo-controlled, parallel-group, outpatient clinical trial comparing JZP150 (4mg) and placebo in individuals with DSM-5 CUD. Participants received motivational interviewing for 2 weeks prior to randomization and had to attempt to quit cannabis within the first week of treatment. Participants received 4 mg of JZP150 or placebo PO QD for 8 weeks during which time they were evaluated weekly for efficacy, safety and tolerability with measures of cannabinoid exposure (self-report and toxicology), problems related to cannabis use, sleep, mood were assessed. In addition, daily video assessments of adherence to study medication, cannabis use and safety were conducted by cellphone. Results: The last subject was randomized on 5/5/2022 meeting the target of 234 subjects (85 female) with 171 completers thus far. The study has been safe and well-tolerated with a low dropout rate (10%). All data collection will be completed by July 2022 with data analysis planned for August 2022. This is the largest clinical trial to date testing the efficacy, safety and tolerability of a FAAH Inhibitor for CUD. Conclusions: JZP150 is safe and well-tolerated. The efficacy results of the study will be discussed. Disclosure: Biohaven: Consultant (Self), Jazz Pharmaceuticals: Advisory Board (Self), Psilocybin Migraine: Patent (Self) 35.3 Safety, Immunogenicity, and Preliminary Efficacy of OXY-sKLH, a Vaccine That Targets Oxycodone, and Exploratory Biomarkers of Opioid Use Disorder and Vaccine Efficacy in Humans Rachel Luba Columbia University and NYSPI, New York, New York, United States Background: Illicit opioid use contributed to the majority of drug-related overdose deaths in 2021. This presentation will highlight new data from a Phase 1a/1b study of OXY-sKLH, an opioid vaccine that targets oxycodone (OXY). We also will describe an independent clinical study to identify biomarkers that can be used to select participants who may be “good responders” to the vaccine. Methods: Unvaccinated individuals with opioid use disorder (OUD; n = 23; 21M, 2F) and non-drug-using volunteers (n = 22; 19M, 3F) were enrolled in our biomarker study. Blood samples were collected to analyze opioid-specific B cell lymphocyte population subsets by antigen-based magnetic enrichment paired with multiparameter flow cytometry, cell sorting paired with sequencing of the B cell receptor, and evaluation of cytokine panels and opioid-specific serum IgG antibodies. The Phase 1a/1b study of OXY-sKLH has enrolled 11 individuals with OUD (10M, 1F). OXY was administered pre- and post-vaccination to examine the efficacy of OXY-sKLH to reduce subjective ratings and physiological responses. The pharmacokinetic (PK) profile of oxycodone also was assessed. Results: The frequency of opioid-specific B cell population subsets was higher in subjects with OUD compared to controls (p < 0.01). The frequency of B cells specific for heroin/morphine (M) were more prevalent than the OXY-specific B cell population. No difference in OXY- and M-specific serum IgG antibodies have been found between groups. Decreases in selected cytokines were observed in those with OUD compared to controls (p < 0.05) and significant correlations were observed between several cytokines and heroin use in those with OUD (p < 0.05). Similar immunoprofiling will be performed in participants in the Phase 1a/1b study. The OXY-sKLH vaccine appears to be safe with only minimal adverse effects. Oxy-specific IgG titers and concentrations are elevated in a subset of participants for > 40 weeks; the PK and preliminary efficacy data will be presented at the conference. Conclusions: Selected immune-related biomarkers, such as opioid-specific B cells and cytokines, may be predictive of OUD. These biomarkers also may be predictive of vaccine efficacy, which would be consistent with our data collected in rodents. Thus far, the vaccine has proven to be safe, and some participants are exhibiting long-lasting antibody levels. Disclosure: BioXcel Therapeutics, Janssen: Contracted Research (Self), Go Medical, Intra-cellular Therapies, Lyndra: Grant (Self), Alkermes: Other Financial or Material Support (Self), Clinilabs, Mallinckrodt, Nektar, Otsuka: Consultant (Self), Opiant: Advisory Board (Self) 35.4 Mechanism of Action of ITI-333, a Novel Modulator of Mu Opioid, Serotonin, and Dopamine and for the Treatment of Opioid Use Disorder Sandra Comer Columbia University and NYSPI, New York, New York, United States Background: New medications are needed to treat opioid use disorder (OUD) by easing somatic symptoms of drug withdrawal and mitigating dysphoria and psychiatric comorbidities that drive opioid use/abuse. We describe properties of ITI-333, a novel molecule combining high affinity binding (Ki < 50 nM) to receptors that individually participate in substance use disorders and psychiatric comorbidities: mu opioid (MOP), serotonin 5-HT2A, and dopamine D1 receptors. Methods: ITI-333 was tested in cell-based assays on recombinant receptors and in male mice and rats for analgesia, and somatic and behavioral signs of opioid withdrawal, heroin reinstatement, tolerance/physical dependence/self-administration, and safety. Data were analyzed by ANOVA. Results: Cell-based assays demonstrate ITI-333 is a biased MOP receptor partial agonist, acting as an antagonist to block effects of high doses of morphine in pain and motor activity models; acting alone as an analgesic in acute and inflammatory pain models. ITI-333 induces cAMP accumulation (agonism) at human recombinant MOP receptors while blocking effects of a full MOP agonist, DAMGO (antagonism). Further, ITI-333 is a biased agonist at MOP receptors, acting as an antagonist on beta-arrestin pathways mediating opioid side effects. ITI-333, (0.01-1mg/kg, SC, n = 10/group), produces naloxone-sensitive analgesia in tail flick (TF) assay, while attenuating morphine analgesia in TF (0.1-1mg/kg, SC, n = 10/group). ITI-333 blocks morphine hyperactivity (32mg/kg, SC, n = 6/group) without affecting spontaneous activity. ITI-333 (0.3, 1, and 3mg/kg, SC) dose-dependently suppresses signs of opioid withdrawal precipitated by naloxone injection in oxycodone-dependent mice (i.e., oxycodone dosed for 8 days; increasing daily doses of 9-33 mg/kg, n = 10/group). Chronic ITI-333 (0.3 or 3mg/kg, SC) does not induce tolerance or physical dependence in rats; acute doses of ITI-333 (0.3 or 3mg/kg, SC, n = 8/group) do not induce GI or pulmonary side effects. ITI-333 (0.003-0.01mg/kg, IV, 8/group) is not self-administered by heroin-maintained rats. ITI-333 was safe and well-tolerated in a first-in-human safety study and continues in clinical development as a treatment for OUD. Conclusions: Data support a role for ITI-333 in mitigating opioid withdrawal symptoms with no significant indications of abuse liability. Disclosure: Intra-Cellular Therapies Inc: Employee (Self), Intra-Cellular Therapies Inc: Stock / Equity (Self) PANEL 36. Memory Mechanisms Relevant to the Development and Maintenance of Posttraumatic Stress Disorder 36.1 Sex-Dependent Role for Prefrontal Cortical Inhibitory Interneurons in the Regulation of Stress-Induced Impairments in Fear Extinction in Rats Stephen Maren Texas A and M University, College Station, Texas, United States Background: Hyperarousal is a core symptom in patients with post-traumatic stress disorder (PTSD). Elevated stress underlies deficits in extinction-based behavioral therapies in PTSD patients. Considerable work indicates that the infralimbic (IL) division of the medial prefrontal cortex (mPFC) is a neural substrate for stress-induced extinction impairments. Here we examine the possibility that stress-induced activation of IL inhibitory interneurons contributes to stress-induced extinction impairments in male and female rats. Methods: Stress-induced activation of mPFC interneurons was assessed by exposing male (n = 12) and female (n = 12) Long-Evans rats to footshock and quantifying parvalbumin (PV) and Fos expression in the IL and prelimbic (PL) cortex. The contribution of PV interneurons to the stress-induced extinction impairment was assessed by chemogenetically inhibiting these cells in male (n = 21) and female (n = 20) rats. We expressed an inhibitory DREADD in PV-Cre transgenic rats prior to fear conditioning and immediate extinction; extinction retrieval was tested 48 hr later. To validate this strategy, a subset of male (n = 14) and female (n = 13) rats received the DREADD ligand CNO or VEH prior to footshock and Fos expression was quantified in DREADD-expressing mPFC cells. The percentage of freezing was used as the behavioral measure of fear. Results: Consistent with our hypothesis, footshock significantly increased Fos expression in IL PV + cells in both sexes. Chemogenetic inhibition of mPFC PV + interneurons had no effect on conditioned freezing during either fear conditioning or immediate extinction. Unexpectedly, this treatment led to a potentiation of freezing during the drug-free extinction retrieval test in male, but not female, rats. Chemogenetic inhibition of mPFC PV + interneurons significantly reduced footshock-induced Fos expression in those neurons, confirming the functional efficacy of the manipulation. Conclusions: These results reveal that mPFC inhibitory interneurons play a sex-specific role in the regulation of extinction under stress. Tonic increases in IL excitability resulting from PV inhibition appear to compromise IL function and impair extinction learning. Disclosure: Nothing to disclose. 36.2 Latent Associative Structures Facilitates Transfer of Learned Fear in Humans Joseph Dunsmoor University of Texas at Austin, Austin, Texas, United States Background: Animals often rely on pre-existing associations to facilitate transfer of new learning. For instance, once a stimulus becomes predictive of an aversive outcome, pre-associated stimuli acquire the capacity to elicit a defensive response, known as sensory preconditioning. In rodents, this form of fear generalization requires the hippocampus and orbitofrontal cortex. Mechanisms of sensory preconditioning in humans are unclear. Insights could offer clues on how fear broadly generalizes to a host of stimuli following aversive events (e.g., trauma). Here, we used a novel sensory preconditioning task during fMRI to investigate latent associative networks involved in fear generalization. Methods: Participants (N = 27) learned that a category (preconditioned stimulus, PS; animals or tool, counterbalanced) predicted a picture of a square, while another PS category (tools or animals, respectively) predicted a circle. Next, one shape (conditioned stimulus, CS + ) was paired with an aversive electrical shock while the other shape (CS-) was unpaired. Subjects then viewed novel exemplars (PS + ) from the category originally paired with the CS + and exemplars (PS-) from the category originally paired with the CS-. Subjects returned 24 hours later for a surprise memory test of PS + /PS- exemplars encoded before and after threat conditioning the previous day. Results: Participants acquired conditioned learning to the CS + , expressed through skin conductance responses and differential (CS + > CS-) activity in the thalamus, insula, and dorsal anterior cingulate cortex (all fMRI activity p < .001 corrected for multiple comparisons at p < .05). During the fear generalization test, there was differential hippocampal and orbitofrontal activity to the PS + > PS- (p < .001, two-tailed t-test). Finally, participants had a 24-hour memory bias for PS + > PS- items encoded prior to fear conditioning (p = .02, two-tailed t-test). Conclusions: Results show how latent associative structures support transfer of learned fear. This extends evidence from rodents on the role of the hippocampus and OFC, and offers insights into key neurocircuity implicated in the higher order transfer of emotional learning. Sensory preconditioning may be a valid but underappreciated model for the widespread and idiosyncratic nature of fear generalization following aversive events, such as trauma. Disclosure: Nothing to disclose. 36.3 Threat-Related Arousal Impacts the Hippocampal Reactivity in PTSD Risk and Related Memory Phenomenology Vishnu Murty Temple University, Philadelphia, Pennsylvania, United States Background: A growing body of research implicates threat-related arousal and hippocampus (HPC) deficits as major determinants of developing post-traumatic stress disorder (PTSD). Research in this domain has often been siloed, treating hyper-arousal and HPC dysfunction independently. Recent work supports a model by which threat-related arousal impairs HPC function, biasing memory away from relational processing. I will present two studies characterizing how threat-related arousal interacts with the HPC to predict PTSD symptoms, and a follow-up study in a normative cohort to unpack how threat-related arousal influences the phenomenology of episodic memory for threatening events. Methods: In Study 1, fMRI markers of HPC threat reactivity, fear-potentiated startle (FPS), and PTSD symptoms 2 weeks following a traumatic event in an emergency room cohort of 117 individuals as part of the AURORA study. Interactions between FPS and HPC threat reactivity were assessed to see how they predict PTSD symptoms. In study 2, threat-related arousal during an in-person haunted house visit and 1-week free recall were collected in a cohort of 42 normative individuals. Interactions between threat-related arousal and free recall content and comprehensibility were characterized. Results: Study 1 showed that decreased HPC threat reactivity was associated with PTSD symptoms two weeks following trauma exposure (n = 117; p < 0.05, Bonferroni-corrected). Further, the relationships between decreased threat reactivity in the HPC and PTSD symptoms was limited to individuals showing high FPS, a marker of hyper-arousal (p < 0.05, Bonferroni-corrected). This study did not evaluate threat’s influence on memory phenomenology. In study 2, we demonstrated that threat-related arousal during an in-person haunted house biased memory recall towards sensory features of the environment rather than action or contextual features (n = 47; p < 0.01). Further, we found that the memories of individuals that showed greater threat-related arousal were rated as less comprehensible and more confusing by a naive set of listeners (n = 200; p < 0.01). Conclusions: Together, our findings characterize how threat influences HPC function, such that arousal biases information processing away from the HPC resulting in greater PTSD symptomology and the construction of more fragmented narratives. Disclosure: Nothing to disclose. 36.4 Successful Memory Encoding in Trauma-Exposed Children Depends on Cortical Sensory Regions, Rather Than the Medial Temporal Lobe Jennifer Stevens Emory University School of Medicine, Atlanta, Georgia, United States Background: Childhood trauma is a primary risk factor for intrusive memories associated with PTSD. The medial temporal lobe (MTL) subserves memory formation and is known to be highly sensitive to damaging effects of stress, but little previous research has addressed MTL function in trauma or stress-exposed children. Methods: Mother-child pairs were recruited through the primary care waiting rooms of a large publicly funded urban hospital. Children ages 8-14 participated in an fMRI task probing MTL-dependent memory encoding, viewing scene stimuli with negative, positive, or neutral affective valence. After a 30-minute delay, children indicated their memory for those scenes in a cued recall task. MTL regions of interest included the amygdala (AMY) and hippocampus (HC). Results: Children showed greater recall for negative and positive scenes, relative to neutral (ps < .001), irrespective of trauma exposure (p > .05). MTL engagement during scene encoding was associated with the enhancing effect of emotion on memory, but the association depended on trauma load (trauma recall enhancement for negative scenes, AMY: F = 6.96, p = 0.02, HC: p = .001; positive scenes, AMY: p = 0.05, HC: p = 0.005). In children reporting fewer traumas, recall was positively associated with MTL engagement, similar to healthy adults in prior work. In children reporting more traumas, recall was negatively associated with MTL engagement. Exploratory analyses showed that higher trauma instead predicted engagement of the left middle temporal sulcus for negative>neutral scenes, and primary visual cortex for positive>neutral scenes, pFWE < .05. Conclusions: MTL engagement in encoding predicted emotion-related memory enhancement only among children who experienced fewer traumas. Those with higher trauma appeared to have preserved recall, potentially explained by greater engagement of alternative routes to memory encoding in visual and multimodal cortex. We posit that this may represent a neurodevelopmental adaptation to a high-trauma environment, such that emotional information is routed toward more stable cortical storage that may influence the earliest phases of sensory processing to help children quickly respond to salient environmental cues. Such adaptations may also decrease memory flexibility and context sensitivity, increasing later risk for intrusive symptoms. Disclosure: Nothing to disclose. MINI PANEL 37. Emerging Intersectional Technologies for Probing Molecular Mechanisms of Neurotransmitter Co-Transmission in Brain and Behavior 37.1 Dissecting the Roles of Dopamine/Glutamate Co-Transmission in Sex Differences Underlying Selective Dopamine Neuron Resilience Zachary Freyberg University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Parkinson’s disease (PD) is characterized by dopamine (DA) neuron loss but some DA neurons are more protected than others. Clues for DA neuron resilience in PD come from a subpopulation of medial VTA DA neurons that co-transmit glutamate. These cells are spared in PD and express the vesicular glutamate transporter 2 (VGLUT2). Additional insights come from sex differences in DA neuron loss as men are more affected, and at earlier ages of onset vs women. Yet, mechanisms for these sex differences and implications for resilience remain unknown. We discovered resilient female DA neurons express more DA neuron VGLUT2 vs males and these sex differences are conserved from flies to rodents to humans (Buck et al 2021). We found that DA neuron VGLUT2 enables DAergic vesicles to tune content during firing (Aguilar et al. 2017). Thus, VGLUT2 permits adequate DA neurotransmission to be maintained in spite of DA neuron loss. Considering the key role of mitochondrial dysfunction in PD, we posit VGLUT2 improves mitochondrial resilience to oxidative stress in DA neurons. Methods: Intersectional imaging/INTRSECT2.0: VGLUT2-cre;TH-flpo mice were injected with Boolean-gated intersectional viruses to selectively label TH + /VGLUT2 + vs TH + only neurons. Cleared brains were imaged via ribbon scanning confocal microscopy (RSCM). Intersectional DA Neuron VGLUT Assay: In flies, we used an intersectional DA neuron VGLUT luciferase reporter to measure sex-specific changes in DA neuron VGLUT expression. Reactive Oxygen Species (ROS) Imaging: We tested DA neuron mitochondrial ROS via the MitoTimer biosensor in both sexes, including after DA neuron VGLUT knockdown with RNAi. All studies were IACUC-approved and included 6+ males and females/group. Results: We combined high-resolution RSCM imaging of INTRSECT-labeled mouse brains with neural network analysis of projections. This identified a pattern of TH + /VGLUT2 + synapses distinct from TH-only neurons. To dissect sex-specific mechanisms for resilience of TH + /VGLUT2 + neurons, a genetic screen in flies showed that mitochondrial metabolism genes are the primary hits for VGLUT2-mediated DA neuron survival in response to insults by DA neurotoxin paraquat (PQ). In line with VGLUT-mediated sex differences, males show 40% more DA neuron mitochondrial ROS and lower VGLUT expression vs females (p = .005). In response to PQ, dVGLUT KD exacerbates mitochondrial ROS and DA neuron loss with males more than females (p < .0001). These data suggest sex differences in VGLUT2 expression underlie DA neuron resilience to mitochondrial stress. Conclusions: DA/glutamate neurons constitute a subpopulation of DA neurons unique from purely DAergic cells. Among these DA/glutamate co-transmitting neurons, levels of VGLUT2 expression mediate sex differences in DA neuron survival by regulating mitochondrial oxidative stress in response to insults. Thus, higher levels of DA neuron VGLUT2 in females diminish overall ROS to boost resilience compared to males. Collectively, our data suggest a key mechanism for VGLUT-mediated resilience is its ability to reduce mitochondrial ROS in DA neurons. Disclosure: Nothing to disclose. 37.2 Intersectional Genetics Tools Reveal Cell-Type Specific Transcriptional Networks of Co-Transmitting Dopaminergic and Glutamatergic Neurons Ryan Logan Boston University School of Medicine, Boston, Massachusetts, United States Background: Accumulating evidence suggests that distinct subpopulations of neurons are capable of co-releasing multiple neurotransmitters to regulate an array of fundamental physiological processes and behaviors. Co-transmitting neuronal subpopulations may be involved in disease-related processes including substance use disorders and neurodegeneration. The advent of new genetic tools enables the investigation of co-transmitting neurons in health and disease with unprecedented resolution. Through collaborative efforts, we have aided in the development and application of novel intersectional genetics approaches for studying multiple subpopulations of neurons in the same brain of the mouse, called intronic recombinase sites enabling combinatorial targeting (INTRSECT 2.0). Our proposal will present new findings on the transcriptional networks in specific subpopulations of cells in the mouse brain that co-transmit dopamine and glutamate and their relevance for drug reward and neuronal resilience. Methods: Male and female mice expressing both Th-Flp (Tyrosine Hydroxylase) and Vglut2-Cre (Vesicular Glutamate Transporter 2) were injected (n = 8 per genotype per sex) with virus cocktail that included AAV8-nEF-CreON-FlpOFF-EYFP (Th + /Vglut2+ cells) and AAV-nEF-CreOFF-FlpON-mCherry (Th+ cells). Following 5-6 weeks of viral expression, mice were sacrificed, and reporter labeled cells were sorted for EYFP, mCherry, and cell-type specific surface markers to parse neurons from microglia (i.e., Cd45/Cd11b). RNA was extracted from sorted cells, then processed for RNA-sequencing, followed by a computational pipeline based on differential expression, pathway enrichment, and gene network module building. Results: Transcriptomics analysis identified ~600 transcripts enriched in Th + /Vglut2+ cells in mouse midbrain (adjusted p < 0.01), with significant associations to presynaptic release and vesicle fusion processing includes SNARE-dependent vesicle fusion (p < 0.05). Many of the sex-specific transcriptional networks were related to metabolism and mitochondrial activity. Gene targets specific to Th + /Vglut2+ cells were prioritized based on cross-species data integration of similar cell types in flies and humans, identifying 200 candidates (AW Fisher meta-analysis of overlap, p < 0.05). These candidates were then screened using flies by the TH-GAL4 system knockouts for locomotor phenotypes related to psychostimulants, opioids, and toxin-induced neurodegeneration. Highly ranked gene candidates from cross-species data integration revealed potential mechanisms in Th + /Vglut2+ neural cells related to drug reward and neurodegeneration, particularly opioid use disorder and Parkinson’s Disease. Conclusions: Our findings use innovative new transcriptomic pipelines developed to employ in INTRSECT-labeled cell populations to specifically define dopamine/glutamate co-transmitting neurons at the level of transcriptional networks. We identified major pathways related to mitochondrial function, reactive oxygen species, bioenergetics, and presynaptic vesicle trafficking and release in TH + /VGLUT2 + neurons of the midbrain, along with the identification of potential upstream regulators and modulators of Vglut2 expression. These candidates may be involved in various disease-related processes in the brain, including substance use disorders and neurodegeneration. Disclosure: Nothing to disclose. 37.3 Effects of Aging on Dopamine-Glutamate Neurons Projecting to the Lateral Entorhinal Cortex Susana Mingote Advanced Science Research Center, New York, New York, United States Background: Dopamine (DA) neurons in the ventral tegmental area (VTA) play a critical role in motivation, learning and memory. Is it well established that DA neurons in the VTA co-express the vesicular glutamate transporter 2 (VGLUT2) and use glutamate (GLU) as a co-neurotransmitter. The GLU co-transmission adds greater dynamic signaling range and heterogeneity to the actions of DA neurons, although its function is still not well defined. Fate-mapping experiments in mice have shown that almost all of DA neurons expressed VGLUT2 during early development. Using intersectional strategies to selective label DA-GLU neurons, we found that the density of DA-GLU neurons is reduced to 30% in adulthood (Mingote et al., 2019). It is not known if the density of VTA DA-GLU neurons continues to decrease with aging and its effects, mental function. Considering that DA-GLU neurons send dense projections to the lateral entorhinal cortex (LEC; Mingote et al., 2015), a key region for spatial memory and particularly sensitive to age-related neurodegeneration, we investigate if the density of DA-GLU terminals in this region was altered in old-aged mice. Methods: We used INTRSECT 2.0 viruses (Fenno et al, 2021) to selectively labeled DA-GLU and DA-only neurons with different fluorescent proteins in 3-, 12- and 24-months old mice. The viruses were injected into the VTA of TH-Flp/+::VGLUT2 cre/+ neurons, and animals were perfused one month after the stereotaxic surgeries for neuronal quantification. The brains were processed for either immunohistochemistry or multiplex in situ hybridization (RNAscope). All studies were IACUC-approved. Results: We found that the number of the DA-GLU neurons in the VTA decreased between 3- and 24-months old mice, while the density of DA-only neurons significantly increased (p < 0.05). In addition, the number of VGLUT2 transcripts in the remaining DA-GLU neurons also significantly decreased in 2-year-old mice (p < 0.05). We then focused of the effects of aging on the projections to the LEC. We found that the density of DA-GLU axons in the LEC significantly decrease by over 60% in 24-months old mice. Conclusions: Our findings suggest that the DA neurons go through a process of neurotransmitter switching during aging, in which DA-GLU neurons become DA-only neurons. In addition, the extensive decrease in the density of DA-GLU axons in the LEC, revealed regional-specific alterations that may contribute to changes in cognitive skills in aging. Disclosure: Nothing to disclose. STUDY GROUP 38. Early Career Scientist Workshop: Tools for Building Your Successful Career Angela Ozburn, Sade Spencer, Nii Addy, Maria Oquendo, Damien Fair, M. Mercedes Perez-Rodriguez Oregon Health & Science University, VA Portland Health Care System, Portland, Oregon, United States Study Group Summary: Few of the research skills learned in our training translate to an independent academic or industry career, where under-represented minority (URM) scientists often face discrimination and additional systemic barriers to success. Despite a 9-fold increase in the number of URMs earning PhDs in the past 2 decades, there has been little progress in recruitment and retention of URM faculty. Moreover, URMs generally receive their first R01 at a later age than do majority awardees, where applications with white PIs have higher probability of being discussed in study section and a 2-fold advantage in funding success (compared with URM PIs). Further challenges exist due to a limited focus on cultivating critical thinking abilities (and other desirable skills e.g., problem solving, project management, persuasive communication, etc.), however it is difficult to self-identify and articulate these proficiencies beyond the training environment. This URM led study group will help fill gaps in the transfer of some of the skills most relevant to embarking upon and maintaining a successful career in science. A labyrinth metaphor can be used to describe the journey of an early career researcher. On the one hand, this is a misleading metaphor because there is no single path to a successful scientific career. On the other hand, like navigating a labyrinth, a successful career requires strategic planning, intuition self-assessment, leadership skills, and time. This study group brings together an esteemed and diverse panel to share strategies they have developed during their journeys. Each panelist is an accomplished researcher in addition to being an effective mentor. Dr. M. Mercedes Perez-Rodriquez, Associate Professor of Psychiatry (Icahn School of Medicine at Mt. Sinai), will introduce the concept of building a business plan to provide a road map for your own scientific career. Dr. Nii Addy, Associate Professor of Psychiatry (Yale School of Medicine) and host of the engaging Addy Hour podcast, will discuss what it means and how to chart a path towards succeeding as your authentic self. Dr. Maria Oquendo, Professor and Chair of Psychiatry (University of Pennsylvania Perelman School of Medicine), will share her journey and provide insight on developing leadership skills for managing teams and projects at all professional levels. Dr. Damien Fair, Professor of Pediatrics and Co-Director of the Masonic Institute for the Developing Brain (University of Minnesota), will introduce self-assessment tools and metrics that can be used to identify areas for further development. Upon the introduction of these tools, Drs. Angela Ozburn (Associate Professor, Oregon Health, and Science University) and Sade Spencer (Assistant Professor, University of Minnesota) will facilitate an interactive discussion between the panelists and the audience members to expand upon these concepts. In alignment with the commitment of ACNP to education and training, this study group provides a unique career development opportunity to focus on skills commonly overlooked in traditional sessions. Deliverables from this study group will include tangible new tools, techniques, and strategies as well as advice for cultivating existing skill sets that can be implemented by a diverse group of early career researchers. Disclosure: Nothing to disclose. PANEL 39. Computational Psychiatry Insights into Cognition and Motivation in Schizophrenia, Bipolar Disorder and Major Depression: Progress from the New CNTRACS Consortium 39.1 Behavioral Markers of Cognitive Impairment across Paradigms in Psychopathology Molly Erickson The University of Chicago, Chicago, Illinois, United States Background: People with serious forms of psychopathology such as schizophrenia and bipolar disorder consistently exhibit impaired performance on a range of cognitive tasks. Using computational modeling, it is possible to estimate functioning in specific aspects of cognition that are shared across these tasks, thus permitting a more nuanced conceptualization of cognitive impairment in these disorders. We predicted that people with mood and psychotic disorders would exhibit core impairments in precision, sustained attention, and memory capacity that are shared across tasks and account for variance in IQ and functional outcome. Methods: People with schizophrenia or schizoaffective disorder (SZ; N = 64), bipolar disorder with psychotic features (BP; N = 42), major depressive disorder (MDD; N = 40), and a group of healthy control participants (HC; N = 76) completed three tasks assessing episodic memory, working memory, and visual perceptual precision. Using computational modeling, precision and attention lapse rate was estimated separately for each task. Memory capacity was also estimated from the working and episodic memory tasks. Principal components analysis was then used to calculate three factor scores (precision, attention lapsing, and memory capacity). Results: SZ participants exhibited trend-level reductions in precision, as well as significantly higher rates of attention lapsing and reduced memory capacity. By contrast, MDD participants exhibited normal precision, sustained attention, and memory capacity, with BP participants showing intermediate impairment. IQ was significantly correlated with the precision factor (r’s = 0.42 to 0.59; p’s < 0.001) and the memory capacity factor (r’s = 0.56 to 0.67; p’s < 0.001) for HC, SZ, and BP. The attention lapsing factor was only correlated with IQ for HC and SZ (r’s = -0.32 to -0.43; p’s < 0.01). None of the factors were significantly correlated with functional outcome. Conclusions: SZ exhibited reduced memory capacity, higher attention lapse rates, and reduced precision across all three tasks, with BP showing intermediate impairment and MDD performing comparably to HC participants. Computational modeling revealed shared cognitive processes across tasks that exhibited significant correlations with IQ, but not functional outcome in the patient groups. Disclosure: Nothing to disclose. 39.2 Can Applying Computational Models to Behavioral Performance Provide Reliable and Valid Information About Specific Dimensions of Cognitive Function? Steven Luck University of California, Davis, California, United States Background: Fitting computational models to behavioral data from cognitive paradigms can allow us to quantify specific aspects of cognitive function, but are the resulting variables sufficiently reliable for use in large-scale studies of psychiatric populations? To address this question, we fit a computational model to data from a working memory task and an episodic memory task, allowing us to separately estimate different factors that contribute to task performance (attention, storage capacity, and precision). Methods: Data from the CNTRaCS project were available for 149-239 men and women (both neurotypical individuals and people with mental illnesses). Participants completed a working memory task (memory loads 1 and 5) and an episodic memory task, both of which required memory for locations in a circular space. A computational model was used to estimate lapses of attention, memory storage capacity, and memory precision. Noncomputational aggregate performance measures were also derived for each task. Results: The split-half reliability was found to be very good (r = .87–.94, p < .0001) for all computational parameters except the estimate of memory precision in the episodic memory task (r = .69, p < .0001). Reliability for most of the computational parameters was nearly as high as reliability for the noncomputational aggregate measures (r = .92–.95, p < .0001). In addition, the pattern of correlations across computational parameters indicated that they measured different aspects of task performance. For example, working memory precision was highly correlated for memory loads of 1 and 5 items (r = .73, p < .0001), but the correlation was much lower between lapses of attention and precision (r = .30–.35, p < .0001). External validity was also good: storage capacity was strongly correlated with the Wechsler Test of Adult Reading for both the episodic memory task (r = .49, p < .0001) and the working memory task (r = .53, p < .0001). Conclusions: These findings show that using computational models to isolate specific aspects of psychological function from behavioral data can yield reliable and valid measures of psychological function. The present models make it possible to distinguish between lapses of attention, insufficient memory storage capacity, and imprecise memory contents, factors that may vary across diagnoses and respond to different treatments. Disclosure: Nothing to disclose. 39.3 Predicting Attention Lapses Across Psychopathology Using EEG Megan Boudewyn University of California, Santa Cruz, California, United States Background: Lapses of attention are thought to contribute to the impairments in performance on a variety of cognitive tasks that have been observed in psychiatric populations. Our goal was to extract and examine neural markers of the attention lapse construct in people with schizophrenia, bipolar disorder and major depression, as well as healthy controls. Following from previous work from our group and others that has connected periods of inattention during cognitive tasks to changes in alpha-band activity (8-13 Hz), a measure derived from EEG recordings, we used pre-stimulus alpha activity as a measure of attention lapses. Our hypothesis is that pre-stimulus alpha power predicts behavioral markers of attention lapsing, thereby accounting for group differences in performance on multiple cognitive tasks. Methods: EEG was recorded while participants with schizophrenia (N = 35), bipolar disorder (N = 25), major depression (N = 35) and demographically matched healthy control participants (N = 35) completed a set of cognitive tasks, including visual perception, episodic and working memory tasks. Logistic linear mixed effects regression models were used to predict response accuracy as a function of diagnostic group and pre-stimulus alpha power. Results: Across tasks, all patient groups showed higher error rates compared to healthy controls (ps<0.05). In addition, increased pre-stimulus alpha activity significantly increased the likelihood of making an error in all groups (ps<0.05). Conclusions: The pattern of results supported our hypothesis, demonstrating that a biological measure of attention lapsing significantly predicts response accuracy across cognitive tasks in all groups. Participants with a psychiatric disorder also showed higher rates of attention lapsing as compared to healthy controls. Overall, the results indicate that attention lapsing contributes to differences in performance across cognitive tasks in a range of major mental health disorders. Disclosure: Nothing to disclose. 39.4 Computational Models Go Online: Opportunities and Pitfalls of Task-Based Epidemiology Angus MacDonald University of Minnesota, Minneapolis, Minnesota, United States Background: Our focus on understanding computational models of performance in serious mental illnesses is complemented by an exploration of performance profiles across the general population. Community samples such as HCP, UK Biobank, ABCD and All of Us will play a central role in understanding psychiatric risk factors by linking adaptive and maladaptive biological mechanisms. To date, such samples show a heavy reliance on self-report questionnaires and computational models of behavioral performance remained under-represented in this revolution. It remains largely unknown how to validly extend online data collection to uncontrolled settings. Methods: Parallel to in-lab task development, we sampled n = 834 participants with valid responses to obtain preliminary data on measures of working memory, episodic memory, and sensory precision in a manner that allowed us to measure the precision of the underlying representation (in all cases as locations along a circle). Participants completed two one-hour sessions, including self-report and validity measures and a subset of behavioral tasks counter-balanced across subjects. In addition to self-reported validity items, tasks included trials to test data quality and attention lapses. Results: Online tasks replicated key in person patterns of performance, in particular neural precision and attentional engagement. Precision measures across tasks were highly correlated as were measures of attentional lapsing, suggesting common underlying behavioral mechanisms. We used conservative split-half cross-validation to establishing relationships with individual differences. Working memory capacity and precision related to the cognitive-perceptual factor of the Schizotypal Personality Questionnaire in both exploratory and validation samples. Attentional lapsing also predicted this factor. Conclusions: While neural precision was drawn upon in diverse functions, differentiating this from attentional impairment remains a crucial goal. Schizotypal traits related to these impairments, and served to validate precautions used in collecting these samples. While the utility of questionnaire-based online studies is increasingly common, these findings show a path for large-scale, task-based epidemiology to link computational parameters to personality traits and psychopathology risk factors. Disclosure: Nothing to disclose. PANEL 40. Disentangling the Neural Mechanisms Underlying Cognitive Control – Relevance to, and Biomarkers of, Dysfunction 40.1 A Role for the Claustrum in Cognitive Control Brian Mathur University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Cognitive dysfunction is a feature of several neuropsychiatric disorders that is characterized by disruptions in cortical networks. Our inability to treat cognitive dysfunction reflects our poor understanding of cortical network biology. A leading model for how widespread cortical network nodes co-activate, into networks is recruitment of a subcortical nucleus that synchronizes cortical network nodes. The subcortical nucleus the claustrum is herein hypothesized to serve this role. Methods: We test this hypothesis using resting-state functional magnetic resonance imaging (n = 51; voxel-wise correction for multiple comparisons FWE p < 0.05) and functional circuit mapping (n = 1,050 neurons; subgraph extraction using permutation test) in mouse of both sexes. In humans we implemented the multi-source interference attention task (MSIT) and experimental pain in (n = 34 healthy subjects of both sexes) and applied linear mixed effect model analyses. Results: At rest, multiple frontal cortical areas are significantly (voxel-wise correction for multiple comparisons FWE p < 0.05) functionally connected with both the claustrum and posterior cortical regions. Using these cortical regions as guides, we find using channelrhodopsin-assisted circuit mapping that these frontal areas connect, via the claustrum, to these posterior cortical regions. In particular, the anterior cingulate and prelimbic prefrontal cortices statistically cluster in their connections with posterior cortices (cohen’s d > 1.66). Probing whether MSIT or experimental pain, which both known to induce synchronous frontoparietal cortical network activity, activate the claustrum we found that stimulus onset at both the MSIT and experimental pain (q = 0.0047) elicit significant claustrum activation in human subjects. Conclusions: These results support a model in which the claustrum is recruited to support frontoparietal cortical networks via common glutamatergic input to frontoparietal cortical network nodes. Disclosure: Nothing to disclose. 40.2 Prefrontal Cortex Parvalbumin Neurons Underlying Attention - Relevance for Neuropsychiatric Disorders Tyler Dexter University of Western Ontario, London, Canada Background: Abnormal oscillatory activity in the prefrontal cortex is a hallmark of neuropsychiatric disorders such as schizophrenia. These abnormalities may arise from dysfunctional parvalbumin interneuron (PVI) function, a class of inhibitory neurons that regulate high-frequency oscillations, and underly the cognitive impairments. Here, we investigated how PVIs in the medial prefrontal cortex (mPFC) of mice contribute to attention under non-pathological conditions and disease states. To model aspects of schizophrenia, we assessed the potential of targeting prefrontal PVIs to improve cognition in a 22q11.2 microdeletion syndrome (22q) mouse model. Methods: To assess attention, we used the touchscreen rodent continuous performance task (rCPT). For Exp 1, male and female mice were bred to express channelrhodopsin (ChR2) on PVIs and implanted with bilateral optic probes in the mPFC. For Exp 2, adult male and female 22q mice and littermate controls were assessed on the rCPT. Additionally, 22q mice were genetically modified for optogenetic control of PVIs. These mice received bilateral injections of a cre-dependent ChR2 virus into the mPFC and were assessed on the rCPT prior to optogenetic stimulation of prefrontal PVI. In vivo optogenetics was used to stimulate prefrontal PVIs at a high (30hz) or low (5hz) frequency during the response phase of the task. Results: Exp 1 shows that 30hz stimulation of prefrontal PVIs significantly improved animal’s ability to discriminate the correct image during the rCPT, while 5hz stimulation significantly impaired these processes. In Exp 2, we observed a significant reduction in rCPT performance in both male and female 22q mice, specifically in the ability to detect and discriminate target stimuli. Finally, preliminary data shows that 30hz stimulation of prefrontal PVIs in 22q mice and controls significantly improves rCPT performance. While 22q mice performed significantly worse than controls at baseline, 30hz stimulation rescued this impairment. Conclusions: We demonstrate that prefrontal PVIs regulate attention in a frequency dependent manner, and that high frequency activation enhances cognitive performance in non-pathological mice. Further, direct targeting of prefrontal PVIs may serve as a therapeutic avenue of interest for improving cognition in neuropsychiatric disorders. Disclosure: Nothing to disclose. 40.3 Mice With an Autism-Associated R451c Mutation in Neuroligin-3 Exhibit Intact Attention Orienting but Atypical Responses to Methylphenidate and Atomoxetine in the Mouse-Posner Task Emma Burrows Florey Institute of Neuroscience and Mental Health, Parkville, Australia Background: Atypical attention orienting has been associated with some autistic symptoms, but the neural mechanisms remain unclear. The human Posner task, a classic attention orienting paradigm, was recently adapted for use with mice, supporting the investigation of the neurobiological underpinnings of atypical attention orienting in preclinical mouse models. The current study tested mice expressing the autism-associated R451C gene mutation in neuroligin-3 (NL3) on the mouse-Posner (mPosner) task following administration of clinically-relevant attention modulators. Methods: Male NL3 and wild-type (WT) mice (n = 8-10 per group) were trained to sustain a nose-poke to a central square on a touchscreen until a validly or invalidly cued target was displayed. The cue was a peripheral non-predictive flash in the exogenous task and a central spatially predictive image in the endogenous task. The effects of methylphenidate (MPH) and atomoxetine (ATO; latin-square design; n = 8-10 per group) on performance were assessed. Mann-Whitney U tests were conducted to compare sessions to criterion in training and genotype and drug effects were analysed by generalised linear latent and mixed models (GLLAMM) with robust standard error estimation and with individual mice treated as random effects. Results: In both tasks, mice were quicker and more accurate in the validly versus invalidly cued trials, consistent with results in the human Posner task (validity). Administration of MPH increased the difficulty of disengaging exogenously oriented attention only in NL3 mice, (reduced accuracy and increased omission errors in invalid trials). MPH enhanced endogenous orienting in both NL3 and WT mice (i.e., increased correct responses and reduced omission errors). In contrast ATO impaired exogenous orienting in both WT and NL3 mice (i.e., decreased response times and increased incorrect responses) and decreased accuracy of endogenous orienting through more omission errors in WT mice only. Conclusions: This study presents the first investigation of attention orienting in a preclinical model of autism and expands our understanding how the NL3R451C mutation may lead to selective alterations in attentional processes. Disclosure: Nothing to disclose. 40.4 Translational Neurophysiological Measures to Evaluate Cognitive Control Following Prenatal Alcohol Exposure in Rodents Jonathan Brigman University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States Background: Fetal Alcohol Spectrum Disorders (FASD) are still the most common type of neurodevelopmental syndrome and cognitive control has been consistently reported to be impaired in affected individuals. The 5 Choice Continuous Performance Task (5C-CPT) has recently been shown to measure attention and cognitive control in rodents and show similar cross-species neural signatures via EEG. Methods: Male and female offspring (12-16 per sex/trt) from a drinking-in-the-dark model (10% EtOH with 0.066% saccharine for 4hrs/day throughout gestation; BAC: ~90mg/dL; controls=0.066% sweetened water, “SAC”). At 8-10 weeks, mice trained to touch white target stimuli. Next, mice underwent stereotactic surgery and fitted with dura-resting skull screws targeting medial prefrontal, parietal, and motor cortices. After recovery non-target trials (5 stimuli presented; withholding of response was rewarded) were added at a 2:1 ratio for 5 days followed by recording at a 5:1 ratio for 12 days. ANOVA was used to examine main effect of treatment, sex, and interaction effects for dependent variables. Results: On 2:1 there was a significant difference for sex and trt, [Session: p = 0.96, Sex: p = 0.04; Trt: p = 0.002; Sex x Trt:, p = 0.06]. The false alarm rate was significantly different for sex and trt with an interaction [Session: p = 0.74, Sex: p < 0.0001; Trt: p < 0.0001; Sex x Trt: p = 0.04]. For 5:1, PAE mice made more false alarms [Session: p < 0.0001, Sex: p = 0.59; Trt: p = 0.001; Sex x Trt: p = 0.25]. EEG revealed high frequency enhancement in correct rejection trials compared to target correct trials [ROI: p = 0.20; Sex: p = 0.54; Trt: p = 0.65; Sex x Trt: p = 0.04] and a strong low frequency enhancement in target-post-error response trials compared to target-post-target [ROI: p = 0.005; Sex: p = 0.31; Trt: p = 0.22 Sex x Trt: p = 0.11]. Conclusions: PAE spared attention on target-only trials while impairing performance when non-targets were added. EEG found an increase in frontal theta in all mice for post-error correction as and increases in posterior beta during choice conflict. Analysis of posterior beta revealed a sex x trt effect with increased power during correct rejection trials in female PAE mice. These results show that 5C-CPT can detect post-error signal changes consistent with human EEG studies and that alterations in cortical activity via EEG may be used as a potential biomarker of PAE. Disclosure: Nothing to disclose. STUDY GROUP 41. Eliminating Mental Health and Substance Use Stigma: The Time is Now Mark Rapaport, Darrell Kirch, Seth Kahan, William Smith, Arthur Evans, Ellen Kahn, Stephenie Larsen, John MacPhee, Christine Moutier, Brandon Staglin, Gahan Pandina Huntsman Mental Health Institute, Salt Lake City, Utah, United States Study Group Summary: Mental health and substance use disorder (SUD) stigma, ignorance, shame, and fear have caused our patients to be marginalized, at times criminalized, and disenfranchised in terms of both psychiatric and physical care. This stigma and prejudice has also led to decreased funding for clinical care, training and research. The study group will discuss employing the social impact model to create a sustained grand challenge to eliminate mental health and substance use disorder stigma. The urgency and opportunity to create a grand challenge will be discussed by Dr. Kirch while the theory and implementation model will be presented by Mr. Kahan, an international expert in leading grand challenges. Dr. Smith will summarize his work on the impact of racism and micro-cultures on not only creating stigma but limiting successful strategies to eliminate stigma. Ms. Kahn will discuss the work that the Human Rights Campaign is doing at the interface between mental health and SUD challenges and the LBTQ + community while Ms. Larsen will demonstrate how Encircle has created safe spaces throughout the Mountain West where LBGTQ + youth can be accepted and receive mental health and SUD care. Mr. MacPhee will describe the exciting work that the JED foundation is doing on high school and college campuses to eliminate stigma and provides evidence-based assessment and resources to our educational system. Dr. Moutier will describe the long-term work that ASFP has done to breakdown the ignorance, shame, guilt and societal judgement that frequently accompanies both people who attempt to kill themselves and the family members of people who die because of suicide. Dr. Evans will describe the thoughtful strategies that the American Psychological Association have implemented in their efforts to decrease stigma. Dr. Pandina will discuss the lifelong work that he and his team has done to battle stigma- from his support of One Mind to the creation of the Science over Stigma movement. While Mr. Staglin will discuss the systematic yet innovative work that he and One Mind have done employing podcasts, video and social media to work on the elimination of stigma. By the end of the study group the participant will have learned about the National Academy’s work on mental health stigma, some of the tremendous work already being done by many individual organizations, why a growing number of people believe that now is the time to create a sustained national movement to eliminate mental health and SUD stigma and prejudice, and how the participant can become involved in this effort. Disclosure: Nothing to disclose. PANEL 42. Developmental Critical Periods for Social Processing: Windows of Vulnerability and Opportunity 42.1 Worth the Risk? Effects of Development and Early Care Quality on Lateral Habenula Involvement in Infant Social Behavior Flexibility Maya Opendak Kennedy Krieger Institute, Baltimore, Maryland, United States Background: Flexible social behavior is critically important during early life, when environmental demands are in constant flux. On the other hand, heightened circuit plasticity during this period also renders the infant uniquely vulnerable to environmental influences that can provide a template for lifelong social behavior. However, the circuit mechanisms linking early experience to lasting social behavior patterns remain unclear. Here, we focus on the ontogeny of the lateral habenula (LHb), a key negative regulator of dopaminergic signaling, in social behavior in typical and perturbed development. Methods: In all studies, we used the Scarcity-Adversity model of Low Bedding (SAM-LB) from PN8-12, in which the dam is given limited nesting materials, with equal #s male and female pups used. Expt 1. Habenulae were dissected at PN18/ PN28 and processed via Western blot. Expt 2. PN18/ PN28 rat pups were injected with 14C 2-deoxyglucose (2-DG, 20 μCi/100 g, s.c.) and then received mild tail shocks (0.5mA) or no shocks, alone or with a social partner present every 5 mins for 40 mins, followed by brain removal for autoradiography. Expt 3. PN3 pups were bilaterally transduced in LHb with pAAV-hSyn-hM4D(Gi)-mCherry or pAAV-hSyn-mCherry. PN18/28 pups underwent peer sociability tests with/without ambient predator odor. Results: We observed an increase in CaMKIIβ expression in PN28 pups compared to PN18 pups, regardless of rearing condition (p < 0.001). Metabolic imaging data showed that post-weaning, social presence and shock (threat) cues were associated with increased 2-DG metabolism in the lateral (p = 0.007) and medial habenula (p = 0.009); these stimuli did not increase Hb engagement at PN18 or in SAM-LB-reared pups. Hb network connectivity varied with rearing, social presence, and threat (p = 0.003). Chemogenetic inhibition of the LHb at PN28 increased social approach when threat odor was present (p = 0.016). SAM-LB was associated with a robust decrease in social approach behavior in both sociability tests at PN28, which could be rescued by LHb inhibition via DREADDs (p = 0.045). Conclusions: Development and early care influence LHb involvement in processing social and threat cues. Whereas weaning typically triggers habenula-dependent inhibition of social approach under threat, early adversity engages LHb-inhibited approach even in the absence of threat. Disclosure: Nothing to disclose. 42.2 Mechanisms Linking Juvenile Social Isolation and Adult Social Behavior Deficits Hirofumi Morishita Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Loneliness is becoming increasingly recognized as a threat to mental health. Juvenile social isolation (JSI) is known to disrupt social behavior in adulthood, but little is known about the neural mechanisms linking these two events. Our previous study suggests that there is a juvenile sensitive period when isolation will induce medial prefrontal cortex (mPFC) abnormalities including parvalbumin positive interneurons (PVIs) in adult mice. Of note, these circuit abnormalities were not present at the end of the isolation, raising the question of when and how JSI-induced social deficits emerge over the course of development. Methods: Developmental progression of JSI-induced social dysfunction was investigated by the 3-chamber sociability test (n = 12-21mice). Affiliative behavior and aggression were also tested among cage mates. To modulate activity of mPFC-PVIs during a re-grouped housing period, male mice were injected with either excitatory DREADD or mCherry at postnatal day (p) 14, isolated for 2 weeks (p21-35) and given CNO in their drinking water for 2 weeks (p35-49). Mice were then subjected to 3 chamber sociability test, open field, elevated plus maze, or light dark box in adulthood (n = 9-10mice). Only male mice were used as female JSI mice did not show sociability deficits. Results: We found that JSI-induced social dysfunction in the 3-chamber test is delayed (not fully emerging until p50) and the sociability deficit, where subjects interact with novel mice, is preceded by negative social interactions between JSI cage mates during the first week after the end of isolation. A chemogenetic modulation of dmPFC-PVI activity for a 2-week adolescent period (p35-49) at the start of re-housing leads to a long-term rescue of sociability (p = 0.045) without altering anxiety or locomotor behaviors. Conclusions: These results suggest that the prevailing “social deprivation model”, where adult social deficits are attributed to disruption of developmental processes occurring during the isolation period, should be supplemented by the “developmental mismatch model”, where social deficits are attributed to disruption of developmental processes occurring after the isolation period. Our study also highlights a novel adolescent sensitive window to prevent the emergence of social behavioral deficits in adulthood. Disclosure: Nothing to disclose. 42.3 Sex Differences in the Impact of Juvenile Stress on Social Behavior and Ventral Hippocampal-Nucleus Accumbens Activity Francis Lee Weill Cornell Medical College, New York, New York, United States Background: Impairment in social behavior is a key feature of many psychiatric disorders. However, less is known about the effects of juvenile stress on the development of circuits mediating social behaviors, especially in females who are more likely to be diagnosed with mood disorders. These studies use animal models for early life adversity to identify sensitive periods in social development. Methods: To quantify development of ventral hippocampus (VH) inputs to nucleus accumbens (NAc), anterograde tracer PHA-L was injected into VH of P23, P30, and P60 male (m) and female (f) mice (n = 6/group). Terminals in NAc were quantified as a measure of VH neuron projection density. Separate male and female mice underwent 10 days of chronic unpredictable stress (CUS) (P22-P31). Three-chamber test and free social interaction (FSI) tests began at P70 (n = 9-11/group). In-vivo calcium imaging was used to record activity in NAc-projecting VH neurons during FSI in adulthood (n = 7–11/group). Results: There was increased fiber density in the NAc at P30 compared to P23 or P60 in males (p < .0001, p < .0001) and females (p = .023, p < .0001). Additionally, females had increased fiber density compared to males at P23 (p < .0001). At P70, in the 3-chamber test, all control (CTL) mice and CUS males spent more time interacting with a social partner than an object while CUS females did not (m: ctl: p < .001, cus: p < .01; f: ctl: p < .0001, cus: p = .854). In FSI, regardless of sex, CUS mice spent less time interacting with a social partner than CTLs (p = .04). For fiber photometry, neural activity was higher 2 seconds post interaction initiation than 0.5 seconds pre for stressed females (p < .001) but not CTLs (p = .162). Additionally, CUS females had increased activity 2 seconds post social interaction relative to CTLs (p = .044). In males, CTL (p = .003) and CUS (p = .001) mice showed increased activity from pre to post social interaction initiation, but there was no difference between groups in the 2 seconds post interaction initiation (p = .894). Conclusions: We identified a juvenile sensitive period for sociability that coincides with changes in VH-NAc connectivity. Females that underwent juvenile CUS show elevated activity in the VH-NAc circuit upon initiation of social interaction. Together, these findings inform our understanding of sex-differences in sensitive windows for social development. Disclosure: Nothing to disclose. MINI PANEL 43. Interactions Between Insulin and Dopamine in the Brain: Implications for Metabolism Across Health and Disease 43.1 Effect of Short-Term Overeating of High Caloric Snacks on Insulin Responsiveness of the Brain in Normal-Weight Men Stephanie Kullmann Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tübingen, Tübingen, Germany Background: Insulin resistance (IR) is the hallmark feature of obesity and type 2 diabetes with detrimental effects in the periphery and the central nervous system. Numerous studies confirm that the disruption of insulin responsiveness in the human brain influences the pathology of metabolic, psychiatric, and neurodegenerative diseases. Insulin action in the brain regulates synaptic plasticity and dopamine signaling. Human brain imaging studies show that central insulin action affects region-specific activity and functional connectivity in the hypothalamus and the mesocorticolimbic circuitry influencing food intake and non-homeostatic feeding for pleasure. It is currently unclear whether short-term overeating hampers insulin sensitivity of the brain in healthy individuals and whether this relates to changes in peripheral metabolism. Methods: Brain insulin sensitivity was assessed by functional magnetic resonance imaging (fMRI) combined with intranasal administration of insulin to the brain before, directly after overeating or control diet, and 1 week afterwards. Food intake and physical activity was recorded during the course of the study. Moreover, participants underwent two oral glucose tolerance tests, whole-body MRI for body fat distribution intrahepatic fat content quantification and performed a dopamine dependent reward learning task. This study was registered under clinicaltrials.gov (NCT03590561). 32 healthy normal-weight male participants were recruited into the study, of which, 29 participants (age range 19 – 27 years, BMI range 19 – 25 kg/m2) completed the study. 18 participants increased their daily caloric intake by 1500 kcal with high caloric snacks (HCD group); 11 participants maintained their habitual diet. Based on the participant specific palatability ratings (HCD group), a nutritionist prepared packages for five days containing 1500 kcal each with different snack (including as example: snickers, brownies, chips, etc.; nutritional composition equivalence: 47-50% Fat, 40-45% Carbohydrates). Results: HCD group showed a significant increase in insulin responsivity in the insular cortex from before to directly after the intervention (T (17) value= 6.43, pFWE<0.05), similarly to persons with obesity from a cross-sectional cohort. Two weeks after the diet, brain insulin action was normalized. Notably, intrahepatic fat significantly increased in the HCD group directly after the hypercaloric diet (mean change of 53%; p = 0.001), which correlated with alterations in brain insulin responsiveness (r = 0.606, p = 0.001). Reward behavior was found corrupted directly after overconsumption with decreased sensitivity for reward (T (27) = -3.6, pboot < .001) and increased sensitivity for punishment (T (27) = 2.6, pboot = .002) in the HCD group. No change in brain insulin action, liver fat content and reward behavior were observed in the control group. Conclusions: We found a significant change in brain insulin action and reward behavior in response to overconsumption of high caloric food in normal-weight men, while body weight and peripheral insulin sensitivity did not show a significant change. Intrahepatic fat, however, significantly increased after the hypercaloric diet and correlated with the altered brain insulin response. In the follow-up visit, brain insulin action was normalized. Disclosure: Nothing to disclose. 43.2 Obesogenic State, Marked by Downregulation of Striatal Dopamine D2-Receptors, is Associated With Changes in Striatal Insulin Receptor Levels and Functioning in Mice Miriam Bocarsly Rutgers NJMS, Newark, New Jersey, United States Background: Understanding the neuronal circuitry underlying feeding behaviors is necessary with estimates suggesting that 40% of adults, globally, are overweight. The central nervous system plays a critical role in overeating and excessive weight gain. While neuronal circuitry underlying food intake is traditionally studied in the context of maintaining homeostatic control of energy balance, amassing evidence supports the idea that control of caloric intake also involves calculations of hedonic value, reward and motivation. Our current research focuses on the role of the striatum, a brain area implicated in reward and motivation, in regulating feeding behaviors. An interesting observation has linked the actions of insulin in the striatum to local dopamine release. Here, in a preclinical model, we demonstrate the effects of a high-fat diet on insulin-mediated striatal dopamine release, identify an interaction between striatal insulin receptors and obesity-related alterations in dopamine D2-receptors, and propose a circuit-level mechanism. Methods: Here we utilized mouse models, including diet-induced obesity and novel transgenic lines, to look at dopamine release in brain slice via fast-scan cyclic voltammetry (FSCV). qPCR was used to determine mRNA expression for dopamine D2 and insulin receptors in the striatum. Data was analyzed using t-test, RM-ANOVA or mixed-effects analysis, as appropriate; p < 0.05 considered significant. Results: Fast-scan cyclic voltammetry shows increased electrically evoked dopamine release in response to insulin in mice showing signs of diet-induced obesity. Postmortem qPCR analysis showed these mice have low levels of striatal dopamine D2 receptor (D2R) mRNA. Using transgenic methods, we designed a mouse model with low striatal D2Rs, and observed mice gained more weight on a standard rodent chow compared to littermate controls. Interestingly, qPCR results showed these mice have increased levels of striatal insulin receptor mRNA, and they showed elevated dopamine release in response to insulin using FSCV. In a separate transgenic model, we eliminated striatal insulin receptors on cholinergic interneurons, and were no longer able to detect a striatal dopamine response to insulin. Of further interest, mice lacking striatal insulin receptors showed increased levels of D2R mRNA. Conclusions: For the first time, we demonstrate a compelling, bidirectional interaction between striatal dopamine D2 receptors and insulin receptors. Further, we demonstrate that this system is dysregulated in a model of obesity, suggesting a novel mechanism by which central reward processing may be perturbed in instances of obesity. Disclosure: Nothing to disclose. 43.3 Can Antipsychotics Induce Insulin Resistance in the Brain? Investigation of the Acute Effects of Olanzapine on CNS Insulin-Mediated Brain Glucose Uptake Using [1-14C]2-Deoxy-D-Glucose (2DG) Autoradiography Sri Mahavir Agarwal University of Toronto, Centre for Addiction and Mental Health, Toronto, Canada Background: Antipsychotics are the cornerstone of treatment in schizophrenia but are associated with an increased risk of type 2 diabetes. Previous studies examining the effects of antipsychotics on insulin and glucose regulation have primarily focused on peripheral pathways. However, insulin receptors and insulin-responsive glucose transporters are widely distributed throughout the brain, and insulin resistance in the brain has been associated with lower cerebral glucose metabolism. Our group has shown previously that olanzapine can inhibit the effect of CNS insulin in rodents to suppress feeding and hepatic glucose production, independently of its weight gain propensity. In this proof-of-concept study, we examined the neural correlates of antipsychotic-induced cerebral insulin resistance as measured by an attenuation in brain glucose uptake in response to CNS insulin administration. Specifically, we used olanzapine (OLA) as the representative antipsychotic given its high metabolic liability and examined the nature and extent of OLA-induced brain insulin resistance. Methods: Male rats were pretreated with OLA (3 mg/kg; dose based on clinical D2 receptor occupancy) or vehicle. [1-14C]2-deoxy-D-glucose (2DG) autoradiography procedures were used to measure cerebral glucose uptake during an intracerebroventricular (ICV) infusion of insulin or vehicle into the third ventricle. The four groups therefore included (central-peripheral) vehicle-vehicle (n = 6), insulin-vehicle (n = 6), insulin-OLA (n = 5), and vehicle-OLA (n = 6). Regions of interests for analysis included the frontal cortex, nucleus accumbens, hypothalamus, hippocampus, amygdala, cerebellum, and dorsal vagal complex, chosen based on their role in metabolism, energy homeostasis, and cognition. Regional tissue radioactivity was quantified on coded films using the MCID Elite system as an index of glucose uptake. Results: Cerebral glucose uptake was statistically significantly different between experimental groups across all investigated brain regions (all p < 0.001). Post hoc analysis using Bonferroni correction revealed that animals in the insulin-vehicle group showed increased cerebral glucose uptake in all brain regions compared to the vehicle-vehicle, vehicle-OLA, and insulin-OLA groups (all p < 0.001). The vehicle-vehicle, vehicle-OLA, and insulin-OLA groups did not differ amongst each other. Conclusions: Intracerebroventricular (ICV) insulin significantly increased cerebral glucose uptake in all regions of interests compared to ICV vehicle. This effect was abolished by peripheral OLA, such that the level of cerebral glucose uptake in the insulin-OLA group was comparable to the control vehicle-vehicle and vehicle-OLA groups. Our results indicate that acute dosing of OLA can block the effect of insulin in multiple areas of the brain. Disclosure: Nothing to disclose. PANEL 44. Functionalizing Common Variants in Schizophrenia 44.3 Systematic Investigation of Allelic Regulatory Activity of Schizophrenia-Associated Common Variants Hyejung Won University of North Carolina, Chapel Hill, North Carolina, United States Background: Schizophrenia is a highly heritable and polygenic neuropsychiatric condition characterized by psychosis, emotional withdrawal, and cognitive deficits. Common variation explains a significant portion of heritability and genome-wide association studies (GWAS) have identified 294 loci. However, it is a substantial challenge to understand the functional impact of these loci because most reside in non-coding DNA with unknown functions. Therefore, the first critical step to bridging the gap between genetic loci and biological underpinning is to identify causal variants and delineate their functional impact. Methods: We built an AAV-based MPRA vector (AAV-MPRA) that comprises a 150bp target sequence with the variant in the center, the minimal promoter, GFP, and a 20bp unique barcode. Using this backbone, we generated an AAV-MPRA library for a computationally predicted credible set of schizophrenia risk variants (5,173 variants) that covers 143 out of 145 loci. The resulting AAV-MPRA library was administered to human neural progenitors (HNPs, N = 10). Two weeks after administering the AAV-MPRA library to HNPs, RNA was extracted from transduced cells and barcoded GFP expression was quantified by RNA sequencing. Results: We identified 440 variants with allelic regulatory effects (FDR < 0.1), covering 103 GWAS loci. Notably, only 11 out of 143 index variants showed regulatory effects, suggesting that perfunctorily focusing on index variants purely based on statistical associations may not correctly pinpoint causal variants. We detected a high concordance between MPRA and eQTLs as 36% of regulatory variants overlapped with the adult brain eQTL data, and 82% of those overlapping variants showed identical regulatory direction. However, ~60% of MPRA-validated variants were not detected in eQTLs. Regulatory variants overlapped with neuronal enhancers more than other cell types, highlighting the cell-type specificity of these variants. However, regulatory and nonregulatory variants did not differ in their enhancer overlap, showing that enhancers are not a predictive factor in distinguishing the two. Conclusions: We demonstrate that MPRA can effectively identify regulatory variants and delineate previously unknown regulatory principles of schizophrenia. Disclosure: Nothing to disclose. 44.4 Genetic Regulation of Caudate Nucleus Transcriptome in Schizophrenia Daniel Weinberger Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: Recent studies of gene expression in human brain in schizophrenia have focused mainly on cortical regions though subcortical nuclei, such as striatum, figure in the circuitry implicated in schizophrenia and its treatment. GWAS have identified a locus that includes DRD2, which is most expressed in striatum, but evidence of a DRD2 mechanism of risk has not been identified. We performed the first comprehensive analysis of the genetic and transcriptional landscape of schizophrenia in human caudate. Methods: In 443 mixed ancestry individuals (154 SCZ, 245 NC, 44 BPD), we identified trans-ancestry eQTLs across genes, transcript, exons, and junctions using TOPMed imputed genotypes and empirical Bayes meta-analysis with multivariate adaptive shrinkage (“mash18”) modeling. We integrated eQTLs analysis, summary based Mendelian randomization (SMR), the latest schizophrenia GWAS (Nature 2022), and transcriptome wide association study (TWAS). Results: We found cis-eQTLs (local false sign rate [lfsr] < 0.05) associated with 23,097 unique genes (protein-coding and noncoding) across all transcript features. Using SMR, we identified 588, 935, 5629, and 2619 unique genes, transcripts, exons, and junctions associated with schizophrenia risk. We identified 553 genes, 1117 transcripts, 4779 exons and 1558 junctions with significant TWAS association (FDR < 0.05) for schizophrenia based on PGC3 GWAS data; 277 (Bonf p-value < 0.05) were unique to caudate compared with other brain regions in schizophrenia TWAS. 174 (Bonf p-value < 0.05) of these 277 genes were not in GWAS significance loci. We found a significant negative association for the DRD2 short (D2s) specific junction (junction 5-7, TWAS FDR < 0.05) and transcript (SMR q-value < 0.05 and HEIDI p-value > 0.01) which replicated in GTEx (SMR p-value < 0.05 and HEIDI p-value > 0.01), implicating under-expression of the D2s with increased schizophrenia risk. We found no association with the DRD2 long isoform. Conclusions: We identify in human caudate many novel genes outside of GWAS loci that may have causal relationships with schizophrenia, confirming that genetic risk involves distributed brain systems mediating diverse information processing streams. The likely mechanism of genetic risk related to DA is compromised presynaptic autoregulation, and thus, a bias towards increased synaptic DA release. Disclosure: Sage Therapeutics: Advisory Board (Self) PANEL 45. Hippocampal Mechanisms of Intergenerational Distress: Interactions Between Structural Stressors and Individual-Level Risk Factors 45.2 Prenatal Maternal Sleep and Infant Offspring Neurodevelopment: Focus on Racial and Ethnic Sleep Disparities and Offspring Risk for ADHD Claudia Lugo-Candelas Columbia University Medical Center, New York State Psychiatric Institute, New York, New York, United States Background: Prenatal maternal sleep may be related to offspring ADHD and difficulties with emotion regulation (ER). Offspring of sleep-restricted dams show decreased hippocampal volumes and self-regulation deficits. As smaller hippocampal volumes and ER difficulties are related to psychopathology risk, maternal sleep may be a novel focus for interventions. Yet, no human study has examined offspring brain in relation to maternal sleep. Further, minoritized racial/ethnic US groups show poorer sleep yet are dramatically underrepresented in prenatal sleep research. We leverage 2 cohorts and present converging data showing racial disparities in prenatal maternal sleep and intergenerational sequelae on offspring’s brain and behavior Methods: Pregnant women reported on prenatal sleep health and on children’s ADHD symptoms, ER, and sleep via the CBCL. In cohort 1 (Puerto Rican dyads), infants underwent MRIs at 4 months, and parents completed CBCLs at 24 months. The Developing Human Connectome pipeline was used to segment T2 images and estimate sub-cortical volumes. In cohort 2 (nation-wide cohort), children were 6 when parents completed the CBCL. Results: Cohort 1 analyses revealed that women who identified as Black reported poorer quality than women who identified as White. For female offspring, poorer prenatal sleep quality predicted smaller hippocampal volumes. Worse sleep quality was associated to greater L amygdala volume in males. Worse maternal sleep quality was related to offspring sleep problems at 24 months. In Cohort 2, prenatal sleep duration and quality - in the 2nd trimester- were related to children’s sleep and ADHD symptoms. Prenatal sleep quality related to worse ER in males only. Conclusions: Prenatal sleep, especially in the 2nd trimester, may be critical to offspring, yet we document race disparities, highlighting the need for interventions aimed at sleep equity. Mechanisms require elucidation, yet males may be more affected in ER, which is associated to amygdala structure and impaired in ADHD. Females may be at higher risk for susceptibility to future adversity, based on hippocampal volumes. As childhood sleep problems carry risk for later negative outcomes (ADHD) we highlight another possible intervention target. We underscore the importance of examining the determinants and intergenerational consequences of prenatal sleep health. Disclosure: Nothing to disclose. 45.3 Maternal, Structural and Genetic Risk Factors for Intergenerational Transmission of Depression Predict Dentate Gyrus Microstructure Milenna van Dijk Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Parental depression puts offspring at risk for depression themselves. I showed in children from the Adolescent Brain and Cognitive Development Study (ABCD) that having a parent AND grandparent further heightens risk: 44% of these children showed psychopathology. Taking hypotheses from rodent work, I found in a well-phenotyped three generational family risk for depression study (3gen) that high risk is associated with lower DG microstructure which predicts future symptoms. To elucidate factors contributing to family risk, I will present unpublished data on associations between genetic and (parental and structural) environmental factors related to family risk and hippocampal morphology and depressive outcomes. Methods: n > 73 individuals (3gen) and n > 4891 (ABCD). Using FreeSurfer and MRtrix pipeline we extracted hippocampal (subfields) mean diffusivity (MD; microstructure). For genotyping (GlobalScreeningArrayV.3) 22740171 SNPs were available. Depression polygenic risk scores (PRS) were calculated from the Broad Depression GWAS summary statistics (Howard 2018). Predictors were area deprivation index (ADI), maternal distress and PRS. Outcomes were hippocampal/DG MD, PHQ-9 (3-gen) or CBCL scores (ABCD). Regressions accounted for family structure and appropriate covariates in a generalized estimating equation framework. Results: Findings are significant at p < 0.05. ADI is associated with higher hippocampal MD, signaling decreased microstructure, which mediates associations between ADI and depressive outcomes. ADI interacts with family risk leading to higher MD specifically in the DG. Maternal distress and PRS individually predict higher DG MD and interact to lead to the highest DG MD. Enrichment analysis showed that the list of genes that most predicted DG MD overlapped most significantly with a gene set involved in neurogenesis, in line with the DG being a unique site of adult neurogenesis. Importantly, across cohorts, higher DG MD predicts future, not current, depressive symptoms and mediates associations between risk factors and symptoms. Conclusions: These results go beyond familial risk to show consistent personal and structural factors that influence hippocampal mechanisms of intergenerational depression to eventually target interventions to those at highest risk and disrupt cycles of transmission. Disclosure: Nothing to disclose. 45.4 Early Life Adversity and Social Withdrawal Behavior in Children – Identifying Risk for Adult Psychopathology Using a Translational Model and Expression-Based Polygenic Scores Patricia Pelufo Silveira McGill University Faculty of Medicine, Montreal, Canada Background: Social withdrawal behavior in childhood has been linked to internalizing problems and increased risk of anxiety disorders and depression later in life. It is known that inhibition of pyramidal neurons in the ventral hippocampus impair normal social behavior (Murugan et al., 2017; Okuyama et al., 2016; Brumback et al., 2017). Methods: To identify the biological underpinnings and predictors of social withdrawal behavior in humans, we used ventral hippocampus RNA sequencing data from rats that naturally varied in their willingness to interact with a co-specific. Data was processed through weighted gene co-expression network analysis (WGCNA), and we identified 4 co-expression modules that significantly correlated with social interaction behavior. Human ortholog genes from these networks were used to inform the calculation of expression-based polygenic scores (ePRS) in different human samples: MAVAN (n = 159), GUSTO (n = 440), and UK BioBank (n = 70,744). Results: In general, variations in these ePRS scores were significantly associated with withdrawal behavior (Child Behavior Checklist, CBCL) in children (betas ranging from 0.39 to 0.11, P < 0.05) and social anxiety/major depressive disorder in adults (betas ranging from 0.13 to 0.04, P < 0.05) in response to early life adversity. Conclusions: Functional genomics translational tools like the one used in this study can help in the identification of individual differences in child behavior that map onto adult psychopathology, especially in response to early adversity. Disclosure: Nothing to disclose. PANEL 46. Targeting Neuroimmune Signaling in Substance Use Disorders 46.1 A Double-Blind, Placebo-Controlled Phase 2a Proof-Of-Concept Study of the Phosphodiesterase Type 4 Inhibitor, Apremilast, for the Treatment of Alcohol Use Disorder Barbara Mason The Scripps Research Institute, La Jolla, California, United States Background: Computational genomic analyses and multiple preclinical studies conducted through the Integrative Neuroscience Initiatives on Alcoholism – NeuroImmune (INIA-N) consortium identified the Phosphodiesterase Type 4 (PDE4) inhibitor, apremilast, as having therapeutic potential for alcohol use disorder (AUD). Apremilast has regulatory approval for the treatment of psoriasis and is thus available for repurposing as a potential therapeutic for AUD. Methods: 51 non-treatment-seeking male and female paid volunteers who met DSM-5 criteria for current AUD ≥ moderate severity were randomly assigned to double-blind treatment with 90mg/d apremilast or matched placebo. The Timeline Followback Interview was used to assess daily intake of standard drinks (~14g alcohol) over an 11-day period of ad libitum drinking. All randomized subjects (N = 51) were included in an intention-to-treat analysis that employed latent growth modeling (LGM) to compare the change in the number of drinks per day and the change in the probability of a heavy drinking day (≥4 drinks females, ≥5 drinks males) in apremilast versus placebo groups. Results: Subjects were 27 males and 24 females, aged 41.2 ± 16.3 years, who met DSM-5 criteria for 6.4 ± 2.3 symptoms, indicating a severe level of AUD. Apremilast showed a significantly (p = 0.025) greater reduction in the number of drinks per day relative to placebo, as well as a greater reduction in the probability of a heavy drinking day (p = 0.030). The LGM predicted an average change of 2.74 drinks per day from day 1 to day 11 for apremilast and 0.48 for placebo and yields a Cohen’s d value of 0.77 which can be interpreted as a “large” effect of apremilast on decreased drinking. For probability of a heavy drinking day, the effect size was 0.39 for apremilast and 0.05 for placebo, with a Cohen’s d of 0.26, or “small-medium.” No serious or unexpected adverse events occurred, were typically mild and were not associated with treatment discontinuation. Conclusions: Significant effects of apremilast on decreasing the number of drinks per day and the rate of heavy drinking relative to placebo support further development of apremilast as a novel treatment for AUD. Results also provide clinical validation of PDE4 inhibition as a therapeutic strategy for AUD. Disclosure: Awakn Life Sciences Corp: Stock / Equity (Self), Imbrium Therapeutics: Advisory Board (Self) 46.2 Targeting Immune Signaling in Genetic Models of Risk for Alcohol and Substance Abuse Angela Ozburn Oregon Health and Science University, VA Portland Health Care System, Portland, Oregon, United States Background: The High Drinking in the Dark (HDID) lines of mice have been selectively bred from a genetically heterogeneous stock (HS/Npt) to achieve high blood alcohol levels in the limited access Drinking in the Dark (DID) task. We leveraged gene expression databases to identify compounds that would result in opposite gene expression signatures of risk for binge drinking and found that our top candidates (terreic acid, pergolide, and apremilast) successfully reduces excessive alcohol drinking. All of these compounds target aspects of immune signaling and have been shown to reduce peripheral inflammatory signaling. Methods: In separate studies, we examined (a) whether the HDID-1 and HDID-2 lines of mice would voluntarily consume the following drugs in a 4 day DID test: nicotine (30, 50, or 70ug/mL), midazolam (150ug/ml), methamphetamine (40ug/ml), and morphine (700ug/ml), (b) whether the HDID lines differed from their founder line, HS/NPT, in consumption levels of these drugs, and (c) whether compounds targeting immune signaling also reduce intake of nicotine or morphine in a 2 day DID (studies are underway to test additional drugs of abuse). Terreic acid (0 or 7.5mg/kg), pergolide (0 or 2mg/kg), and apremilast (0 or 40mg/kg) were administered intraperitoneally 1 hr prior to DID. All studies include male and female mice, where (a) and (b) included n = 7-13 mice/sex/drug/dose/strain. Results: (a) We observed no genotype differences in methamphetamine intake, but significant differences in nicotine, midazolam, and morphine intake. Both HDID lines drank significantly more midazolam than their founders (p’s<0.05), providing strong support for shared genetic contribution to binge ethanol and midazolam intake. Nicotine and morphine data suggested a divergence of genes captured between the HDID lines during the selection process, with HDID-2 mice, but not HDID-1 mice, consuming more of both drugs than HS/NPT mice (p’s<0.05). (b) Terreic acid, pergolide, and apremilast significantly reduced nicotine (p’s<0.01) and morphine (p’s<0.01) intake in male and female HDID-1 and -2 mice. Conclusions: Together, this work tells us that increasing anti-inflammatory signaling reduces excessive substance use in unique animal models that exhibit construct validity, face validity, and predictive validity for AUD and SUD. Disclosure: Nothing to disclose. 46.3 The Role of the Interleukin-6 (IL-6) System in Alcohol-Induced Amygdalar and Cortical Synaptic Dysfunction Marisa Roberto The Scripps Research Institute, La Jolla, California, United States Background: Neuroimmune pathways such as interleukin-6 (IL-6) regulate brain function to influence complex behavior, and their dysfunction is associated with alcohol use disorder (AUD). Thus, we studied the synaptic mechanisms underlying the alcohol-induced adaptation of IL-6 at GABA and glutamate synapses in the central nucleus of the amygdala (CeA) and prelimbic region of the medial prefrontal cortex (PL). As IL-6 receptor (IL-6R) antibodies are FDA approved to treat inflammatory illnesses, we tested whether they decrease excessive ethanol drinking. Methods: We induced ethanol dependence in C57BL/6J mice using chronic intermittent ethanol vapor/2 bottle choice paradigm. We used 1) electrophysiology to record spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) in CeA and PL pyramidal neurons, 2) in situ hybridization (ISH) to determine cell-type expression of IL-6 and 3) behavioral pharmacology to measure drinking (IL6R antibody, Bioxcell: i.p., 200 ug/day for 5 days). Results: We found that acute application of IL-6 modulates spontaneous CeA GABA release (i.e., sIPSC frequency) in a sex-dependent manner. In males, IL-6 (50 ng/ml) increased the mean sIPSC frequency in the majority (7/10) of naïve CeA neurons. This effect was reversed in dependent males, where IL-6 decreased sIPSC fequency by 26 ± 3% (p < 0.01; n = 10/6 mice). IL-6 had a split effects (increase and decrease) on sIPSC frequency in the non-dependent males without affecting amplitude or kinetics of any groups. In females, IL-6 had split effects on both the mean sIPSC frequency and amplitude, without altering kinetics. In male mPFC, IL-6 decreased glutamate release in the naïve (by 31 ± 4%, p < 0.01; n = 11/6 mice) but had split effects in the non-dependent and dependent males. In contrast, IL-6 had split effects on the sEPSC frequencies in the naïve and non-dependent females but decreased glutamate release in dependent females (to 77 ± 11%; p = 0.094; n = 7/5 mice). ISH revealed that glial cells contribute most to the ethanol-induced IL-6 adaptive changes. Systemic administration of an IL-6R antibody reduced excessive drinking in dependent females. Conclusions: Collectively, our data identify prominent sex differences in the mechanism by which the IL-6 system regulates synaptic transmission in the CeA and PL, and that chronic ethanol alters IL-6’s modulatory function. Disclosure: Nothing to disclose. 46.4 Interleukin-33 as a Neuroimmunophysiological Mediator of Excessive Alcohol Intake Regina Mangieri The University of Texas, Austin, Texas, United States Background: Excessive alcohol consumption and dependence are associated with increased excitatory synaptic activity and depressed membrane excitability of nucleus accumbens (NAc) neurons. Previously there was little evidence to link alcohol-induced NAc neuroplasticity to neuroimmune activity, but we hypothesized they may be related. Interleukin (IL)-33 is a pleiotropic cytokine that regulates excitatory synapses and its expression is associated with risk for heavy drinking. Here, we validated IL-33 as possible mediator of excessive alcohol drinking. Methods: Male mice were exposed to ethanol vapor or air and brain slices were prepared for electrophysiology 24 hr into withdrawal; whole-cell patch clamp recordings were collected from NAc neurons following incubation + /- IL-33 (50 ng/mL, > 30 min). Alcohol drinking by male mice was assessed in the every-other-day, 24-hr, 2-bottle choice (2BC, 15% alcohol vs. water) model of excessive drinking; IL-33 (10 µg/kg, i.p.) or vehicle was injected 4 hr prior to 2BC. NAc Il33 expression was measured in whole tissue and an astrocyte RNA-enriched fraction. Results: (a) IL-33 mimicked the effects of ethanol exposure in the Air group (increased excitatory synaptic event frequency + reduced membrane excitability) and had an entirely different pattern of effects in the Ethanol Vapor group (n = 13-20 cells/treatment/condition). (b) When IL-33 injections began on the 1st day of 2BC (10 mice), IL-33 suppressed ethanol and total fluid intake; following cessation of treatment, preference and intake returned to vehicle control levels (n = 10). (c) When mice (n = 10) first established excessive intake prior to treatment, IL-33 transiently suppressed ethanol and total fluid intake; following cessation of treatment, there was a marked escalation of ethanol preference and intake (~140% pre-treatment). (d) NAc Il33 expression (n = 4 mice) was 2-fold enriched in the astrocyte fraction. Conclusions: Astrocytes are the primary source of IL-33 in the NAc, while the functional activity of NAc neurons is responsive to IL-33. Both the physiological and behavioral consequences of IL-33 treatment varied with ethanol experience. These results provide proof-of-concept that IL-33 signaling is altered by alcohol exposure and engagement of IL-33 signaling may be a form of astrocyte-neuron crosstalk that promotes drinking. Disclosure: Nothing to disclose. PANEL 47. New Characterization of the Phenotype of Bipolar Disorder in Older Adults From Data of an International Research Consortium 47.1 Bipolar Symptoms, Somatic Burden and Functioning in Older-Age Bipolar Disorder: A Replication Study From the Global Aging & Geriatric Experiments in Bipolar Disorder Database Project Martha Sajatovic Case Western Reserve University, Cleveland, Ohio, United States Background: The Global Aging and Geriatric Experiments in Bipolar Disorder Database (GAGE-BD) project pools archival datasets on older age BD (OABD). An initial Wave 1 (W1) analysis found both manic and depressive symptoms reduced with age. This replication analysis used the same methods with a new Wave 2 (W2) dataset. Methods: This cross-sectional analysis examined the association between BD symptoms and age (Aim 1) and the contribution of symptoms and somatic burden to functioning and age moderation (Aim 2). BD symptoms were assessed with the Young Mania Rating Scale (YMRS) and depressive symptom severity grouped into 4 ordinal categories. Somatic burden was grouped into 8 categories and functioning was assessed with the Global Assessment of Functioning (GAF). Results: Compared to W1, the W2 sample was younger (p < 0.001), less educated (p < 0.001), had more BD symptoms (p < 0.001), lower functioning (p < 0.001) and fewer somatic conditions (p < 0.001). In the full W2, with education considered, there was a negative association between age and YMRS. In the OABD subgroup, older age was associated with reduced mania severity (p < .01). In contrast to our hypothesis, greater age was associated with more severe depression in the full W2 (p < .001) even adjusting for sex and education; also, the case in the OABD subgroup (p < .05) Older age was paradoxically associated with fewer somatic comorbidities in full W2 (p < 0.001) but not related to comorbidities in the OABD subgroup. Older individuals had lower GAF scores (p < .02 full W2, p < .01 OABD). Controlling for gender and education, more severe BD symptoms (mania p < .001, depression p < .001) and greater comorbidity (p < .002) were associated with worse functioning. In the full W2, age moderated the association of depression with GAF such that older individuals showed a stronger relationship (p = .01); similarly true in the OABD subgroup (p = .01). Conclusions: This W2 analysis suggests older age is associated with less severe mania, more severe depression, and lower somatic comorbidity. Functioning was worst among older individuals, especially those with more severe depression and comorbidity. Discrepant findings in this replication analysis could be due to W2 having more severe BD symptoms vs. W1. It could be that age ameliorates depression when levels are mild but exacerbates them when levels are more moderate or severe. Disclosure: Nuromate, Otsuka, International Society for Bipolar Disorders (ISBD): Grant (Self), Alkermes, Otsuka, Sunovion, Janssen, Lundbeck, Teva, Clinical Education Alliance, Health Analytics: Consultant (Self), Springer Press, Johns Hopkins University Press, Oxford Press, UpToDate, American Physician’s Institute (CMEtoGo, Psychopharmacology Institute, Novus, American Epilepsy Society, American Society of Clinical Psychopharmacology, American Academy of Child and Adolescent Psychiatry, Neurocrine: Other Financial or Material Support (Self) 47.2 Categorization, Harmonization, and Consensus Essential Elements for Multi-Site Data Collection and Analysis of Older-Age Bipolar Disorder Phenotypes Lisa Eyler University of California, San Diego, San Diego, California, United States Background: Rigorous research on the unique clinical presentation of older-age bipolar disorder (OABD) patients is imperative to guide prevention and treatment efforts. Previous work was limited by small samples from single institutions. The Global Aging and Geriatric Experiments in Bipolar Disorder (GAGE-BD) study integrates and harmonizes archival data from sites across the world and has provided a testbed for development of novel methods and recommendations for prospective studies. Methods: Based upon our systematic review of measures used in 53 studies of OABD, and upon data dictionaries and numeric data from 19 cohorts comprising over 1400 individuals with and without bipolar disorder (BD), we developed an ontology of data domains, subdomains, and sub-subdomains for classifying OABD-relevant variables. We then created a computerized procedure, implemented in both Python and RedCAP, used by trained data analysts to classify each variable according its domains/subdomains and to indicate harmonization-relevant characteristics. In addition, we conducted a Delphi survey of 26 global OABD experts to reach consensus about the essential variables to include in a prospective multi-site study of OABD. Variables with >80% consensus, found frequently in the literature and in GAGE-BD contributing cohorts, and/or covering important domains/subdomains from our ontology were compiled into a set of essential data elements. Results: Our ontology consists of 20 domains (e.g., demographics, current illness severity, current pharmacological treatment, physical health, cognitive), 102 subdomains, and 129 sub-subdomains. The computerized classification procedure was efficient and the output dataset has been used to identify which datasets contain relevant variables and how they can be harmonized. Our set of essential data elements comprises 62 variables covering 15 key domains; of particular relevance for OABD studies are measures of comorbidity, cognition, and functioning. Conclusions: Using rigorous scientific methods, we developed and employed an ontology, variable categorization procedure, and set of essential data elements to guide prospective data collection in OABD. This work will help better characterize OABD and also benefit the growing number of international consortia that study mood disorders using archival and/or prospective data. Disclosure: Nothing to disclose. 47.3 Characterizing the Brain Phenotype of Bipolar Disorder in Older Adults: Results of an International Multimodal Neuroimaging Investigation Luca Villa University of Oxford, Oxford, United Kingdom Background: Bipolar disorder (BD) in older adults has been associated with severe clinical outcomes including cognitive impairment and increased risk for dementia and high risk for suicide. However, the differences in brain circuitry that may underlie BD in later life are not known and have been relatively neglected by past neuroimaging literature, which has often relied upon relatively small sample sizes (N ≤ 65). This talk will detail novel work from the GAGE-BD consortium, investigating structural differences within the brain associated with BD during later life, using the largest neuroimaging sample yet of older adults with BD (OABD). Methods: Data was collected from the GAGE-BD consortium, and the sample was comprised of OABD (N = 378) and healthy controls (HCs; N = 304). Group differences in cortical thickness, cortical surface area, and subcortical gray matter volume were investigated, and a subsample was also analyzed using diffusion tensor imaging data from OABD (N = 85) and HCs (N = 92), as well as a separate replication dataset of OABD (N = 29) and HCs (N = 47), investigating differences in fractional anisotropy. Group interactions with age and age of BD onset were also investigated to study age-related differences in brain structure. Results: OABD showed lower cortical thickness than HCs within prefrontal, insula, and temporal cortices (pfdr = .02-.003). OABD also showed lower corpus callosum fractional anisotropy than HCs (pfwe = .03), which was replicated (pfdr = .04-.002). Age of onset emerged as a key factor in identifying separable phenotypes within OABD, with negative relationships between age and cortical thickness observed only in OABD with later onset (pfdr = .03). Conclusions: The data suggest BD in later life is characterised by substantial gray matter reductions in regions integral for emotion processing and reduced white matter integrity in regions key to interhemispheric communication. These novel findings have implications for the mechanisms underlying past research associating a later onset of BD with greater cognitive impairment and may be pertinent to the greater risk for dementia associated with BD during later life. Overall, these results provide an unprecedented view of the underlying neurobiology involved in BD during later life, having implications for our understanding of the illness’ onset, outcomes, and treatment. Disclosure: Nothing to disclose. 47.4 Epigenetic GrimAge Acceleration and Cognitive Dysfunction in Bipolar Disorder Jair Soares University of Texas Houston Medical School, Houston, Texas, United States Background: Among biological markers of aging, epigenetic aging estimates based on DNA methylation levels have been recently proposed and shown to be accelerated in both blood and postmortem brain of patients with BD, although no study has been performed investigating the clinical drivers and implications of the epigenetic lifespan predictor GrimAge. The goal of this study was to investigate the relationship between blood GrimAge and GrimAge acceleration (AgeAccelGrim) with clinical, functioning, and cognitive outcomes in patients with BD and controls. Methods: We measured genome-wide DNA methylation levels and predicted GrimAge and AgeAccelGrim in a sample of N = 153 patients with BD (37.0 ± 11.2 years, 71.9% female) and N = 50 healthy controls (35.5 ± 10.4 years, 68% female) matched for demographic variables. Generalized linear models were employed to compare AgeAccelGrim between groups and identify its association with multiple clinical outcomes while controlling for demographic variables and blood cell proportions (CD4 + T-lymphocytes, monocytes, granulocytes, and B-cells). Adjusted p-values were derived via false discovery rate (FDR) correction for Type I error. Results: BD diagnosis was significantly associated with a higher AgeAccelGrim compared to controls (β = 0.197, p = 0.009, 21.8% higher), with significant group-dependent interactions found between AgeAccelGrim and blood cell proportions. In patients, five predictors demonstrated a significant relationship with AgeAccelGrim: short-term affective memory (β = -0.078, p = 0.030; -7.6%), short-term non-affective memory (β = -0.088, p = 0.018; -8.5%), inhibition (β = 0.064, p = 0.046; +6.6%), problem solving (β = -0.067, p = 0.034; -6.5%), and not currently smoking (β = -0.392, p < 0.001; -32.5% lower than smokers). No significant interactions were found between AgeAccelGrim with functioning, acute mood symptoms, current medication status, current lithium use, number of total hospitalizations, and BD subtype. Conclusions: Our results suggest that accelerated epigenetic aging, as measured by AgeAccelGrim, is increased in BD and significantly associated with smoking, blood cell proportions, and cognitive dysfunction. Future studies investigating the biological underpinnings of these associations are warranted. Disclosure: Compass Pathways, Relmada, Mind Medicine: Contracted Research (Self), Alkermes, Boeringer Ingelheim, J and J, Merck: Advisory Board (Self), Atai: Stock / Equity (Self) PANEL 48. The Genome Gut Microbiome and Metabolome Inform About Heterogeneity in Anxiety Depression, and Treatment Outcomes 48.4 Mapping the Peripheral Metabolome to Alterations in Functional Connectivity of Brain Networks in Major Depression Boadie Dunlop Emory University School of Medicine, Atlanta, Georgia, United States Background: Major depressive disorder (MDD) is characterized by heterogeneity of clinical presentations and biological disturbances. Peripheral metabolomic signatures differ across clinical dimensions of several CNS disorders and characterize the biological effects of treatments. However, whether peripheral metabolomic patterns impact CNS functioning, or can serve as peripheral markers of brain states has received very little study due to the paucity of datasets with both neuroimaging and metabolomic measurements. Methods: Peripheral metabolomic profiles and resting state functional magnetic resonance imaging (fMRI) were acquired at baseline study visits and repeated after 12 weeks of antidepressant medication or cognitive behavior therapy. Metabolomic analyses were conducted on the Gas Chromatography Time-of-Flight Mass Spectrometry (GC-TOF) non-targeted platform that enables detection of over 170 primary metabolites. fMRI data was analyzed using bilateral seeds for the affective network (subcallosal cingulate cortex) and the executive control network (dorsolateral prefrontal cortex). Results: Concentrations of individual peripheral metabolomic constituents, including glutamate, serotonin, indoxyl sulfate, and acylcarnitines, mapped to differential connectivity in both the affective and executive control networks. Dorsolateral prefrontal cortex connectivity within the executive control network correlated with peripheral blood concentrations of short-chain (positive correlation) and medium-chain (negative correlation) acylcarnitines. Changes in peripheral glutamate concentration positively correlated with affective network connectivity with supplementary motor region positively correlated with glutamate concentrations, and affective connectivity with anterior insula, anterior cingulate cortex, and premotor cortex correlated with peripheral concentrations of indoxyl sulfate (a gut-microbiome-derived metabolite of tryptophan). Conclusions: The peripheral metabolome can meaningfully inform about the pathobiology of MDD. Heterogeneity of clinical symptoms may be explained in part by variability in blood metabolites. These results open the door to developing treatments to target peripheral metabolic abnormalities that may contribute to psychiatric symptoms. Disclosure: Myriad Neuroscience, Aya Biosciences: Advisory Board (Self), Sage, Cerebral Therapeutics, Otsuka: Consultant (Self), Compass Pathways, Usona, Boehringer Ingelheim, Contracted Research (Self) STUDY GROUP 49. Perceptual Dysregulation Across Disorders: Toward a Computationally Defined Psychiatric Nosology Sahib Khalsa, Albert Powers, Daniel Javitt, Karen Quigley, Guido Frank, Sarah Stern, Martin Paulus, Jamie Feusner, Joost Haarsma, Sarah Garfinkel, Negar Fani, Guillermo Horga Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Study Group Summary: An accurate understanding of the environment and one’s place in it is critical for survival. Increasingly, formal computational models have been developed to quantitatively capture how organisms from rodents to humans encode and update neural representations of external and internal states. Abnormalities in these processes and their corresponding neural circuits have also been linked to the development of a range of exteroceptive and interoceptive forms of psychopathology. For example, inappropriate updating and an overweighting of expectations have been shown to correspond to severity of delusions and hallucinations, respectively. Similarly, anxiety has recently been suggested to depend on interoceptive inference, whereby physiological inputs maladaptively influence inferences about one’s own states of affect and arousal. Because inferential abnormalities and their corresponding symptoms cut across diagnoses, an understanding of psychopathology rooted in these factors would inherently transcend diagnostic boundaries. One possible benefit of such models is that they may more deeply inform the biological basis of symptom generation, maintenance, and resolution. This is because they are grounded in an understanding of sensory and perceptual neurocircuitry that has been well-delineated over the past 50 years. Lastly, both forward and backward translation to test new interventions based on these factors is made possible by the fact that formal neural process models for inference have been proposed and tested in both clinical and preclinical studies. The goal of this workshop is to discuss the relevance of perceptual processing to symptom formation. We have assembled a group of experts in perceptual inference across ages, academic ranks, diverse backgrounds, institutions, and continents, to discuss how commonalities and differences in various aspects of perceptual inference may inform the construction of a psychiatric nosology based more explicitly on formal models of neurophysiology. We envision a cross-generational conversation with established and emerging leaders in sensory and computational neurobiology and across psychiatric and neurological disorders to tackle pressing questions: How can we disentangle bottom-up from top-down processes? How do similarities or differences between exteroceptive and interoceptive inference inform the pathophysiology of psychiatric disorders? How can we instantiate better predictive coding models of dimensional psychopathology that surpass correlational modeling? What can we learn from disorders that exhibit interoceptive deficits, such as schizophrenia and autism spectrum disorders? How can interoceptive deficits in neurological disorders (e.g., allostatic-interoceptive network dysfunction in frontotemporal dementia, autonomic dysfunction in Parkinson’s, fatigue manifestations in multiple sclerosis, body symptoms in functional disorders) inform the pathophysiology of psychiatric disorders? How can neural process models of interoception translate to better tests and treatments for disorders which are commonly assumed to exhibit interoceptive deficits, such as eating disorders, anxiety disorders, PTSD, and substance use disorders? Will such models offer common strategies to refine or improve diagnostic assessment, treatment development, and prognostic prediction across all forms of mental health disorders? Disclosure: Nothing to disclose. PANEL 50. The Role of Kappa Opioid Receptors in Mediating Stress-Related Deficits in Reward Processing and Their Potential as Novel Therapeutics for Disorders Involving Anhedonia 50.1 Kappa Opioid Control of Dopamine Circuits and Changes With Stress Elyssa Margolis University of California, San Francisco, California, United States Background: Actions of endogenous opioid peptides at kappa opioid receptors (KORs) on dopamine neurons of the ventral tegmental area (VTA) contribute to anhedonia and related neuropsychiatric disorders. KOR agonist actions on the dopamine neurons are necessary and sufficient for their aversive signaling. KORs are expressed in most dopamine neurons, yet we found KOR activation inhibits somata that project to the medial prefrontal cortex (mPFC) or amygdala, but not nucleus accumbens. Understanding how KORs modulate dopamine neurons in different behavioral states will facilitate appropriate therapeutic targeting of specific KOR- and dopamine-related disorders. Methods: Whole cell electrophysiology recordings were made in VTA neurons in acute brain slices from slices from control or foot shock (0.5 s of 0.8 mA shock per min over 10 min) stressed male rats. Place conditioning was performed in 3 chamber boxes (two paired and one neutral context). Rats received 4 pairings per context with intra-VTA bilateral 0.5 uL containing 36 ng U69,593 or vehicle. Results: Where in naive (n = 50 neurons) or sham (n = 7 neurons) treated rats VTA neurons responded with either a hyper polarization or no response to the KOR agonist U69,593 (1 uM), in recordings from foot shock stressed rats approximately one third of VTA neurons tested responded with a depolarization (n = 8/22 neurons, p = 0.00013 naive vs stressed, permutation test). This switch persists for over 3 days after a single stressor. Brief incubation of VTA slices in corticotrophin releasing factor (CRF; 200 nM for 5 min) was produced a similar KOR switch. CRF specifically caused the switch in dorsal mPFC-projecting VTA neurons. Stress also eliminated intra-VTA U69,593 induced conditioned place aversion (sham difference score -210 + /- 50 sec, n = 11; shock difference score 90 + /- 60 sec, n = 10; one way ANOVA df = 1, f = 14.7, p = 0.001). Acute peripheral pain also generates a switch in KOR signaling (n = 4/15 VTA neurons). Conclusions: These observations show that aversive stressors generate a change in KOR signaling in a subset of dopamine neurons: KOR activation becomes excitatory. Elevated dopamine release in the dorsal mPFC can impair executive function leading to poor decision making, decreased motivation, and increased impulsivity. Thus, this KOR switch may be one maladaptive response by which stress impairs cognitive function. Disclosure: BlackThorn Therapeutics, Contracted Research (Self) 50.4 A Phase 2a Randomized, Double-Blind, Placebo-Controlled Study Investigating the Efficacy and Safety of the Kappa Opioid Receptor Antagonist JNJ-67953964 (Aticaprant) as Adjunctive Treatment for Major Depressive Disorder Mark Schmidt Janssen Research and Development, LLC, Beerse, Belgium Background: Aticaprant (JNJ-67953964), previously known as Cerecor (CERC)-501 and LY2456302, is a small molecule, high-affinity, selective kappa opioid receptor (KOR) antagonist. KORs and their native ligand dynorphin are localized in areas of the brain that mediate reward and stress behaviors and therefore may play a key role in mood, stress, and addictive disorders. Methods: This was a multi-center, double-blind, placebo-controlled, randomized, parallel-group study in subjects with MDD who have had an inadequate response to SSRI/SNRI treatment. The study consisted of a screening period and an 11-week treatment phase. The treatment phase consisted of 3 periods: a double-blind placebo (PBO) lead-in after which subjects were randomly assigned to receive aticaprant or to continue PBO for 6 weeks. Subjects who completed the double-blind treatment period entered a PBO withdrawal period. The primary endpoint was the change from baseline on the MADRS over the 6-week treatment period in subjects who did not respond to PBO during the lead-in period. Secondary endpoints included the Snaith-Hamilton Pleasure Scale (SHAPS) as an anhedonia measure. Results: A total of 184 participants were enrolled: 166 for the full ITT population and 169 in the safety population. The mean (SD) age was 42.6 (12.7) years, 72.3% were female. All participants but 1 had anhedonia (SHAPS ≥ 20) at baseline. The mean (SD) baseline MADRS at treatment baseline was 25.3 (7.86). The mean change from baseline in MADRS at Week 6 was -9.7 (8.02) for aticaprant and -6.6 (8.57) for PBO. The estimated LS Mean difference between aticaprant and PBO was -3.1 with 1-sided 80% CI upper limit of -2.21 (p = 0.002, observed effect size=0.36). In a pre-specified subgroup analysis, the magnitude of the overall antidepressant efficacy was greater in participants with elevated anhedonia compared with those with less severe anhedonia. 47.1% of participants on aticaprant and 35.7% of participants on PBO experienced at least one TEAE. The most common were headache 11.8%/7.1% and diarrhea 8.2%/2.4% for aticaprant/PBO. Pruritus was reported by 5.9% in the aticaprant group, none on PBO. Changes in clinical labs and vital signs were minimal. Conclusions: Adjunct aticaprant treatment of subjects with MDD results in significantly greater reduction on the MADRS compared to PBO and the safety profile is favorable. Disclosure: Janssen Research and Development: Employee (Self) MINI PANEL 51. Explore/Exploit Dilemma and Psychopathology 51.1 Explore-Exploit Tradeoff and Encoding of Reinforcement in the Human Dorsal Stream Alexandre Dombrovski University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Organisms face a difficult tradeoff between exploiting, known good options and exploring potentially better alternatives. Prior research has shown that, when exploring and exploiting a few discrete options, we rely on sampling and reinforcement histories encoded in our basal forebrain and prefrontal cortex. Additional demands, however, arise when we rapidly move though a continuous space, choosing when to harvest a reward, not unlike a primate moving through a forest. We show that exploration/exploitation in this environment relies on dynamic maps in the human dorsal stream, comprised of the caudal subnetwork encompassing the temporo-occipital MT + and the caudal posterior parietal cortex (PPC) and the rostral subnetwork encompassing rostral PPC and frontal ventral and dorsal premotor areas and the frontal eye fields (FEF). Methods: Studies 1 (fMRI) and 2 (MEG) employed Cohen and Frank’s clock task and a previously validated reinforcement learning model of exploration-exploitation (Hallquist and Dombrovski, Cognition, 2019). Results were replicated in Study 3 (fMRI). We used a new multi-level modeling network for neural signals (Dombrovski et al., Nat Commun, 2020). Results: BOLD signal in the dorsal attention network (DAN) and posterior cortical oscillations revealed reward signals evolving faster than in Skinnerian conditioning yet integrating a longer reinforcement history than a working memory (WM) buffer (AIC difference for neural model comparison >100 at all time points and locations). Exploitation scaled with updates to the PPC dynamic map encoding the total number of valuable options and spatially specific reward prediction errors for the chosen option (all p_fdr < .01). These spatially specific updates modulated posterior beta1/alpha oscillations 500-800ms post-reinforcement (p_fdr < .01) and BOLD throughout the frontal and parietal DAN nodes (p_fwe < .05). Exploration scaled only with reward prediction error signals in the rostral DAN subnetwork (p_fdr < .01). Finally, midline frontoparietal early theta oscillations were modulated by outcome valence (p_fdr < .01). However, theta activity was not spatially specific. Contrary to previous reports, it did not scale parametrically with reward prediction errors and did not facilitate exploration. Conclusions: Dynamic reward-laden posterior parietal map enable humans to exploit rapidly emerging opportunities and likely depend on beta1/alpha oscillations. Decisions to explore are made later in the dorsal visuo-motor transformation stream, in fronto-parietal regions close to the somatomotor interface. These observations are in line with Cisek’s affordance competition theory of dynamic decision-making. Disclosure: Nothing to disclose. 51.2 Failure to Resolve the Explore-Exploit Dilemma in Borderline Personality Disorder: Relationships With Dimensional Psychopathology and Suicidal Behavior Michael Hallquist University of North Carolina, Chapel Hill, North Carolina, United States Background: People with borderline personality disorder (BPD) become deadlocked in dysfunctional social interactions and fail to explore constructive alternatives. In a crisis, their search for solutions is often narrow and ineffective. Psychodynamic and cognitive-behavioral accounts of these phenomena have heuristic value but make no specific predictions about information processing and neural dynamics. Our prior theoretical work demonstrated that as successful decision-makers explore a large space, they manage cognitive load and converge on the best solution by selectively remembering preferred options and forgetting the rest. Methods: Our preliminary study of exploration in 115 individuals with BPD (36 high-lethality suicide attempters [BPD_HL], 48 low-lethality suicide attempters [BPD_LL], 31 non-attempters with BPD [BPD_NON]) and 53 psychiatrically healthy controls (HC) examined the relationship between exploration, BPD, history of suicidal behavior, and dimensions of psychopathology representing risk factors for suicide. Participants completed the clock task in the fMRI scanner. Our behavioral analyses employed the SCEPTIC computational model and multi-level frequentist models. We examined whether borderline personality, stability traits (neuroticism, conscientiousness, agreeableness) and history of suicidal behavior were associated with ineffective exploration and a failed transition to exploitation. A subsample of participants completed an ecological momentary assessment (EMA) study (N = 115: 25 BPD_NON, 34 HC, 38 BPD_HL, and 18 BPD_LL). We examined the relationship between behavior and prospectively observed suicidal ideation using multi-level structural equation modeling. Results: Individuals with BPD vs. HC engaged in narrow exploration and this pattern was particularly pronounced in BPD_HL and BPD_LL (chi2(3) = 13.07, p < .0045). High-lethality suicide attempts were further associated with a weaker win-stay/lose-shift pattern indicating the general learning deficit seen in our previous studies of serious suicidal behavior (chi2(3) = 27.69, p < 10^-5). Maladaptive levels of stability traits (neuroticism, agreeableness, conscientiousness) were associated with a similar learning deficit, but their effects did not explain the relationship between learning and suicide attempts. In the EMA study, impaired learning on the explore/exploit task predicted increased prospective suicidal ideation (beta = -0.222, p = 0.01, CI: -0.400, -0.027). Conclusions: Ineffective, narrow exploration of available options may underlie impaired social problem-solving and facilitate suicidal behavior in individuals with personality pathology. Suicidal behavior is additionally associated with basic learning deficits. Disclosure: Nothing to disclose. 51.3 Maladaptive Exploration and Avoidance in Anxiety Vanessa Brown University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Avoidance is a central clinical impairment in anxiety disorders, yet the development, maintenance, and reduction of avoidance is poorly understood. Conceptualizing clinical avoidance as a maladaptive resolution of the explore-exploit dilemma enables avoidance to be more precisely defined and studied through the lens of computational psychiatry. In decision-making, the explore-exploit dilemma defines the tradeoff between 1) exploring novel, potentially better options with 2) exploiting known good options when the outcomes of options are uncertain. We examined how humans resolved the explore-exploit dilemma in complex environments that capture real-life demands as well as how anxiety relates to maladaptive behavior in these environments. Methods: In Study 1, participants of both genders (N = 95) completed a probabilistic exploration task requiring adaptive resolution of the explore-exploit dilemma under varying levels of cognitive demands (environment size, memory demands). Participants’ behavior was compared to simulated behavior from random and value-maximizing agents using computational models of choice and exploration. In study 2, participants of both genders (N = 192) completed the probabilistic exploration task as well as two other tasks (horizon and grid tasks) measuring explore-exploit behavior while navigating environments with aversive or rewarding outcomes. Anxiety was measured using well-validated self-report measures (DASS and OASIS) and behavior was analyzed using multilevel computational and regression-based models of choices. Results: In Study 1, participants explored more overall than chance (difference in directed exploration parameter from hierarchical Bayesian computational model vs. random agent: median=2.48, 97.7% of MCMC samples > 0). With greater cognitive demands, participants explored less but did not become more random (effect of memory demands: median = -2.80, 95.5% < 0; environment size: median = -3.92, 99.8% < 0). In Study 2, participants reporting higher anxiety showed less adaptive exploration, particularly in aversive environments, resulting in worse performance and greater likelihood of more aversive outcomes (multilevel logistic regression predicting effects of anxiety on adaptive exploration measure from horizon task: z = -3.41, p < 0.001; multilevel linear regression predicting effects of anxiety on adaptive exploration measure on grid task: t = -7.63, p < 0.001; interaction with valence: t = -7.58, p < 0.001). Anxiety did not affect changes in exploration with cognitive demands on the probabilistic exploration task, ruling out an inability to cope with cognitive demands on the tasks as a possible explanation for poor exploration in anxiety (all z values < 2.0). Conclusions: Our data show that humans can adaptively navigate complex environments to effectively resolve the explore-exploit dilemma in a resource-rational manner. In anxiety, this ability is degraded, particularly in aversive environments, and may lead to maladaptive avoidance of uncertain, potentially aversive outcomes. Disclosure: Aya Technologies: Consultant (Self) MINI PANEL 52. Measuring Mental Health and Brain Health in the Era of Health Data Science 52.2 Measuring Anomalous Self-Experiences Using Natural Language Processing Techniques in Open Ended Speech Samples for Individuals at Risk for Psychosis Agrima Srivastava Icahn School of Medicine at Mount Sinai, New York City, New York, United States Background: Schizophrenia patients tend to have a heightened awareness of internal experiences, in which aspects of oneself are often experienced as similar to that of external objects. Anomalous Self Experience is measured using Clinical scale and, in this study, we used Natural Language Processing (NLP) to capture themes of anomalous self-experience in spoken language during open-ended interviews of early psychosis (EP) and clinical high risk (CHR) patients Methods: Speech was elicited during open-ended interviews from 129 Healthy Controls, 178 CHR, and 65 EP patients. The Bidirectional Encoder Representations from Transformers (BERT) model provides contextualized embeddings and was used to vectorize all the “I” sentences from the open-ended interviews and items in the Inventory of Psychotic-like Anomalous Self-Experiences (IPASE) scale. A Logistic Regression classifier was used to test the efficacy of using the vectorized sentences with “I” tokens in separating the patient group from Healthy Controls. Anomalous Self Experience was modeled by measuring the semantic similarity between the vectorized “I” sentences and items from the IPASE scale Results: Logistic Regression Classifier was able to separate the vectorized “I” sentences of CHRs with HC (AUC = 76.52) and EP with HC (AUC = 77.77). The CHR participants were found to be significantly closer to the reference IPASE sentences than healthy controls (s = 0.55, p < 0.01). Participants having EP were also found to be significantly closer to IPASE as opposed to healthy controls (s = 0.32, p < 0.01). Conclusions: Training a classifier using context-aware embeddings of sentences having the “I” tokens in conjunction with similarity to the IPASE reference items can help us separate patients having disturbances of self-awareness and consciousness. The current subjects under study do not have their IPASE ratings and that is one of the limitations of our study. In the future, we need to test the ground truth of our work by predicting the ratings of the IPASE scale from the text obtained from the spoken language during open-ended interviews of CHR and EP patients. Disclosure: Nothing to disclose. 52.3 Gaussian Mixture Model Class Profiles of Mobile Phone Typing Behaviors are Associated With Risk for Bipolar Disorder John Zulueta University of Illinois, Chicago, Illinois, United States Background: The goal of the BiAffect study is to develop models of psychopathology using data derived from mobile phone typing kinematics. Previous work from our group has demonstrated the feasibility of using such data to model measures of mood and cognitive function. In this study, we build on this work and identify different classes of user types based on mobile phone keyboard typing characteristics and demonstrate that Mood Disorder Questionnaire (MDQ) screening status is significantly associated with class membership. Methods: Phone usage data was obtained for participants enrolled in the BiAffect study from March 2018 to May 13, 2022. Only participants with at least 4 weeks of data were included (n = 440). Features that had been found to be relevant in previous models of mood and cognition were calculated, and a gaussian mixture model was fit to these data using the mclust package for R with the number of classes selected by using Bayesian information criteria (BIC) score. The data from the subset of participants who provided birth and gender and who had completed the MDQ (n = 252) were then used to fit stepwise multinomial regression models to predict class membership. Models were compared using ANOVA, and the Nagelkerke pseudo-R2 was calculated for each model. Results: Using BIC selection criterion, the optimal GMM model was a 5-class solution with ellipsoidal, variable volume, equal shape and orientation clusters. The BIC of this solution was 26,1383.18, and the next highest BIC was 26,134.25 for a 6-component solution with ellipsoidal, variable volume, variable shape, equal orientation clusters. The classes of the model vary in terms of their typing speed, session length, rate of backspace usage, and time to both initiate a backspace and resume typing after entering a backspace. Compared to the null model explaining class membership, the age only multinomial model was a significantly better fit Chi.Sq(4, 568) = 122.1, P < .001, Pseudo- R2 = 0.37; adding gender did not improve fit Chi.Sq (4, 566) = 1.9, P = 0.75, Pseudo- R2 = 0.38; and adding MDQ status improved fit Chi.Sq (4, 554) = 12.0, P = 0.018, Pseudo-R2 = 0.41. Positive MDQ status was associated with increased odds of membership in 2 classes. One of these classes was characterized as having typing sessions with the largest amount of characters typed very quickly, and the other class is notable for having relatively slower typing rates than the class with the most similar character count and error rate but decreased odds of association with a positive MDQ. Conclusions: Mobile phone keyboard typing behaviors can be classified into discrete classes. Controlling for age and gender, an elevated risk of bipolar disorder as indicated by a positive screen on MDQ is associated with membership into specific classes suggesting the existence of trait level differences that can be detected via analysis of phone usage metadata. Disclosure: Reckitt Benckiser Group PLC: Employee (Spouse), Reckitt Benckiser Group PLC: Stock / Equity (Spouse) PANEL 53. Progress Toward Clinical Applications of Genetically Encoded Neuromodulation 53.1 Theranostic Ultrasound-Mediated Blood-Brain Barrier Opening and Viral Gene Delivery With a Short-Pulse Sequence Alec Batts Columbia University, New York, New York, United States Background: Focused ultrasound (FUS) in conjunction with systemically administered microbubbles is a noninvasive, safe, and reversible strategy for targeted drug and gene delivery to the brain. We previously developed a technology called theranostic ultrasound (TUS) which combines focused, therapeutic pulses and microbubble cavitation imaging into a single, repurposed diagnostic ultrasound transducer configuration and have since characterized the feasibility for gene delivery by TUS-mediated blood-brain barrier opening (BBBO) in mice using a novel pulse sequence for bilateral BBBO. Methods: A P4-1 diagnostic imaging phased array (1.5 MHz transmit frequency, 1.0 MPa derated peak-negative pressure), was operated by a custom Verasonics MATLAB script to employ the rapid alternating steering angles (RASTA) pulse sequence for bilateral recombinant viral vector-mediated gene delivery in male C57BL6/J mice at two systemic doses: low (1.0e10 gc/mouse) and high (6.5e10 gc/mouse). The adeno-associated virus (AAV) construct consisted of the AAV9 serotype with a CAG promoter and GFP transgene and was systemically injected via the tail vein. The effects of 1.5, 5, and 10-cycle transmit pulse lengths were evaluated by quantification of GFP expression and concentration with fluorescence microscopy and ELISA, respectively. Results: Quantification of GFP fluorescence area 4 weeks after BBBO with TUS revealed significant increases in GFP fluorescence area with pulse length between the 5-cycle and 10-cycle pulse length groups (p = 0.0441) and between the 1.5-cycle and 10-cycle pulse length groups (p = 0.0023) at the low AAV dose. At the high dose, significant increases were observed between the 1.5-cycle and 10-cycle pulse length groups (p = 0.0414). ELISA results confirmed observations from fluorescence microscopy, where significant increases in GFP concentration were detected within all pairwise comparisons in both dose groups except for the 1.5 vs. 5-cycle comparison in the low dose group (all p < 0.05). Conclusions: AAV9 delivery with TUS RASTA presented a novel bilateral gene delivery platform within a single diagnostic imaging array configuration. Such a platform could provide a computationally flexible alternative to existing preclinical ultrasound-guided focused ultrasound (USgFUS) systems currently employed for viral delivery to the brain. Disclosure: Nothing to disclose. 53.2 Preclinical Validation of a Novel Chemogenetic Therapeutic Strategy for Opioid Use Disorder Lucas Sjulson Albert Einstein College of Medicine, Bronx, New York, United States Background: There is an urgent need to develop new treatments for opioid use disorder. Here we describe the development of a novel chemogenetic treatment strategy using a mutant Low-Affinity Mu Opioid Receptor (LAMuOR, pronounced “L’Amour”) in dopaminergic cells of the ventral tegmental area (VTA). We tested the hypothesis that LAMuOR would suppress exogenous opioid-induced dopamine release and eliminate the reward value of opioids. Methods: We injected Cre-dependent AAV-LAMuOR (10^7, 10^8, and 10^9 viral genomes (vg)) or negative control AAV into the VTA of DAT-IRES-Cre mice unilaterally (n = 6 per group, three males and three females), as well as AAV-hSyn-dLight into the ipsilateral nucleus accumbens. We then used fiber photometry to measure dopamine release in response to IP fentanyl or cocaine. In the second experiment, we injected AAV-LAMuOR into the VTA bilaterally (n = 6 per group, three males and three females) and tested these animals behaviorally for open field locomotion, sucrose preference, and oxycodone consumption in a two-bottle choice paradigm. Results: The 10^8 vg group showed a blunted dopamine response to fentanyl (p = 0.02), and the 10^9 vg group showed a reduction below baseline (p = 0.015). However, the response to cocaine was intact. IP fentanyl increased open field locomotion in the control and 10^7 vg groups (p < 0.01), but the 10^8 vg group showed no change (p = 0.43), and the 10^9 vg group showed a significant decrease (p < 0.01). There were no differences in sucrose preference (p = 0.8). Finally, the 10^8 and 10^9 vg groups showed decreased oxycodone consumption in two-bottle choice (p = 0.031). We did not find significant differences between male and female mice. Conclusions: These results suggest that LAMuOR may be a promising chemogenetic treatment strategy by which a single treatment could permanently eliminate the rewarding properties of opioids and confer lifelong protection against opioid use disorder. Disclosure: Nothing to disclose. 53.3 Negative Feedback Chemogenetic Neuromodulation: Targeted Habenular Expression of an Excitatory Cocaine-Sensitive Chemogenetic Receptor Decreases Cocaine Self-Administration Mike Michaelides National Institute on Drug Abuse, Baltimore, Maryland, United States Background: Pharmacological interventions and neuromodulation represent promising strategies for treatment of substance use disorder (SUD). However, such approaches can have undesirable side effects because they can influence general purpose motivational processes. Chemogenetics is a neuroscience technology that permits drug-controlled neuromodulation of discrete neural circuit pathways by relying on the use of engineered receptors, which are introduced in a cell type of interest, and are inert until activated by a cognate chemical agonist. Such receptors can be used in a negative feedback process to blunt drug abuse, either by reducing reward or increasing aversion solely during drug intake. To examine the feasibility of such a strategy, we expressed a novel cocaine-sensitive chemogenetic receptor (coca5HT3) into the lateral habenula (LHb), referred to as the brain’s “anti-reward” center, in rats and examined their propensity to self-administer cocaine using the intravenous drug self-administration (IVSA) procedure. Methods: Adult male Long Evans rats were injected with an adeno-associated virus (AAV), AAV-hSyn-coca5HT3-ires-mCherry into the LHb (AP: -3.8; ML: ± 0.8; DV: -4.8, bregma) (n = 10) or underwent sham surgery (n = 15). Rats were then trained to self-administer food in daily operant sessions using a fixed ratio 1 (FR1) schedule followed by FR5 testing. Rats then underwent surgery for jugular vein catheterization and began daily cocaine IVSA (0.5 mg/kg/infusion) training sessions for 10 days, initially on FR1 and then on FR5 for the last 5 days. Rats were then tested for IVSA on an FR5 schedule using different cocaine unit doses (0.0625, 0.125, 0.25, 0.5, and 1 mg/kg/inf) presented in randomized order. Rats were then euthanized and assessed for LHb coca-5HT3 expression. Results: Rats expressing coca5HT3 in LHb did not differ from controls in food SA or cocaine IVSA training but showed significantly lower active lever presses (p = 0.01), infusions (p = 0.005), and intake (p = 0.01) of cocaine in the dose-response phase of the IVSA. Conclusions: Expression of an excitatory cocaine-sensitive chemogenetic receptor in the LHb led to a significant decrease in cocaine IVSA supporting the notion that negative feedback chemogenetic receptor activation by addictive drugs may provide a promising new gene therapy approach to selectively treat SUDs. Disclosure: Redpin Therapeutics, Attune Neurossciences: Contracted Research (Self) PANEL 54. Positive Psychology Interventions for Depression and Their Mechanisms of Action 54.3 Amygdala Neurofeedback During Positive Memory Recall for Treatment Resistant Depression Kymberly Young University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Previous work has shown that patients suffering from depression can increase their amygdala response while recalling positive memories via real-time neurofeedback training, and these changes are associated with reductions in depressive symptoms and normalization of processing biases. This study investigated if this intervention might also be effective in patients suffering from depression who do not respond to standard psychological and pharmacological interventions, i.e., treatment resistant. Methods: 16 participants received 5 neurofeedback sessions training them to increase their amygdala activity while recalling positive memories. Outcomes measures were depressive symptoms assessed by BDI scores up to 12 weeks follow-up, changes in autobiographical memory specificity, and, neurofeedback success, operationalized as percent signal change in the amygdala from initial baseline to final transfer run. Results: Neurofeedback success was significant; participants succeeded in increasing their amygdala activity. There was a main effect of visit on BDI scores, indicating a significant decrease in depressive symptomatology starting at the fourth neurofeedback session and lasting up to 12 weeks follow-up. There was also a significant increase in the percent of positive specific autobiographical memories recalled. Conclusions: Although preliminary and in need of a control group, these results suggest that amygdala neurofeedback could constitute an interesting avenue as a new non-pharmacological, non-invasive intervention for patients with treatment resistant depression. Disclosure: Nothing to disclose. 54.4 Hippocampal Cells Multiplex Positive and Negative Engrams Steve Ramirez Boston University, Boston, Massachusetts, United States Background: The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory. Recent studies have demonstrated vast structural and functional heterogeneity along the dorsal-ventral axis of the hippocampus, and while much is known about how the dorsal hippocampus processes spatial-temporal content, much less is known about whether or not the ventral hippocampus (vHPC) partitions positive and negative experiences into distinct sets of cells. Methods: Here, we utilize activity-dependent and transgenic approaches to tag positive and negative hippocampus-mediated engrams in a within-subject manner. Moreover, we used a combination of RNA sequencing, immunohistochemical approaches, and projection-specific optogenetic perturbations to catalogue the molecular, cellular, and behaviorally relevant landscape of positive and negative engrams. Results: The combination of transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence-specific engrams in the vHPC demonstrated two partially segregated cell populations and projections that respond to positive and negative experiences. Next, using an RNA sequencing approach, we find that vHPC positive and negative engram cells display distinct transcriptional programs compared to a neutral engram population. Additionally, while optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive preference or avoidance, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), drives preference and avoidance (N = 8-10 mice in all groups; p < 0.01 for all findings). These terminals can also undergo a “switch” or “reset” in their capacity to drive either, thereby demonstrating their flexible contributions to behavior. Conclusions: We conclude that the vHPC contains genetically, cellularly, and behaviorally distinct populations of cells processing positive and negative memory engrams. Together, our findings provide a novel means by which to visualize multiple engrams within the same brain and point to their unique genetic signatures as reference maps for the future development of new therapeutic strategies. Disclosure: Nothing to disclose. STUDY GROUP 55. Biology of Social Determinants of Mental and Cognitive Health Dilip Jeste, Dolores Malaspina, Steven Cole, Kara Bagot, Joan Luby, Faith Dickerson, Tarek Rajji La Jolla, California, United States Study Group Summary: Social determinants of mental and cognitive health (SDoMCHs) are non-medical factors such as early childhood environment, education, poverty, social isolation, and racial and other forms of discrimination, that significantly impact health and healthcare disparities. Research into biology of SDoMCHs is an exciting new frontier. Adverse SDoMCHs like poverty and racism produce pro-inflammatory effects that may cause accelerated aging. However, potentially reversible epigenetic processes may alter ability to cope with social stressors, thus increasing a person’s vulnerability vs. resilience to future environmental stressors. HPA axis and microbiome gut brain axis influence behavior and “social brain” across the lifespan. Structural racism refers to ways in which societies foster discrimination via inequitable systems that reinforce discriminatory values and resource distributions. Maternal racism- and poverty-related distress is found to result in adverse intrauterine environment and immune system dysfunction, resulting in premature births and lower birth-weight infants among people of color, and subsequent segregation in impoverished neighborhoods with environmental pollutants, leading to higher rates of disease and death. This study group will discuss challenges to advancing basic and clinical research on biological mechanisms underlying effects of SDoMCHs across the lifespan, and developing bio-psycho-sociaI interventions. The participants have studied the effects of neurobiology of social isolation (Jeste), early life adversity affecting lifelong gene expression (Malaspina), early life influences of SDoMCHs on neuroimmune interactions (Cole), biological effects of structural racism from intrauterine to built environments in people of color (Bagot), biological embedding of poverty in a developing brain (Luby), and interventions modifying the effects SDoMCHs on neuroplasticity through neurostimulation (Rajji), and unique aspects of microbial diversity in Costa Rican centenarians (Tomás). We will discuss several issues.What novel approaches can advance studies of biology of SDoMCHs? How to untangle complex networks of biobehavioral influences including reciprocal interactions among social status, psychological experience, stress biology, and immune cell gene regulation? What neurobiological mechanisms are involved in accelerated aging underlying premature mortality in people with cumulative adverse effects of SDoMCHs beginning in early childhood? In contrast, how can higher microbial diversity in Costa Rican blue zone, and greater abundance of A. muciniphila in mice with longer lifespan, lead to discovery of probiotics to promote healthy aging and longevity? What are the implications of intrauterine adversity leading to structural changes in fetal brain for developing preventive interventions during pregnancy to enhance fetal development? How do SDoMCHs modulate brain plasticity, facilitating or hindering brain stimulation effects, and how can they be used to develop personalized neurostimulation strategies? What techniques can help implement primordial illness prevention by mitigating adverse SDoMCHs at public health level, and promote positive determinants like resilience at individual level? How can we use known basic biology of social isolation to develop behavioral vaccines to enhance positive social connections? Disclosure: Nothing to disclose. PANEL 56. Norepinephrine in Motivated Behavior: How Neuronal Activity, Cell Types, Circuits, and Receptors Interact to Influence Stress, Reward, Eating, and Choice 56.1 Natural Locus Coeruleus Dynamics During Feeding Natale Sciolino University of Connecticut, STORRS, North Carolina, United States Background: The ability of the brain to integrate internal physiological drives, such as hunger and satiety, with an ever-changing environment is essential for survival. It is well-known that noradrenergic neurons of the locus coeruleus (LC-NE) play a key role in modulating diverse physiological and behavioral states¬ such as arousal, sensory processing, stress, and attention. Upon the presentation of salient environmental stimuli, including non-noxious and aversive events, LC-NE neurons are robustly activated. Much less is known about the role of LC-NE neurons in the regulation of feeding and the integration of internally driven motivational states, such as hunger and satiety. Methods: Here, we combined in vivo fiber photometry calcium imaging, optogenetics, and chemogenetics with behavioral and metabolic approaches to test the hypothesis that endogenous LC-NE activity is dynamically modulated during feeding in the mouse model system. Results: We demonstrate that the activity pattern of LC-NE neurons is enhanced during food approach and suppressed during feeding behavior in fasted mice. The approach-related LC-NE neuronal responses were completely abolished later in the session when mice had consumed more food, whereas the consummatory-related neuronal responses were slightly attenuated, indicating that satiety state influences food-related LC activity. Further, we found that chemogenetic activation of LC-NE neurons suppressed feeding without altering metabolism. Activation of LC-NE neurons also resulted in modest weight loss (2% loss). Using optogenetics, we found that feeding is suppressed when LC-NE neurons are activated by either a brief stimulation paired with feeding or stimulation over a longer duration. Lastly, we identified an LC-lateral hypothalamus circuit that suppresses feeding and elicits aversion and anxiety-like behavior. Conclusions: In context with the broader LC literature, our findings suggest that LC-NE neurons are involved in the modulation of feeding by integrating both external cues (e.g., anxiogenic environmental cues) and internal drives (e.g., satiety). Disclosure: Nothing to disclose. 56.2 Decoding Locus Coeruleus Heterogeneity in Arousal and Stress Michael Bruchas University of Washington - Seattle, Seattle, Washington, United States Background: Previous studies have revealed a pericoerulear (peri-LC) group of GABAergic neurons that have been relatively uncharacterized, but thought to modulate arousal by inhibiting the locus coeruelus noradrenergic system. Methods: We defined the functional connectivity between those neurons and the LC. We performed single-nuclei RNA sequencing and spatial transcriptomics to investigate the molecular features of both LC and peri-LC cell types. Finally, we used a compliment of strategies to record and manipulate, the activity of peri-LC GABAergic neurons during arousal-related behaviors. Results: We found monosynaptic connectivity between the peri-LC and NE cells groups. Next, we measured neuronal activity in the peri-LC GABAergic neurons using fiber photometry (n = 14 mice), and found varying responses to different arousing stimuli, including predator odor exposure (p < 0.0001; paired t-test), foot shock (p < 0.0001; paired t-test), open field test (OFT) (p < 0.0001; paired t-test), and novel object test (p < 0.001; paired t-test). Additionally, optogenetic activation of peri-LC GABAergic neurons (n = 7 ChR2 mice; n = 10 control) induced pupillary constriction (p < 0.05, unpaired t-test) and decreased locomotion (genotype main effect p < 0.001; frequency of stimulation p < .01; two-way ANOVA with Dunnett’s multiple comparison test), suggestive of decreased arousal; chemogenetic silencing of these neurons (n = 9 DREADD mice; n = 8 control) decreased center time in the OFT (p < .01; unpaired t-test), time on the light side in light-dark box test (p < .001; unpaired t-test), and open arm time in an elevated zero maze (EZM) (p < .05; unpaired t-test). Then, we examined the transcriptional profiles of LC-NE (n = 1,324) and peri-LC GABAergic (n = 11,182) neurons using single-nuclei RNA sequencing, mapped these molecular features using PIXELseq (spatial transcriptomics). In particular, we found neuropeptide-cognate receptor pairs, including Pnoc/Oprl1, Penk/Oprd1, and Tac1/Tacr1 (>10%), denoting potential mechanisms of LC modulation by the peri-LC and using photometry to show that peri-LC subpopulations respond differently to various arousing stimuli. Conclusions: We report that the peri-LC GABAergic neurons consist of transcriptionally heterogeneous groups which integrate diverse inputs to modulate arousal, stress, and avoidance behaviors. Disclosures: Neurolux, Inc: Board Member (Self), Kypha, Inc: Consultant (Spouse) 56.3 NE-One’s Guess: Noradrenergic Influences on Decision Making Elena Vazey University of Massachusetts, Amherst, Massachusetts, United States Background: Frontal cortex regions are critical for goal-directed choices and action selection. They achieve these functions with input from ascending neuromodulators, including norepinephrine (NE) released from the locus coeruleus (LC). One region of interest for behavioral choice is premotor cortex (M2), a region heavily innervated by LC-NE where neural correlates of behavioral choice can be identified. Methods: We used a β adrenergic antagonist (propranolol) and recorded from M2 while Long-Evans rats (n = 10 female and n = 9 male) performed a simple decision-making task, the two alternative forced choice task. We analyzed data from 347 well isolated single units from M2 in both sexes. In a separate group of rats (n = 4 of each sex) we used RNAscope to evaluate β1 and β2 adrenergic receptor density in M2. Results: Both male and female rats performed the decision task well. Using a sparsely trained linear classifier we identified the rapid onset of distinct and highly accurate neural patterns associated with behavioral choice in M2 elicited by cue presentation and maintained in M2 through to the completion of a lever press. Blocking β adrenergic signaling did not impact basal firing properties but greatly reduced the decoding in M2, particularly in in females. The disruption of choice representation in M2 was associated with reduced suppression of irrelevant choice signals. Alongside this disruption of neural encoding, we saw a marked increase in decision time and response times, in addition to a reduction in task participation and an increase in omitted responses, particularly in females. We used RNAscope to determine if these differences in β adrenergic regulation of decision-making behavior and neural signaling could be explained by differences in adrenergic receptor density. We found that females had higher levels of β2 adrenoreceptor expression in M2 and that these receptors were strongly expressed on GABAergic interneurons. Conclusions: Blocking β noradrenergic signaling impairs decision making behavior in a sex-dependent manner, with females more sensitive to cognitive disruption of choice behavior. Blocking β adrenergic signaling led to a disruption of choice signals in M2 by preventing suppression of irrelevant choice signals. In complement, females showed higher density of β2 adrenergic receptor expression than males on interneurons in M2. Disclosure: Nothing to disclose. 56.4 Novel Roles of Alpha2a-Adrenergic Heteroceptors in Stress and Reward Danny Winder Vanderbilt University, Nashville, Tennessee, United States Background: Alpha2a-adrenergic receptor (alpha2a-AR) agonists are candidate substance use disorder therapeutics due to their ability to recruit noradrenergic autoreceptors to dampen stress system engagement. However, we recently found that postsynaptic alpha2a-ARs are required for stress-induced reinstatement of cocaine-conditioned behavior. Methods: We utilized a variety of approaches in FosTRAP (Targeted Recombination in Active Populations) mice to define an ensemble of cells activated by the alpha2a-AR partial agonist guanfacine (“Guansembles”) in the bed nucleus of the stria terminalis (BST/BNST), a region key to stress-induced reinstatement of drug seeking. Male and female mice were used in these studies. Results: We define BNST “Guansembles” and show they differ from restraint stress-activated cells. Guanfacine produced inhibition of cAMP-dependent signaling in Guansembles, while chronic restraint stress increased cAMP-dependent signaling. Guanfacine both excited and inhibited aspects of Guansemble neuronal activity. Further, while some stressors produced overall reductions in Guansemble activity, active coping events during restraint stress and exposure to unexpected shocks were both associated with Guansemble recruitment. Using viral tracing, we define a BNST Guansemble afferent network that includes regions involved in the interplay of stress and homeostatic functions. Finally, we show that activation of Guansembles produces alterations in behavior on the elevated plus maze consistent with task-specific anxiety-like behavior. Conclusions: Overall, we define a population of BNST neurons recruited by alpha2a-AR signaling that opposes the behavioral action of canonical autoreceptor alpha2a-AR populations and which are differentially recruited by distinct stressors. Moreover, we demonstrate stressor-specific physiological responses in a specific neuronal population. Disclosure: Nothing to disclose. PANEL 57. Gene, Splicing and Isoform Regulation in the Developing Human Brain Informs Novel Psychiatric Genetic Mechanism 57.2 MicroRNA-eQTLs in the Developing Human Neocortex Link miR-4707-3p Expression to Brain Size Jason Stein University of North Carolina, Chapel Hill, North Carolina, United States Background: Genome-wide association studies (GWAS) have identified many genetic loci influencing human behavior, cognition, and brain structure. Expression quantitative trait loci (eQTL) data is often used to link non-coding brain-trait associated loci with genes that putatively mediate their effects. Brain eQTL studies are most often conducted in bulk adult post-mortem tissue and are focused on measuring mRNA expression levels from protein-coding genes. Though these methods have been successful in linking a subset of non-coding brain-trait associated loci to genes, there may be multiple mechanisms by which a single locus influences a complex trait, and many loci are still unlinked to genes. This suggests other types of RNAs, unmeasured in previous eQTL studies, may be mediating the genetic associations. Methods: We profiled the expression of miRNAs across fetal cortical tissue samples from donors between 14 and 21 gestation weeks using high-throughput small-RNA-sequencing. Following TOPMed mixed-ancestry imputation, 12.4 million genetic variants were combined with the expression of 907 known and novel miRNAs across 212 fetal cortical tissue samples to perform a local-miRNA-eQTL analysis. Results: We quantified expression of 907 miRNAs, discovering 111 novel early brain-expressed miRNAs, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and head size phenotypes, where the miRNA expression increasing allele was associated with decreased head size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells led to increased proliferative and neurogenic gene markers, indicating miR-4707-3p modulates progenitor proliferation. Conclusions: Using small-RNA-sequencing, we reveal robust miRNA expression across cortical tissue during mid-gestation, a stage and tissue which has not previously been captured in previous eQTL studies using standard RNA-sequencing techniques. Integrating miRNA-eQTLs with existing GWAS yielded discovery of a miRNA modulating developmental fate decisions that alter human brain size. Disclosure: Nothing to disclose. 57.3 Cell-Type-Specificity of Isoform Diversity in the Developing Human Neocortex Informs Mechanisms of Neurodevelopmental Disorders Luis de la Torre-Ubieta David Geffen School of Medicine at UCLA, Los Angeles, California, United States Background: Human brain development is under tight molecular genetic control and the recent advent of single cell genomics has revolutionized our ability to elucidate the diverse underlying cell-types and states. Although RNA splicing is highly prevalent in the brain and has strong links to neuropsychiatric disorders, including autism (ASD) and schizophrenia, previous work has not systematically investigated the role of splicing and transcript-isoform diversity in human brain development at single-cell resolution. Methods: We leverage single molecule long-read sequencing (PacBio IsoSeq) to deeply profile the full-length transcriptome of >7000 individually barcoded single-cells in the developing human neocortex at mid-gestation from 3 independent donors. Results: We identify 173,783 unique isoforms, corresponding to 22,391 genes expressed across 16 distinct cell-type clusters. Remarkably, we find that only 27.4% of these isoforms were previously known, and 17.5% of known genes harbor completely novel exons. Novel exons are observed in high-confidence ASD risk genes and are strongly enriched for neuropsychiatric genome-wide association (GWAS) signal. Excitatory neuron lineages exhibit the greatest isoform diversity, and we uncover myriad novel isoform-switching events occurring during the transition from mitotic radial glia progenitors to maturing excitatory neurons. Furthermore, isoform-based single cell clustering identifies previously uncharacterized cell subtypes, including a migrating excitatory neuron cluster associated with genetic risk for schizophrenia. Conclusions: This work highlights the important contribution of transcript-isoform diversity in cellular identity and cell fate specification in the developing neocortex and illuminates novel risk mechanisms for neuropsychiatric disease. Disclosure: Nothing to disclose. 57.4 Cross-Population, Cell-Type Informed Atlas of Gene, Isoform, and Splicing Regulation in the Developing Human Brain Michael Gandal University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Genome-wide association studies (GWAS) have identified hundreds of loci associated with neurodevelopmental psychiatric disorders, yet our understanding of the risk genes and mechanisms underlying these associations remains a major challenge. Strong enrichment for psychiatric genetic risk among fetal brain regulatory elements has prompted several recent efforts to characterize gene expression regulation in the developing human brain through expression quantitative trait loci (eQTL) for integration with GWAS. Yet, previous integrative efforts have been hindered by the small scale of individual studies, differences in ancestral composition, and lack of investigation of cell-type-specific mechanisms. Methods: Here, we uniformly process and systematically mega-analyze gene-, isoform-, and splicing-QTLs, as well as cell-type specific QTLs from 9 deconvoluted cell-types, in the developing human brain across 682 donors spanning European (N = 292), African American (N = 164), and Admixed American ancestries (N = 145). Genotypes were imputed into the TOPMed multi-ethnic reference panel. Gene and isoform expression was quantified using salmon and splicing patterns were assessed using leafcutter. Results: We identified 17,847 genes harboring a significant QTL, including 2,095 (11.7%) only detectable within a cell-type-specific context. Network-based integration of fetal single-cell chromatin accessibility further mapped specific cell-types for 18.4% of eQTL containing genes. Cross-population fine-mapping significantly improved resolution to detect underlying candidate causal variants, and joint statistical fine-mapping with GWAS identified high confidence colocalized SNP-gene pairs for nearly two-fold more schizophrenia GWAS loci than observed in the adult brain. In parallel, transcriptome-wide association prioritized dozens of new candidate risk mechanisms underlying SCZ and BD. Finally, we build a comprehensive set of developmentally regulated gene and isoform co-expression networks capturing unique genetic enrichments for psychiatric disorders. Conclusions: Together, this work provides a comprehensive, cross-population view of genetic regulation in the developing human brain and its contribution to complex developmental and psychiatric traits. Disclosure: Nothing to disclose. PANEL 58. Single Cell Technologies to Understand Brain Function at the Cell and Circuit Level in Animals and Humans 58.1 Developmental, Lineage and Local-Context Effects on Brain Cell Types Revealed by Single Cell Sequencing Fenna Krienen Princeton University, Princeton, New Jersey, United States Background: Marmosets are New World monkeys that share fundamental aspects of brain cell type composition with other primates including humans. Marmosets are an emerging model for translational neuroscience and genomic research, due to their small size, rapid reproductive cycle, and evolutionary relationship to humans. Another major practical advantage of the marmoset compared to other primates is its relatively small brain and lower levels of myelination, which facilitate quantitative spatial mapping and reconstruction of cell type morphology. Methods: We performed single nucleus RNA sequencing of 2.4 million brain cells sampled across 11 cortical locations and 7 subcortical structures (hypothalamus, amygdala, brainstem, cerebellum, basal forebrain, striatum, thalamus) in the adult marmoset brain (n = 10, 5 female). We used latent factor analysis and gene set enrichment analysis to learn factors that underlie shared and distinct transcriptomic signals across neuron types. GABAergic interneuron types in the forebrain were spatially profiled and quantitated using single molecule FISH (smFISH; RNAscope) across whole sagittal sections (4 sections per type). RNAscope puncta were quantitated with the TissueFAXS Viewer Software. We reconstructed the morphology of a subset of these types with GFP AAV labeling. Results: We resolved 288 neuronal and non-neuronal types across brain structures. Within cortex, excitatory neurons, inhibitory neurons, and astrocytes all exhibited regional differential expression with moderate sharing across neuron types and very little between neurons and glia. Hierarchical clustering of expression profiles revealed unexpected relationships between neuron types across phylogenetically distinct brain structures. smFISH exhibited gradients in proportions of target types and intensity within interneuron populations of cortex and striatum. Using AAV GFP sparse labeling combined with smFISH, we visualized the morphology and spatial distribution of major forebrain interneuron types, including the recently discovered primate specific TAC3+ interneuron population for the first time. Conclusions: We used single nucleus RNA sequencing, viral labeling and smFISH to provide novel insights into the diversity and sharing of neural types and states across major structures of the primate brain. Disclosure: Nothing to disclose. 58.3 Integrated Single Cell and Spatial Transcriptomic Analysis in Reward Circuitry of the Human Brain Kristen Maynard Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: Given the close relationship between brain structure and function, assigning gene expression to distinct anatomical subdivisions and cell populations within reward structures of the human brain improves our understanding of these regions. Emerging approaches like spatial transcriptomics can quantify RNA transcripts within tissue architecture thereby retaining both anatomical and transcriptome-scale molecular information. Combined with single nucleus RNA-sequencing (snRNA-seq), integrative spatial maps of topographically organized cells can be generated. Methods: To further our understanding of the molecular architecture across key nodes of reward circuitry, we used the 10x Genomics Visium and single cell gene expression platforms to generate spatial maps of gene expression in the dorsolateral prefrontal cortex (DLPFC) of the adult human brain (n = 10 neurotypical donors). We also performed snRNA-seq in the human nucleus accumbens (NAc) and habenula (Hb, n = 7 neurotypical donors) to generate a molecular atlas of cell types in these regions critical for reward processing. Results: Using unsupervised clustering, we provide a data-driven molecular neuroanatomical atlas of the DLPFC across its anterior-posterior axis (n = 113,927 spots, n = 30 sections). We defined unique gene expression signatures for unsupervised spatial domains at different resolutions (k = 9, 16, 28). We added cellular resolution to this molecular atlas by performing spot deconvolution with snRNA-seq data from spatially adjacent tissue sections from the same donors. In parallel, we generated snRNA-seq data in the NAc and Hb and identified transcriptionally distinct cell types, including subpopulations of medium spiny neurons in NAc and lateral and medial populations in Hb. Finally, we evaluated the enrichment of genes associated with neuropsychiatric disorders in discrete spatial domains and cell populations across these three regions. Conclusions: We define the first unsupervised molecular neuroanatomical atlas of neurotypical DLPFC. We also provide the first molecular profiles of distinct cell types in the human NAc and Hb. By integrating these data with neuropsychiatric gene sets, we provide novel insights into how genetic risk for psychiatric disorders maps to underlying brain structure and function. Disclosure: Nothing to disclose. 58.4 Pharmacogenomic Discovery in Villages of Neural Cells From a Cohort of Individuals With Severe Psychiatric Disorders Ralda Nehme Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States Background: How the genetic variation associated with psychiatric disorders affects cells and their properties is unknown. To understand this, we used neurons and astrocytes derived from iPSCs from individuals with severe psychiatric disorders and studied the dynamic responses of cells to perturbations of their physiology. Methods: In the cell village approach, cells from many donors are mixed, processed, and scored for phenotypes together, eliminating variability. Each cell is then assigned to its donor-of-origin using combinations of SNPs ascertained in the sequencing data. We generated cell villages of iPSCs, neuronal progenitors, neurons, and astrocytes from a cohort of 44 individuals (males and females) including 20 with psychiatric disorders. We subjected these villages to pharmacological perturbations including cholesterol regulators (Simvastatin, 10uM; Atorvastain, 10uM; and Efavirenz, 10uM) and the antipsychotic Clozapine (30uM), and performed scRNAseq and Gene Set Enrichment Analysis on treated and untreated villages. Results: When exposed to Clozapine both neurons and astrocytes upregulated cholesterol biosynthesis genes. How Clozapine increases expression of cholesterol genes is unknown, yet clinical data suggests schizophrenia patients have impaired cholesterol biosynthesis, and those treated with Clozapine display higher blood lipid levels. Increasing cholesterol synthesis in the brain may thus explain why Clozapine is so effective in these patients. Conversely, statin treatments drove discreet changes in each cell type. When exposed to simvastatin and atorvastatin, but not Efavirenz, neurons upregulated genes involved in cholesterol biosynthesis. In astrocytes, however, simvastatin and atorvastatin significantly affected viability and upregulated genes involved in cytoskeletal matrix organization and cell migration and downregulated pathways mediating synaptic homeostasis and cell adhesion, possibly via regulating GTPases such as RHOA and RHOB, both upregulated in astrocytes. Conclusions: Our findings suggest Clozapine treatment increases the expression of cholesterol synthesis genes in both neurons and astrocytes, suggesting a possible mechanism of action, while statin treatment alters GTPase signaling in astrocytes, impacting cell adhesion and synapse integrity, and reducing overall viability. Disclosure: Nothing to disclose. PANEL 59. Broadcasting Cholinergic Control of Memory Across Time: How Slow Can You Go? 59.2 State-Dependent Spatiotemporal Dynamics of Cholinergic Signaling in the Cortex Jessica Cardin Yale Medical School, New Haven, Connecticut, United States Background: Classical views suggest that variation in cortical activity associated with behavioral state fluctuations reflects the brain-wide, homogeneous influence of ascending neuromodulatory systems such as acetylcholine (ACh). However, recent studies indicate substantial diversity of cholinergic neurons and the spatiotemporal relationships between cholinergic signaling and neuronal activity remain unknown. We used mesoscopic imaging across the neocortex of awake mice to quantify relationships between behavioral state, cortical activity, and cholinergic signaling. Methods: To simultaneously monitor neuronal activity and cholinergic signaling, we expressed the red fluorescent calcium indicator jRCaMP1b and the green fluorescent ACh sensor ACh3.0 throughout the brain. We used mesoscopic imaging of both reporters through the intact skull of mice that were head-fixed and freely running on a wheel (n = 6). We compared across distinct behavioral states: quiescence, high facial movement, and locomotion. Results: Both ACh and calcium signals exhibited spatially heterogeneous, spontaneous fluctuations across the cortex. ACh activity varied across cortical areas (p < 0.001) and differed by state (p = 0.015, two-way repeated measures ANOVA, n = 6 mice per group). Inter-areal correlations in ACh signaling were heterogenous and state-dependent (adjusted p < 0.01, Benjamini-Yekutieli FDR corrected permutation test for each pair, n = 6 mice). Cholinergic coupling to neuronal activity varied with area and state, with increased correlation between the two signals broadly across the cortex (adjusted p < 0.01) during high facial motion but a selective reduction in frontal correlations during locomotion (adjusted p < 0.01; Benjamini-Yekutieli FDR corrected permutation test for each pair, n = 6 mice). Cholinergic signaling prior to, but not during, the stimulus in a visual detection task was predictive of trial-by-trial performance in expert but not naive animals (n = 4 mice). Conclusions: We find state-dependent spatiotemporal fluctuations in cholinergic signaling. The relationship between cholinergic modulation and cortical activity is spatially compartmentalized, state-dependent, and exhibits plasticity during learning. Disclosure: Nothing to disclose. 59.3 Functional Consequences of Lateral Entorhinal Cortical Cholinergic Dysfunction With Age Mala Ananth National Institutes of Health, Bethesda, Maryland, United States Background: Alterations to basal forebrain cholinergic circuits are a hallmark of cognitive impairment, however the time-course and relevance of these changes are not well understood. Cholinergic projections to the entorhinal cortex (EC) are of particular interest, as the EC has been identified as a seed of vulnerability early in aging. Using the EC as a model of early cortical vulnerability, we investigated the role of the cholinergic system in EC function and dysfunction with age. Methods: In parallel studies across humans and rodents, we examined the relationship between cholinergic system health and EC function. We used Positron Emission Tomography (PET) imaging with [18F]VAT, a presynaptic cholinergic ligand that serves as a marker of cholinergic synapse integrity, to compare EC [18F]VAT binding between healthy volunteers vs. those that presented with cognitive impairment. In rodents, we used confocal imaging to compare cholinergic terminal density in the EC in animals with and without aging pathology. Using behavioral assays, we assessed the consequences of age on EC function. To better understand mechanisms underlying EC dysfunction with age, we used activity dependent markers to determine whether engagement of the EC or EC-projecting cholinergic neurons during behavioral tasks was impaired with age. Results: We found that participants with cognitive impairment had lower EC VAT binding compared to healthy, cognitively intact counterparts. This significantly corresponded with lower function on cognitive tasks that engage EC. We were able to model this phenomenon in rodents. We found lower EC cholinergic projection density corresponded with lower EC function on a behavioral task, and preceded cell body loss. Additionally, we found that aged animals had fewer activated neurons in the EC and fewer activated cholinergic neurons in the basal forebrain than young animals performing an EC-engaging task. Conclusions: Using a cross-species approach, we find important parallels in the involvement of the cholinergic system in EC function and dysfunction with age. Intact cholinergic circuits are critical for normal EC function. Disruptions to these circuits correspond with loss of EC function found early in aging. Ongoing studies investigate factors that make this circuit particularly vulnerable with age. Disclosure: Nothing to disclose. 59.4 Septohippocampal Cholinergic Inputs and Spatial Working Memory are Sustained by Ongoing Adult Neurogenesis Alex Dranovsky Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Adult neurogenesis is reduced during aging and impaired in disorders of stress, memory, and cognition though its normal function remains unclear. Moreover, a systems level understanding of how a small number of young hippocampal neurons could dramatically influence brain function is lacking. We examined whether adult neurogenesis sustains hippocampal connections cumulatively across the life span and identified a slowly emerging major reorganization of septohippocampal cholinergic inputs following disruption of adult hippocampal neurogenesis. Methods: Single and dual label retrograde tracing and brain clearing were used to identify and characterize cholinergic reorganization after ablation of neurogenesis. Fluorescent acetylcholine sensor GRAB-ACh3.0 and microdialysis were used in awake behaving mice for studies of cholinergic function. Behavioral pharmacology and DREADDs were used for loss and gain of function and rescue experiments. Some studies used both male and female mice while others used only males. N = 5-14. Results: Long-term suppression of neurogenesis as occurs during stress and aging resulted in an accelerated decline in hippocampal acetylcholine signaling (p = 0.0007) and a slow and progressing emergence of profound working memory deficits (p = 0.0009). These deficits were accompanied by compensatory reorganization of cholinergic dentate gyrus inputs with increased cholinergic innervation to the ventral hippocampus and recruitment of ventrally projecting neurons by the dorsal projection. Conclusions: Our study demonstrates that hippocampal neurogenesis supports memory by maintaining the septohippocampal cholinergic circuit across the lifespan. It also provides a systems level explanation for the progressive nature of memory deterioration during normal and pathological aging and indicates that cholinergic innervation is structurally and functionally malleable by experience. Disclosure: Nothing to disclose. PANEL 60. Prazosin for Neuropsychiatric Disorders Beyond PTSD: Efficacy Signals for Alcohol Use Disorder, for Chronic Posttraumatic Headache, and for Disruptive Agitation in Alzheimer’s Disease 60.2 Prazosin Decreases Alcohol Consumption in Active-Duty Soldiers With Alcohol Use Disorder and Elevated Cardiovascular Parameter Murray Raskind University of Washington, Seattle, Washington, United States Background: Excessive noradrenergic signaling contributes to aversive symptoms of alcohol withdrawal that interfere with abstinence or reduced hazardous use. Prazosin is an alpha-1 adrenoreceptor (AR) antagonist that reduces noradrenergic signaling. Methods: One hundred two active-duty soldiers already participating in command-mandated Army outpatient alcohol treatment were randomized to also receive the brain-penetrant alpha-1 adrenergic receptor antagonist prazosin or placebo for 13 weeks. Study drug was titrated up based on tolerability to a maximum possible dose of 4 mg at 10AM, 6 mg at 3PM and 10 mg at bedtime. Alcohol consumption outcome measures were standard drink units (SDUs) per day averaged over each week, % days any drinking per week, and % days heavy drinking per week. Results: Mixed Models Repeated Measures (MMRM) analysis was performed. Although baseline alcohol consumption was markedly reduced by the pre-randomization outpatient alcohol treatment program, addition of prazosin treatment still produced a greater slope of decline in SDUs per day compared to placebo (p = 0.02). Pre-planned subgroup analyses were then performed in soldiers with elevated baseline cardiovascular measures consistent with increased noradrenergic signaling. In soldiers with elevated standing heart rate (n = 15), prazosin reduced SDUs per day (p = 0.01), % days drinking (p = 0.03), and % days heavy drinking (p = 0.001) relative to placebo. In soldiers with elevated standing systolic blood pressure (n = 27), prazosin reduced SDUs per day (p = 0.04) and tended to reduce % days drinking (p = 0.056). Prazosin also reduced depression symptoms and incidence of emergent depressed mood compared to placebo (p = 0.05 and p = 0.01, respectively). During the final four weeks of prazosin vs. placebo treatment that followed completion of Army outpatient AUD treatment, alcohol consumption in soldiers with elevated baseline cardiovascular measures increased in those receiving placebo but remained suppressed in those receiving prazosin. Conclusions: These results extend reports that higher pre-treatment cardiovascular measures predict prazosin efficacy for AUD and suggest potential prazosin usefulness for relapse prevention. Disclosure: Nothing to disclose. 60.3 Randomized Controlled Trial of Prazosin for Prophylaxis of Posttraumatic Headaches Cindy Mayer VA Northwest Mental Illness Research, Education, and Clinical Center, Seattle, Washington, United States Background: Prazosin is an alpha-1 adrenoreceptor antagonist that reduces noradrenergic signaling peripherally and centrally. Encouraging results from an open-label trial in which prazosin substantially reduced headache (HA) frequency in military veterans following mild traumatic brain injury (mTBI) provided the rationale for this pilot randomized controlled trial (RCT) of prazosin for prophylaxis of posttraumatic headache (PTH). Methods: A 22-wk parallel group pilot RCT was performed in 48 military veterans and active-duty soldiers with mTBI-related PTH. Study design was based on IHS consensus guidelines for RCTs for chronic migraines. Following a pre-treatment baseline period, participants with at least 8 qualifying HA days per 4 wks were randomized 2:1 prazosin to placebo. Study drug was titrated as tolerated over 5 wks to a maximum possible dose of 5 mg am and 20 mg at bedtime. Participants were maintained on the achieved dose for 12 wks. The primary outcome measure was change in frequency of qualifying HA days per 4 wks. Key secondary outcome measures were % of participants achieving at least 50% reduction in HA days per 4 wks and change in Headache Impact Test-6 (HIT-6) scores, an indicator of HA-related disability. Other outcome measures were the PTSD Checklist-Military (PCL-M) and the Insomnia Severity Index (ISI). Results: Logistic mixed effects regression analysis demonstrated significantly greater reduction of HA days per 4 wks over time in the prazosin (N = 32) compared to placebo (N = 16) arms, p < 0.01. Declines of HA days per 4 wks from baseline to final 4 wks were almost twice as large in the prazosin compared to placebo condition ([mean ± SEM] 18.3 ± 1.6 to 6.4 ± 1.3 vs.18.6 ± 2.3 to 12.0 ± 2.6). There was a significantly larger percentage of participants with >50% reduction in HA days per 4 wks with prazosin compared to placebo (70% ± 8 vs. 29% ± 12, p < 0.05) and a significantly larger reduction of HIT-6 scores in the prazosin vs. placebo conditions (61.0 ± 1.5 to 55.1 ± 1.5 vs. 63.6 ± 2.1 to 64.2 ± 2.1, p < 0.05). Reductions in PCL-M and ISI scores also favored prazosin (p = 0.05 and p < 0.05, respectively). Conclusions: This pilot RCT provides an efficacy signal for prazosin for PTH prophylaxis. A larger RCT of prazosin for PTH prophylaxis that includes civilian participants following non-military mTBI is needed to confirm and extend these promising results. Disclosure: Nothing to disclose. 60.4 Prazosin for Disruptive Agitation in Alzheimer’s Disease: PEACE-AD Elaine Peskind VA Puget Sound Health Care System, Seattle, Washington, United States Background: Disruptive agitation is a major source of distress to patients and caregivers and is a common precipitant of long-term care placement. High central nervous system (CNS) noradrenergic signaling at the alpha-1 adrenoreceptor (AR) in Alzheimer’s disease (AD) appears to contribute to disruptive agitation. Prazosin, a CNS active alpha-1 AR antagonist, was effective for reducing disruptive agitation and was well tolerated in our previous single site pilot study. Methods: In this multi-site randomized controlled trial (RCT) in which recruitment was substantially handicapped by the COVID-19 pandemic, participants were randomized to prazosin or placebo using a 2:1 permuted block randomization. Prazosin was titrated over 4 weeks to a maximum possible dose of 4 mg mid-morning and 6 mg at bedtime based on tolerability and persistent agitation. Adverse events and orthostatic blood pressure and heart rate were monitored. Primary outcome measure was the ADCS-Clinical Global Impression of Change-Agitation (CGIC-A) targeting disruptive agitated behaviors. Secondary outcomes were the 17-item Neuropsychiatric Inventory (NPI), Cohen Mansfield Agitation Inventory (CMAI), ADCS-Activities of Daily Living (ADCS-ADL) for severe dementia, and total number study days completed. An exploratory outcome was the NPI 5-item subscale reflecting agitation. Results: Thirty-five participants were randomized 2:1 to prazosin or placebo for 12 weeks. Mixed Models Repeated Measures analysis was performed. There were no significant differences in the CGIC-A or total NPI scores. However, reduction in CMAI score significantly favored prazosin (p = 0.04) and the 5-item NPI Agitation subscale numerically favored prazosin. The adverse event (AE) profile was as anticipated for prazosin; AEs that occurred in >5% of prazosin participants and >2X the occurrence in the placebo group included syncope, dizziness, nausea, and somnolence. Conclusions: This study provides a modest signal for prazosin efficacy for disruptive agitation in AD. Both the efficacy and safety data were limited by the small N, particularly in the placebo group. A larger multi-center study of prazosin for moderate-severe disruptive agitation in home- and long-term care-residing Alzheimer’s disease patients is necessary to extend these results. Disclosure: Nothing to disclose.	36456692	PMC9714389	NO-CC CODE	2022-12-06 23:15:02	no	Neuropsychopharmacology. 2022 Dec 1; 47(Suppl 1):1-62	utf-8	Neuropsychopharmacology	2,022	10.1038/s41386-022-01483-2	oa_other
==== Front Genes Immun Genes Immun Genes and Immunity 1466-4879 1476-5470 Nature Publishing Group UK London 190 10.1038/s41435-022-00190-8 Correction Publisher Correction: Role of DAMPs in respiratory virus-induced acute respiratory distress syndrome — with a preliminary reference to SARS-CoV-2 pneumonia http://orcid.org/0000-0002-0090-0629 Land Walter Gottlieb [email protected] 12 1 German Academy for Transplantation Medicine, Munich, Germany 2 grid.11843.3f 0000 0001 2157 9291 Molecular ImmunoRheumatology, INSERM UMR_S1109, Laboratory of Excellence Transplantex, University of Strasbourg, Strasbourg, France 1 12 2022 11 © The Author(s), under exclusive licence to Springer Nature Limited 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Subject terms Infectious diseases Immunology ==== Body pmcCorrection to: Genes & Immunity 10.1038/s41435-021-00140-w, published online 17 June 2021 Abstract When surveying the current literature on COVID-19, the “cytokine storm” is considered to be pathogenetically involved in its severe outcomes such as acute respiratory distress syndrome, systemic inflammatory response syndrome, and eventually multiple organ failure. In this review, the similar role of DAMPs is addressed, that is, of those molecules, which operate upstream of the inflammatory pathway by activating those cells, which ultimately release the cytokines. Given the still limited reports on their role in COVID-19, the emerging topic is extended to respiratory viral infections with focus on influenza. At first, a brief introduction is given on the function of various classes of activating DAMPs and counterbalancing suppressing DAMPs (SAMPs) in initiating controlled inflammation-promoting and inflammation-resolving defense responses upon infectious and sterile insults. It is stressed that the excessive emission of DAMPs upon severe injury uncovers their fateful property in triggering dysregulated life-threatening hyperinflammatory responses. Such a scenario may happen when the viral load is too high, for example, in the respiratory tract, “forcing” many virus-infected host cells to decide to commit “suicidal” regulated cell death (e.g., necroptosis, pyroptosis) associated with release of large amounts of DAMPs: an important topic of this review. Ironically, although the aim of this “suicidal” cell death is to save and restore organismal homeostasis, the intrinsic release of excessive amounts of DAMPs leads to those dysregulated hyperinflammatory responses—as typically involved in the pathogenesis of acute respiratory distress syndrome and systemic inflammatory response syndrome in respiratory viral infections. Consequently, as briefly outlined in this review, these molecules can be considered valuable diagnostic and prognostic biomarkers to monitor and evaluate the course of the viral disorder, in particular, to grasp the eventual transition precociously from a controlled defense response as observed in mild/moderate cases to a dysregulated life-threatening hyperinflammatory response as seen, for example, in severe/fatal COVID-19. Moreover, the pathogenetic involvement of these molecules qualifies them as relevant future therapeutic targets to prevent severe/fatal outcomes. Finally, a theory is presented proposing that the superimposition of coronavirus-induced DAMPs with non-virus-induced DAMPs from other origins such as air pollution or high age may contribute to severe and fatal courses of coronavirus pneumonia. This article is part of the Special Issue “Immunology of cell death in cancer & infection”, Guest Editor: Professor Abhishek D. Garg, Katholieke Universiteit Leuven, Belgium. It was unintentionally published in issue 22-3 (2021). You can access the article via this link: https://www.nature.com/articles/s41435-021-00140-w. We apologise for the inconvenience.	36456661	PMC9714390	NO-CC CODE	2022-12-02 23:24:43	no	Genes Immun. 2022 Dec 1; 23(8):245	utf-8	Genes Immun	2,022	10.1038/s41435-022-00190-8	oa_other
==== Front Nat Med Nat Med Nature Medicine 1078-8956 1546-170X Nature Publishing Group US New York 2093 10.1038/s41591-022-02093-7 Research Briefing Effect of air pollution on the human immune system 1 12 2022 12 © Springer Nature America, Inc. 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Inhaled particulates from environmental pollutants accumulate in macrophages in lung-associated lymph nodes over years, compromising immune surveillance via direct effects on immune cell function and lymphoid architecture. These findings reveal the importance of improved air quality to preserve immune health against current and emerging pathogens. Subject terms Translational research Lymph node https://doi.org/10.13039/100000050 U.S. Department of Health & Human Services \| NIH \| National Heart, Lung, and Blood Institute (NHLBI) HL145547 ==== Body pmcThe question The world population is aging, and the majority of healthcare costs, morbidity and mortality that are associated with diseases concern individuals in the sixth decade of life and older1. Thus, we need to improve our understanding of the underlying mechanisms that exacerbate disease susceptibility in elderly individuals. As starkly observed in the SARS-COV-2 pandemic, elderly individuals have increased susceptibility to respiratory infections as well as other lung diseases, such as chronic obstructive pulmonary disease and cancer2. This susceptibility is attributed to senescent changes in immune cells, which result in systemic inflammation and functional impairments in adaptive immunity3. However, the immune system is localized in mucosal and lymphoid tissues throughout the body, and the effect of aging on local immune responses has not been well-studied. The lung has continuous exposure to the environment, and the effect of this exposure on the immune system over age is not known. In this study, we investigated the role of atmospheric particulate matter in lung-associated immunity over the human lifespan. The discovery In our studies of human tissue immunity that used samples that were obtained from deceased human organ donors4, we consistently observed that lymph nodes (LNs) associated with the lungs were black in color, owing to the presence of black particulate matter, whereas gut-associated LNs were the expected beige color, with no particulates. Thus, we began to investigate the effect of particulates on immune cells and LN architecture in these different LN sites using quantitative imaging and cellular and functional assays. We found that atmospheric particulate matter accumulated with age specifically in lung-associated LNs but not in gut-associated LNs, and this accumulation increased remarkably after age 40 (Fig. 1). Because lymphatic vessels connect LNs and tissues, one might expect that black particulates would disperse across other types of tissue-draining LNs; however, the fact that particulates became entrapped in lung-associated LNs suggested local effects of particulate matter on lung immunity. We found that particulates were contained within a specific subset of macrophages that was located in the T cell zone of the LN and not within follicles. Importantly, particulate-containing macrophages exhibited reduced activation, impaired production of pro-inflammatory cytokines and significantly reduced phagocytic capacity, whereas macrophages in the same LN that did not contain particulates did not exhibit these functional alterations. Particulate accumulation in lung-associated LNs further led to age-associated alterations in LN structural integrity, owing to the disruption of B cell follicles and lymphatic drainage. These results show that inhaled particulates have direct and cumulative effects on innate and adaptive immune processes that take place in the lymphoid organs that carry out immune surveillance of the lungs and respiratory tract.Fig. 1 Lung-associated lymph nodes accumulate carbon particulates with age. a, Gross appearance of lung-associated LNs (LLNs) over age. b, Brightfield confocal images of human LLNs and mesenteric LNs (MLNs) obtained from organ donors of various ages. Images of whole LN sections were reconstructed from 70–400 individual 20X images; magnified images show areas in white boxes. Black regions show particulate contents. © 2022, Ural, B. B. et al. The interpretation Our study shows that pollutants in our environment have a direct and detrimental effect on the human immune system, and specifically the immune organs that are associated with the respiratory tract. LNs filter impurities and coordinate the clearance of harmful antigens and pathogens, but over decades the LNs connected to the lungs become clogged with particulates, and as a result they are not able to carry out essential functions of host defense and immune surveillance. In addition, the effects of pollutants are cumulative and can in part account for the worse outcome of respiratory infections in elderly individuals compared with younger populations. Our study raises important questions concerning the mechanisms by which particulates are contained by specific macrophage subsets in the LNs. It is unknown whether these macrophages are resident in the LN or whether they are derived from lung macrophages that migrate to the associated LN. It will be interesting to assess whether targeting certain macrophage populations may facilitate the clearance of particulate matter. Moreover, we showed that particulates impair the phagocytic capacity that is mediated by scavenger receptors that are expressed by macrophages. However, the effects of particulates on other pathways for phagocytosis of pathogens and cellular debris remain to be established. In addition, it will be important to understand the full effect of inhaled particulates in the resident immune cell populations in the lung itself, which is an area of ongoing investigation. Basak Burcu Ural and Donna L. Farber Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, USA. Expert opinion “While the approach undertaken here is classical in its methodology, having access to human material from large numbers of donors can be very challenging and has to be commended. The manuscript is very well-written, and the data are very interesting, robust and well-presented.” Thomas Marichal, Liège University, Liège, Belgium Behind the paper In 2011, we set up a tissue resource from human organ donors, with the goal of investigating tissue-resident immune cells in mucosal sites and lymphoid organs5. During the course of these studies, we observed that lung-associated LNs were black in donors with no history of smoking, whereas LNs in other sites, such as those associated with the gut, exhibited a beige color that is typical of leukocytes. We investigated this phenomenon further and found that the black color was due to carbon particulates, which showed increased accumulation with age. Our ability to control for particulates, age and location through the sampling of LNs from different sites in donors of all ages enabled us to uncover a mechanism for how environmental pollutants interact with the immune system. D.L.F. From the editor “This work by Ural and colleagues stood out because it examined the effects of age on human immune system function using LN samples, rather than blood samples as is common, and because it offered new insight into how environmental pollutants may alter immune activity. The results will hopefully motivate further research into how carbon emissions and other forms of pollution affect immune responses.” Editorial Team, Nature Medicine This is a summary of: Ural, B. B. et al. Inhaled particulate accumulation with age impairs immune function and architecture in human lung lymph nodes. Nat. Med. 10.1038/s41591-022-02073-x (2022). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Dzau VJ Inouye SK Rowe JW Finkelman E Yamada T Enabling healthful aging for all - The National Academy of Medicine grand challenge in healthy Longevity N. Engl. J. Med. 2019 381 1699 1701 10.1056/NEJMp1912298 31633895 2. Schneider JL The aging lung: physiology, disease, and immunity Cell 2021 184 1990 2019 10.1016/j.cell.2021.03.005 33811810 3. Frasca D Blomberg BB Inflammaging decreases adaptive and innate immune responses in mice and humans Biogerontology 2016 17 7 19 10.1007/s10522-015-9578-8 25921609 4. Farber DL Tissues, not blood, are where immune cells function Nature 2021 593 506 509 10.1038/d41586-021-01396-y 34035530 5. Thome JJ Spatial map of human T cell compartmentalization and maintenance over decades of life Cell 2014 159 814 828 10.1016/j.cell.2014.10.026 25417158	36456836	PMC9714391	NO-CC CODE	2022-12-02 23:24:44	no	Nat Med. 2022 Dec 1;:1-2	utf-8	Nat Med	2,022	10.1038/s41591-022-02093-7	oa_other
==== Front Neuropsychopharmacology Neuropsychopharmacology Neuropsychopharmacology 0893-133X 1740-634X Springer International Publishing Cham 36456693 1484 10.1038/s41386-022-01484-1 Abstracts Collection ACNP 61st Annual Meeting: Poster Abstracts P1 - P270 1 12 2022 12 2022 47 Suppl 1 63219 © The Author(s), under exclusive licence to American College of Neuropsychopharmacology 2022 issue-copyright-statement© American College of Neuropsychopharmacology 2022 ==== Body pmc December 4-7, 2022 Phoenix, Arizona Sponsorship Statement: Publication of this supplement is sponsored by the ACNP. All content was reviewed and selected by the Program Committee, which held full responsibility for the abstract selections. Only disclosures for presenting authors are listed. Asterisks in the author lists indicate presenter of the abstract at the annual meeting. Abstract numbers do not correlate to poster number assigned for presentation at the Annual Meeting. P1. Biosignatures in LLD and AD Using Circulating Brain-Derived Exosomes Erica Vieira, Etienne Sibille, Sanjeev Kumar, Ana Paula Mendes-Silva, Madison Bak, Tarek Rajji, Breno Diniz Centre for Addiction and Mental Health, Toronto, Canada Background: Chronic inflammation and neurodegeneration are well-characterized pathogenic factors in depression and dementia, especially late-life depression and Alzheimer’s Dementia (AD), and a potential therapeutic target for treatment. One important mechanism linking inflammatory and neurodegenerative biomarkers with specific cellular activation and communication is the release of extracellular vesicles (EVs). EVs are nano-sized vesicles, have specific membrane proteins, and contain nucleic acid (microRNA) and protein cargo. EVs can be released from cells under various conditions, including chronic inflammation and stress, demonstrating significant age-dependent differences in their pro-inflammatory profile. In addition, brain cells, such as neurons and astrocytes, release EVs that can be extracted from plasma samples. The EVs can be divided into three groups based on size. The most studied vesicle is the exosome, ranging from 30 to 150 nm. The exosomes are considered cell-specific vesicles, and the evaluation of their content can provide detailed information about the biological changes of specific cell types than the evaluation of whole plasma content. Exosomes from brain cells, such as neuron- (NDEs) and astrocytes-derived exosomes (ADEs), can easily cross the blood-brain barrier and be identified in the periphery. The NDEs and ADEs role is a new research field and an intricate pathway involving the crosstalk between the CNS, the neuroendocrine, and the immune systems. While studies have advanced in AD, the role of exosomes in LLD is poorly investigated. In addition, LLD is multifactorial and could be a prodromal state associated with neurodegenerative diseases, including AD, frontal, temporal dementia (FTD), and vascular dementia (VD). Therefore, the signature of the exosomes could share common pathways and molecules between LLD and AD. There are no studies in the literature comparing NDEs and ADEs in those two neuropsychiatric illnesses until the present moment. The aim of this project is to characterize biosignatures in NDEs and ADEs (cell-specific) and plasma (non-cell-specific), creating a molecular profile for LLD and AD. Methods: Therefore, 46 LLD subjects, 25 AD patients, and 34 healthy elderly controls were recruited, matched by age and gender. After the psychiatric evaluation, the blood was collected and centrifuged to obtain the plasma-free platelet. The sample was collected and stored at -80°C. We used the kit vFC™ vesicle flow cytometry for counting and sizing vesicles. Results: Individuals with LLD presented lower levels of NDE and ADE compared to controls. For ADE, the opposite was demonstrated in AD Patients, with 2 times more ADE than CT and LLD. Next, we evaluate 49 pro-inflammatory cytokines and neurodegenerative protein levels in total exosomes and plasma samples. There was an overlap in the production of 32 proteins when comparing plasma and exosome evaluation. Twelve proteins were identified only in the plasma sample, and 5 only in the exosomes. CXCL2, GDNF, NF-light, GFAP, DR3, and IL-4 receptor levels in the exosomes increased compared to plasma, showing a cell-specific driving response via exosomes. Overlapping the protein evaluation in the exosomes from LLD and AD individuals, 14 proteins were common for both disorders, 15 were exclusive in LLD exosomes, and none of the proteins evaluated were exclusive in the exosomes of AD participants. Conclusions: These preliminary conclusions reinforce the importance of the exosomes in cellular communication, and the common biomarkers shared in LLD and AD. This mechanism can contribute to the crosstalk between brain cells and the periphery as a window to directly evaluate the molecular pathology of LLD and AD. Keywords: Late-life Depression, Alzheimer’s Disease, Extracellular Vesicles, Inflammatory Markers, Neurodegeneration Disclosure: Nothing to disclose. P2. Profiling the Human Anterior Hippocampus With Spatially-Resolved Transcriptomics Stephanie Page, Anthony Ramnauth, Madhavi Tippani, Erik Nelson, Heena Divecha, Elizabeth Pattie, Thomas Hyde, Leonardo Collado Torres, Kristen Maynard, Stephanie Hicks, Keri Martinowich Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: The three-layered archicortex of the hippocampal formation (hippocampus) has unique patterns of gene expression, morphology, physiology, and connectivity that developmentally change across the lifespan. In rodents and non-human primates, the hippocampus is one of the only brain regions in which neurogenesis has been described throughout the lifespan. Due to its structure and circuitry, the region has been linked to a number of critical behavioral functions, including regulation of learning, memory and mood. Hippocampal neurons are organized in densely packed layers according to dendrite-axon polarity, which reflects the intrinsic connectivity of the hippocampal circuitry. Generally, afferent projections to the hippocampus are received in the dentate gyrus (DG), which then transmits through synaptic relays in the cornu ammonis (CA) areas, with the final efferent projections transmitting information out of the hippocampus via the subiculum (SUB). Synaptic relays within the hippocampus are major sites of structural and functional plasticity across the lifespan that regulate critical functions related to learning, memory, mood and stress regulation. Many important plasticity-related transcripts are localized to the dendritic compartment, and their transcription, transport out of the nucleus, and translation within the dendritic compartment is tightly regulated, both developmentally and by neuronal activity. Recent publications have generated cell-type specific molecular profiles of the human hippocampus in adults and across the lifespan using single-nucleus RNA sequencing; however, these resources lack spatial resolution within the hippocampus and lose transcriptomic information from the cytosolic compartment. Additionally, despite extensive characterization of the functional importance of postnatal neurogenesis in rodent DG, indisputable evidence of adult neurogenesis in the human DG remains elusive and its persistence throughout the lifespan remains controversial. Given the tight correlation between spatial structure and function in the hippocampus, and the particular importance of transcripts localized to the synaptic compartment, molecular profiling technologies with the capacity to address these gaps in our knowledge would be highly useful. We previously generated spatial transcriptomic maps of human dorsolateral prefrontal cortex that captured laminar structure from the white matter to the pial surface, and which included both nuclear and cytosolic gene expression. These data were made publicly available in the form of interactive browsers for the neuroscience community to interrogate the data (Maynard, Collado-Torres, 2021), and here, we describe the generation of a similar resource in the human hippocampus, which also includes developmental stages from infant to adult. Methods: We used the 10x Genomics Visium Spatial Gene Expression platform, which combines spatially-resolved transcriptomic profiles with high resolution histological images to generate spatial transcriptomic maps of the anterior human hippocampus, analogous to the ventral hippocampus of the rodent, across the lifespan. To first characterize the organization of the adult hippocampus, we scored the tissue blocks and used multiple arrays to fully capture all major subfields (CA1-4, DG, and SUB) in adult donors (N = 9 neurotypical donors, both males and females, 159744 spots from 32 capture areas). Concurrently, to investigate hippocampal development over the postnatal lifespan specifically in the DG, we generated spatially-resolved, transcriptome-wide gene expression profiles focusing on this region from neurotypical donors spanning the human lifespan: infant, teen, adult and elderly donors (N = 3 donors, 10,503-12,552 spots per group, 12 total capture areas). Results: We applied spatially-informed, unsupervised clustering algorithms across all donors to identify gene expression signatures that define spatial domains. Then, we compared these spatial domains to independently-derived, manually labeled annotations of anatomically-defined hippocampal subfields, which were visually guided by histological staining and expression of known marker genes. We compared gene enrichment across spatial domains, and identified postnatal developmental markers mapping to a number of biological functions including neurogenesis, plasticity, and apoptosis, among others. To make these data available to the community and most effectively support studies of hippocampal function, we generated a user-friendly, interactive web app for data exploration. Conclusions: We generated spatially-resolved transcriptomic profiles of the adult human hippocampus that span all major subfields, and within the DG specifically, across the lifespan, which identified both canonical and novel patterns of spatial gene expression. Spatially-resolved techniques to identify molecular profiles within the context of the underlying tissue architecture is particularly important given the tight structure-function relationship of the cytoarchitecture within the developing and adult hippocampus. This resource provides a rich source of information about spatial gene expression in the hippocampus across the lifespan, which can be mined for information that contributes to the understanding of the etiology of human-specific neurodevelopmental disorders and neurodegenerative diseases where the hippocampus is implicated. Keywords: Hippocampus, Spatial Transcriptomics, Dentate Gyrus Disclosure: Nothing to disclose. P3. Sex Moderates the Impact of Development Stress and Age on Mitochondrial Function Gladys Shaw, Molly Hyer, Amy Wegener, Samya Dyer, C. Christina Mehta, Igho Ofotokun, Gretchen Neigh* Virginia Commonwealth University, Richmond, Virginia, United States Background: Mitochondrial dysfunction may drive alterations in neural function and behavior and is influenced by aging and early life experiences. Here, male and female rodents were used to determine the influence of sex, age, and adolescent stress on synaptic mitochondrial function and predictive value of peripheral markers. Methods: C57Bl/6NTac mice were subject to chronic repeated predation stress (CRPS) for 15 days during their adolescent (PND36-50) and early adult (PND57-71) stages. In addition, impact of aging was assessed in Wistar rats. In both studies, synaptic mitochondrial respiration was assessed using the SeahorseXFe24 instrument in the hippocampus (HPC) and prefrontal cortex (PFC). Peripheral markers of inflammation, reactive oxygen, and mitochondrial function were measured. Results: Stress history did not change oxygen consumption rate (OCR) of synaptic mitochondria in the PFC of either sex (p > 0.05). Stress history decreased OCR in the HPC (p = 0.0045) of males, but increased OCR in the HPC (p < 0.0001) of females. With regard to aging influences on mitochondrial function, in males, age altered overall OCR within synaptic mitochondria of PFC (p < 0.0001) and HPC (p < 0.0001). Compared to young and aged males, middle-aged males had reduced basal respiration (p < 0.0001), maximal respiration (p = 0.0006), proton leak (p = 0.0003), ATP production (p < 0.0001), and spare capacity (p = 0.005). Western blot densitometry indicated that aged males had the highest density of mitochondria (p = 0.01). In females, PFC OCR was elevated in middle-aged females compared to young (p = 0.03) and aged (p < 0.0001) females. Middle-aged females had higher basal respiration (p < 0.0001), maximal respiration (p < 0.0001), ATP production (p < 0.0001), and spare capacity (p < 0.0001) in both HPC and PFC than other ages. Density of synaptic mitochondria in aged females was reduced (p = 0.004). Sample size for CRPS study was 10 per group for each sex. Sample size for the aging study was 6 per group per sex per age. Test statistics for results presented above are included here. Stress history decreased OCR in the HPC (F(1,132) = 8.340, p = 0.0045) of males, but increased OCR in the HPC (F(1,108) = 17.68, p < 0.0001) of females. With regard to aging influences on mitochondrial function, in males, age altered overall OCR within synaptic mitochondria of PFC (F(2,180) = 32.96, p < 0.0001) and HPC (F(2,180) = 60.31, p < 0.0001). Compared to young and aged males, middle-aged males had reduced basal respiration (F(2,30)=13.02; p < 0.0001), maximal respiration (F(2,30)=9.52; p = 0.0006), proton leak (F(2,30)=10.54; p = 0.0003), ATP production (F(2,30)=26.63; p < 0.0001), and spare capacity (F(2,30)=6.44; p = 0.005). Western blot densitometry indicated that aged males had the highest density of mitochondria (F(2,12)=6.85; p = 0.01). Middle-aged females had higher basal respiration (F(2,30)=21.63; p < 0.0001), maximal respiration (F(2,30)=26.17; p < 0.0001), ATP production (F(2,30)=20.59; p < 0.0001), and spare capacity (F(2,30)=29.88; p < 0.0001) in HPC and PFC than other ages. Conclusions: Although both males and females displayed changes in synaptic OCR as a function of early life stress and age, the patterns of the changes differed by sex. Mitochondrial dynamics within the males showed no region specific differences, yet female dynamics were significantly lower in the HPC than the PFC in all dynamic measurements. Ongoing work suggests that estrogen signaling within the mitochondria is a critical factor in these differences and reflected in peripheral metrics. Keywords: Mitochondrial Respiration, Sex Differences, Synapse, Prefrontal Cortex, Hippocampus Disclosure: Nothing to disclose. P4. Defining Neuropeptide Signals That Slow Cognitive Aging Emily Leptich, Rachel Arey* Baylor College of Medicine, Houston, Texas, United States Background: Cognitive decline is a major deficit that arises with age in humans. Though human life expectancy is increasing, there are currently no treatments to effectively treat or prevent age-related memory decline, creating a growing public health burden. Previous work in mammals and our own work in C. elegans has revealed a correlation between maintaining memory function with age and activity of the memory regulating transcription factor cAMP response-element binding protein (CREB). However, CREB is ubiquitously expressed throughout the brain and body; therefore, identifying tissue- and cell-type specific activators of CREB would reveal promising targets for the development of novel therapeutics that age-related memory loss. We recently found that activation of the highly conserved Gαq signaling pathway in a single sensory neuron (AWC) in C. elegans could enhance molecularly conserved, CREB-dependent long-term memory (LTM) in young animals as well as slow age-related LTM deficits. Maintenance of LTM ability with age by elevated Gαq signaling was found to be due to cell non-autonomous, but cell-specific regulation of CREB activity in “memory center” neurons (AIM) in the worm. We recently determined that LTM phenotypes observed due to increased Gαq signaling resulted from increased neuropeptide release by the AWC sensory neurons. However, the identities of these memory-promoting neuropeptides, their downstream signaling pathways, and their role in age-related cognitive decline remain unknown. Methods: C. elegans maintenance: Animals were maintained under standard laboratory conditions and fed the E. Coli strain OP50 ad libitum. Synchronized populations for behavior and lifespan assays were generated by standard hypochlorite treatment. RNAi Treatment: Standard RNAi by feeding was performed to achieve gene knockdown. Briefly, HT115 E. Coli expressing RNAi was fed to C. elegans. To knock down genes specifically in adulthood, animals were fed RNAi at the L4 larval stage, after terminal nervous system differentiation. To achieve RNA-knockdown selectively in neurons, experiments were performed in a transgenic C. elegans strain (egl-30(js126);LC108 [punc-119::sid-1]) that overexpresses a double stranded RNA transporter in all neurons. Behavior Assays: Standard positive olfactory association assays were performed. These assays pair the neutral odorant butanone with food (E. coli) so that animals form a positive butanone association. Learning and memory were assayed as a training-dependent increase in preference for butanone as measured by population chemotaxis assays (~100 animals per assay) to obtain a chemotaxis index. Memory performance was calculated by Performance Index (Chemotaxis_Index(trained) - Chemotaxis_Index (naive/untrained). For all behavioral assays, 10-15 replicates were used, and either one- or two-way ANOVA followed by Bonferroni post-hoc tests were performed. Lifespan Assays: Standard C. elegans lifespan assays were performed in the context of RNAi treatment. For each condition, three biological replicates of ~100 worms were used. Data was analyzed by log-rank (Mantel-Cox) method in Kaplan-Meier survival analysis. Results: We find that genetic manipulations that specifically increase neuropeptide secretion from the AWC neuron in C. elegans slow age-related deficits in associative learning and LTM (p < 0.05). Longevity analysis revealed that increased neuropeptide secretion for the AWC, has no detectable effect on lifespan (p > 0.05), suggesting that the maintenance of learning and memory ability observed is due to increased neuronal healthspan. In order to identify these healthspan promoting signals, we generated a list of approximately 40 candidate AWC-expressed neuropeptides using publicly available genomics datasets, many of which have no previously known role in associative behavior or aging. We are performing an RNAi-based screen in the worm to determine if specific neuropeptides are necessary for the neuropeptide-dependent enhanced learning and memory ability of animals with elevated Gαq signaling. We have identified novel peptide signals, including insulin-like peptides (ins) and neuropeptide-like proteins (nlps), that promote associative learning and memory, and in ongoing studies, we are examining other peptides of interest and their role in regulation of CREB activity. Conclusions: Here we have found that enhancing the activity and secretion of specific neuropeptides slows age-related deficits in associative learning and memory. Identifying new molecules that regulate CREB in a cell-specific manner will be of broad interest as the effects of increasing CREB activity have memory-promoting effects in both invertebrates and mammals, and may present new therapeutic targets for the treatment of cognitive decline. Keywords: CREB, Aging, Neuropeptides Disclosure: Nothing to disclose. P5. Exercise Parameters That Open a ‘Molecular Memory Window’ for Cognitive Enhancement Shine Light on Key Memory Mechanism in the Adult, Aging, and Alzheimer’s Disease Brain Ashley Keiser, Tri Dong, Enikö Kramár, Christopher Butler, Siwei Chen, Dina Matheos, Joy Beardwood, Ameer Al-Shammari, Yasaman Alaghband, Vanessa Alizo Vera, Nicole Berchtold, Sharmin Shanur, Agatha Augustynski, Pierre Baldi, Carl Cotman, Marcelo Wood University of California, Irvine, Irvine, California, United States Background: The ability to learn, consolidate and retrieve information is critical for everyday life and this ability begins to decline with normal aging and is severely exacerbated by Alzheimer’s Disease (AD). Basic research and clinical trials universally demonstrate the benefits of exercise for cognitive function. However, age and physical disability-related factors that may affect individuals throughout the lifespan progressively limit and reduce engagement in exercise and associated cognitive benefits. Defining the mechanisms by which exercise leads to cognitive enhancement throughout the lifespan will hold therapeutic benefit for individuals of all ages and abilities. Methods: In this study, we use exercise as an approach to unlock a novel understanding of the molecular mechanisms that enable and drive consolidation of memory. We identify and utilize specific exercise parameters that allow memory consolidation to occur under inadequate, subthreshold learning conditions, as well as parameters where these cognitive benefits are maintained for specific durations of time. Specifically, adult male mice underwent 14 days of initial exercise, received a sedentary delay period (0-2 weeks), and a brief 2-day period of reactivating exercise, followed by 3 min inadequate, subthreshold training in an object location memory (OLM) task. Those parameters were then used to examine hippocampal long-term potentiation (LTP) using theta burst stimulation in the schaffer collateral pathway. To begin to define the mechanism responsible for driving long-term memory formation and maintaining cognitive benefits, we utilized an unbiased RNA-sequencing approach on dorsal hippocampal tissue taken during memory consolidation, 1 hour following training, in mice receiving the same exercise parameters above. To examine the role of an identified gene target ACVR1C, a type 1 receptor for the TGF-β family of signaling molecules in hippocampus-dependent learning and synaptic plasticity, we used intra-hippocampal delivery of AAV1-ACVR1C point mutant constructs that either enhance or disrupt function and allowed it to express for two weeks before onset of behavior. Next, sedentary mice were trained using either a subthreshold (3 min) or standard (10 min) OLM task and memory was tested the following day. The same mice from behavioral studies were used to assess the impact of Acvr1c manipulation on hippocampal LTP. Given misregulation of the TGF-β pathway that occurs with age and in AD patients, we next aimed to determine whether enhancing Acvr1c function would regulate long-term memory formation and synaptic plasticity in aging and AD mouse hippocampus and ameliorate impairments. First, we examined whether Acvr1c declines with age. Dorsal hippocampus was obtained from 3 and 20 mo. female and male C57BL/6J mice and processed for RT-qPCR. Additionally, Acvr1c transcripts per million (TPM) values from an RNA-Seq data set obtained through the MODEL-AD consortium were analyzed from 4, 8 and 12 mo. C57BL/6J and 5xFAD female and male mice. To determine whether enhancing ACVR1C function in dorsal hippocampus would ameliorate impairments in memory and LTP in 18 mo. C57BL/6J and 5xFAD males, aforementioned methods were applied. Results: We demonstrate that 14 days of voluntary wheel-running facilitates hippocampus-dependent memory (One-way ANOVA, Group: (F(6,64) = 8.13, P < 0.0001; Tukey test: P < 0.001, 14D vs. Sed) and synaptic plasticity (One-way ANOVA, Group: (F(6,89) = 22.22, P < 0.0001; Tukey test: P < 0.0001, 14D vs. Sed) in adult mice compared to sedentary, effects which can be maintained and re-engaged with brief 2-day re-introduction to exercise following a sedentary delay (Behavior: Tukey test: P < 0.05, LTP: P < 0.0001, 2-day re-introduction vs SED). We identify a gene coding for a type 1 receptor for the TGF-β family of signaling molecules, Acvr1c as one of few genes (including Bdnf) showing up-regulation in the hippocampus under exercise conditions that enable the formation of long-term memory and synaptic plasticity. We find that disrupted ACVR1C function under adequate learning conditions in adults impairs memory (t(17) = 4.65, P = 0.0002) and synaptic plasticity (t(18) = 3.512, P = 0.0025). Conversely, overexpression of ACVR1C enables learning under inadequate training conditions in adults (t(18) = 3.303, P = 0.004) and enhances LTP (t(14) = 3.953, P = 0.0014). We find Acvr1c levels decrease in the hippocampus with age in C57Bl/6J (t(26) = 2.72, P = 0.01) and 5xFAD female and male mice (Three-way ANOVA, Age: (F(2,48) = 54.95, P < 0.0001; Tukey test: P < 0.0001 12 mo. vs. 4 mo.), and demonstrate that Acvr1c over-expression ameliorates age and AD-associated impairments in memory (18 mo. C57BL/6J: (t(12) = 2.350, P = 0.036) and synaptic plasticity (18 mo. C57BL/6J: (t(14) = 3.953, P = 0.001), 18 mo. 5xFAD: (t(10) = 9.653, P = 0.001). Conclusions: Using a novel exercise-based approach that allows memory formation to occur under inadequate, subthreshold training conditions, we identify ACVR1C as a fundamental driver and bidirectional regulator of memory formation and synaptic plasticity in the adult brain. We also demonstrate a key role for ACVR1C in the aging- and AD-brain. These data suggest that promoting ACVR1C through exercise or pharmacological intervention may protect against age and AD-associated cognitive impairment, providing a potentially powerful and novel disease modifying treatment strategy for AD. Keywords: Memory, Exercise, Long Term Potentiation, Hippocampus, Alzheimer’s Disease Disclosure: Nothing to disclose. P6. A Comparison of Pathways Associated With Protein Aggregation in Alzheimer’s Disease and Schizophrenia Leslie Nucifora, Christopher Ross, Russell Margolis, Gwenn Smith, Robert Sweet, Frederick Nucifora Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Alzheimer’s disease (AD) is the most common neurodegenerative disorder, characterized universally by cognitive decline, and heterogeneously by emotional and behavioral changes. Between 40-60% of AD patients experience psychotic symptoms. Recent studies suggest greater cognitive impairment and more rapid cognitive decline in AD patients with psychosis (AD + P). Schizophrenia (SCZ) affects 1% of the population and is characterized by positive and negative symptoms, as well as cognitive deficits. Psychosis, in the form of hallucinations and delusions, is the most prominent symptom. SCZ is also a heterogeneous disorder in its clinical presentation and progression. The pathological hallmark of AD is protein aggregation. Protein aggregation likely disrupts important proteins and pathways that can lead to the symptoms of AD, including psychosis and cognitive decline. We have demonstrated protein aggregation in a subset of patients with SCZ. In addition, we have shown using olfactory neurons, that SCZ patients with aggregation have more pronounced cognitive impairment than SCZ patients without aggregation as a mechanism. Further, we and others have previously shown that the protein products of rare genetic variants linked with SCZ in unique pedigrees can aggregate and disrupt the function of critical proteins required for the proper functioning of the cell. While protein aggregation has been linked to neuronal cell death in AD, the protein aggregation we observe in SCZ likely leads to cellular dysfunction and not profound cell loss. While the mechanisms leading to aggregation in both diseases may be partly distinct, there are likely similarities, with proteins that are prone to aggregate overlapping between diseases and relating to the common clinical phenotype of psychosis and/or cognitive impairment. Therefore, we hypothesize that there will be proteins and pathways in AD that will overlap with some of those identified in SCZ patients with protein aggregation as a mechanism, providing insight into common pathways and mechanisms associated with AD and SCZ. Methods: A comparative proteomic study of protein mass spectrometry data obtained from insoluble proteins biochemically isolated from Alzheimer’s disease as reported in a previous proteomic study (Hales et al., 2016) and schizophrenia postmortem brains. Proteome Discoverer was used to analyze the proteomic data to maximize the number of proteins identified. Panther pathway analysis was used analyze the enriched protein data to determine proteins and biological processes most relevant to protein insolubility in AD and SCZ. Results: We have obtained promising preliminary data through a comparative proteomic investigation of the insoluble proteins in a SCZ subtype, characterized by protein aggregation, with proteins identified in a previous similar proteomic insolubility study in AD, that suggests overlapping proteins and pathways between the two neuropsychiatric disorders. These results suggest a common proteomic signature in AD and SCZ. Conclusions: By comparing the aggregated proteins in AD to aggregated proteins in SCZ, a disease characterized by psychosis and cognitive deficits, we can better understand common pathways implicated in these disorders. Keywords: Alzheimer’s Disease, Schizophrenia (SCZ), Proteomics Disclosure: Nothing to disclose. P7. Sex-Specific Association of Amyloid Precursor Protein With Microglial Activation and Modulation by Apolipoprotein E Genotype in Major Depressive Disorder Nunzio Pomara, Chelsea Reichert Plaska, Davide Bruno, Jaime Ramos-Cejudo, Ricardo S. Osorio, Amanda Heslegrave, Anilkumar Pillai, Bruno Imbimbo, Henrik Zetterberg, Kaj Blennow Nathan S Kline Institute, New York University, Orangeburg, New York, United States Background: Female sex and depression are both associated with increased risk for Alzheimer’s disease (AD) but the mechanisms for the increased risk remain poorly understood. Increased brain amyloid deposition and microglial activation have both been implicated in AD and depression. However, to our knowledge, there are no studies which have examined if sex- and depression-related effects on amyloid β-peptide (Aβ) dynamics and microglia activation may contribute to increased risk for AD associated with female sex and depression. Non-amyloidogenic proteolytic cleavage of amyloid precursor protein (APP) by α-secretase results in release of one secreted form of APP (sAPPα), whereas the amyloidogenic cleavage by β-secretase releases a C-terminally truncated form of APP (sAPPβ) plus Aβ. Both sAPPα and sAPPβ have been reported to induce microglia activation. These observations prompted us to examine the relationship between sex, depression, and cerebrospinal fluid (CSF) levels of sAPPα and sAPPβ. CSF levels of the secreted ectodomain of triggering receptor expressed on myeloid cells 2 (sTREM2), which has emerged as a specific marker of microglial activation, was also examined. Additionally, we also investigated the potential influence of APOE genotype on these biomarkers. Methods: Fifty-one cognitively intact subjects (31 subjects with major depressive disorder and 20 age-matched healthy controls) aged 60 years and older, completed a 3-year longitudinal study and an optional lumbar puncture. Independent-samples Mann-Whitney-U tests were used to examine group differences by diagnosis, sex, and APOE genotype for sAPP-α and sAPPβ. Results: There were no significant differences between subjects with late-life major depression (LLMD) and Controls in mean (± SEM) CSF sAPPα (551 ± 48 vs 616 ± 50 pg/mL) or sAPPβ (164 ± 15 vs 187 ± 14 pg/mL) levels. Irrespective of diagnosis, females had significantly higher levels of both sAPPα (p = 0.016) and sAPPβ (p = 0.010) compared to males. sAPPα and sAPPβ were significantly correlated with each other (rho = 0.930, p < 0.001). Mean CSF sTREM2 concentrations were 3507 ± 490 pg/mL in LLMD subjects and 5096 ± 640 pg/mL in Controls (p = 0.075). In the whole sample, sTREM2 correlated with both sAPPα (rho = 0.462, p = 0.001) and sAPPβ (rho = 0.504, p < 0.001). When examining the relationships as a function of diagnosis, sAPPα was found to be associated with sTREM2 in Controls only (rho = 0.474, p = 0.035) while sAPPβ correlated with sTREM2 in LLMD only (rho = 0.370, p = 0.048). When examining the relationship as function of sex, we found that sTREM2 significantly correlated with both sAPPα (rho = 0.629, p = 0.002) and sAPPβ (rho = 0.673, p = 0.001) in females but not in males (sAPPα: rho = 0.310, p = 0.109; sAPPβ: rho = 0.341, p = 0.076). Finally, as a function of APOE genotype, significant associations were found only for APOEe4 non-carriers. sTREM2 correlated with both sAPPα (rho = 0.567, p < 0.001) and sAPPβ (rho = 0.579, p < 0.001) in the e4 negative group, but not for e4 positive (sAPPα: rho = 0.182, p = 0.516; sAPPβ: rho = 0.193, p = 0.491). Conclusions: We did not find significant differences in mean CSF sAPPα and sAPPβ levels between Controls and LLMD or between APOE ε4 carriers and non-carriers. We found significant associations of sTREM2 with both sAPPα and sAPPβ in females but not in males. sAPPα and sAPPβ correlated significantly only in non-APOE ε4 carriers but not in APOE ε4 subjects. These observations suggest that APP expression may influence neuroinflammatory response mainly in female subjects and in non-APOE ε4 carriers and should thus be explored as a potential molecular therapeutic target for the prevention and treatment of AD in these populations. Keywords: CSF sTREM2, sAPPβ, sAPPα, Microglia, Depression Disclosure: Nothing to disclose. P8. The Pink Brain Project: Cognitive and Immunological Effects of Yoga Compared to Memory Training in Older Women at Risk for Alzheimer’s Disease Adrienne Grzenda, Prabha Siddarth, Helen Lavretsky UCLA, Los Angeles, California, United States Background: Subjective cognitive decline (SCD) and mild cognitive impairment (MCI) accompanied by cardiovascular risk factors (CVRFs) are known risk factor for developing dementia. Yoga has been identified as a safe practice with positive effects on cognitive functions in healthy elderly and older adults at risk for cognitive decline. While yoga commonly involves postures (=asanas), focus on breath (=pranayama), postural alignment, and movement, brief meditative practices are also beneficial for brain health and cognition. Kirtan Kriya (KK), a yogic meditation, is associated with improved mood, cognition, and quality of life in SCD and MCI. Methods: We conducted a randomized, controlled trial to assess the efficacy of Kundalini yoga paired with Kirtan Kriya meditation (YOGA) and memory enhancement training (MET) on mood and cognitive functioning in a group of older women with CVRFs and SCD (clinicaltrials.gov=NCT03503669). INTERVENTIONS: The YOGA intervention consisted of weekly, 60-minute in-person classes with a certified instructor for 12 weeks. In addition, each participant received a CD containing a 12-minute guided KK recording for daily homework practice. MET involved 12 weekly in-person group classes with 12 minute daily homework exercises. OUTCOMES: Memory performance was the primary outcome. A delayed recall domain score was computed from the Hopkins Verbal Learning Test, Wechsler Memory Scale, and Rey-Osterreith Complex Figure 3-min Delay tests. An executive function domain score was calculated from the Stroop Interference and Trails B tests. Secondary outcomes included depression (BDI, Beck Depression Inventory), anxiety (HAM-A, Hamilton Anxiety Rating Scale), stress (PSS, Perceived Stress Scale), resilience (CD-RISC, Connor-Davidson Resilience scale), subjective memory (MFQ, Memory Functioning Questionnaire; Factor 1=Frequency of Forgetting; Factor 2: Seriousness of Forgetting), and health-related quality of life (SF-36, 36-Item Short Form Survey). Outcomes were measured at 12-weeks and 24-weeks follow-up. BIOLOGICAL SAMPLES: Peripheral whole blood samples were collected at baseline, 12-weeks, and 24-weeks follow-up for RNA sequencing and cytokine/chemokine assays. RNA-SEQ: RNA Sequencing was performed on 2 Illumina NovaSeq 6000 S4 lanes for 2x150 cycles in paired-end mode, generating 150 bp sequencing reads (mean ~30 million reads/sample). Differential gene expression was tested using negative binomial generalized linear models (DeSeq2 and edgeR, R 4.1). CYTOKINE/CHEMOKINE ASSAY: Human 38-plex magnetic cytokine/chemokine kits (EMD Millipore, HCYTMAG-60K-PX38, Burlington, MA) were used per manufacturer’s instructions. STATISTICAL ANALYSIS: Outcomes (clinical and cytokine/chemokine concentrations) were analyzed using a mixed effects general linear model (SAS 9.4), including intervention group, time, and the interaction between time and intervention group. Age, sex, and education were used as covariates (only for cognitive outcomes). Post-hoc analyses determined the significance of specific pair-wise group differences and within-group changes. Effect sizes (Cohen’s d) were calculated for clinical outcomes. Results: CLINICAL OUTCOMES: A total of 79 participants (YOGA = 40; MET = 39) were randomized and 63 completed the 24-week follow-up (YOGA = 65% completion rate; MET = 95%; χ2(1)=10.9, p < 0.001). Mean age of participants was 66.5 (SD = 9.2) years and mean MMSE was 28.4 (SD = 1.4). At 12-weeks and 24-weeks follow-up, both interventions demonstrated improvement in frequency of forgetting (MFQ-Factor 1). Between group differences were not significant (F(1, 76)=0.2, p = 0.7). At 24-weeks, YOGA participants demonstrated between- and within-groups improvements in seriousness of forgetting/MFQ-Factor 2 (YOGA mean change (SD) = 0.65 (1.25), t(76)=2.1, p = 0.04; MET mean change (SD) = -0.31(1.35), t(76)=-0.9, p = 0.4; F(1, 76)=4.9, p = 0.03; effect size (95% confidence interval)=-0.73 (-1.26, -0.19)). YOGA participants demonstrated between- and within-groups decline in delayed recall scores at 24-weeks (YOGA mean change (SD) = -0.31(0.37 t(76)=-3.8, p = 0.0003; MET mean change (SD = 0.02 (0.55), t(76)=0.5, p = 0.6; F(1, 76)=10.3, p = 0.002; effect size (95% confidence interval=0.19 (-0.32, 0.70)). Executive functioning, however, showed no between- or within-groups differences (F(1, 76)=0.8, p = 0.4). GENE EXPRESSION: Compared to MET, at 12- and 24-weeks follow-up, YOGA uniquely modulated targets related to interferon signaling and innate and adaptive immunity. CYTOKINE/CHEMOKINE CONCENTRATIONS: Compared to YOGA, MET participants displayed higher Eotaxin-1 levels (F(2,67)=3.94, p = 0.02) at 12- and 24-weeks follow-up. Conclusions: At 24-weeks follow-up, YOGA yielded a significant, large effect size improvement in subjective cognitive impairment compared to MET. On a transcriptional level, YOGA is a robust mediator of psycho-neuro-immune pathways, including suppression of several typically pro-inflammatory molecules. Eotaxin-1 levels increased over time in MET but not YOGA participants. Eotaxin-1 levels have been shown to increase with age and cognitive decline. These results suggest clinical and biological benefits of YOGA for SCD in menopausal women at risk for Alzheimer’s disease due to cardiovascular disease, linking changes in cognition to anti-inflammatory effects of yoga. Keywords: Clinical Trial, Cognitive Decline, Transcriptomics, Neuroimmunology, Prevention of Alzheimer’s Disease Disclosure: Nothing to disclose. P9. Network Controllability: A Potential Targeting Approach for TMS With Important Considerations for Aging Lysianne Beynel, Lifu Deng, Courtney Crowell, Moritz Dannhauer, Susan Hilbig, Hannah Palmer, Alex Brito, Angel Peterchev, Bruce Luber, Sarah Lisanby, Greg Appelbaum, Simon Davis National Institute of Mental Health, Bethesda, Maryland, United States Background: Repetitive Transcranial Magnetic Stimulation (rTMS) is a non-invasive brain stimulation technique widely used in both clinical practice and research studies, due to its ability to modulate neuronal activation. However, rTMS suffers from an overall moderate efficacy, and improving targeting approaches may significantly increase rTMS effects. Indeed, while early studies used imprecise scalp-based coil placement to define the stimulation target, more recent studies rely, instead, on fMRI results for neuronavigated targeting. This is done either by having participants perform a task and extracting the peak activation within a specific region of interest, or by extracting the region with strongest connectivity value during a task or resting state scan. While these approaches have enhanced rTMS efficacy, they rely on identifying bivariate relationships between a single seed and target node, even though the brain consists of assemblies of large-scale networks that communicate in a highly multivariate fashion. This problem is especially relevant for rTMS targeting in older adults, as the brain undergoes a number of large-scale network changes. Thus, a central challenge is in identifying a relevant multivariate network measure that summarizes these complex network dynamics and their age-related change. One such measure is network controllability, which measures the strength of influence of one cortical node on the global network. Central to this approach, is the hypothesis that applying energy (e.g., via rTMS) to nodes with high modal controllability might push the brain into hard-to-reach states. We recently demonstrated that young adult participants benefited the most from rTMS when the stimulation site coincided with the node of higher modal controllability. The current study focused on how controllability changes with age, and how these changes can impact the effectiveness of rTMS in aging populations. Methods: Imaging data from 52 young adults (23.8 ± 4.8 years old, 29 females) and 29 older adults (67.9 ± 5.7 years old, 17 females) were used. After neuroimaging (including diffusion-weighted imaging), subjects received active or sham rTMS over the left dorsolateral prefrontal cortex or the left parietal cortex during four subsequent visits. DWI data were preprocessed using a standard DWI processing pipeline with FSL and MRtrix software, to generate a high-dimensional structural connectome in each participant. The number of white matter streamlines connecting each pair of regions was used to compute the modal controllability of each node. Modal controllability values were then compared between the two groups: across the whole brain and for four networks of the Yeo atlas. In addition, since modal controllability is derived from DWI data, we also computed the degree, the number of links connecting one node with every others, to test whether the results were specific to controllability or were simply a reflection of structural connectivity changes with aging. Results: Results demonstrated significant modal controllability differences due to aging. Specifically, paired sample t-tests showed that older adults have significantly higher modal controllability for each of the four tested networks (DMN, Fronto-Parietal, Visual and Somoto-Motor) compared to younger adults (p < 0.001 for all comparisons, CIs = [0.009; 0.02]; [0.01; 0.03]; [0.01; 0.02]; [0.02; 0.03] for each network respectively). Young adults were found to have higher modal controllability in parietal and medial prefrontal regions, as expected by network controllability theory which states that most of those nodes are in cognitive control network. However, older adults did not display this pattern and instead had higher modal controllability within the SFG and premotor regions. Finally, the degree analysis did not reveal any significant differences associated with aging in those regions of interest, providing evidence that the results were specific to controllability. Conclusions: Taken together these results suggest that while network controllability can be used as a potential targeting approach for rTMS, and has been associated with some promising results, some important consideration needs to be given regarding the effect of aging on what targets should be chosen for maximum effect. Contrary to assumptions based on executive control, for older adults the modal controllability nodes were not found in the fronto-parietal network but instead in sensory and premotor regions. Further, the fact that aging differences on controllability were not explained by structural connectivity changes reinforces the idea that this measure reflects more than just connectivity. Keywords: rTMS, Diffusion Tensor Imaging (DTI), Aging Disclosure: Nothing to disclose. P10. Disentangling the Relationship Between Age, Sleep, and Alcohol Consumption: Examination Among Healthy Community-Residing Men and Women Christian Garcia, Ben Lewis, Sara Nixon University of Florida, Center for Addiction Research and Education, Gainesville, Florida, United States Background: Epidemiological evidence demonstrates that the prevalence of current drinking among older adults has increased dramatically over the previous two decades. The significance of this change is amplified by the fact that aging is generally accompanied by increased vulnerability to alcohol’s negative effects on multiple biobehavioral processes, including sleep. Notably, regardless of alcohol use, sleep problems are commonly reported in even healthy aging adults. Thus, the intersection of age, alcohol use and sleep constitute a significant area of study. Here, we examined relationships between sleep and alcohol use in a sample of healthy, community-residing older adults, focusing on potential sex differences in these effects. Together, evolving patterns of alcohol use, shifts in populations age distributions, and robust data regarding the sensitivity and relevance of sleep to well-being, reinforce the relevance of this area of study. Methods: 56 (36 women) healthy current drinking adults between the ages of 65 and 90 (M = 74.02 ± 5.86) were recruited to take part in an initial study examining potential effects of psychosocial stressors on alcohol use. For our current objective, we focus on sleep disruption/problems. Composite scores for typical (QFI) amount of absolute alcohol consumed was derived. Participants completed the Pittsburg Sleep Quality Index (PSQI), a self-report measure of sleep to evaluate subjective sleep quality with global scores greater than 5 reflecting poor sleep. General linear models were used to assess the relationship between sleep disturbance and alcohol use (dependent measure). Sex and its potential interaction with sleep was included in the model. Additional analyses utilizing NIAAA criteria for low risk and binge drinking were also conducted. Results: Significant effects of sex [F(1,108)=6.76, p = .01] and sleep disturbance [F(1,108)=16.07, p < .0001] were noted. As expected, men endorsed greater alcohol consumption than women. Contrary to our initial expectations, higher indices of poor sleep predicted less alcohol use. However, these effects are qualified by a significant interaction between sleep and sex [F(1,108)=4.10, p = .05]. Interrogation of the interaction revealed a stronger negative relationship between higher levels of sleep disruption and lower alcohol among men (r = -0.44, p = .004) than for women (r = -.27, p = .02). There were no sex differences in rates of adherence to low risk drinking guidelines (p = .11). Higher indicators of sleep disruption predicted greater likelihood of adherence to proposed low risk guidelines [F(1,108)=4.04, p = .05], with no sex by sleep interaction. Higher indices of sleep disruption were also positively related to numbers of binge drinking episodes [F(1,108)=11.36, p = .001]. The presence of a significant interaction between sex and sleep quality [F(1,108)=11.55, p = .001] for binge drinking episodes shows a robust negative relationship among men (r = -.45, p = .003), compared to that for women (r = .15, p = .20). Conclusions: Taken together, our results highlight differences in drinking related to sleep quality/disturbances offering compelling evidence that poor sleep may be predictive of less alcohol use and thus less likelihood of exceeding proposed limits for low risk drinking in healthy community-residing older adults. Inversely, older adults exceeding NIAAA levels for low risk and binge drinking generally reported better sleep. Thus, while unexpected, these results may reflect unique socio-demographic characteristics in our sample (e.g., well-educated, predominantly white non-Hispanic, high income). Additionally, observed sex-specific differences partially align with current evidence suggesting more robust associations between use patterns and sleep in men overall. Given the dramatic increase in the aging population, rise in drinking prevalence, and common reports of severe sleep deficits/disruptions among older groups, disentangling patterns of use and how they may interact with sleep and other biobehavioral processes remains an important area of future research. Keywords: Sleep Disturbances, Older Adults, Alcohol Disclosure: Nothing to disclose. P11. Longitudinal Impact of Midlife Metabolic Health on Memory Function: Role of Sex and Reproductive Aging Kyoko Konishi, Sarah Aroner, Anne Remington, Harlyn Aizley, Jill Goldstein Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, United States Background: In early midlife, men and women undergo different aging processes. In addition to chronological aging, women undergo reproductive aging whereby they experience a depletion of sex steroid hormones, including estradiol. Estradiol is not only neuroprotective but regulates glucose metabolism and mitochondrial function, including oxidative stress production. With menopause, there is a 15-20% decrease in glucose metabolism and mitochondrial function. As such, menopause presents a window of vulnerability in women that may increase susceptibility to neurodegeneration and cognitive impairment. Here, we assessed the longitudinal impact of metabolic and mitochondrial function, in relation to sex and reproductive aging, on memory performance in early midlife. Methods: Participants were assessed at three time points in midlife: at ages 40-50, 45-55, and 55-63 years old. 103 participants (48M:55F) underwent metabolic and memory assessments at ages 40-50. At ages 45-55, as part of a larger cohort study, the same participants underwent a 5-year follow-up assessment of verbal and associative memory and mitochondrial function was assessed using 8-hydroxydeoxyguanosine (8-OHdG), a measure of oxidative DNA damage. Finally, in a subsequent 8-year follow-up study, 76 participants (32M:44F) from the larger cohort, now ages 55-63 years old, completed the same memory tasks to assess memory decline in midlife. Generalized estimating equations adjusting for age, education, baseline memory performance, and follow-up years were used to assess associations between metabolic function and memory outcomes. Models were run both stratified by sex and with the inclusion of an interaction term. Results: At ages 40-50, there were no significant relationships between metabolic health and memory measures in both men and women (p > 0.05). However, women with prediabetic/diabetic levels of HbA1c (>=5.7) at ages 40-50 performed significantly worse on memory tasks 5 years later compared to those with lower levels of HbA1c (b = -6.32, p < 0.01) and differed significantly from men (p < 0.01). In men, we did not find any associations between HbA1c levels and memory performance. Examining the impact of menopause, we found that women with higher HbA1c levels who transitioned to postmenopause during the follow-up period had worse memory performance 5 years later (b = -1.22, p < 0.01) compared with those who remained in pre/perimenopause (p = 0.05). In addition, specifically in women, poor metabolic health (HbA1c) at ages 40-50 was also related to higher levels of oxidative DNA damage (8-OHdG) measured 5-years later (b = 1.04, p = 0.01; men: b = 0.17, p = 0.54). Elevated levels of 8-OHdG at ages 45-55, in turn, were related to greater verbal memory decline over the subsequent 8-year follow-up period (b = -1.54, p = 0.01), again specifically in women (b = -4.12, p = 0.02; women vs. men: p = 0.05). In women, effects again were predominantly driven by those who transitioned from pre/peri- to postmenopause during the 8-year follow-up period (b = -5.92 p < 0.0001) compared to those that were postmenopausal at both time points. Conclusions: Results suggest that midlife metabolic health is related to mitochondrial function and has a greater longitudinal impact on memory performance in women compared to men. Further, results suggest that midlife metabolic health may impact memory decline in women during the transition through menopause. However, over time, the healthy menopausal brain may compensate, resulting in attenuation of memory decline. Keywords: Memory, Sex Differences, Menopause, Oxidative DNA Damage, Metabolic Function Disclosure: Nothing to disclose. P12. Metabolic Factors Underlying Individual Differences in Cognition and Brain Structure in Healthy Postmenopausal Women Julie Dumas, Jenna Makarewicz, Olivia Nop, Isabel May, James Boyd, Alexandra Potter, Alexander Conley, Brian Boyd, Brittany Bosko, Paul Newhouse University of Vermont Larner College of Medicine, Burlington, Vermont, United States Background: The cognitive changes that occur at menopause have not yet been connected to late life risk for pathological aging and Alzheimer’s disease (AD). Understanding the neurobiological factors related to individual differences in cognition at menopause is critical for understanding normal cognitive aging and identifying risk factors for pathological aging in women. We used data from a subset of subjects from the Cholinergic Health After MenoPause (CHAMP) Study (clinicaltrials.gov NCT04129060) to examine how blood pressure, cholesterol, and blood glucose influenced relationships between brain structure and cognitive performance in healthy postmenopausal women. Methods: At screening all women gave blood samples, had vital signs measured, and completed neuropsychological assessments including Repeatable Battery for the Assessment of Neuropsychological Status (RBANS). On the first study day of the larger clinical trial, subjects had a brain MRI where structural and functional scans were obtained. Structural MRI data were analyzed with FreeSurfer version 7.1.1 to extract volumes for hippocampal subfields as well as limbic regions including the basal forebrain, fornix, hypothalamus, and mamillary bodies that were then corrected for total intracranial volume. We examined relationships between brain structure, performance on the neuropsychological assessments, and the metabolic measures including systolic and diastolic blood pressure, fasting glucose and hemoglobin A1C, and HDL and LDL cholesterol. Results: Forty-five women contributed data to this analysis and were an average age of 59.3 years (SD 5.1 y). They were all postmenopausal, medically healthy, and cognitively normal as defined by our study enrollment criteria. Inclusion criteria allowed medication for controlled high blood pressure (nine women), hyperlipidemia (four women), and Type 2 diabetes (one woman). We examined relationships between variables of interest and then used partial correlations to examine the cognition and brain structure relationships that remained after controlling for the metabolic factors. First, age was correlated with systolic blood pressure (r = .22, p = .03) but not other metabolic, cognitive, or brain volume data. By contrast, metabolic measures that were consistently positively correlated with cognitive performance and brain volume from the limbic regions and hippocampal subfields included systolic blood pressure, hemoglobin A1C, and LDL. We used a partial correlation approach to examine relationships between neuropsychological performance and brain structure after controlling for metabolic factors. We found consistent relationships between immediate memory from the RBANS and the volume of brain regions in the limbic areas including the left mamillary body (r = .51, p < .001) and left fornix (r = .38, p = .02) after controlling for systolic blood pressure, hemoglobin, and LDL. Relationships remained between immediate memory and the right hippocampal body (r = .52, p < .001) and right hippocampal tail (r = .48, p < .003) after controlling for systolic blood pressure, LDL, and hemoglobin A1C. Relationships that were somewhat weaker were also observed between delayed memory on the RBANS with some of the hippocampal subfield measures when controlling for systolic blood pressure, hemoglobin A1C, and LDL (ps < .05). Conclusions: These data show that controlling for some metabolic variables allowed us to observe relationships between memory performance and limbic and hippocampal volumes in healthy, cognitively normal, postmenopausal women. These relationships were observed at smaller levels before controlling for metabolic measures. We interpret these findings as evidence that there are relationships between cognitive performance and brain volumes of brain regions relevant for those cognitive processes that are stronger after controlling for subclinical metabolic factors. These data have implications for understanding cognition and brain relationships in women after menopause and highlight the importance of examining metabolic processes that influence brain aging. These data also highlight the need for good control of blood pressure, cholesterol, and blood sugar in women as they age. Keywords: Menopause, MRI, Cognition Disclosure: Nothing to disclose. P13. Regional Grey Matter Volume Predicts Symptom Improvement in a Randomized Clinical Trial of Tai Chi in Geriatric Depression Hanadi Ajam Oughli, Beatrix Krause-Sorio, Prabha Siddarth, Michaela Milillo, Lisa Kilpatrick, Linda Ercoli, Katherine Narr, Helen Lavretsky University of California - Los Angeles, Los Angeles, California, United States Background: Geriatric depression (GD) is associated with significant medical comorbidity, cognitive impairment and brain atrophy, premature mortality, and suboptimal treatment response. More efficacious treatment to improve mood, cognition and quality of life in GD are therefore needed. Mind-body interventions like Tai Chi are promising adjunct treatments to antidepressants. We tested the effect of three months of Tai Chi vs. health education control on grey matter volume (GMV) change and whether baseline GMV predicted symptom and cognitive outcome differentially in the two groups. Methods: Forty-nine older adults over 60 (38 women, 11 men) diagnosed with major depressive disorder undergoing concurrent antidepressant treatment were randomized to either Tai Chi training (N = 26) or health education control (N = 23). We administered clinical scales for depression and anxiety and participants were evaluated with a comprehensive neuropsychological battery at baseline and follow-up. In addition, participants underwent an MRI scan at baseline and follow-up. We used general linear models (GLMs) to test time-by-group interactions on these clinical and cognitive scores. Freesurfer version 6.0 was used to process T1-weighted images and perform voxel-wise whole-brain GLMs of group-by-time on symmetrized percent GMV change. Furthermore, we tested group differences in the relationship between baseline GMV and symptom change. All models included age and sex as covariates. Results: The groups did not differ in demographics or clinical scores at baseline (p > .05) and there were no group differences in symptom change from baseline to follow-up (p > .11). There were no group differences in treatment-related GMV change. However, while lower baseline GMV in several clusters in the Tai Chi group was associated with larger improvements in anxiety, the health education control group did not show such effects. This pattern was similar for depressive symptoms in a cluster in the right precuneus. Conclusions: Three months of Tai Chi may be insufficient time to lead to clinical improvements and structural brain changes in GD. However, individual GMV may predict symptom improvements. Longer trials are needed to investigate the long-term effects of Tai Chi on clinical symptoms and neuroplasticity. Keywords: Late Life Depression, Geriatric, Cognition, Neuroimaging Disclosure: Nothing to disclose. P14. Speech Biomarkers for Delirium and Cognitive Impairment in Hospitalized Older Adults Sunny Tang, Yan Cong, Gwenyth Mercep, Mutahira Bhatti, Grace Serpe, Valeria Gromova, Majnu John, Mark Liberman, Liron Sinvani Feinstein Institutes for Medical Research, Glen Oaks, New York, United States Background: Up to 50% of hospitalized older adults (75+) experience delirium, which is associated with increased morbidity, mortality, and resource utilization, as well as a 12-fold increased risk for new-onset dementia. Yet, 75% of delirium remains undiagnosed. Few studies have systematically studied speech and language disturbances in hospitalized older adults with delirium, and to our knowledge, no previous study has attempted to objectively quantify these phenomena with computational methods. The objectives of this study were to 1) systematically evaluate and describe speech and language disturbance in hospitalized older adults with and without delirium, 2) evaluate whether automated speech analysis could be feasibly implemented for acutely ill hospitalized patients, and 3) explore whether computational speech and language features could be used to diagnose patients with delirium. Methods: Hospitalized older adults aged 75 years and older (n = 33) were recruited across three medicine units at an academic hospital. Patients with documented diagnosis of dementia were excluded. Delirium assessment was completed by trained research assistants and verified by a delirium expert (LS), and included: Orientation, 3-item recall, confusion assessment method (CAM) long form; and Richmond Agitation and Sedation Scale (RASS). Diagnosis was based on these datapoints and the DSM-5 criteria for delirium. Clinical ratings for language disturbance were completed using the Scale for the Assessment of Thought Language and Communication (TLC). Audio recordings were collected for open-ended prompts, a paragraph reading, and fluency tasks, then transcribed verbatim. Recordings and transcripts were processed separately for each task using an automated pipeline to extract acoustic (prosody and voice quality, speaking tempo and pauses) and textual features (semantic coherence, dysfluencies and speech errors, lexical characteristics, parts-of-speech, speech quantity). To reduce the feature space, we first selected only the features which showed a trend-level correlation with the total CAM score (p < 0.10), then visually inspected correlation plots to remove redundancy. The final feature set included 26 measures. Group effects were compared using ANOVA and chi-squared tests, correlations were measured with Pearson coefficients, and category fluency totals were compared to normative data using one-sided t-tests. We used binomial elastic net regression models to predict delirium status (delirium(-) vs. delirium(+)), training on the full dataset with 10-fold internal cross-validation. Results: Of the 33 participants, 10 met criteria for delirium (Delirium(+)) and 23 did not (Delirium(-)). The Delirium(+) group scored significantly higher on total TLC score (p = 0.05, d = 0.81) and incoherence (p = 0.001, d = 1.41). Participants with delirium scored lower on category fluency compared to those without delirium (p = 0.02, d = -0.97), and both groups scored lower than the normative population (Delirium(-): p < 0.001, d = -1.22; Delirium(+): p < 0.001, d = -2.20). Higher CAM score was correlated with total TLC score (r = 0.41, p = 0.02), incoherence (r = 0.58, p < 0.001), loss of goal (r = 0.36, p = 0.04) and lower category fluency (r = -0.41, p = 0.02). Delirium status was predicted with demographics alone, demographics and clinical speech ratings, demographics and computational speech/language features, or all of these. The model with demographics and computational speech/language features performed best, classifying delirium status with accuracy of 78%, kappa of 0.4, and area under the curve of 0.90. All 23 Delirium(-) participants were correctly identified, in addition to 8/10 Delirium(+) participants. In this model, presence of delirium was most highly predicted by speech errors during paragraph reading, transcribed symbols (including punctuation, restarts, and incomplete words) in the open-ended narratives, use of determiners in the family narrative task, and semantic diversity (lexical ambiguity) of words given during the fluency tasks. Absence of delirium was most highly predicted by filled pauses during the family narrative task (e.g., “um,” “uh”), use of adverbs in open-ended narratives, use of adjectives during the picture description, and variance in jitter (fluctuations in voice amplitude) during fluency tasks. Conclusions: Hospitalized older adults with delirium demonstrate significant impairments in speech and language. In particular, delirium is associated with incoherence, loss of goal, and decreased category fluency scores. Hospitalized older adults without delirium may also demonstrate a subtle cognitive impairment relative to the normative population, as reflected in lower category fluency scores. Automated speech and language analysis was feasibly completed in the acute care setting, and computational features were highly informative for predicting delirium status, providing a proof of concept for this approach. Keywords: Delirium, Older Adults, Natural Language Processing (NLP), Cognitive Impairment, Automated Natural Speech Analysis Disclosures: Winterlight Labs: Consultant, Contracted Research (Self); North Shore Therapeutics: Board Member, Founder (Self) P15. Biological Aging in Alcohol Use Disorder: A Multi-Method Investigation in Blood and Brain Lea Zillich, Metin Cetin, Elisabeth M. Hummel, Gabriel R. Fries, Josef Frank, Fabian Streit, Jerome C. Foo, Marion M. Friske, Georgy Bakalkin, Anita C. Hansson, Rainer Spanagel, Greg Sutherland, Consuelo Walss-Bass, Dirk A. Moser, Marcella Rietschel, Stephanie H. Witt Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Background: Alcohol Use Disorder (AUD) is associated with increased mortality and a variety of diseases, such as neurodegenerative diseases and cancer. Biological aging is defined as the gradual deterioration of biological functions, whereas chronological age describes the time since birth. Accelerated biological aging could provide an explanation for the increased mortality and morbidity in AUD. There are multiple biomarkers to estimate biological aging (BioAge-markers), such as epigenetic clocks, telomere length (TL), and mitochondrial DNA copy numbers (mtDNAcn), which are also used as a marker for inflammation. Epigenetic clocks estimate different age-related phenotypes, such as chronological age (Horvath), biological age (Levine), and telomere length based on DNA methylation levels. The acceleration of aging can be calculated by regressing chronological age on epigenetic age and analyzing the residuals. So far, no analysis has investigated multiple BioAge-markers in the context of AUD in peripheral blood and it is unclear how they relate to AUD in other tissues. While AUD has an impact on the entire body, addiction is thought to develop in the brain. Recent studies point towards changes in DNA methylation and gene expression in neuroinflammation-related pathways in the brain, which could be related to mechanisms of biological aging. TL declines naturally with aging, although the effect is much smaller in the brain than in whole blood leukocytes because there are fewer cells that differentiate/maturate. At the same time, increased levels of neuroinflammation in AUD could lead to a reduction in telomere length and altered mtDNAcn (and levels of heteroplasmy). It is hypothesized that biological aging is accelerated in AUD and that this effect is more pronounced in whole blood than in brain samples. Methods: BioAge-markers were measured in two cohorts of individuals with AUD and controls. The first cohort consisted of 179 individuals, 84 of which had a diagnosis of AUD, in whom whole blood samples were collected at the start of withdrawal treatment. The second cohort consisted of postmortem human brain samples. The brain tissue was obtained from the New South Wales Brain Tissue Resource Centre at the University of Sydney. A total of 91 samples from Brodmann Area 9 (N cases = 43), 94 from the caudate nucleus (N cases = 44), and 63 from the ventral striatum (N cases = 33) were included in the present analysis. For both cohorts, telomere length and mtDNAcn were measured by quantitative real-time polymerase chain reactions (qRT-PCR) and methylation levels were analyzed with the Illumina Infinium EPIC BeadChip. Levine’s, Horvath’s, and the telomere epigenetic clocks were calculated using the R package methylclock. For Levine’s and Horvath’s clocks, the age acceleration, controlled for cell-type composition, was used as an outcome, while the raw estimate was used for estimated telomere length to ensure comparability with measured TL. We performed linear regression analyses with the BioAge-markers as outcomes and included sex, AUD status, an AUD statusage interaction term, smoking, and age (for measured and estimated TL and mtDNAcn) as covariates. To explore the association between the BioAge-markers, we calculated Pearson correlation between the markers in each sample. Results: In peripheral blood, we identified significant associations between the BioAge-markers and AUD status, all pointing towards accelerated biological aging in AUD. All BioAge-markers were associated with chronological age in the expected directions and small to medium correlations between the BioAge-markers were observed. The highest correlation was observed for measured and estimated telomere length with r = 0.26. In postmortem human brain samples, a significant association of measured TL and AUD (p = 0.047) and a trend for the AUDage interaction (p = 0.061) was observed in the caudate nucleus. Only small correlations between the BioAge-markers were observed in postmortem brain samples. Conclusions: The present study is the first to investigate telomere length, epigenetic clocks, and mtDNAcn from postmortem brain and whole blood samples in individuals with AUD compared with healthy controls. We found evidence for increased biological aging in AUD, as shown by significant associations between epigenetic age acceleration, decreased telomere length and decreased mtDNAcn with AUD in whole blood. The investigation of biological aging in AUD seems to be most appropriate in blood samples. The present study cannot distinguish whether there is no AUD-related increase in biological age in the brain or whether BioAge-markers do not capture age acceleration equally well in brain tissue. Levine’s and Horvath’s epigenetic clocks are designed to be multi-tissue biomarkers and should therefore perform similarly in brain tissue. Although the telomere clock was trained on DNA methylation in blood, we observed only a small association between estimated and measured telomere lengths in the present study. Further studies investigating brain and blood tissue from the same individual are needed to draw conclusions about the overlap of molecular mechanisms of biological aging in blood and brain. Keywords: Biological Aging, Alcohol Use Disorder, Telomere Length, Mitochondrial DNA Copy Numbers, Epigenetic Clock Disclosure: Nothing to disclose. P16. Cholinergic Integrity in Postmenopausal Women Measured by [18F]FEOBV PET: Relationship to Age, Cholinergic Anatomy, Cognitive Performance and Alzheimer’s Disease Risk Factors Paul Newhouse, Alexander Conley, Tonnar Castellano, Brian Boyd, J. Patrick Begnoche, Sepideh Shokouhi, Brittany Bosko, Julie Dumas Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: Women appear at higher risk for Alzheimer’s disease. This increased risk may be linked to loss of estradiol (E2) support to basal forebrain cholinergic systems. The cholinergic system has been implicated in many aspects of the cognitive effects shown after E2 administration including attention, working memory, and effort-demanding tasks such as verbal memory. The Cognitive Health After MenoPause (CHAMP, NCT04129060) study is examining cholinergic functional integrity in normal postmenopausal women by measuring working memory performance, functional brain activation, cholinergic basal forebrain structure, and uptake of the cholinergic PET radiotracer [18F]-fluoroethoxybenzovesamicol ([18F]-FEOBV) and whether individual differences in AD biomarkers including amyloid and tau fluid markers (plasma, CSF), amyloid PET, and markers of neurodegeneration (nFL) are related to cognition, brain activation after anticholinergic challenge, cholinergic anatomy and functional integrity. Methods: One hundred twenty healthy cognitively normal postmenopausal women aged 50-70 years undergo a multimodal assessment including sleep and activity measures, cognitive performance, blood/plasma for genetics and AD biomarkers, and lumbar puncture for CSF biomarkers. In addition, participants undergo MRI scan during anticholinergic drug challenge (mecamylamine) to examine working memory-related brain activity as well as cholinergic basal forebrain structure/volume. Participants underwent a 30 min static PET scan starting three hours after injection with 6.5 MCi ±10% of [18F]-FEOBV. White matter mask from T1 MRI was applied to the [18F]-FEOBV PET as reference region for the calculation of the standardized uptake value rations (SUVRs) in global GM composite ROI (parietal, frontal, temporal, occipital, cingulate, hippocampus) as well as within individual ROIs. Basal forebrain (BFB) volumes for each hemisphere were extracted from T1 MRI for the sub regions Ch1-3 and the Nucleus Basalis of Meynert (NBM; Ch4). We examined how regional and whole-brain [18F]-FEOBV PET uptake correlated with age, Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) cognitive performance and amyloid PET using florbetapir. Results: Preliminary data show that ten healthy postmenopausal women (mean age: 57.3 ± 5.1 years) have thus far completed cholinergic PET. Global SUVR was observed to decrease with increasing age (r = -0.52, p < .1). Larger GM volume of the cholinergic NBM for both hemispheres LH: r = .51, RH: r = .65, p = .06) was associated with higher global [18F]-FEOBV SUVR. Global [18F]-FEOBV uptake was positively associated with global cognition as measured by the RBANS Total (r = 0.41). Finally, [18F]-FEOBV uptake was lower in participants who showed clinically elevated amyloid levels by florbetapir PET. Assessment of the relationship of cholinergic integrity via [18F]-FEOBV, BFB volume and cholinergic functional imaging to reproductive and menopause history and AD biomarker data is continuing. Conclusions: Decline of cholinergic integrity post menopause may increase the risk of future cognitive decline and [18F]-FEOBV may be a useful marker of future cholinergic decline along with AD-related biomarkers. The relationships between cholinergic integrity and other predictors in this pilot sample support the hypothesis of a menopausal increase in risk for AD-related pathology in some women, which may be accelerated by loss of cholinergic tone through estradiol depletion following the menopause transition. Keywords: Acetylcholine, Women’s Health, Alzheimer’s Disease, Positron Emission Tomography Imaging Disclosure: Nothing to disclose. P17. Insulin Resistance and Accelerated Cognitive Aging Katie Watson Lin, Fahim Abbasi, Thalia Robakis, Alison Myoraku, Isaac Satz, Natalie Rasgon* Stanford University School of Medicine, Palo Alto, California, United States Background: Insulin resistance is an early sign of metabolic dysfunction with the potential to lead to neuropsychiatric sequelae in the long term. Early detrimental effects of IR on brain function have been demonstrated with both indirect and direct measures of IR. However, the trajectory of pathological brain aging and its mediators and moderators are not well-defined. In order to identify whether insulin resistance in otherwise healthy young and middle-aged adults is associated with preclinical signs of neuropsychiatric impairment, we recruited 126 overweight but nondiabetic, non-depressed individuals who completed an insulin suppression test for direct measure of insulin resistance as well as a battery of cognitive and neuropsychiatric measures. We describe the study design and cohort and the relationships among baseline metabolic function and cognitive and psychiatric outcomes, including both subtle cognitive changes as measured by neuropsychiatric testing, and subclinical mood symptoms. Methods: Participants were recruited through flyers distributed on-campus and in communities surrounding Stanford University. Potential participants were screened for the following eligibility criteria: 23 to 61 years of age, body mass index (BMI) of 21 to 41 kg/m2, at least 12 years of education and a Mini Mental Status Exam (MMSE) score of >27, and adequate visual and auditory acuity for cognitive testing. Insulin-mediated glucose uptake was directly measured by a modified and validated version of the insulin suppression test (IST). The plasma glucose and insulin concentration values during the last 30 min of the infusion were averaged to determine the steady-state plasma glucose (SSPG) and steady-state plasma insulin (SSPI) concentrations, respectively. Plasma insulin, leptin, and cortisol were measured at the Core Laboratory for Clinical Studies at Washington University School of Medicine. Average relative telomere length was measured by quantitative PCR using a method adapted from the original published method by Cawthon et al. and expressed as the ratio of telomere abundance vs. a single copy gene (human b-globin) abundance (T/S ratios). Depressive symptoms were rated using the 24-item Hamilton Depression Rating Scale (HDRS). A cognitive battery of four hippocampus-linked tests was used in all participants in a fixed or randomized order. Results: Insulin resistance was associated with impaired performance on a visuomotor task (Purdue Pegboard) as well as increases in subclinical symptoms of depression. Furthermore, the direct measure of insulin resistance (SSPG concentration), but not fasting plasma insulin, fasting plasma glucose, or BMI, was associated with higher levels of depressive symptoms in subjects without clinical depression. Only the association of SSPG concentration with depressive symptoms passed multiple testing correction. The association of SSPG concentration with depressive symptoms was robust to adjustment for age, education, BMI, and fasting glucose. Telomere length, obtained as a measure of chronic physiologic stress or allostatic load, showed weak negative correlations with SSPG and fasting glucose, and stronger ones with age and dyslipidemia. Conclusions: This study of young and mid-life adults explores premorbid changes in metabolic function, mood, and cognition that may presage the development of overt psychiatric illness or cognitive decline. We found that even in this population of young, healthy but overweight adults, there is a range of insulin sensitivity, where greater degrees of insulin resistance were associated with higher BMI, hypertriglyceridemia, low HDL cholesterol, and increased circulating levels of fasting insulin, C-peptide, and leptin. Furthermore, higher degrees of IR were associated with greater degrees of subclinical depressive symptoms and with impaired performance specifically in a visuomotor task, though not in cognitive assessments across the board. Additionally, the significance of the association between IR and both visuomotor performance and subclinical depressive symptoms were attenuated when BMI and fasting glucose were included in the model, suggesting etiological overlap among these factors. We hypothesize that cognitive and psychiatric declines related to IR likely have a long subclinical prodrome that precedes overt impairment by many years. These data provide a baseline by which this hypothesis may be tested in future analyses. Keywords: Insulin Resistance, Telomeres, Cognitive Decline Disclosure: Nothing to disclose. P18. Effects of Chronic Oral THC Self-Administration on Working Memory Across the Lifespan Barry Setlow, Sabrina Zequeira, Emely Gazarov, Alara Guvenli, Erin Berthold, Takato Hiranita, Lance McMahon, Abhisheak Sharma, Christopher McCurdy, Jennifer Bizon University of Florida, Gainesville, Florida, United States Background: Individuals over the age of 65 have become the fastest-growing demographic of cannabis users. As the number of older adults in the US is expected to reach 90 million by 2050, it is imperative to understand the potential cognitive impacts of cannabis/cannabinoid use in this age group. Cannabis and cannabinoids such as delta-9-tetrahydrocannabinol (THC, the major psychoactive component of cannabis) generally tend to impair cognitive performance, but almost all studies of cannabis and cannabinoids have been conducted in young adult subjects. Given that many aged individuals already exhibit cognitive deficits, it is important to determine how cannabis/cannabinoids affect cognition in this population. Methods: To address this issue, we evaluated the effects of chronic oral THC self-administration on performance in a working memory task in rats. Young adult (5 months, n = 20) and aged (23 months, n = 18) Fischer 344 x Brown Norway F1 hybrid rats of both sexes were trained in operant chambers on a delayed response working memory task that depends critically on the prefrontal cortex. In this task, rats were trained in daily 60 min sessions (over 100 trials/day) to remember the left/right position of a response lever over short (0-24 s) delays to earn food rewards. Upon reaching stable performance on the task, rats were given 3 weeks of daily 60 min access to either plain gelatin or gelatin containing 1.0 mg/kg THC in their home cage in the afternoons, while testing in the working memory task continued in the mornings. In a second experiment, behaviorally-naïve aged rats were given daily access to either plain gelatin or gelatin containing 1.0 mg/kg THC in their home cage, and blood samples were collected after 3 weeks for analysis of circulating inflammatory factors. Behavioral data were analyzed via multi-factor ANOVA, with drug condition, age, and sex as between-subjects variables and working memory delay as a within-subject variable. Cytokine data were analyzed via Welch’s t-tests. Results: As expected, among rats that consumed plain (control) gelatin, aged rats performed worse than young on the working memory task (F(1,30)=10.38, p = .003). More importantly, there was an age x drug condition x delay interaction, such that THC had distinct effects in young adult vs. aged rats, particularly at long delays (F(6,180)=2.45, p = .03). Focused comparisons of the effects of THC in each age group showed that among young adults, rats that consumed THC gelatin performed comparably to rats that consumed plain gelatin (no main effects or interactions involving drug condition). In contrast, aged rats that consumed THC gelatin performed significantly more accurately than rats that consumed plain gelatin (F(1,14)=5.01, p = .04), particularly at long retention delays (F(6,84)=2.76, p = .02). There were no sex differences in the effects of THC consumption in either young adult or aged rats (no interactions involving sex and drug condition). In the second experiment, rats that consumed THC gelatin for 3 weeks had reduced levels of some pro-inflammatory cytokines (e.g., TNFα, t(19.99 = 16.61, p < .001) and increased levels of some anti-inflammatory cytokines (e.g., IL-10, t(19.99)=6.55, p < .001). Conclusions: These findings suggest that at advanced age, chronic cannabis/cannabinoids may have beneficial effects on at least one aspect of executive function (working memory). In addition, the oral route of administration (which is used frequently by older adults) and the fact that consumption was voluntary indicate that the THC dose employed here is within a tolerable range that does not produce significant adverse effects. Finally, given that alterations in the peripheral inflammatory environment have been associated with age-related cognitive impairments, the fact that chronic THC appears to attenuate some aspects of inflammatory signaling suggests one mechanism by which it may exert beneficial effects on working memory. Keywords: Cannabis, Aging, Working Memory, Rat, THC Disclosure: Nothing to disclose. P19. Ketogenic Diet Interacts With Estrogen to Restore Memory in Aging Females Tyler Cox, Cobb Karinne, Patel Kesha, Murphy Caitlyn, Horovitz David, Moats Jacqueline, Dufala Haley, Enos Reilly, Hollis Fiona, Joseph McQuail University of South Carolina, Columbia, South Carolina, United States Background: Women typically live to older ages than men and are more likely to be afflicted by Alzheimer’s disease (AD). Indeed, the influence of biological sex and sex hormones is the focus of ongoing study to determine the mechanisms that contribute to worse than-expected cognitive outcomes in aging women and to identify interventions that are appropriate to protect cognition in a sex-dependent manner. The ketogenic diet has emerged as a potential intervention to treat central and peripheral signs of AD, which include defective brain glucose metabolism and elevated resting blood glucose. However, it is not clear the degree to which normally aging males and females experience the same cognitive benefits of the ketogenic diet and whether age-related reductions in circulating estrogens, which typifies menopause, interacts with effects of this diet. Consequently, we investigated the effects of a ketogenic diet on cognition in normally aging male and female rats (Study 1) or the effects of diet in a surgical model of menopause with and without hormone replacement (Study 2). Methods: In Study 1, male and female Fisher 344/Brown Norway F1 hybrid rats were obtained from the National Institute of Aging at 6 or 24 months of age. Rats were assigned to consume sex-adjusted, calorie-matched diets that provide most calories from carbohydrates (control diet) or medium-chain triglycerides (ketogenic diet). A subset of rats was included as ad libitum-fed controls, with unrestricted access to normal rat chow, to differentiate effects of calorie-restriction from diet composition. (n = 7-10/sex/age/diet). After 6 weeks on diet, during which time body weight and blood levels of glucose and β-hydroxybutyrate were monitored continuously to confirm nutritional status, all rats were characterized in the Morris water maze (MWM) using an 8-day place-learning, spatial reference memory protocol to evaluate hippocampus-dependent cognition. For Study 2, 18-months-old female rats were ovariectomized (OVX) and implanted subcutaneously with silastic implants to release estrogen (E2) or inert vehicle (OIL), before assignment to specialized diets (n = 10-12/hormone/diet) and behavioral testing as described in Study 1. Results: In Study 1, sex, age, and diet all significantly influenced spatial memory; males were overall better than females, aged performed worse than young, and calorie-restriction and ketogenic diet improved memory relative to ad libitum controls. There was also a trend towards a sex-by-age-by-diet interaction. Follow-up comparisons revealed that young males exhibited better spatial memory following calorie restriction but in older males, only ketogenic diet improved memory compared to ad libitum-fed controls. In females, calorie-restriction improved spatial memory, but only young females, not aged females, showed better memory after consuming ketogenic diet. The loss of benefit conferred by consumption of ketogenic diet could relate to changes in circulating E2 as we determined in Study 2 that replenishing E2 to middle-aged rats after OVX led to ketogenic diet-related enhancement of memory that was not evident in OIL-treated controls. Further, replenishing E2 to animals consuming the calorie-matched control diet did not improve performance relative to OIL. Conclusions: We conclude that dietary interventions, including calorie restriction and ketogenic diet, are practical and effective interventions that protect against age-associated cognitive decline. However, the efficacy of certain diets to preserve memory varies between males and females over the lifespan. Aging females do not show the same cognitive benefits of ketogenic diet that was observed in younger females or aged males. Positive effects of ketogenic diet in aging females can be observed after administration of E2, suggesting that this diet might confer cognitive advantages in the context of hormone replacement therapy initiated close to the onset of menopause in middle-aged women. Collectively, this insight could be used to optimize and personalize dietary guidelines for subpopulations of older individuals at risk for memory loss and AD. Keywords: Ketogenic Diet, Declarative Memory, Sex Differences, Brain Aging, Alzheimer’s Disease Disclosure: Nothing to disclose. P20. Neuroadaptive Acetylcholinesterase Regulation in Stress and Cognitive Aging Vinay Parikh, Alyssa Kniffin, Charlotte Bavley, Miranda Targum, Joanna Severino, James Flowers, Debra Bangasser, Mathieu Wimmer Temple University, Philadelphia, Pennsylvania, United States Background: Synaptic release of the neurotransmitter “acetylcholine (ACh)” has been implicated in the neuromodulation of cognitive capacity. Our previous research has shown that a reduction in ACh transmission in cortical networks increases cognitive vulnerability in aging. However, the mechanisms that regulate age-related changes in cholinergic transmission and individual differences in cognitive aging remain unknown. Acetylcholinesterase (AChE) is a hydrolytic enzyme which promotes ultra-fast cholinergic signaling in brain circuits. Here we sought to identify the role of synaptic (AChE-S) and readthrough (AChE-R) variants in AChE regulation and cognition in aging. These variants are reported to be altered in age-related neuropathologies. Because stress exposure has previously been shown to alter AChE expression and psychological stress is a risk factor for age-related cognitive decline, we also examined the effects of chronic variable stress (VS) on AChE expression and catalytic activity. Methods: Young (3 mo) and aged (22 mo) rats of both sexes were trained in an operant attention task that requires the animals to discriminate between signaled and non-signaled cues for subsequent reward. After attaining performance criterion, rats were subjected to a distractor test session following which brain tissues from the prefrontal cortex (PFC) and hippocampus (HPC) were microdissected for AChE mRNA and protein isolation. The impact of VS on AChE biochemistry was investigated in another cohort of adult male and female rats. For these studies, the AChE activity was assessed in both salt-soluble and detergent soluble fractions that contain different proportions of AChE-R encoding monomeric (G1) and AChE-S encoding tetrameric (G4) subunits, respectively. Results: In general, aged rats performed poorly on the distractor test session as compared to their young counterparts (F(1,28)=15.17; p < .001). However, their performance remained highly variable with some animals exhibiting significant impairments (p < .001 vs age-unimpaired) while others performed at par with the young animals (p > .43). Quantification of mRNA expression using RT-qPCR revealed an age-dependent reduction in total AChE expression in the PFC (p < .05) that was primarily driven by the S variant. Interestingly, the average ratio of R/S variants was found to be higher in aged rats in both brain regions (PFC: 1.27 vs.1.01 in young; HPC: 1.93 vs. 1.02) illustrating a transcriptional shift. Our stress experiments revealed sex-specific differences with higher AChE activity in subcellular fractions rich in G1 and G4 subunits in females from the PFC (p < .04). However, VS reduced AChE activity in detergent-soluble fraction regardless of sex (F(1,19)=30.65; p < .001). Moreover, a sex x VS interaction was observed (F(1,18)>3.72; p < .05) depicting higher AChE-R subunits in males and lower AChE-S subunits in females. Conclusions: Age-related reduction in AChE-S (more prominent synaptic variant) may possibly reflect a neuroadaptive role in preserving cholinergic function by reducing ACh hydrolysis. This interpretation also parallels reduced catalytic activity in the detergent soluble fraction mostly rich in the G4 isoform in VS-exposed rats. However, an increase in AChE-R variant during stress or aging may offset this protective effect. Collectively, these data indicate that an imbalance in AChE-R/S variants may underlie cognitive vulnerability in aging and accelerated cognitive decline with stress. Moreover, stress-induced compensatory changes in cholinergic signaling and its impact on age-related changes in cognitive processes may differ between sexes. Keywords: Aging, Cognition, Stress, Acetylcholinesterase, Splice Variants Disclosure: Nothing to disclose. P21. In Vivo Calcium Imaging Reveals Sex Differences in Ventral Hippocampal Activity in Alzheimer’s Disease Mice Holly Hunsberger, Kameron Kaplan, Lainey Toennies The Chicago Medical School at Rosalind Franklin University, North Chicago, Illinois, United States Background: Neuropsychiatric disturbances, such as depression and anxiety, are observed in 90% of Alzheimer’s disease (AD) patients and are frequent in those at risk for AD. We’ve previously shown that anxiety is prevalent at an earlier age in female APP/PS1 AD mice and that women with anxiety and amyloid burden transition more quickly to dementia than men in the ADNI human cohort. We’ve also shown that there are sex-specific circuit and brain-wide network changes in AD mice at 6 months of age. Additionally, early-stage AD patients often exhibit hyperexcitability throughout the hippocampus which correlates to memory impairment and eventually worsening of the disease. Here, we aimed to determine how and when this neuronal activity is changed in aging and AD mice. Methods: Male and female control and APP/PS1 mice at 2 and 6 months of age were injected with a AAV-syn-GCaMP8f virus and a GRIN lens was implanted targeting the ventral hippocampus (n = 4-6). After 3 weeks of recovery, mice were run through open field, elevated plus maze, and contextual fear conditioning to assess anxiety-like behaviors and cognition. During each task, we recorded calcium activity using Inscopix software and analysis programs. Data were analyzed using ANOVA, with repeated measures when appropriate. Tukey was used for all post-hoc comparisons. Alpha was set to 0.05 for all analyses. Data are expressed as means + /- SEM. Results: We found sex-specific changes in neuronal activity and behavior. Females exhibited earlier anxiety and cognitive decline as previously reported (p < 0.05). Male AD mice did not exhibit increased anxiety-like behavior in these tasks relative to controls. However, male AD mice exhibited changes in neuronal activity compared to controls depending on time of day in the elevated plus maze (p < 0.001). Additionally, this increase in calcium transients correlated with increased anxiety behavior in males (p < 0.05). We are actively continuing these experiments to add a 6-month age group to determine how aging impacts the neuronal excitation/inhibition balance. Conclusions: Both men and women with an AD diagnosis will develop cognitive decline, but the mechanisms driving this decline might be different. Therefore, it is essential to understand the differences in disease progression in order to create personalized therapeutics. Our initial studies provided a snapshot of the sex-specific mood and circuit changes throughout the brain, but with our in vivo calcium imaging system we are now able to view dynamic brain changes while the animal ages and track individual cellular activity across different behavioral paradigms. These results provide evidence that brain hyperexcitability starts earlier in males while females show more behavior changes early in the disease. Keywords: Alzheimer’s Disease, Anxiety, Sex Differences, In Vivo Calcium Imaging, Hippocampus Disclosure: Nothing to disclose. P22. Prelimbic Correlates of Fear Memory Generalization in Endocannabinoid 2-Arachidonoylglycerol Deficient States Luis Rosas-Vidal, Saptarnab Naskar, Megan Altemuse, Sachin Patel Northwestern University, Chicago, Illinois, United States Background: Post-traumatic stress disorder (PTSD) is a psychiatric disorder that develops following exposure to a traumatic event. Its lifetime prevalence is estimated to be 6.8%. Fear responses to stimuli that were previously present during a traumatic experience enables survival. However, in PTSD, patients may experience generalization of their fear responses even to otherwise safe stimuli. The endocannabinoid (eCB) system is a retrograde neurotransmitter system that has been implicated in regulating fear and anxiety. 2-arachidonoylglycerol (2-AG), one of the major centrally active eCB lipids, is thought to mediate resiliency to traumatic experiences. Here we aim to explore if 2-AG is involved in regulating fear generalization and characterize how prelimbic neurons are involved in fear discrimination and how 2-AG mediates this process. Methods: We used a mice model of fear-conditioning in combination with systemic injections of a drug (DO34) that blocks synthesis of 2-AG. Male mice were exposed to 8 tone presentations (CS+), each co-terminating with a brief electric foot-shock. The following day (Day2), mice were injected with DO34 (50 mg/kg) intraperitoneally 2 hours prior a memory recall test conducted by exposing mice to alternating presentations of 2 novel tones (NT) and 2 CS + tones. Fear responses were measured by quantifying the percentage of time during tone presentations that the mice exhibit freezing (immobility except movement required for breathing). For our in vivo recording experiments, mice were injected with a viral vector expressing the calcium indicator GCaMP7f in the prelimbic cortex (PL) and a miniature GRIN lens above PL. Mice were allowed to recover and were habituated to having a miniaturized microscope attached to a baseplate sitting over the lens. Following habituation, mice were conditioned to tones as described above while simultaneously recording calcium activity using the miniaturized microscope. The following day, mice were injected with either vehicle or DO34 and were exposed to NT and CS + tones as described above. The recorded imaging data was processed and individual calcium traces were extracted using CNMFe segmentation algorithm. Peritone histograms were generated from the Z-scored data. Neurons with post-shock responses exceeding ± 3 Z-scores in any 2 consecutive 1s bins during the 10s post-shock period were considered to have significant excitatory or inhibitory responses ((+) responsive and (-) responsive, respectively). SVM decoder analysis was performed on peri-event extracted traces to assess if NT and CS + were decodable from each other. Results: To address if 2-AG is involved in regulating fear generalization, mice were injected with DO34 prior to fear recall (n = 19 and 16, vehicle and DO34 respectively). Mice injected with DO34 showed a significant increase in freezing only to novel tone presentations (all p’s<0.0338), but not to the CS + , suggesting that blocking 2-AG signaling enhances fear generalization to novel tones. To address how fear generalization is represented at the neural level and how this representation is modified by reductions in 2-AG, we repeated our fear conditioning experiment while recording changes in single cell calcium activity within PL. Our imaging data shows that DO34 increased average neuronal activity to novel tones (n = 636 and 670 neurons, vehicle and DO34 respectively). Focusing on neurons that significantly change their activity to the tone did not reveal any differences in magnitude between groups for (+) responsive (n = 114 and 204 neurons, vehicle and DO34 respectively). Interestingly, the proportion of neurons that respond to both CS + and novel tones was significantly larger for the DO34 group (10.62 % vs 18.36 %; chi square statistic= 7.3072, p = 0.00687) while the proportion of neurons that respond to the CS + was smaller (32.23 % vs 20.82 %; chi square statistic= 12.771, p = 0.000352. Thus reducing 2-AG signaling leads to increased generalization and this in turn is associated with an increased proportion of PL neurons that signal equally to both CS + and novel tones. We hypothesized that the increase in fear generalization may be due to a loss in the ability to discriminate between the NT and CS + by PL neurons. Surprisingly, using an SVM decoder we found that NT and CS + can be decoded from each other and in fact the decoder accuracy increases in the DO34 group (96.0% for Veh, 99.5% DO34, 43.3% shuffled control; all p’s< 0.0002) . Conclusions: Our present data suggests that 2-AG signaling may be required for maintaining the specificity of fear memories. Furthermore, reducing 2-AG leads to a larger proportion of PL neurons that fail to discriminate between CS + and novel tones. Interestingly, while there is an increase in the proportion of PL neurons that respond to both tones, the coding characteristics for NT and CS + are distinct enough that they can be decoded from each other. Thus, while the proportion of PL tone responsive neurons reflect generalization/discrimination the characteristic of the neuronal responses to NT and CS + are decodable from each other and independent of generalization state. All this may suggest that generalization may be driven by inputs to PL that are sensitive to 2-AG deficient states rather than arising in PL itself. Keywords: Fear Conditioning, Prelimbic, Miniscope, in Vivo Calcium Imaging, Endocannabinoids Disclosure: Nothing to disclose. P23. Stress Cues Exposure Induced Excitatory Plasticity in the Pentapartite Synapse Stephen Walterhouse, Devki Bhatt, Michael Meyerink, Ritchy Hodebourg, Anna Kruyer, Michael Scofield, Peter Kalivas, Lindsay McDonald, Constanza Garcia-Keller Medical College of Wisconsin, Milwaukee, Wisconsin, United States Background: Converging epidemiological studies indicate that a history of acute life-threatening events increases the incidence of post-traumatic stress disorder (PTSD), and a diagnosis for PTSD carries 30-50% comorbidity with substance use disorders (SUDs). Thus, patients with comorbid PTSD/SUDs have greater drug use severity and show poorer treatment outcomes than patients diagnosed with either alone. Using a rodent model, we found that a single stressful event experienced 3 weeks prior can enhance drug intake and trigger a number of enduring adaptations within corticostriatal synapses of the nucleus accumbens core (NAcore), which resemble drug-induced adaptations. We recently found that pairing the stressful event with a novel odor (stress conditioned stimulus, stress CS) elicits a conditioned stress response and recapitulates some of the behavioral and physiological aspects of PTSD. Previous publications have shown that drug-associated cue presentation evokes transient increases in the pentapartite synapse, including the pre and postsynaptic neurons, astrocytes, microglia, and the extracellular matrix (ECM). Given the overlap between the enduring adaptations produced by acute restraint stress and withdrawal from drug use, we hypothesize that the exposure to a stress CS elicits synaptic plasticity in the pentapartite synapse in NAcore. Moreover, medium spine neurons (MSNs) constitute 90-95% of the neurons in the NAcore and are chemically coded into two subtypes that selectively express D1 or D2 dopamine receptors. These two populations appear to subserve distinct behavioral functions, with D1 activation generally promoting behaviors and D2 activation inhibiting behaviors. However, the effect on stress CS exposure on accumbal population remains elusive. Methods: Male and female Sprague Dawley and Long Evan rats were restrained for 2 hours in plexiglas cylinders and exposed to an odor that became a stress CS, or sham animals were left in new home-cage boxes exposed to an odor (neutral stimulus - NS). Three weeks after the stressful experience animals were exposed to a cage that contained bedding in one corner and stress CS or NS on the opposite corner (noxious object which to be buried). After completing the defensive burying task, animals were then euthanized, perfused and tissue was processed for the following analysis: 1) spine morphology studies using DiI lipophilic colorant, 2) matrix metalloproteinase (MMP) activity was quantified using zymography gel microinjections, 3) astrocyte morphology studies using GFAP-hM3dq-mCherry virus, and 4) microglia/macrophage populations expressing IBA-1 were semi-quantitatively analyzed by immunohistochemistry. Additionally, we recorded single cell Ca2+ dynamics in D1- and D2-MSNs from freely moving rats (D1 or D2-cre rats) using a miniature microscope (nVista) and virally expressed Cre-dependent Ca2+ indicator (GCaMP8M) during defensive burying task and extinction to stress CS. Results: In all, we have observed key features of stress CS-induced changes in pentapartite synaptic plasticity. Stress CS exposure is associated with synaptic potentiation in NAcore, quantified by an increase in dendritic spine head diameter and spine density, and increased matrix metalloprotease-9 activity that catalyzes proteins from the extracellular matrix (ECM). Stress CS exposure also induced down-regulation of astroglial glutamate transporters (GLT-1) and retraction of astrocyte synaptic coverage compared to control and stress NS animals. Also, stress CS animals had induced changes in microglia morphology, as well as the number and volume of IBA-1+ cells compared to control and stress NS animals. Furthermore, we observed differential changes in Ca2+ dynamics in D1 vs D2-MSN during the defensive burying task and extinction to stress CS. Conclusions: These data suggest that the neuroadaptations and calcium dynamics that coincide with 15 min defensive burying task may be correlated with the synaptic plasticity in NAcore that lead to differential stress coping responses. Previous data from the lab has shown that stress CS exposure did not induce sucrose-seeking in sucrose-trained rats, thus we hypothesize that the plasticity described here is potentially indicative of pathological features of stress disorder. Keywords: Post Traumatic Stress Disorder, Nucleus Accumbens, Calcium Imaging Disclosure: Nothing to disclose. P24. Cell-Type Specific Induction of Cyclo-Oxygenase-2 in Layer II/III Prefrontal Cortical Neurons Mediates Stress-Induced Anxiety Phenotypes in Mice Robert Fenster, Kenneth McCullough, Sergey Naumenko, Yan Li, Andrew Thompson, Claudia Klengel, Joy Otten, Shu Dan, Torsten Klengel, Vadim Bolshakov, Kerry Ressler McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: The ability of the medial prefrontal cortex (mPFC) to exert top-down control of behavior is affected by stress. Stress is also known to produce transcriptional responses that influence long-term behavioral adaptations. The molecular response of mPFC to stress is incompletely understood, however, in part because of the region’s cellular heterogeneity. There is great need for novel therapeutic targets for stress-induced behavioral disorders. Methods: We used single nuclear sequencing (InDrops) to sequence over 100,000 nuclei from mPFC after a stress-inducing Pavlovian fear conditioning assay (n = 8 groups of n = 2-3 male mice) and identified cell-type specific differentially expressed genes after fear conditioning and fear expression. We have used fluorescent in situ hybridization (FISH) and immunohistochemical techniques to confirm markers for cell clusters found with single nuclear sequencing (n = 4-5 male mice). We have used a combination of pharmacology, cre/lox recombination, and slice electrophysiology with behavior to characterize candidates functionally. Results: We identified Ptgs2, encoding cyclo-oxygenase 2, as an important candidate that is upregulated in layer II/III excitatory neurons after stress. Specifically, Ptgs2 was transiently upregulated with shock-induced fear learning and fear expression, along with Bdnf, Nptx2, and Lingo1, in a layer II/III neuronal population marked by the neuronal excitatory gene Slc17a7 and cell-type specific neuropeptide Penk. These dynamic cell-type specific expression patterns identified with snRNAseq were validated with quantitative fluorescent in situ hybridization. Using a pharmacological approach, we found that systemic lumiracoxib, a selective Ptgs2-inhibitor, led to a significant reduction in fear expression (2-way ANOVA, Drug effect, p = 0.03). Furthermore, genetic ablation of Ptgs2 in mPFC led to reduced stress-induced anxiety-like behaviors in the elevated plus maze (Unpaired student’s t-test, p = 0.02). Layer II/III neurons expressing virally-induced Cre in a floxed Ptgs2 background exhibit baseline depolarization compared with non-transduced neighboring cells (Resting membrane potential, unpaired student’s t-test, p = 0.002). Conclusions: Together these findings suggest that Ptgs2 is expressed in a dynamic, cell-type specific way in Layer II/III Penk+ neurons in mPFC, and that its role in prostaglandin and /or endocannabinoid regulation within these neurons may be an important mediator of stress-related anxiety behavior. Keywords: COX-1 and COX-2, Anxiety and PTSD, Medial Prefrontal Cortex, Mouse Models, Single-nucleus RNA Sequencing Disclosure: Nothing to disclose. P25. A Link Between Social Buffering of Acute Stress and Tumor Necrosis Factor Gina Kemp, David Stellwagen McGill University, Montreal, Canada Background: The social buffering of stress occurs when an animal displays a reduced stress response in the presence of a conspecific. Few studies have investigated the physiological changes that underlie and accompany this phenomenon, but the immune system has emerged as a common denominator in the social buffering of stress. In rodents, socializing has yielded positive outcomes for wound healing and stroke recovery. Indeed, it is well studied that tumor necrosis factor (TNF), a key cytokine, plays an important synaptic role as a neuromodulator. We have recently demonstrated mechanistically that TNF mediates stress-induced plasticity (Kemp et. al., In Press - Molecular Psychiatry, 2022). In this original poster, I present emerging evidence for links between socializing and TNF in the context of acute stress. Methods: All described experiments were conducted according to the Canadian Council for Animal Care as reviewed and approved by the Montreal General Hospital Facility Animal Care Committee. C57BL/6J mice between 8 and 16-weeks-old were exposed to two stress paradigms: forced swim stress and restraint stress. After both paradigms, animals were either single housed, or group housed in new cages with their stressed littermates. Unstressed control groups were age matched and were subjected to the same housing and handling conditions without the exposure to stress paradigms. After 24 hours, the behavioral output of the acute-stress response was tested using the light-dark box test (10 min total) (Crawley and Goodwin, 1980). Time spent in the light box inversely correlates with anxiety-like behavior. The biochemical output of acute-stress response was also measured 24 hours post-stress: mice were euthanized by isoflurane, blood samples were collected by cardiac puncture, and bilateral ventral hippocampal tissue (vHPC) was excised to measure the biochemical output of acute stress. TNF levels were measured using the mouse TNF ELISA kit (eBioscience, Mouse TNF alpha ELISA Ready-SET-Go! Kit #88-7324). Samples were standardized to total protein-input measured by a bicinchoninic acid assay (BCA) kit from Thermo Fisher Scientific (#23227). Samples also were analyzed for other cytokines using a Milliplex panel (Mouse High Sensitivity T-Cell, Millipore, St. Charles, MO, USA) and analyzed using the Bio-Plex™ 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Serum corticosterone (CORT) was measured by ELISA kit (Cayman Chemical, cat. #501320). Results: Anxiety-like behavior was increased in single-housed males subjected to forced swim stress (FST) (student t-test; p = 0.0115, N = 9-10 / group), but there was no comparable phenotype detected in females (student t-test; p = 0.5976, N = 7-8 / group). The described phenotype in males had already been shown to be accompanied by a significant increase in TNF (Kemp, In Press). Furthermore, single housing on its own in this context does not increase anxiety-like behavior nor TNF levels (Kemp, In Press). Intriguingly, unlike single-housed mice, group-housed mice post-FST did not show an anxiety-like phenotype (student t-test, p = 0.5066, N = 10-11 / group). This observation was also replicated using restraint stress (one-way ANOVA F (2, 31) = 7.440, p = 0.0023, Tukey’s post hoc analysis of control versus single-housing post-RS p = 0.0047, control versus group-housing post-RS p = 0.9680, single-housing post-RS versus group-housing post-RS p = 0.0073, N = 11-12 / group). Additionally, group-housed animals post-stress showed a reduction in vHPC TNF (student t-test p = 0.0439). The levels of four other cytokines, namely IL-1b, IL-10, IL-12 and MIP-2, were also significantly increased in socialized animals post-stress when probed using a cytokine multiplex (all one-way ANOVAs were significant; Tukey’s post hoc analysis of control versus group-housing post-stress for IL-1b p = 0.0356, IL-10 p = 0.0432, IL-12 p = 0.0331, and MIP-2 p = 0.0403, N = 4-5 / group) When we tested the levels of corticosterone (CORT), preliminary data showed that TNF levels better correlate with anxiety-like behavior than CORT. Indeed, there was a main effect of prior exposure to stress on serum CORT levels (24 hours post-stress; two-way ANOVA F (1, 10) = 8.551, p = 0.0152, N = 3-4 / group) and there was no difference between group-stress and single-stress animals (p = 0.9952). Conclusions: We report a moderating effect of housing conditions post-stress on anxiety-like behavior and TNF cytokine levels in the vHPC. Indeed, socially housed animals post-stress do not display an anxiety-like phenotype and show an active suppression of vHPC TNF levels compared to single-housed animals post-stress. Furthermore, we show an induction of other cytokines in the absence of anxiety-like behavior post-stress. Therefore, these findings contribute to an ongoing scholarly discussion whether resilience and susceptibility are two sides of the same coin or if are they two different phenomena with distinct active mechanisms for each. Indeed, our results point to active molecular mechanisms involved in the social buffering of stress as a form of resilience. Further investigation of these mechanisms could provide us with a path to characterize the molecular underpinning of resilience. Keywords: Social Stress Buffering, Cytokines, Corticosterone, Anxiety-Like Behavior, Tumor Necrosis Factor Disclosure: Nothing to disclose. P26. Traumatic Brain Injury Increases Contextual Fear Generalization, Which is Paralleled by Hippocampal Memory Trace Dysfunction and Can Be Decreased by (R,S)-Ketamine Administration Josephine McGowan, Liliana Ladner, Claire Shubeck, Amanda Anquiera-Gonzalez, Christina LaGamma, Juliana Tapia, Ariana DeFrancesco, Tzong-Shiue Yu, Steven Kernie, Christine Ann Denny Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Traumatic brain injury (TBI) is a debilitating neurological disorder caused by an impact to the head by an outside force. TBI results in persistent cognitive impairments, including fear generalization, which is defined as the inability to distinguish between aversive and neutral stimuli. Fear generalization can be assayed in mice using a contextual fear discrimination (CFD) paradigm, which tests fear behavior in learned aversive versus neutral contexts. Methods: To identify the fear generalization neural ensembles altered by TBI, we utilized ArcCreERT2 x enhanced yellow fluorescent protein (eYFP) mice, which allow for activity-dependent labeling and quantification of memory traces. ArcCreERT2 x eYFP mice were administered either a sham surgery or the controlled cortical impact (CCI) model of TBI. One month following surgery, mice were injected with 4-OHT to open up a tagging window and administered a 1-shock contextual fear conditioning (CFC) paradigm. Mice were then administered an additional 5 days of CFD and euthanized following exposure to the aversive (A) or neutral (B) contexts. Dentate gyrus (DG) neural activity was quantified. (n = 13-15 mice / group). In a separate set of studies, we determined if administering (R,S)-ketamine 1 hour after a CCI would decrease fear generalization in TBI mice. (n = 6-8 mice / group). Results: TBI mice exhibited increased fear generalization (e.g., lack of discrimination between the aversive and neutral context) in the CFD paradigm (ANOVA: Context x Time p = 0.0464; Context p = 0.6064; Time p < 0.0001). This is in contract to the sham mice, who discriminated between the aversive and neutral context (ANOVA: Context x Time *p < 0.0001; Context p < 0.0001; Time p < 0.0001). TBI did not alter the number of eYFP+ DG cells activated during fear encoding (ANOVA: Treatment x Context p = 0.7645; Treatment p = 0.7926; Context p = 0.9476) or the number of c-fos+ DG cells during memory retrieval (ANOVA: Treatment x Context p = 0.3028; Treatment p = 0.3138; Context p = 0.3166). Sham mice exhibited a significant increase in the percentage of co-labeled DG eYFP + /c-fos+ cells in the aversive context when compared with the neural context. However, TBI mice exhibited comparable percentages of co-labeled DG eYFP + /c-fos+ cells in both contexts (ANOVA – Treatment x Context p = 0.4171; Treatment p = 0.2120; Context p = 0.0030). Finally, previous data in our lab have shown that a single administration of (R,S)-ketamine (30 mg/kg) prior to a stress can buffer against learned fear and facilitate CFD. Here, we report that administering (R,S)-ketamine (30 mg/kg) 1 hour after CCI decreased TBI-induced fear generalization mice (ANOVA – Context x Drug p = 0.4111; Context *p = 0.0030; Drug p = 0.1670). Conclusions: Our data show that 1) TBI results in increased fear generalization; 2) this behavior is paralleled by altering fear memory traces in the DG; and 3) this behavioral deficit that can be reversed with post-impact subanesthetic administration of (R,S)-ketamine. This work enhances our understanding of the neural basis of fear generalization in individuals with TBI and reveals potential avenues for further translational research. Keywords: TBI, (R,S)-Ketamine, Fear Generalization, Memory Engram Cell, Hippocampus Disclosure: Nothing to disclose. P27. Projection-Targeted Photopharmacology Reveals Anxiolytic Function of Presynaptic mGluR2 in Cortical-Amygdala Circuits Joseph Stujenske, Hermany Munguba, Vanessa Gutzeit, Ashna Singh, Noelle Eghbali, Melanie Kristt, Daniel Shaver, Sonal Thukral, Francis Lee, Johannes Broichhagen, Conor Liston, Joshua Levitz Weill Cornell Medical College, New York, New York, United States Background: Anxiety disorders are the most common psychiatric disorders, affecting about 30% of US adults during their lifetime. Anxiolytic medications primarily work through serotoninergic or GABAergic signaling, while there are no approved treatments that work via a principally glutamatergic mechanism. Metabotropic glutamate receptor 2 (mGluR2) is mainly localized presynaptically and together with mGluR3 mediates feedback inhibition of glutamate release. mGluR2/3 mixed agonists have been shown to mediate anxiolytic effects in humans through an unknown mechanism. In this study, we describe distinct anxiolytic roles of mGluR2 at medial prefrontal (mPFC) and insular (IC) projections to the basolateral amygdala (BLA). Methods: Using adeno-associated viruses in a transgenic Grm2-Cre mice, mGluR2+ projectors to the BLA were labeled. The specific roles of mPFC and IC projections in anxiety-related behavior were characterized using optogenetic techniques and fiber photometry. The specific function of presynaptic mGluR2 was probed using a novel photopharmacological tool, photoswitchable orthogonally and remotely tethered ligands, to specifically activate mGluR2 in prefrontal or insular terminals. Conventional and photopharmacological manipulations were performed during in vitro electrophysiology recordings to characterize its function in synaptic transmission and in vivo during behavioral assays that elicit various forms of anxiety-related avoidance reactions. N = 5-20 male mice per group. Results: Systemic administration of a mGluR2/3 mixed agonist (LY37) decreased spatial avoidance, while a mixed antagonist (LY34) increased avoidance. LY37 infused into the BLA also mediated anxiolysis. mGluR2 was found to be enriched in projectors to the BLA from the mPFC and IC. Neuronal activation of both cell populations was observed during anxiety-related assays using fiber photometry. In vitro, 89% (n = 19) and 100% (n = 14) of BLA principal neurons were activated by mGluR2+ projections from the mPFC and IC, respectively. This activation was acutely dampened by mGluR2 photoactivation, while long-term depression was induced by prolonged photoactivation. Acute photoactivation of mGluR2 in mPFC, but not IC, terminals decreased spatial avoidance, and prolonged photoactivation led to anxiolysis at 4 hours but not 7 days post-light. In contrast, acute photoactivation of mGluR2 in IC, but not mPFC, led to diminished social avoidance and avoidance of predator urine. Photoactivation of mGluR2 in either projection decreased the latency during novelty-suppressed feeding. Conclusions: We describe both distinct and overlapping anxiolytic functions of mGluR2 in mPFC and IC projections to the BLA. mGluR2 represents a promising therapeutic target for the treatment of anxiety. Keywords: Photopharmacology, GPCR, Metabotropic Glutamate Receptor 2 (mGluR2), Medial Prefrontal Cortex, Basolateral Amygdala Disclosure: Nothing to disclose. P28. Development of an Improved Oral Tablet Formulation of BNC210, a Negative Allosteric Modulator of the Alpha 7 Nicotinic Acetylcholine Receptor, Suitable for Evaluation as an Acute Treatment for Social Anxiety Disorder Elizabeth Doolin, Dharam Paul, Julia Crossman, Michael Odontiadis, Susan O’Connor, Errol DeSouza* Bionomics Ltd., Cambridge, Massachusetts, United States Background: The DSM-5 describes Social Anxiety Disorder (SAD) as a marked fear or anxiety about one or more social situations in which the individual is exposed to possible scrutiny by others. Examples include social interactions (e.g., having a conversation, meeting unfamiliar people), being observed (e.g., eating or drinking), and performing in front of others (e.g., giving a speech). SAD is a chronic disorder which may also include severe episodes of acute anxiety such as those precipitated by performance-type events. A safe and effective, fast-acting therapy would be of benefit in situations like these. The only FDA-approved drugs for SAD are select antidepressants (fluvoxamine, sertraline, paroxetine, venlafaxine) which are not suitable for acute treatment due to their slow (2-4 weeks) onset of action. While no drugs have been approved to treat acute SAD, beta blockers and benzodiazepines (BZDs) are used off label. However, beta blockers mainly address physical symptoms (flushing, increased heart rate, trembling), while the serious safety concerns associated with BZDs have restricted their use. BNC210 is a novel drug acting through negative allosteric modulation of the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) and has received FDA Fast Track designation for acute treatment of SAD and other anxiety disorders. It has been administered to more than 400 participants in Phase 1 and 2 clinical studies and lacks the severe side effects associated with the drugs that many patients with SAD are currently taking. BNC210 has demonstrated anxiety-lowering effects on human behavior and on neural correlates of anxiety in acute dosing clinical trials (using a liquid suspension formulation of BNC210 given with food to facilitate absorption). In a CCK-4 challenge model of panic attack in healthy volunteers, single doses of BNC210 significantly reduced panic symptom number and intensity using the Panic Symptom Scale (PSS). In Generalized Anxiety Disorder (GAD) patients performing the Emotional Faces Task during fMRI, single doses of BNC210 significantly reduced two neural correlates of anxiety; amygdala activation caused by viewing emotional faces and connectivity between the anterior cingulate cortex and the amygdala (this connection is strong in pathological anxiety). In the same study, BNC210 significantly reduced threat avoidance in a behavioral task and reduced state anxiety (measured using the State-Trait Anxiety Scale) in the GAD patients. BNC210 is currently being evaluated in a Phase 2 study for the acute treatment of SAD (PREVAIL Study, NCT05193409). Using a new oral tablet formulation of BNC210 with improved absorption characteristics over the previously used suspension formulation, single doses of BNC210 (225 mg or 675 mg) are being compared to placebo (n = 50 per group) on reducing anxiety provoked by a speaking challenge as measured using the Subjective Units of Distress Scale (SUDS) in patients with SAD. Methods: A novel solid tablet formulation of BNC210 was developed and contains a spray dried dispersion of BNC210 to improve the solubility of the compound. During the development process, several prototype formulations were compared in in vitro dissolution assays and the lead formulations were compared in dog pharmacokinetic (PK) studies. The final selected formulation was then evaluated in two single oral dose human PK studies to compare the PK properties of the tablet with the suspension formulation (300 mg BNC210, fed vs fasted), and then to determine PK parameters and dose linearity with ascending doses of the new tablet (300 to 1,200 mg BNC210, fasted). Results: The first human PK study showed that the final formulation developed for the BNC210 tablet overcame the food effect of the liquid suspension. At a 300 mg dose, the liquid suspension showed a marked increase in mean Cmax (2.3-fold) and mean AUC (3.5-fold) values following a high fat meal compared to fasted, whereas at the same dose, there was little impact on these tablet PK parameters whether given with or without food (mean Cmax was marginally reduced by 12% and mean AUC was increased by only 27% in the presence of a high fat meal). Furthermore, with the tablet, the median time to reach maximal plasma concentration (Tmax) was reduced from 4 hours with a high fat meal to 1.75 hours in the fasted state. The PK parameters for single dose administration of the tablet showed dose linear exposure over the range of 300 mg to 1,200 mg BNC210 in the fasted condition, and the median Tmax was 0.75 to 1.75 hours at the doses evaluated. Conclusions: BNC210 has been reformulated into a tablet with improved PK properties, potentially making it suitable for PRN use in an outpatient setting for the treatment of SAD. Drug exposure is no longer dependent on concomitant food intake, allowing for maximal plasma concentrations to be reached after approximately 1 to 1.5 hours after dose administration. This means that BNC210 could be taken just prior to an anticipated anxiety-provoking event such as a performance or social event. Additionally, plasma concentrations remain at reasonable levels for several hours after maximum concentration is reached, suggesting that repeat dosing may not be needed. Topline data from the ongoing BNC210 Phase 2 study for the acute treatment of SAD (PREVAIL Study, NCT05193409) are expected in late 2022/early 2023. Keywords: Social Anxiety Disorder, Acute Treatment, Phase II Clinical Trial, BNC210 Disclosure: Nothing to disclose. P29. Pharmacometrics Analysis and Drug Reformulation of BNC210 to Optimize Its Evaluation in a Phase 2 Trial in PTSD Patients Elizabeth Doolin, Dharam Paul, Susan O’Connor, Paul Rolan, Julia Crossman, Michael Odontiadis, Errol DeSouza* Bionomics Ltd., Cambridge, Massachusetts, United States Background: Currently approved therapeutics for Post-Traumatic Stress Disorder (PTSD) have adverse effects and limitations that signify an unmet medical need for more effective and better-tolerated therapies. BNC210 is a first-in-class negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) in development for the treatment of trauma- and stressor-related disorders and anxiety disorders. Extensive characterization in nonclinical and clinical studies has revealed diverse properties with potential to treat PTSD symptoms and some of the known pathophysiology underlying PTSD. BNC210 has received FDA Fast Track designation for the treatment of PTSD and other trauma- and stressor-related disorders and is currently being evaluated in a monotherapy Phase 2b trial in ~200 patients with PTSD (ATTUNE Study, NCT04951076). BNC210 was previously evaluated in a Phase 2 PTSD trial (RESTORE Study, NCT02933606) where participants received BNC210 (150, 300 or 600 mg) or placebo b.i.d., in a liquid suspension formulation that needed to be taken with food for optimal absorption. No significant separation from placebo was measured on the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) Total Symptom Severity score over the 12-week treatment period. However, positive information from the trial data included: statistically significant effects on depression symptoms (CAPS-5 Criterion D) at early time points, and no trend for increased adverse events, cognitive impairment, nor suicidal ideation or worsening behavior, with treatment. Population pharmacokinetics (PK) estimated drug plasma exposures (AUC) in the participants and revealed that the liquid suspension gave lower than projected exposure in the outpatient setting, a finding which triggered the development of a new, oral tablet formulation of BNC210. Pharmacometrics modelling was performed to explore an exposure-response (PK/PD) relationship for BNC210 based on the estimated AUCs and observed CAPS-5 scores. Methods: Population PK model: Plasma samples were obtained from participants on the RESTORE study at 3 timepoints over the 12-week period and BNC210 concentrations were measured. Using these data, a population PK analysis was performed, and AUC values were estimated from a one compartment PK model that allowed for time varying bioavailability. PK/PD model: A PK/PD analysis was performed to evaluate the exposure-response relationship for BNC210 estimated AUC values and CAPS-5 Total Symptom Severity scores as a continuous direct effect. The effect was modelled on the logit scale which bound it not to be larger than the highest possible score or lower than the smallest possible score. Effects were evaluated using three models: linear, Emax and power function. Reformulation: Spray dried dispersion technology was applied to develop an oral solid dose tablet formulation of BNC210 with improved solubility that could overcome the limitations of the liquid suspension, specifically, a strong food effect and non-linear absorption. Three human PK studies were conducted to compare tablet PK parameters to the suspension, establish dose linearity with single ascending doses, and to determine a dosing regimen that could be used in future PTSD clinical trials. Results: Population PK model: The model indicated that the estimated BNC210 AUC values in the RESTORE Study outpatient trial were lower than projected, and the higher BNC210 dose levels were associated with decreasing bioavailability over time. After 12 weeks of treatment the estimated achieved exposure for the 600 mg b.i.d. dose group was ~50% lower than previously measured in an in-clinic 7-day PK study. PK/PD model: An inhibitory Emax model, including estimates of inter-individual variability produced the best PD model fit and predicted an exposure-response relationship for CAPS-5 Total Symptom Severity scores (p-value<0.01), where higher AUC values were related to a larger effect. The model predicted that the AUC90 (90% of the maximum drug effect) was ~25,000 ng.hr/mL which corresponded to a predicted difference from placebo on the CAPS-5 scale in the range of a potentially clinically meaningful result. Reformulation: In single dose human PK studies, the BNC210 tablet overcame the food effect of the suspension formulation e.g., a 300 mg dose (fed and fasted) achieved respective mean values for Cmax of 1,528 and 1,727 ng/mL, and AUC values of 14,000 and 11,000 ng.hr/mL, and exposure was dose linear over the range of 300 to 1,200 mg. A multiple dosing PK study (900 mg of the tablet formulation b.i.d.) achieved more than the target exposure of 25,000 ng.hr/mL for further evaluation of BNC210 in a PTSD study. Conclusions: The population PK model showed that exposure from the BNC210 liquid suspension formulation was insufficient to achieve efficacy in the RESTORE PTSD study where estimated mean AUC at steady state for the 600 mg b.i.d dose group was only 10,900 ng.hr/mL at Week 12. However, the pharmacometrics model predicted the potential for BNC210 to have benefit in PTSD provided that adequate blood levels could be achieved. These analyses, as well as the development of the new tablet formulation, justified further evaluation of BNC210 in PTSD patients and provided a basis for optimal design of the ongoing Phase 2b trial to demonstrate efficacy in PTSD (ATTUNE Study, NCT04951076); top line data are expected in mid-2023. Keywords: PTSD, Phase II Clinical Trial, Exposure-Response Model, Reformulation, BNC210 Disclosure: Nothing to disclose. P30. Altered Heartbeat-Evoked Neural Responses During Peripheral Adrenergic Stimulation in Generalized Anxiety Disorder Charles Verdonk, Adam R. Teed, Evan J. White, Xi Ren, Jennifer L. Stewart, Martin P. Paulus, Sahib S. Khalsa Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Altered perceptual processing of signals originating from within the body (i.e., interoceptive dysfunction) has been linked to anxious psychopathology. In a recent pharmaco-fMRI study we showed that individuals with generalized anxiety disorder (GAD) exhibit autonomic hypersensitivity evidenced by elevated heart rates, heightened interoceptive awareness of cardiorespiratory sensations, increased anxiety, and a blunted neural response localized to the ventromedial prefrontal cortex during low level beta-adrenergic stimulation (Teed et al (2022)). Here, we examine whether the peripheral adrenergic modulation of cardiac signals with isoproterenol differentially affected the heartbeat evoked potential (HEP), an electroencephalogram (EEG) marker of neural activity in response to heartbeats, in females with GAD versus healthy comparison females (HC). Methods: Simultaneous EEG-fMRI data from this crossover randomized clinical trial were collected in 52 adult females (26 GAD, 26 HC matched on age and body mass index (BMI), the same study population as in Teed et al (2022)) during administration of intravenous bolus infusions of isoproterenol (0.5 and 2.0 micrograms, μg) and saline, each administered twice in a double-blind fashion within a single scanning session. Participants continuously rated the perceived intensity of their cardiorespiratory sensations by rotating an MRI-compatible dial throughout each infusion. EEG and electrocardiogram (ECG) signals were recorded during each 240-second infusion using an MRI-compatible 32-channel scalp EEG system including simultaneous single lead ECG. During offline processing, data were band-pass filtered between 0.3 and 30 Hertz, and the EEG data was referenced to the common average signal. Cardiac R-wave peaks were detected from the ECG signal for every heartbeat using a semi-automated approach. EEG data were epoched in an event-related manner, from -100 milliseconds (ms) to 650 ms after the ECG R-peak, with the cardiac R-wave serving as the temporal reference. We minimized several cardiac-related artifacts in the EEG signal, including the cardiac field artifact and the ballistocardiogram artifact, using the optimal basis set approach followed by application of independent component analysis to remove residuals of the ballistocardiogram artifact. Baseline correction was performed by subtracting the mean of the 100 ms window preceding the epoch of statistical analysis from the entire grand-averaged HEP signal. We implemented a data-driven statistical analysis approach, cluster-based permutation, to compare HEP amplitudes during isoproterenol and saline infusions in individuals with GAD vs HC in the time range [100-600 ms] after the R peak. Results: The GAD group (mean age: 26 ± 7 years, BMI: 25.8 ± 4.8) and HC group (mean age: 24 ± 5 years, BMI: 24.1 ± 3.2) did not differ on age or BMI. During the peak response period of the 0.5-μg isoproterenol dose, the GAD group showed changes in HEP amplitude that significantly differed from the HCs at right centro-parietal electrodes; specifically, the HEP amplitude during the peak response period was more positive in GAD (Cohen d = 1.18; P < 0.001) within a latency from 216–272 ms after the R-peak. During the early recovery period, HEP amplitudes were more negative within a latency from 528-592 ms after the R-peak (Cohen d = 0.90; P < 0.01). In addition, relative to the HC group, the GAD group reported significantly greater increases in cardiorespiratory sensation intensity during the 0.5-μg dose of isoproterenol (Cohen d = 0.80; P < 0.01), that were non-significantly correlated with changes in HEP amplitude at a trend level across all participants (peak response: r = -0.25, P = 0.08; early recovery: r = -0.27, P = 0.05). During saline infusion, the GAD group also exhibited a larger HEP amplitude in right fronto-central electrodes (Cohen d = 0.97; P < 0.001) within a 92-ms window starting 224 ms after the R-peak. Control analyses showed that the altered HEP amplitudes observed in GAD vs HC were not due to cardiac-related confounding factors, such as differences in heart rate or T-wave amplitude. At the transdiagnostic sample level, correlational analyses of physiological and psychological indices revealed that HEP amplitudes were significantly correlated in opposing directions with the anxiety subscale of the PROMIS Negative Affect questionnaire (0.5 μg isoproterenol: r = 0.46, P < 0.001; saline: r = -0.42, P < 0.01) and with the physical concerns subscale of the Anxiety Sensitivity Index questionnaire (0.5 μg isoproterenol: r = 0.47, P < 0.001; saline: r = -0.34, P < 0.01). No significant HEP changes or multilevel associations were identified at the 2 μg dose. Conclusions: In this study GAD individuals showed altered HEPs and heightened cardiac interoceptive awareness, which is consistent with autonomic hypersensitivity during low levels of peripheral adrenergic stimulation. This and previous results support the hypothesis of increased bottom-up sensitivity to adrenergic arousal signals in the disorder. In addition, the increased HEP responses observed during saline infusion suggest that GAD individuals may also exhibit a top-down sensitivity toward heartbeat signals, perhaps associated with abnormal cognitive processing of interoceptive signals. Taken together, our findings further support the notion that GAD individuals exhibit a peripheral autonomic hypersensitivity which contributes to dysfunctional cardiac interoception in this condition. Keywords: Generalized Anxiety Disorder, Interoception, Heartbeat Evoked Potential, Cardiac Sensation Disclosure: Nothing to disclose. P31. Darigabat Reduces Acute Psychological and Physiological Panic and Fear Symptoms Induced by CO2 Inhalation in Healthy Participants Stacey Versavel, Rachel Gurrell, Ih Chang, Ann Dandurand, Sridhar Duvvuri, Amy Giugliano, Gina Pastino, Theresa Pham, Gabriel Jacobs, Koshar Safai Pour, Rob Zuiker, Raymond Sanchez, John Renger Cerevel Therapeutics, Cambridge, Massachusetts, United States Background: Panic disorder (PD) is a serious condition that may cause significant psychological and physical distress. PD is associated with dysregulation in fear and anxiety neurocircuitry involving serotonin, norepinephrine, and gamma-amino-butyric-acid (GABA) pathways, with current pharmacotherapy consisting mainly of drugs that target these systems, such as selective serotonin reuptake inhibitors and benzodiazepines (BZDs). However, no one class of drugs adequately addresses both the subjective (e.g., fear, dread) and somatic (e.g., tachycardia) symptoms of PD. As such, a significant proportion of patients with PD have incomplete or partial response to available treatments with a low likelihood of remission and a significant probability of relapse. Given there have been no new approved drugs for the treatment of PD since 2005 there is a need for innovative novel treatment options. There is substantial nonclinical evidence that α2/3 subunit–containing GABAA receptors are responsible for the anxiolytic effects of benzodiazepines, and that the α1 subtype is associated with the sedative properties. Darigabat was rationally designed to have non-sedative anxiolytic potential by selectively enhancing the effect of GABA at α2/3/5 GABAA receptor subtypes, while sparing activity at α1, and is in development for the treatment of neurological and psychiatric disorders. To characterize the potential panicolytic effect of darigabat, CO2 inhalation was applied as an experimental model to reliably induce panic and fear in healthy participants. The model is sensitive to pharmacological manipulation by anxiolytics, allowing investigation of pharmacodynamic effects of drugs in early clinical development. Methods: This randomized, double-blind, placebo- and active-controlled trial assessed the panicolytic effect of multiple doses of darigabat on panic and fear symptoms evoked by CO2 inhalation in healthy participants (NCT04592536). Only individuals sensitive to the anxiogenic effects of the 35% CO2 double-breath inhalation at screening were eligible for randomization. In this two-period, two-sequence partial crossover design, each eligible participant was randomized to receive either placebo and one of three active treatments in 3 separate cohorts (n = 18-20/cohort) for 8 days: cohort 1 darigabat 25 mg BID, cohort 2 alprazolam 1 mg BID, and cohort 3 darigabat 7.5 mg BID. Darigabat was titrated to achieve the target maintenance dose on Day 5. On Day 8 of each crossover period, a CO2 challenge was performed at 3 hours after dosing. Alprazolam was used as a positive control to establish assay sensitivity. With each participant’s placebo period serving as their own control, the change in panic and fear symptoms measured before and immediately after CO2 inhalation using the Panic Symptom List-IV total score (PSL-IV; primary endpoint) and fear visual analog scale (VAS Fear; secondary endpoint) were assessed. The PSL-IV contains 13 items derived from those listed for panic disorder in the Diagnostic and Statistical Manual of Mental Disorders, version 4 (DSM-4) covers aspects of the PD experience including the psychological (such as fear of dying) and the physiological (such as palpitations and gasping for breath. Cardiovascular parameters (blood pressure, and heart rate) were objectively measured continuously using a non-invasive finger-cuff-based hemodynamic monitoring system (Finapres) for approximately 15 mins prior to and following CO2 challenge to determine the effects of drug treatment on the physiological panic and fear response (secondary endpoint). While the trial was not prospectively designed for formal hypothesis testing with statistical power, nominal P values for each comparison are presented. Steady state pharmacokinetic samples were obtained at 2 and 4 hours after dosing on Day 8. Results: In the primary outcome measure PSL-IV total score on Day 8, the darigabat 7.5 mg and 25 mg BID treatment groups demonstrated a 3.9-point (P = 0.036) and 4.5-point (P = 0.008) improvement versus placebo, respectively. In the secondary outcome measure VAS Fear, the 7.5-mg and 25-mg BID treatment groups demonstrated a 12.8-point (P = 0.026) and 7.8-point (P = 0.282) improvement versus placebo, respectively. Compared with placebo, alprazolam 1 mg BID exhibited outcomes in line with expectations, with placebo-adjusted improvements of 1.6-points (P = 0.286) and 0.9-points (P = 0.876) on PSL-IV total score and VAS Fear on Day 8, respectively. The results on the individual PSL-IV items indicated a broad reduction of both the psychological and physiological symptoms experienced as a result of CO2 inhalation. Both doses of darigabat attenuated the transient increase in blood pressure induced by CO2 inhalation on Day 8, suggesting darigabat reduces the physiological response associated with CO2 challenge. Alprazolam did not attenuate the physiological response associated with CO2 challenge. Plasma concentrations of darigabat were consistent with previous trials and estimated to achieve approximately 50% and 80% receptor occupancy at α2-containing GABAA receptors at 7.5 mg and 25 mg BID, respectively. Darigabat was generally well tolerated. Conclusions: This trial demonstrated the panicolytic potential of darigabat, based on reduction of acute panic and fear symptoms in a validated, experimental clinical panic model in healthy participants. These data warrant the further evaluation of darigabat in patients with anxiety disorders. Keywords: GABA-A, Positive Allosteric Modulators, Panic, Novel Therapeutics, Experimental Methods Disclosures: Cerevel Therapeutics: Employee(Self) Eliem Therapeutics: Employee (Spouse), vZenium: Founder (Spouse) P32. Insights From Rodent Work: Can Environmental Contaminants Contribute to the Development of Anxiety Disorders? Mauricio Caceres Chacon, Sian Rodríguez Rosado, Gabriela Hernández Busot, Alexdiel Figueroa Pérez, Hector Haddock Martínez, Melissa Rivera López, Osmarie Martínez Guzmán, Demetrio Sierra Mercado University of Puerto Rico, San Juan, Puerto Rico Background: The increase in the diagnosis of mental health disorders such as anxiety has created a need to evaluate for external factors that might contribute to the development of these disorders. Recently, epidemiological studies have begun to show links between exposure to environmental contaminants and anxiety disorders. One possible contaminant is the extensively used herbicide, glyphosate, which has been identified in food and water sources. Glyphosate was initially considered safe for mammals because it acts by inhibiting a metabolic route almost exclusive to plants. However, a correlation has been seen between the increased diagnosis of anxiety and the use of glyphosate. Glyphosate, as other possible contaminants, is regulated by the Environmental Protection Agency (EPA). The EPA has established a chronic reference dose of 2.0 mg/kg of glyphosate to have no detrimental effects on health. However, the effect of glyphosate at this dose on the development of anxiety has not been studied. Therefore, we aim to evaluate the effect of glyphosate on anxiety-like and exploratory behaviors. Moreover, given that the amygdala and prefrontal cortex have both been shown to play an important role in the expression and modulation of anxiety and exploration, we aim to evaluate these brain regions for neuronal activity to identify a mechanism for any behavioral changes. Methods: Male rats (n = 13) were given water containing glyphosate ad libitum for 16 weeks. Water consumption was measured weekly, and water was prepared for a target dose of 2.0mg/kg daily of glyphosate. Control rats (n = 12) received filtered drinking water. Anxiety-like behaviors were assessed after 10 weeks of exposure in an elevated plus maze (EPM). Exploratory response to novelty was measured using an open field with a novel object in the center (NOET) after 14 weeks, as well as an auditory startle response test (ASR, 5 repetitions of a novel tone in a familiar context) after 16 weeks. Rats were then sacrificed, and brain tissue was fixed and collected. Immunohistochemsitry for c-Fos was performed in brain slices containing amygdala, as well as the prelimbic (PL) and infralimbic (IL) cortices of the prefrontal cortex to evaluate for neuronal activity. Student’s t-test was used for analysis of behavioral results and immunohistochemistry analysis. Results: In the EPM, glyphosate decreased the time spent in the open arms (glyphosate: 51.4; control: 108.9; t22 = 2.98; p = 0.0069), indicating increased anxiety. In the NOET, glyphosate decreased the time spent interacting with the novel object (glyphosate: 45.34; control: 97.9; t22 = 2.73; p = 0.0122), consistent with decreased exploration. Moreover, glyphosate increased the percent time spent freezing in the presence of a novel tone (glyphosate: 14.38; control: 5.94; t23 = 2.094; p = 0.0475), suggesting increased startle to a novel, neutral stimulus. Brain tissue analysis showed that glyphosate did not affect neuronal activity in PL (glyphosate: 270.5; control: 382.0; t10 = 1.365; p = 0.2023), IL (glyphosate: 201.4; control: 253.3; t10 = 1.059; p = 0.3147) nor amygdala (glyphosate: 98.83; control: 100.3; t10 = 0.1318; p = 0.8978). Conclusions: Contaminants, such as glyphosate, even at doses considered safe by the EPA, can increase anxiety-like behaviors and decrease exploration in rats. These finding support the idea that environmental contaminants should be evaluated for detrimental effects on mental health. Further exploration of the mechanism by which contaminants, such as glyphosate, causes behavioral changes is needed. Future directions include performing immunohistochemistry on brain regions involved in anxiety-like behaviors such as the bed nucleus of the stria terminalis and ventral hippocampus. Keywords: Anxiety, Mental health Disorders, Animal Models, Environmental Risk Factors Disclosure: Nothing to disclose. P33. Relationship Between Emotion Regulation Styles and Fear Conditioning in Trauma Exposed Veterans With and Without PTSD Morgan Bartholomew, Thomas Metzler, Thomas Neylan, Sabra Inslicht UCSF, San Francisco VA Medical Center, San Francisco, California, United States Background: Effortful emotion regulation strategies such as cognitive reappraisal and expressive suppression share common neural mechanisms with fear learning and extinction (Birthe Macdonald, 2020). Disrupted fear learning and extinction processes are thought to support severity of symptoms of post-traumatic stress disorder (PTSD). In healthy populations, use of cognitive reappraisal is associated with reduced acquisition of fear learning and greater extinction learning and recall (Andrea Hermann, 2014). Other studies have not demonstrated a relationship between explicit ER strategies and fear conditioning/extinction learning (Haruka Kitamura, 2022), suggesting that further consideration of individual factors is needed. PTSD has been associated with increased use of expressive suppression, as well as alterations in fear conditioning, extinction, and extinction retention (A J Khan, 2021; Inslicht et al., 2013; Orr et al., 2000). Further characterization of the relationship between ER processes and fear learning processes in trauma exposure individuals may help us target skills to increase the effectiveness of PTSD interventions. Methods: A laboratory study was conducted in trauma-exposed men and women with and without PTSD to examine the impact of emotion regulation styles on fear conditioning and extinction. Tendencies towards using cognitive reappraisal and expressive suppression were assessed via the Emotion Regulation Questionnaire. Participants underwent a fear conditioning, extinction, and retention procedure adapted from Orr et al. (Orr et al., 2000) and previously described (Inslicht et al., 2013), which took place over the course of three sessions. All psychophysiology testing sessions took place in a dedicated psychophysiology laboratory. The UCS was a 500 ms electric pulse, which ranged from .5 to 5.0 mA, as previously determined by the participant to be “highly annoying but not painful”. During the first session, participants underwent a habituation phase during which participants were presented with 5 of each of the colored circles (to be CS + and to be CS-) in the absence of the shock (UCS). This was followed by the fear acquisition phase, in which each presentation of the CS + was followed by a 500 ms shock and the CS– was not. The fear extinction session took place 72 hours following the first session. During this session, participants were presented with 10 non-reinforced presentations each of the CS + and CS − . One week following extinction, extinction retention session took place. Participants were shown 4 non-reinforced presentations each of the CS + and CS − . Results: PTSD diagnosis was found to moderate the relationship between cognitive reappraisal and differential SCR response to CS + and CS- during acquisition (z = 2.23, P = 0.02). Individuals without PTSD showed a greater negative relationship between cognitive reappraisal and differential SCR, such that greater cognitive reappraisal predicted reduced differential SCR during fear acquisition (z = -2.00, P = 0.05). Individuals with PTSD did not demonstrate a significant relationship between cognitive reappraisal and differential SCR (z = 0.49, P = 0.62). PTSD did not moderate the relationship between expressive suppression and differential SCR response during extinction learning, as predicted (P = 0.33). However, there was a main effect of expressive suppression on differential SCR during extinction learning, such that greater expressive suppression predicted greater differential SCR during extinction retention (z = 2.06, P = 0.04). Group differences in use of cognitive reappraisal and expressive suppression were not observed between individuals with and without PTSD diagnosis. Conclusions: The current analysis adds to evidence that cognitive reappraisal predicts reduced fear acquisition. Further, our data suggest that expressive suppression predicts maintenance of fear responding in the presence of safety cues, suggesting a failure to encode the inhibitory association between CS + and shock during extinction learning is related. This suggests that common mechanisms are being impacted by these two, conceptually distinct, facets of emotion – explicit emotion regulation and fear conditioning. Contrary to hypothesis, differences in ER strategies between PTSD groups were not observed in our sample and PTSD diagnosis did not moderate the relationship between expressive suppression and extinction retention. Several methodological considerations may explain this finding, including that our control group consisted of individuals who had been previously trauma exposed but had never met criteria for PTSD. Future work should include inclusion of a non-trauma exposed control group, to further clarify these relationships. Keywords: Fear Conditioning and Extinction, Emotion Regulation, PTSD Disclosure: Nothing to disclose. P34. Parallel Dopamine Circuit Dynamics in Chronic Stress-Induced Behavioral Outcomes Carole Morel, Sarah Montgomery, Long Li, Emily Teichman, Barbara Juarez, Romain Durand-de Cuttoli, Nikos Tzavaras, Scott Russo, Eric Nestler, Erin Calipari, Allyson Friedman, Ming-Hu Han Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Comorbidity is more the norm than the exception, where more than 93% of Medicare dollars are used for patients who suffer from comorbidities in the United States. Anxiety, anhedonia, and depression are highly prevalent comorbid symptoms in human neuropsychiatric profiles. This co-occurrence is associated with complex symptomatology, greater treatment resistance, chronicity, disability, and higher suicide attempt rates in comorbid patients. However, the shared or segregated mechanisms remain largely unknown. Clinical and preclinical studies implicate the ventral tegmental area (VTA) dopamine circuits in the emergence of anxiety, anhedonia and depressive phenotypes. The VTA dopamine neurons project widely throughout the brain, including to emotion-related brain regions, such as the cortex, the nucleus accumbens (NAc), and the amygdala (AMG). Here we aim to identify the parallel alterations of the dopaminergic circuits and how they control singular anxiety or co-occurring anxiety, anhedonia and depressive-like behaviors following chronic stress exposure. Methods: We use a chronic social defeat stress paradigm (CSDS), known to induce individual and complex behavioral stress-related outcomes, to capture singular or co-occurring measures of anxiety, anhedonia and depressive-like behaviors in mice. Following CSDS, we assess social behaviors, reward sensitivity and processing, and approach/avoidance behaviors. We employ viral strategies and transgenic mice to isolate VTA dopamine circuits and isolate VTA dopamine neurons projecting to the AMG from VTA dopamine neurons targeting the NAc. We then combine neural circuit-probing techniques with in vivo fiber photometry and electrophysiological approaches to define the physiological characteristics and dynamics of the VTA dopamine circuits emerging in response to CSDS exposure. We also use optogenetics to selectively link VTA circuit activity with the distinct stress-induced behavioral outcomes. Results: We first observe that CSDS induces depressive-like behaviors (ANOVA, P < 0.01, n = 40), alterations in cognitive function (ANOVA, P < 0.05, n = 28), also and anxiety-like behaviors (Kruskal-Wallis, P < 0.01, n = 40) that emerge independently from the depressive-like phenotype (Spearman r = 0.07; r = 0.06, P > 0.05, n = 25). Our circuit-probing approaches showed that VTA-AMG and VTA-NAc dopamine circuits emerge from two distinct neuronal populations. In line with our previous studies revealing that VTA-NAc dopamine neuron hyperactivity encodes social avoidance behaviors, while VTA-AMG dopamine neuron hypoactivity is linked with anxiety-like behaviors, here we identify that CSDS alters the VTA-AMG and VTA-NAc circuit dynamics differently. We define that the activity of the VTA-AMG circuit selectively regulates anxiety-like behaviors following social stress exposure but not social avoidance behaviors, while the VTA-NAc circuit dynamics regulate reward processing and social behaviors (ANOVA, P < 0.05, n = 11). We further observed distinct VTA-NAc and VTA-AMG extrinsic and intrinsic physiological alterations when compared to control mice (ANOVA, P < 0.01, n = 10-12). Finally, our physiological studies establish that anxiety-like behaviors are associated with reduced VTA-AMG circuit activity and increased AMG neuron excitability (Kruskal-Wallis, P < 0.01, n = 8-10; ANOVA, P < 0.05, n = 10-12). Conclusions: Over 50% of patients suffering from anxiety or major depressive disorders report a history of other mental illnesses. The complex and variable symptomatology that emerges following chronic stress exposure -a major risk factor for triggering psychiatric disorders- challenges the single brain circuit/ single phenotype preclinical framework. Here, our results show that chronic social stress exposure induces opposite cellular and physiological alterations in parallel VTA circuits. Our studies identify VTA-AMG circuit reduced activity as a common biomarker and treatment target for anxiety-like behaviors across complex symptomatology such as singular or co-occurring anxiety with depressive-like phenotypes. Our studies thus provide insight for future clinical studies exploiting the distinct dopamine circuits as pharmacological targets for anxiety, anhedonia, and depressive disorders. Keywords: Anxiety, Depression, Dopamine Circuits, In Vivo Fiber Photometry, Electrophysiological Approaches Disclosure: Nothing to disclose. P35. Ventral Hippocampal Input to the Infralimbic Cortex is Necessary for the Effects of Extinction on Set Shifting After Chronic Stress Denisse Paredes, David Morilak The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States Background: Psychiatric disorders such as post-traumatic stress disorder and major depressive disorder are characterized by deficits in cognitive flexibility. Exposure therapy can be effective in reversing cognitive deficits in these patients. Fear extinction in rodents bears similarity to exposure therapy. Extinction reverses chronic stress-induced deficits in cognitive flexibility on the attentional set-shifting test (AST), a medial prefrontal cortically-mediated executive process. Extinction requires the activity of pyramidal neurons in the infralimbic cortex, and BDNF-initiated signaling cascades to reverse stress-induced impairments in set shifting. However, the circuit mechanisms governing the extinction-mediated BDNF plasticity in the infralimbic are unknown. The ventral hippocampus plays a key role in regulating infralimbic activity during extinction learning, and plasticity in the ventral hippocampus is necessary for extinction memory consolidation. In these experiments, we investigated the role of ventral hippocampal (vHipp) input to the infralimbic cortex (IL) for the effects of extinction after chronic stress in reversing cognitive deficits in male and female rats. Methods: To selectively inhibit or activate ventral hippocampal input to the infralimbic, we injected either AAV- CaMKIIa-hM4D(Gi)-mCherry or AAV-CaMKIIa-EGFP into the ventral hippocampus. Immediately prior to extinction, rats received bilateral microinjections of CNO into the IL to silence ventral hippocampal terminals in the IL. In separate experiments, stressed rats received AAV-CaMKIIa-Gq in the ventral hippocampus (and CNO in the IL) to activate ventral hippocampal terminals in the IL in lieu of extinction. 24 hours after treatment, rats were tested on the AST to assess cognitive flexibility. Results: Our results demonstrate that chemogenetically silencing pyramidal cell input from the vHipp to the IL blocks the phosphorylation of the BDNF receptor TrkB, and prevents the effects of extinction in reversing stress-induced cognitive deficits (n = 10-12/group, p < 0.01). Further, we demonstrate that activating vHipp input in the IL, in the absence of extinction, is sufficient to reverse stress-induced deficits in set shifting (n = 6-12/group, p < 0.01). Importantly, the effects of activating vHipp terminals in the IL are dependent on BDNF signaling, as local infusion in the IL of a neutralizing antibody prevented these beneficial effects. Conclusions: These findings suggest vHipp-driven BDNF signaling in the IL is critical for extinction to counteract the deleterious cognitive effects of chronic stress. Keywords: Extinction, Ventral Hippocampus, Infralimbic Cortex, BDNF Disclosure: Nothing to disclose. P36. A Prospective Evaluation of Fear Conditioning During COVID-19 in Persons With Trauma History Sabra Inslicht, Morgan Bartholomew, Connie Fee, Thomas Metzler, Thomas Neylan University of California, San Francisco, San Francisco, California, United States Background: The fear conditioning model has been widely used to explain hallmark features of PTSD, including intrusive recollections and hyperarousal symptoms that underlie chronic physiological alterations in sympathetic and neuroendocrine activity. Enhanced fear conditioning and impaired extinction have been associated with PTSD in cross sectional laboratory studies. While it has been suggested that “conditionability” is trait-like and reflects learning mechanisms that occur during or soon after threatening traumatic experiences, little work has validated this prominent explanatory model of PTSD during real-life ongoing traumatic stress. The theoretical model would predict that individuals who are more conditionable or have impaired extinction would be more stress reactive to the real-world threat that many have experienced during the COVID-19 pandemic. Our overall objective was to test the ecological validity of the fear conditioning model by examining whether fear conditioning responses prior to the pandemic predict stress reactivity during COVID-19. We predicted that greater fear conditioning (higher differential skin conductance responses to CS + vs CS- stimuli during acquisition) and decreased extinction (higher differential skin conductance responses to CS + vs CS- stimuli during extinction) would be associated with greater stress related to COVID-19. Methods: We conducted a longitudinal follow up study of a pre-existing cohort of PTSD and trauma-exposed non-PTSD participants who previously completed fear conditioning experiments. Extensive data were previously collected from 208 men and women (50% female); ages 18-65. PTSD status was previously determined by DSM-IV criteria using the Clinician Administered PTSD Scale (CAPS) administered prior to the pandemic. Participants previously underwent a fear conditioning and extinction procedure (involving shock as the unconditioned stimulus) which took place over two study visits, separated by 72 hours. Follow up measures examined COVID-19 exposure, including self-report of illness in self, family, close contacts, severity of illness, and stress reactivity related to COVID-19 with the Impact of Events Scale, Ruminative Responses Scale, Yale Brown Obsessive-Compulsive Scale, and Perceived Stress Scale. Hierarchical linear regressions at each phase (fear conditioning, fear extinction) were performed separately with differential skin conductance (CS + vs CS-) predicting stress symptoms during COVID-19. Results: Smaller differential responses during fear conditioning were associated with greater obsessive and compulsive symptom severity scores (Beta = -.17, p < .05). Smaller differential responses during fear extinction were associated with greater COVID-19-related intrusions (Beta = -.29, p = .07), ruminations, Beta = -.35, p < .05) and perceived stress (Beta = -.41, p < .05). The coefficients for all other variables showed effects in the same direction, although were not significant. Conclusions: Our findings suggest that pre-pandemic responses during fear conditioning and extinction in the lab prospectively predict stress reactivity during the pandemic. Contrary to initial predictions, we found that a smaller differential skin conductance response to CS + and CS- cues, primarily during fear extinction, measured prior to the pandemic were broadly associated with greater stress reactivity during the pandemic across multiple measures. One possible explanation is that decreased stimulus discrimination, or the awareness to adequately differentiate between danger and safety cues, may lead to increased generalized threat and heightened contextual anxiety. Further evaluation of whether the fear-based model predicts the persistence of stress responses over time and as threat changes will provide valuable information on mechanisms underlying recovery. This information will provide the basis for the development of strategies to support and protect populations that are vulnerable to the ongoing and future similar crises. Keywords: Anxiety and PTSD, Fear Conditioning and Extinction, COVID-19 Disclosure: Nothing to disclose. P37. Neural Correlates of Implicit Regulation of Emotional Conflict in Post-Traumatic Stress Disorder and Mild Traumatic Brain Injury Mira Milad, Zhenfu Wen, Isabel Moallem, Michelle Jeffers, Esther M. Blessing, Duna Abu-Amara, Amit Etkin, Mohammed R. Milad, Charles R. Marmar New York University School of Medicine, New York, New York, United States Background: Functional neuroimaging studies have previously reported dysfunctional activation and connectivity within cortical and subcortical structures in post-traumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI). The aberrant neural activations within these circuits is likely to contribute to emotion dysregulation, a key pathology in both disorders. Implicit regulation of emotional conflict is an important component of emotion regulation that has not been fully studied in these disorders. Using functional MRI data from 305 participants that underwent the emotional conflict task, we examined the neural correlates of emotional conflict detection and its implicit regulation in patients with PTSD, mTBI and their comorbidity. Methods: The 305 participants analyzed in this study included 50 with full or subthreshold PTSD, 93 with mTBI, 29 with full or subthreshold PTSD + mTBI, and 133 healthy comparison. In the emotional conflict task, participants were asked to name the emotion expressed on a face while ignoring an overlaying emotional word. Congruent trials were defined as trials where the emotional expression on the face (either fear or happy) and word (FEAR and HAPPY) match. Incongruent trials were defined as trials where the face and word do not match. Two primary contrasts were considered. One represented the detection of presence or absence of conflict between the face and the word (incongruent vs. congruent trials). Another contrast represented the evaluation and regulation of conflict (incongruent preceded by an incongruent trial vs. incongruent preceded by a congruent trial). Three analytic strategies were used. The first tested activations induced by the task pooling across all participants. For this analysis, we used FWE correction at the voxel level across the brain. In the second set of analyses, we compared healthy controls versus all diagnoses combined. For the third set, we compared healthy controls versus separate diagnoses (HC vs. PTSD, HC vs. TBI, HC vs. PTSD and PTSD + TBI, and HC vs. PTSD + TBI). We used a whole-brain voxel level threshold of p < 0.001 and FWE correction at the cluster level for the second and third analyses. Results: Across all participants, conflict detection induced robust activations within the dorsal anterior cingulate (dACC), thalamus, and bilateral insular cortices (IC). Absence of conflict induced activations in the ventromedial prefrontal cortex, middle frontal cortex and precuneus. Regulation of conflict induced activations within the sensorimotor area, middle frontal gyrus, right IC, and precentral gyrus. For the second set of analyses, during the detection of conflict, compared to HC the diagnosed group exhibited significantly higher activation in the left hippocampus. The HC group showed higher activations within the dACC, precuneus, and thalamus during the evaluation of conflict. No significant between-group differences were observed during the regulation of conflict. For the last set of analyses, we did not detect any statistically significant between-group differences. While no significant differences in activation were noted within the amygdala and rostral anterior cingulate in any of the contrasts analyzed, functional connectivity analyses seeding the hippocampus and dACC revealed significant connectivity with the amygdala and the rACC across all participants. Conclusions: When encountering emotionally conflicting cues, our data suggest that multiple cortical and subcortical neural nodes are critical for processing sensory and emotional information to maintain emotional homeostasis. These regions include the thalamus, hippocampus, amygdala, precuneus, anterior cingulate, insular, ventral and medial prefrontal cortices. A subset of these neural nodes showed dysregulated activations in participants with PTSD, mTBI and PTSD + mTBI, suggesting aberrant detection and evaluation of conflict. A surprising finding in in the last set of analyses is the absence of significant results from the comparison of the separate diagnoses to the healthy participants. This might be attributed to one of three possibilities: 1) patient populations examined maintain intact neural circuits to detect and regulate emotional conflict 2) that the task and/or current neuroimaging tools are unable to detect subtle neural differences pertinent to the detection and/or processing of emotional conflict, or 3) insufficient power. Given that we did observe significant findings when combining all diagnosed participants, and given recent neuroimaging studies suggesting the need for ~100 subjects per group, we speculate that insufficient power is the most likely explanation. Collectively, however, our results replicate and strongly affirm the role of several neural nodes in the implicit regulation of emotional conflict. Keywords: PTSD, Conflict Monitoring, TBI, Functional MRI (fMRI) Disclosure: Nothing to disclose. P38. Neuroticism and Cortisol Levels in Response to the COVID-19 Pandemic Predict Post-Traumatic Stress Symptoms in Children: A Biopsychosocial Approach to Identify At-Risk Children Alexe Bilodeau-Houle, Catherine Raymond, Marie-France Marin* Université du Québec à Montréal, Research Center of the Montreal Mental Health University Institute, Montréal, Canada Background: Although a majority of individuals will be exposed to a potentially traumatic event at some point in their life, only a significant minority will develop post-traumatic stress disorder (PTSD). The lifetime prevalence is estimated to be 9%, affecting twice as many women as men. In addition, this effect is particularly pronounced starting from puberty onwards. To optimize prevention, detection, and treatment plans, a wealth of research has been conducted to identify the psychological and biological factors that may render an individual more vulnerable to developing PTSD when confronted with a traumatic event. On a psychological level, it is well established that various personality traits can modulate one’s risk to develop the pathology, such as anxiety and neuroticism. At a biological level, various systems have been examined, notably the hypothalamic-pituitary-adrenal (HPA) axis, for which the main end product is cortisol (a major stress hormone in humans). Most studies have shown lower cortisol levels in patients suffering from PTSD. For a long time, low HPA axis functioning was thought to result from the pathology, but more recent evidence suggests that dysregulated patterns of cortisol prior to trauma exposure is a risk factor for the development of the pathology. In children, very few studies have assessed the relationship between HPA axis functioning prior to trauma and the development of PTSD. Further, the few studies conducted on the subject suggest a hyperactivation of the HPA axis. Due to its unprecedented nature, the recent COVID-19 pandemic has been a highly stressful event that has induced significant psychological distress in the population. Moreover, important individual differences have been observed. Using a longitudinal design, we took advantage of the pandemic context to assess whether cortisol levels assessed before and during the first few months of the first lockdown could predict post-traumatic stress symptoms in children and whether psychological traits could moderate this effect. Methods: 92 children who previously took part in one of our laboratory-based experiments between 2017-2019 and were free of physical and mental health problems were re-contacted in May 2020 to participate in this longitudinal study on psychological distress during the pandemic. In June 2020 (T1), 68 children (39 girls, 29 boys) aged between 9 and 14 provided a 6 cm hair sample, which was then divided into two segments of 3 cm. These two hair segments allowed us to capture cumulative cortisol levels that were secreted 1) 3 months before the onset of the first lockdown in Quebec, Canada (December 2019 to March 2020) and 2) during the first 3 months of the first lockdown (March 2020 to June 2020). From these two segments, we calculated the percentage change in cumulative cortisol levels between the beginning of and prior to the pandemic. At T1, children also filled out the Big Five Questionnaire for Children (BFQ-C) to assess neuroticism. Post-traumatic stress symptoms were assessed four times, every three months (June 2020-T1; September 2020-T2; December 2020-T3, and March 2021-T4), using the Children’s Revised Impact of Event Scale (CRIES). Given that anxiety symptoms are highly correlated with post-traumatic stress symptoms, anxiety symptoms were also measured at each timepoint (T1 to T4) using the State-Trait Anxiety Inventory for Children (STAI-C; state scale). Thereafter, state anxiety scores were added as covariates to the model. Results: We conducted a linear mixed-effects model including time, neuroticism, and cumulative cortisol percent change, as well as their interaction as fixed effects. Sex and trait anxiety were used as covariates in the model. The analyses revealed a significant interaction between time, neuroticism, and cumulative cortisol percent change. Specifically, a decrease in cortisol levels early in the pandemic was associated with increased post-traumatic stress symptoms for children with heightened levels of neuroticism at T1 and T2 (n = 67, F(3, 172) = 3.51, p = .017, R2m = .24, R2c = .59). Conclusions: These results suggest that insufficient activation of the HPA axis when faced with a stressful event, combined with high levels of neuroticism, may represent a risk factor for the short-term development of post-traumatic stress symptoms in healthy children. This highlights the importance of considering both psychological and biological factors when striving for a better understanding of the pathology. Gaining insight into the predictors of psychological distress in healthy children when confronted with a highly stressful enduring stressor might be informative for the early identification of at-risk children during future crises. Keywords: Cortisol, COVID-19, Children, Post-Traumatic Stress Symptoms, Neuroticism Disclosure: Nothing to disclose. P39. Gray Matter Association With Extinction-Induced Neural Activation in Patients With Anxiety Disorders Noor Nassar, Zhenfu Wen, Mohammed Milad New York University Grossman School of Medicine, Manhattan, New York, United States Background: The failure to appropriately retain extinguished fear is a key feature of clinical anxiety. Thus, exploring the neural correlates that underlie the extinction of conditioned fear is critical to advance our understanding of the psychopathology of anxiety disorders. Initial neuroimaging data reveal some connections between clinical symptoms and structural measures of several sub/cortical regions. However, the specific impact of structural size and the correspondence between cortical thickness and changes in brain activation remain a space for greater exploration. Here, we performed an analysis using voxel-based morphometry (VBM) to assess differences in gray matter volume (GMV) and its associated brain activations during fear extinction memory recall between healthy controls and patients with anxiety disorders. Methods: We analyzed structural images from 170 participants, including 75 controls and 95 with anxiety disorders, who underwent a two-day threat conditioning and extinction paradigm. The VBM analysis was performed using the computational anatomy toolbox (CAT12). We first assessed the main effect of group (HC vs. ANX) in GMV using a voxel-wise linear regression analysis, with total intracranial volume, age, and sex as covariates. For regions that showed significant group difference in GMV, we further examined whether the abnormal GMV of anxiety disorders correlates with brain activation during extinction memory recall. We examined GMV and brain activations of several region-of-interests (ROI) that were thought to be critical in threat acquisition and its extinction, including the amygdala, hippocampus, insular cortex, dorsal anterior cingulate cortex (dACC), and ventromedial prefrontal cortex (vmPFC). An initial criterion of voxel-level p < 0.005, and a small volume family-wise error (FWE) correction pFWE<0.05 were used to detect significant clusters. A whole-brain analysis was also conducted with a criterion of voxel-level p < 0.001 and cluster-level pFWE < 0.05 to identify significant brain regions. Results: Relative to the healthy controls, decreased GMV in the anterior hippocampus (small-volume correction pFWE<0.05), and increased GMV in the right dorsolateral prefrontal cortex (dlPFC, whole-brain cluster-level pFWE<0.05) were noted in patients with anxiety disorders. The GMV differences between control and anxiety disorders were associated with differing brain activations during extinction memory recall. Specifically, in healthy controls, hippocampus volume positively correlated with ventromedial prefrontal cortex (vmPFC) activation (small-volume correction pFWE<0.05), while in patients with anxiety disorders, hippocampus volume negatively correlated with dACC activation (small-volume correction pFWE<0.05). Additionally, dlPFC volume positively correlated with activations of dACC, pre- and post-central gyrus, and supramarginal gyrus (whole-brain cluster-level, all pFWE<0.05) in healthy controls but not in patients with anxiety disorders. Conclusions: The data presented suggest that difference in functional activations during extinction recall may be associated with differences in GMV. There lies a correspondence between GMV of one region and activation of a region it subserves or interacts with. As such, disruptions to the functioning of regions corresponding with extinction retention (i.e., vmPFC) may be due to both, decreased cortical volumes of that region and decreased activations of the region due to GMV-dependent associations of another region (i.e., hippocampus). The associations between GMV and extinction-related activation represent an additional level of nuance to the impact of GMV in psychiatric psychopathology and therapeutic responses. Keywords: Gray Matter Volumes, fMRI, Anxiety, Threat, Anxiety Disorders, Functional and Structural MRI Disclosure: Nothing to disclose. P40. Hippocampal Resting-State Functional Connectivity in Posttraumatic Stress Disorder: Preliminary Results From the PGC-ENIGMA PTSD Working Group Cecilia Hinojosa, Courtney C. Haswell, C. Lexi Baird, Mohammed S.E. Sendi, Rajendra A. Morey, Sanne J.H. van Rooij Emory University School of Medicine, Atlanta, Georgia, United States Background: Alterations in the neurobiology of individuals with PTSD compared to controls have been found in the threat neurocircuitry, including the amygdala and hippocampus. Studies have observed abnormal resting-state functional connectivity (rs-FC) between regions underlying the threat neurocircuitry compared to controls, such as a weaker anticorrelation between the amygdala and rostral anterior cingulate cortex (rACC) and weaker connectivity between the hippocampus and regions of the prefrontal cortex. However, these studies were very small (n < 25), and larger sample sizes are needed for more unbiased analyses. Methods: As part of the PGC-ENIGMA PTSD Working Group, seed-based resting-state fMRI data were centrally preprocessed using a standardized pipeline (HALFpipe) for N = 3007. Next, data were visually inspected for quality using the Linux command slicesdir by three independent raters (CAH, CCH, SVR). Data were excluded from the analysis for artifacts or low signal (N = 387). Across 22 collection sites, resting-state fMRI data was available for N = 794 PTSD and N = 1092 Control participants for the right amygdala, N = 797 PTSD and N = 1097 Control participants for the left amygdala, and N = 796 PTSD and N = 1096 Control participants for the right and left hippocampus. Group-level whole-brain seed-based analyses were conducted on the right and left amygdala and hippocampus using SPM12 using a p < 0.005 and FWE-corrected cluster threshold. Results: PTSD participants compared to Controls showed more positive rs-FC between the right amygdala and subgenual ACC (sgACC; peak, MNI: x = -8, y = 14, z = -22; p < 0.001, k = 625, FWE cluster-level corrected, p = 0.001) and similarly, the left amygdala and sgACC (peak, MNI: x = -8, y = 18, z = -22; p < 0.001, k = 286, FWE cluster-level corrected, p = 0.109). PTSD patients compared to Controls showed more positive rs-FC between the right hippocampus with thalamus/brainstem (peak, MNI: x = 6, y = -18, z = -14; p < 0.001, k = 555, FWE cluster-level corrected, p = 0.002), and similarly, between the left hippocampus and thalamus/brainstem (peak, MNI: x = 0, y = -24, z = -18; p < 0.001, k = 573, FWE cluster-level corrected, p = 0.002). Conclusions: Using the largest centrally analyzed resting-state dataset for PTSD to date, our results illustrate greater rs-FC between the amygdala and sgACC and between the hippocampus and thalamus in PTSD patients compared to controls. Previous studies using smaller samples, and both rs-FC and threat-related task-based designs, have suggested an aberrant functional relationship in PTSD participants within the amygdala and regions of the ventromedial prefrontal cortex (vmPFC). Our preliminary findings provide further evidence of the inadequate regulatory relationship between these two regions using the largest study to utilize resting-state data. Regarding our hippocampal findings, increased rs-FC between the hippocampus and thalamus could contribute to altered threat processing in PTSD. These findings are important as they can help guide treatments targeting these aberrant connections, such as neuromodulation. Keywords: Resting-State Functional Connectivity, Posttraumatic Stress Disorder, Amygdala, Hippocampus Disclosure: Nothing to disclose. P41. Greater Social Cognition-Related Right Temporal Pole Activation in World Trade Center Responders With PTSD - Preliminary Evidence Saren Seeley, Zoe Schreiber, Maya Verghese, Tomasina Leska, Laurel Morris, Leah Cahn, Erno Hermans, Robert Pietrzak, M. Mercedes Perez-Rodriguez, Adriana Feder Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: The ability to accurately ‘read’ and respond to others is key in developing and maintaining a supportive social network. People with posttraumatic stress disorder (PTSD) often report low access to social support and more difficulty in interpersonal relationships. Further, recent neuroimaging findings suggest that social cognition deficits could affect how people with PTSD perceive threat cues. However, few neuroimaging studies have investigated social cognition in trauma-exposed adults across dimensions of resilience (i.e., presence or absence of psychopathology) and degree of exposure to the event. In this study, we sought to examine social cognition-related brain activation during a mentalizing task in symptomatic individuals with PTSD, compared both to highly resilient and lower-exposed controls. We hypothesized that the PTSD group would show lower social cognition-related BOLD activation in hypothesized regions of interest relative to both lower-exposed and resilient control groups. Methods: As part of a larger, multimodal study of PTSD and resilience in World Trade Center (WTC) responders, we recruited 74 adults who experienced the 9/11/2001 attacks on the WTC, and/or were involved in post-9/11 recovery work in New York City 2001-2002. Stratified sampling aimed to recruit participants in three groups: (1) PTSD (any number of WTC -related exposures; met full criteria for lifetime WTC-related PTSD based on the Clinician-Administered PTSD Scale for DSM-5 [CAPS-5], with persistent clinically significant PTSD symptoms in the past month); (2) highly resilient (four or more WTC-related exposures linked to risk for PTSD; no current or lifetime psychopathology); and (3) lower-exposed (three or fewer WTC exposures; no current or lifetime psychopathology). The target sample size in this ongoing study is N = 105 (CDC/NIOSH # U01OH011473). Participants attended a neuroimaging session in which they completed an in-scanner version of the Reading the Mind in the Eyes social cognition task. On each trial, participants had to decide which one of two words shown corresponded to a cropped photograph of a person’s face. Word pairs were either social (e.g., “apprehensive”, “pensive”) or non-social (e.g., “dark hair”, “looking left”). The Social > NonSocial contrast represented the effect of social cognition, rather than general perceptual and decisional functions. fMRI data preprocessing used fMRIPrep 21.0.2 to generate individual motion-, susceptibility distortion, and slice timing-corrected echoes, which were subsequently denoised using TE-dependent independent components analysis (TEDANA 0.0.12), and finally smoothed with a 4-mm FWHM Gaussian kernel. Four of the 74 participants were excluded due to excessive motion (n = 3) or technical error (n = 1), leaving a final n = 22 in the PTSD group, n = 31 in the resilient group, and n = 17 in the lower-exposed group. Results: Linear models identified a main effect of trial type (Social vs. NonSocial) on task performance: mean reaction time was slower (F = 36.3, p < .0001) and participants made more errors (F = 248.2, p < .0001) on Social trials. There was no main effect of group on mean reaction time (F = 1.49, p = .22) or accuracy (F = 2.85, p = .064) and no interaction with trial type. For the fMRI data, Social > NonSocial contrast images at the single-subject level were aggregated for the group-level analysis. A whole brain 1-sample t test for the Social > NonSocial effect identified clusters matching a priori ROIs related to social cognition, including medial prefrontal cortex, temporal poles, inferior frontal gyrus pars orbicularis, and precuneus/posterior cingulate cortex, using a threshold of pFWE = .05 and k = 50. There was a main effect of group for the right temporal pole cluster (50 16 -32), F(2,67) = 3.98, r2 = .08, p = .023. Contrary to hypothesis, Social > NonSocial BOLD signal in the right temporal pole cluster was significantly greater in the PTSD group compared to the resilient (p = .027) and lower-exposed control (p = .011) groups, while control groups did not differ significantly (p = .491). The continuous measure of PTSD severity (CAPS5-Past Month scores) also predicted higher Social > NonSocial right temporal pole activation, F(1,68) = 6.92, p = .01). Conclusions: Our findings are consistent with prior research implicating structural and functional differences in the right temporal pole in individuals with PTSD. They also provide additional support for social cognition as an emerging but important area for future investigations of the neural processes involved in psychological factors linked to risk or resilience after traumatic stressors. Keywords: PTSD, Post Traumatic Stress Disorder, Social Cognition, Functional MRI (fMRI) Disclosure: Nothing to disclose. P42. Open-Label Accelerated 1-Hz rTMS Pilot Targeting Intraparietal Sulcus in Generalized Anxiety Disorder Nicholas Balderston, Marta Teferi, Lily Brown, Desmond Oathes, Yvette Sheline University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Repetitive transcranial magnetic stimulation (rTMS) is a promising avenue to develop novel treatments for anxiety. Despite this promise, the primary development pathway for anxiety has been to adapt protocols designed for depression treatment. Although these disorders are highly comorbid, the underlying neural mechanisms may differ, leading to mixed efficacy among anxiety patients. Accordingly, there is a critical need for novel neuromodulatory protocols that specifically target the neural circuits affected by anxiety. Additionally, such protocols should be developed using dimensional measures that reliably capture behavioral phenotypes associated with network dysfunction. Methods: The purpose of this study is to provide preliminary evidence to determine the safety and efficacy of parietal stimulation for anxiety. The current results are from an ongoing open label pilot targeting the right IPS in Generalized Anxiety Disorder (GAD) patients. Patients received week of accelerated 1 Hz rTMS (5 days, 8 sessions/day, 600 pulses/session) at 100% of resting motor threshold. Anxiety was measured at study start and ~76 hours post rTMS using threat of unpredictable shock. Anxiety was quantified as the increase in startle magnitude during the threat periods compared to the safe periods (anxiety potentiated startle; APS). Results: At the time of writing, this ongoing study has 5 enrolled patients with 3 completers. Using an intent-to-treat analysis, APS was reduced from pre to post rTMS with a moderate effect size (cohen’s d = 0.51). Conclusions: Although preliminary and qualitative, these results offer promising support for future studies aimed at treating GAD with parietal rTMS. These results are consistent with our previous work showing that the IPS exhibits hyperexcitability and hyperconnectivity during unpredictable threat, and that reducing this hyperexcitability with 1 Hz stimulation can reduce anxiety, as indexed by APS. Together with this previous work, the current results suggest that the IPS may be a key region for anxiety expression and a prime target for inhibitory neuromodulation. Future clinical trials should replicate this work using appropriate blinding, adequate control conditions, and a large enough sample size to ensure adequate power (N > 70/group). Keywords: rTMS, Generalized Anxiety Disorder, Startle Disclosure: Nothing to disclose. P43. Impact of Trauma Type on Neural Mechanisms of Threat Conditioning and its Extinction Isabel Moallem, Zhenfu Wen, Mira Hammoud-Milad, Edward Pace-Schott, Mohammed Milad NYU Langone, New York, New York, United States Background: Associative learning theories suggest that psychopathology following trauma exposure might arise from dysfunctions in the neural circuits underlying threat conditioning and extinction learning. Within the context of threat and fear extinction, prior studies in posttraumatic stress disorder (PTSD) have consistently reported impaired activations within regions of the medial prefrontal cortex, insular cortex, hippocampus, amygdala, and various parietal and temporal cortical regions. To our knowledge, none of the studies to-date have been able to evaluate the impact of different types of trauma exposure on the neurobiology of threat and fear extinction due to limited power. In this study, we combined data from 3 studies to examine whether trauma type (violent vs nonviolent) differentially impacts the neurobiology of threat conditioning and its subsequent extinction mechanisms. Methods: We analyzed data from 207 trauma-exposed individuals, with or without PTSD diagnosis, who underwent an established 2-day threat conditioning, extinction learning, and extinction recall paradigm. We analyzed skin conductance responses (SCR) and functional magnetic resonance imaging (fMRI) data. Reported traumatic events were categorized as either violent or nonviolent. The violent category included experiencing or witnessing: combat, physical/sexual assault or abuse, resulting in total sample of n = 126 in this category—we refer to this group as the violent trauma-exposed (VTE) group. The other 81 participants fell into the nonviolent trauma-exposed (NVTE) group. This category included experiencing or witnessing: accidental injury, natural disaster, unexpected death. To further refine our analyses within the VTE group, we subdivided this cohort into sexual (n = 55) vs nonsexual trauma (n = 71) categories. For the fMRI data analyses, we first examined regions that were thought to be critical for conditioning and extinction, including amygdala, hippocampus, insular cortex, ventromedial prefrontal cortex, and dorsal anterior cingulate cortex (small-volume correction, family-wise error [FWE] corrected). For whole-brain analysis, we used a voxel-level threshold of p < 0.005 and cluster-level pFWE<0.05. Results: For the SCR analyses, the VTE group showed significantly higher SCR to the extinguished conditioned stimuli relative to the NVTE group (p < 0.01), suggesting impaired extinction memory recall in the VTE group. No other statistically significant SCR differences were noted between these two groups or within the sub-groups analyses (all ps>0.10). In the fMRI analyses, we observed significantly increased activations within the posterior insular cortex and visual cortices (cluster-level pFWE<0.05) during threat conditioning and significantly decreased activations in the dorsal anterior cingulate cortex, inferior parietal cortex, and posterior insular cortex during late extinction learning (cluster-level pFWE<0.05). The dysfunctional activations during late extinction in the VTE group are consistent with impaired extinction. As for the sub analyses focusing on the sexual vs. nonsexual VTE, the group in the nonsexual VTE exhibited higher activation in the rostral anterior cingulate cortex during early conditioning (cluster-level pFWE<0.05), and higher activations in the inferior parietal insular, and temporal cortices during extinction memory recall (cluster-level pFWE<0.05). Conclusions: We report findings showing that participants who experienced violent types of trauma demonstrated impaired fear extinction at the SCR level and dysfunctional activations within neural nodes important for attention and sensory processing of threat cues during threat acquisition and during the extinction learning. We did not observe any significant dysfunctional activations within neural circuits involved in threat detection or emotion regulation (i.e., amygdala, hippocampus) in any of our analyses, including sexual vs. nonsexual trauma groups. These null findings suggest that, for the most part, violent trauma, regardless of it being sexual or nonsexual, appears to have comparable impact on the neural circuits of threat detection and emotion regulation. Violent vs. nonviolent trauma, however, seems to have a more differential impact on brain circuits related to attention and perception. Keywords: PTSD, fMRI, Trauma Disclosure: Nothing to disclose. P44. Neural Correlates of Treatment Response Differ Between CBT and Active Therapy Control in Adolescents With Anxiety Disorders: Evidence From an Approach-Avoidance Conflict Paradigm Cecilia Westbrook, Michael Schlund, Greg Siegle, Jennifer Silk, Neal Ryan, Erika Forbes, Dana McMakin, Philip Kendall, Anthony Mannarino, Cecile Ladouceur University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States Background: Cognitive-behavioral therapy (CBT) is a gold-standard intervention for pediatric anxiety disorders. Part of its efficacy derives from exposure therapy, in which children approach feared stimuli and learn to decrease their anxiety in a graded fashion. Despite its clinical track record, 20-40% of patients fail to respond to CBT (Kendall and Peterman, 2015), which could be explained in part by reduced approach motivation needed to engage with feared stimuli. The purpose of this study was to investigate neural correlates of treatment success for CBT using data from a previously-published RCT (Silk et al., 2018), which compared the treatment of youth with anxiety disorders with CBT to an active therapy control, child-centered therapy (CCT), including comparisons with youth without psychiatric diagnoses (NPD). We examined data from an Approach-Avoid Conflict fMRI task (Schlund et al., 2010), hypothesizing that compared to NPD, anxious youth would have greater avoid-related activity, but those who responded to treatment would have greater approach- and approach-avoid conflict activity at baseline. Regions of interest included ventral and dorsal striatum, amygdala, dorsal ACC and anterior insula. Methods: A total of 97 youth with an anxiety disorder (generalized, social, separation) (ANX) age 9-14 randomized 2:1 to CBT (N = 67) or CCT (N = 30) and had usable imaging data; 38 age- and sex-matched NPD youth also had usable data. Youth underwent a baseline assessment using a standardized DSM-IV diagnostic interview (K-SADS-PL) and a measure of anxiety severity (the Pediatric Anxiety Rating Scale (PARS), then a baseline MRI, then had 16 sessions of CBT or CCT therapy prior to a second assessment and MRI. Treatment response was defined as a reduction of 35% or more in PARS score. MRI data were preprocessed using fmriprep (https://fmriprep.org/en/stable/) and analyzed using FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki). Multiple comparison correction was done using cluster-based random field theory with a cluster forming threshold of z = 3.1 and a corrected cluster p-value threshold of 0.05. All analyses controlled for age and sex. Results: The majority of ANX youth (N = 67) responded to treatment. Contrary to our hypotheses, there were no differences in approach-, avoid- or approach-avoid conflict-related brain activity between anxious and NPD youth, nor did activity predict treatment response either at baseline or in pre-post brain activity changes. However, when we examined the interaction between therapy group and treatment response, there were significant activations for the approach condition in bilateral dorsolateral and medial PFC, right dorsal striatum, bilateral precuneus/posterior cingulate, bilateral posterior insula, inferior parietal, superior temporal and occipital cortices. For the avoid contrast, the interaction term produced activations in bilateral dlPFC, bilateral vlPFC, left amygdala, left ventral and dorsal striatum, bilateral superior and inferior parietal cortices, bilateral temporal cortices, and occipital cortex. Visualization of these interactions revealed that treatment non-response in the CBT group corresponded to decreases in approach and avoid-related activity, whereas treatment response corresponded to either increases or no change in activity. The CCT group demonstrated the opposite pattern, such that treatment non-response corresponded to increases in activity, while treatment response corresponded to decreases or no change in activity. Conclusions: Although the majority of ANX youth responded to treatment, patterns of change in neural activity differed between treatment types, suggesting that mechanisms of treatment efficacy may be unique to CBT. Change in brain activity related to both approach and avoidance motivation, both within and outside of defined regions of interest, indicates that both neural systems are relevant to treatment of anxiety in youth but activate differentially in exposure-based relative to supportive modes of treatment. Keywords: Adolescent Anxiety, Task fMRI, Approach/Avoidance, Cognitive Behavior Therapy, RCT Disclosure: Nothing to disclose. P45. Real-Time fMRI Neurofeedback of Left Dorsolateral Prefrontal Cortex During Emotional Cognitive Control: A Randomized Controlled Trial Timothy McDermott, Tsuchiyagaito Aki, Ramirez Sam, Mallory Cannon, James Touthang, Masaya Misaki, Robin Aupperle Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Real-time fMRI neurofeedback (rt-fMRI-nf) is an endogenous neuromodulation approach whereby individuals self-regulate their own neural activity in the moment using strategies related to a particular construct of interest. Cognitive control deficits in an emotional context negatively impact people with mental health disorders, including those with depression, anxiety, and PTSD. The left dorsolateral prefrontal cortex (dlPFC) has been shown to be a centrally important region for facilitating emotional cognitive control. Using a double-blind randomized controlled trial design, this study tested a novel rt-fMRI-nf protocol that sought to increase left dlPFC blood-oxygen level dependent activity during emotional cognitive control and improve self-report and behavioral measures of emotional cognitive control. Methods: Healthy adult participants (N = 70; 42 females; mean age = 31.2 years) enrolled in the study and were randomized to either active (n = 37; left dlPFC) or sham (n = 33; left postcentral gyrus) single-session rtfMRI-nf (clintrials.gov identifier: NCT04543513). Feedback was given from a 7-mm sphere region-of-interest centered on coordinates in left dlPFC or left postcentral gyrus, respectively. During neurofeedback, participants focused on executive functioning tasks (e.g., mental math, word list recall, rehearsing to-do list) while distracting negative emotional words were presented, which engaged emotional cognitive control. Single-session neurofeedback consisted of five rt-fMRI-nf runs, starting with practice, followed by three training runs, and lastly a transfer run without feedback. Participants also completed self-report [Patient-Reported Outcomes Measurement Information System (PROMIS): Self-Efficacy for Managing Emotions] and behavioral measures [emotional Stroop task (EST) interference effect] of emotional cognitive control prior to rt-fMRI-nf and at one-day follow-up. The primary outcome was left dlPFC activity during neurofeedback, and secondary outcomes were self-report/behavioral measures of emotional cognitive control. A series of linear mixed-effects models (LMEs) were conducted to examine if active rt-fMRI-nf, compared to sham, led to greater changes for each outcome measures [fixed effects: group, time, group-by-time interaction; random effect: subject id]. We also examined protocol tolerability and subjective emotional distress associated with the negative words using a post-neurofeedback survey with 1-10 ratings. Results: LMEs showed a significant group-by-time interaction on left dlPFC activity (p = .031; η2 = .021), and this interaction was such that active neurofeedback led to greater left dlPFC activity in run 1 (p = .030; d = .66) and run 2 (p = .042; d = .62), but not for run 3 (p = .82) nor the transfer run (p = .30). LMEs showed no significant effects on either self-report or behavioral measures of emotional cognitive control (p’s > .46). Note that these self-report/behavioral measures were significantly inversely correlated with each other (p < .001; r = -.41), indicating that those with higher PROMIS: Self-Efficacy for Managing Emotions scores had lower emotional interference on the EST. Participants across conditions described the protocol as being tolerable with minimal distress, and they were unable to identify their rtfMRI-nf condition (p = .83). While the overall level of emotional distress reported in reaction to the negative words shown during neurofeedback was quite low across both groups (M = 1.57 on a 1-10 scale), participants in the active group reported significantly lower distress (M = 1.32) compared to the sham group (M = 1.84; p = .039; d = -.51). Conclusions: These findings support the feasibility of left dlPFC rt-fMRI-nf and identify potentially acute effects on left dlPFC activity and subjective emotional distress. However, overall results indicate that rt-fMRI-nf had an initial but not a sustained effect on left dlPFC activity, and thus, they present complexity and nuance to consider when using rt-fMRI-nf to modulate neural activity and improve psychological function. The sustained impact of the current dlPFC neurofeedback protocol may have been limited by participant fatigue associated with a long protocol, individual variability in left dlPFC architecture, and diminishing challenge of the cognitive tasks over time. Future studies are needed to optimize the cognitive strategies used, the dlPFC target, and the timing and duration of the neurofeedback protocol, as well as to determine whether dlPFC neurofeedback may have differential impacts for clinical populations. Keywords: Non-invasive Neuromodulation, Functional MRI (fMRI), fMRI Negative Affective Stimuli, Cognitive Enhancement, Real-Time fMRI Neurofeedback Disclosure: Nothing to disclose. P46. Behavioral Activation and Exposure Therapy Improve Anxiety Symptoms and Alter Neural Processing of Approach-Avoidance Conflict Hannah Berg, Timothy McDermott, Elisabeth Akeman, Jessica Santiago, James Touthang, Kelly Cosgrove, Mallory Cannon, Ashley Clausen, Namik Kirlic, Ryan Smith, Christopher Martell, Kate Wolitzky-Taylor, Michelle Craske, James Abelson, Jerzy Bodurka, Martin Paulus, Robin Aupperle Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Individuals with anxiety disorders often engage in maladaptive decision-making when faced with approach-avoidance conflict (AAC), a behavioral pattern that is addressed in exposure-based therapy (EXP) and behavioral activation (BA). Identifying mechanisms by which these treatments promote adaptive behavior may guide the development of process-specific treatment approaches. In the present study, we examined behavioral and neural responses to AAC before and after BA and EXP in a sample of adults with generalized anxiety disorder (GAD). Methods: Participants were randomized to complete 10 weekly sessions of either BA or EXP. Symptoms were assessed pre-treatment, at each treatment session, and post-treatment with the GAD-7, PROMIS Anxiety and PROMIS Depression scales. Individuals who completed at least 7 out of the 10 therapy sessions were considered treatment completers and included in clinical outcome analyses. Treatment completion and treatment response, defined as at least 30% reduction in GAD-7 scores from pre- to post-treatment, were compared across treatment arms using logistic regressions. Linear mixed-effects regressions were conducted for symptom scores with time (all timepoints) and treatment arm (BA, EXP) entered as predictors. Before and after treatment, participants completed an AAC task during fMRI. In the task, participants chose how much to approach or avoid based on explicit cues indicating the most likely outcomes associated with each position along a horizontal runway onscreen. On each trial, an approach decision could result in an aversive outcome (negatively-valenced images and sounds), an appetitive outcome (earning 2, 4, or 6 cents), or both (i.e., an AAC trial). Treatment completers who completed the AAC task pre- and post-treatment, and had useable fMRI data, were included in analyses of behavior and brain activity during the AAC task. We conducted linear mixed-effects regressions for the effects of time (pre-, post-treatment), treatment arm, and trial-type (conflict with 2, 4, or 6 cents) on runway position and response time across AAC trials. Computational parameters reflecting emotional conflict (i.e., aversiveness of threats relative to rewards) and decision uncertainty, both of which have been previously found to be elevated in anxious individuals, were extracted from approach and avoidance behavior, and we conducted linear mixed-effects regressions for the effects of time and treatment arm on these parameters. Next, neural responses (blood-oxygen-level-dependent [BOLD] signal) were extracted for a priori regions of interest, including dorsolateral prefrontal cortex (dlPFC), amygdala, anterior cingulate, anterior insula, and striatum responses to the decision phase; and striatum responses to picture presentation and reward receipt. We conducted linear mixed-effects regressions for each region of interest, with time, treatment arm, and trial-type (aversive; appetitive; conflict with 2, 4, or 6 cents) as predictors, and neural responses as the outcome variable. Age, sex, and education were included as covariates in all group-level regressions. Results: Of the 102 participants enrolled in the study, 70 completed treatment (38 BA, 32 EXP), reflecting comparable completion across treatment arms (p = 0.127). A majority of treatment completers responded to treatment in both BA (66%) and EXP (59%) with comparable response across treatment arms (p = .761). Effect sizes on GAD-7 scores pre- to post-treatment were large for both BA (d = -1.14) and EXP (d = -0.97). Pre- to post-treatment contrasts did not differ across treatments for GAD-7 scores (p = .540), but did differ across treatments for PROMIS Anxiety (p = .039, d = .13) and PROMIS Depression scores (p = .029, d = .14), in both cases reflecting greater symptom improvement in BA compared to EXP. 49 participants (29 BA, 20 EXP) were included in behavioral and fMRI analyses. Following both treatments, reaction times decreased (p = .008, ηp2 = .03) and, unexpectedly, approach behavior also decreased (p = .048; ηp2 = .02) on AAC trials. The computational parameter reflecting emotional conflict increased following treatments (p = .017, ηp2 = .12); the decision uncertainty parameter did not significantly change following treatments (p = .162). Analyses of neural responses revealed that BA, more than EXP, was associated with increased left dlPFC engagement during decision-making (time by treatment-arm interaction: p = .020; ηp2 = .01), and both treatments were associated with increases in striatal response to image outcomes (p = .037, ηp2 = .03) and monetary reward receipt (p = .015, ηp2 = .04). Conclusions: BA and EXP are shown to be effective treatments for GAD. Increased activation in left dlPFC during decision-making and increased striatal engagement during emotionally salient outcomes may reflect the neural basis of a process by which BA and EXP influence approach-avoidance conflict decision-making. One interpretation is that, with treatment, individuals increase dlPFC-mediated effortful deliberation during AAC decision-making, and increase striatally-mediated encoding of salient outcomes, both in the task and in real-world scenarios. Increases in left dlPFC activity were particularly robust in BA, potentially due to BA’s focus on deliberately engaging in values-driven behavior. Further research is warranted to investigate whether psychological treatments can be optimized by targeting specific decisional processes. Keywords: Anxiety, Generalized Anxiety Disorder, fMRI, Decision Making, Approach-Avoidance Conflict Disclosure: Nothing to disclose. P47. Sex-Specific White Matter Microarchitectural Alterations in Preadolescent Youth With Anxiety Disorders Nakul Aggarwal, Do Tromp, Daniel Pine, Lisa Williams, Ned Kalin University of Wisconsin - Madison, Madison, Wisconsin, United States Background: Anxiety disorders (ADs) emerge in childhood and early adolescence and are among the most common psychiatric illnesses. With the onset of puberty, females are twice as likely as males to have ADs. Considerable research has implicated alterations in the white matter (WM) microstructure of prefrontal-limbic tracts, as well as other WM regions, in the pathophysiology of ADs. These findings are of interest because WM consists primarily of myelinated axons, and myelin plays an important role in mediating optimal neuronal communication. However, few studies have examined anxiety-related WM alterations in youth and in relation to sex. To further examine associations between childhood ADs, WM microstructural integrity, and sex, we analyzed diffusion tensor imaging (DTI) data from 295 preadolescent youth with and without ADs. Methods: Preadolescent youth with ADs (social, generalized, and/or separation AD) and healthy controls were enrolled between ages 8-12 at 2 study sites (UW-Madison, NIMH) and completed a DTI scan on a 3T-scanner. The final sample (n = 295; 201 females) included 163 children with ADs and 132 controls. Tractography- and voxel-based analyses examined the main effect of ADs, as well as the AD by sex interaction, for WM metrics [fractional anisotropy (FA), radial diffusivity (RD), mean diffusivity (MD), and axial diffusivity (XD)] in seven bilateral tracts of interest and in WM across the whole brain. Results: Results demonstrated widespread anxiety-related alterations in DTI metrics across multiple WM regions. Critically, anxiety-related effects demonstrated a significant interaction with sex, such that AD-related FA reductions and RD increases were observed exclusively in boys and not in girls. Preadolescent boys with ADs exhibited decreased FA and elevated RD relative to healthy control boys (P-FWE < 0.05) across various WM regions throughout the brain, in association (UF, EC, IFO, SLF, ST, ILF), commissural (CC), projection (CR, IC), and brainstem (CST, CP, ML) tracts, while no group differences were seen in girls. Conclusions: This study constitutes the largest cross-sectional DTI study of childhood anxiety to date. The results show that childhood ADs are associated with broadly distributed alterations in WM microarchitecture across the brain, and, importantly, this relationship is evident only in boys. These findings – particularly the combination of reduced FA and increased RD – point to the possibility that WM microstructure and myelination processes may be related to the underlying pathophysiology of childhood ADs, laying the foundation for future work exploring the viability of WM microstructure as a treatment target for novel therapies. Keywords: Childhood Anxiety, White Matter Microstructure, Diffusion Tensor Imaging (DTI), Myelination, Sex Differences Disclosure: Nothing to disclose. P48. Brain Regions Conferring Psychological Resilience: A Meta-Analysis Andrew James, Allison Kuehn, Clinton Kilts University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States Background: Neuroimaging investigations in psychiatry have historically sought to identify neurobiological mechanisms placing individuals at risk for developing psychiatric disorders. But a growing interest has emerged for identifying neurobiological mechanisms that promote psychological resilience, a MeSH term (D055500) defined in 2009 as “The human ability to adapt in the face of tragedy, trauma, adversity, hardship, and ongoing significant life stressors.” To address this growing interest, we conducted a systematic review and neuroimaging meta-analysis of brain regions which may promote psychological resilience. Methods: We conducted a systematic review of 4 bibliographic databases (PubMed, Embase, PsychInfo, and Web of Science) for journal articles including keyword “resilience” and a functional neuroimaging technique. (See poster for exact, database-specific search queries.) These searches yielded 498 unique articles. 429 articles were excluded for reasons including a non-psychological definition of resilience (e.g., cognitive resilience following neurosurgery), not recruiting a resilient sample, and not providing neuroimaging data or coordinates, yielding 69 articles included for analysis. This search also yielded 42 review articles whose bibliographies were searched for relevant articles: these articles encompassed an additional 2205 articles of which 78 met inclusion criteria, for 69 + 78 = 147 unique articles. All elements of the systematic review – including reason for exclusion, sample size, and brain coordinates – were memorialized in the non-proprietary reference manager Zotero for rigor and reproducibility. Custom python code then extracted articles’ ROI coordinates in Talairach or MNI atlas space for activation likelihood estimation (ALE) meta-analysis by GingerALE. The 147 studies yielded 499 foci more significantly associated with the resilient groups than with the healthy control and/or patient groups. GingerALE was performed with uncorrected p = 0.001 and 100 permutations for FWE p = 0.01. All code available by request. Results: The ALE meta-analysis associated three regions with psychological resilience: right amygdala (ALE = 0.060, Z = 7.76), right amygdala (ALE = 0.056, Z = 7.41), and dorsal anterior cingulate (ALE = 0.032, Z = 5.04). Post-hoc examination of studies contributing to each region found that the amygdalae were largely comprised from studies of PTSD (~45%) and depression (~30%), while the anterior cingulate was predominantly comprised from studies of PTSD (38%) and schizophrenia (38%). Conclusions: The amygdalae and anterior cingulate play critical roles in diverse cognitions including threat detection, error detection, and emotion regulation. Notably, amygdala and anterior cingulate hyperactivity has been associated with both PTSD and MDD. Our findings suggest these regions play more complex roles in promoting risk for and resiliency against psychiatric disorders than previously thought. Future work will seek to identify disorder-specific predictors of risk and/or resilience. Keywords: Risk and Resilience, Human Neuroimaging, Emotional Regulation Disclosure: Nothing to disclose. P49. Altered Default-Mode (DMN)-Frontoparietal (FPN) Network Connectivity Following Mindfulness-Based Cognitive Therapy (MBCT) in PTSD Patients Linked to Treatment Response - A Randomized Controlled Trial With Active Comparator Anthony King, Chandra Sripada, Israel Liberzon, K. Luan Phan The Ohio State University, Columbus, Ohio, United States Background: Posttraumatic stress disorder (PTSD) is common, can be chronic and debilitating, and has high human and economic costs to individuals, families, and society. While existing trauma exposure-based psychotherapies show high efficacy, many PTSD patients decline trauma-focused therapy, and meta-analyses report 30-50% of PTSD patients treated with trauma-focused therapy do not show clinically meaningful improvement, suggesting “one size may not fit all”. Accumulating evidence finds clinically significant efficacy of 8 wk group mindfulness-based approaches like Mindfulness-based Cognitive Therapy (MBCT) for acute depression, anxiety, and PTSD, in addition to original target of relapse prevention for depression and substance use disorders, warranting more research on efficacy for PTSD and potential neural mechanisms. We reported in 2016 that a mindfulness-based intervention for PTSD led to increased resting state functional connectivity (rsFC) between posterior cingulate cortex (PCC, key node of Default Mode Network, DMN) and dorsolaterasl prefrontal cortex (dlPFC, key node of frontoparietal network, FPN); and mindfulness-associated increased PCC-dlPFC rsFC has since been independently replicated by 3 laboratories. In 2017 we registered hypotheses with NCCIH (R61 “Go-criteria”) that MBCT - compared to an active Mind-body comparator, progressive muscular relaxation (PMR) – would lead to increased PCC-dlPFC rsFC, and both MBCT and PMR would lead to decreased PCC-insula rsFC (which is increased in PTSD). Methods: We conducted a randomized controlled trial (RCT) with PTSD patients comparing MBCT to an active mind-body control (PMR, designed to be structurally similar to MBCT but no mindfulness instruction) using cluster randomization (blinded to assignment during recruitment). Functional MRI (3T GE scanner) scans were collected at intake and within 3 weeks post-treatment. We consented 161 persons from the community, and enrolled 77 with PTSD diagnosis by CAPS-5 and intake fMRI scan. Due to COVID pandemic, 22 patients were lost to follow-up, and interventions were shifted to remote delivery (“Zoom”), and 41 participants completed the RCT with both pre- and post-therapy fMRI scans. Assessments (CAPS-5 and HAM-D) were conducted by blinded assessors, and CGIs obtained by consensus of clinical team, and the PI and analysts were blinded. Self-report measures (PCL-5, decentering, PTSD cognitions, rumination, etc) were conducted using web-based REDCap. fMRI paradigms including resting state (8 min), a previously validated self-referential task, and a contextual processing task. We assessed rsFC using a PCC seed in CONN toolbox, and used a flexible ANOVA in SPM12 testing a time (pre- vs post-therapy) by treatment (MBCT vs PMR) interaction term. Results: We report results from our rsFC data testing our registered hypotheses that MBCT leads to increased PCC-dlPFC and decreased PCC-insula rsFC. Both MBCT and PMR led to clinically meaningful improvements in PTSD (MBCT mean 8.9 point CAPS-5 reduction, Hedge’s g = 1.0, p < .001, PMR 10.1 point CAPS-5 reduction, g = 1.0, p < .001), no significant between treatment effect. Both interventions were well-tolerated and had completion rates (5+ sessions) >80%, Rates of “responders” (CGI-I 1–very much or 2-much improved) were 48% MBCT and 50% PMR. In the PCC-seed rsFC ANOVA interaction Z-map we found a cluster in right dlPFC/ BA 10 MINI:(30, 48, 12), F = 20.91, Z = 3.90, k = 94 voxels, SVC pFWE=0.021. Test of the change score (pre- vs post delta) in the extracted dlPFC (5 mm sphere) beta values found significantly greater (p = .002) increase in rsFC in the MBCT group compared to the PMR group (Hedges g = 1.03). Furthermore, change in PCC-dlPFC rsFC was greater in MBCT responders vs non-responders (p < .05, g = 0.57). Both MBCT and PMR led to decreased PCC-insula rsFC (pre vs post extracted aal insula g = -0.35 in MBCT, g = -0.53 in PMR, p < .05). Conclusions: We found evidence supporting our registered hypothesis (R61 “Go-criteria”) that MBCT but not PMR lead to an increase in PCC-dlPFC rsFC (altered DMN-FPN cross-network connectivity). This is also consistent with recently reported findings from independent research groups. Furthermore, the increase in PCC-dlPFC rsFC was significantly related to clinical improvement (blinded CGI-I scores for PTSD) in the MBCT group only. These data further support PCC-dlPFC FC as a potential treatment target mechanism of mindfulness interventions, potentially related to increased volitional meta-cognitive attention and emotional regulation of spontaneous distressing thoughts. Both MBCT and PMR decreased PCC-insula rsFC, suggesting a general mechanism related to improvement of PTSD symptoms. Keywords: Mindfulness, PTSD, Default Mode Network (DMN), Frontoparietal Network, Insula Connectivity Disclosure: Nothing to disclose. P50. Validation of an Updated Brain Circuit to Decode the Neural Signature of Threat Conditioning and Fear Homeostasis Zhenfu Wen, Jörgen Rosén, Fredrik Åhs, Sara Lazar, Edward Pace-Schott, Elizabeth Phelps, Joseph LeDoux, Mohammed Milad New York University, New York, New York, United States Background: Threat conditioning and extinction is one of the primary translational models for understanding anxiety and trauma-related psychopathology. The ‘threat circuit’, which mainly includes the medial prefrontal cortex, insular cortex, hippocampus, and amygdala, is thought to play a key role in the threat acquisition and its extinction. Recent evidence, however, suggests that the processing of threating and fearful stimuli is highly distributed across multiple neural systems. In this study, we combined functional MRI data and machine-learning methods to identify and validate distributed neural representations of stimuli coding threat or safety. We then used external datasets from multiple paradigms to test the specificity of the newly identified circuit. Methods: Task-based functional MRI data from a total of 1455 participants were analyzed in the present study. We used the neural activations within and beyond the ‘threat network’ to distinguish conditioned stimuli associated with threat (CS+) or safety (CS-) across experimental phases of threat conditioning and extinction. We evaluated the performance of the decoding models using cross-validation on a discovery dataset (n = 420) and tested the generalizability of the models using two external datasets (n = 126 and 94). We then estimated predictive weight of each voxel and identified brain regions that significantly contributed to the decoding of the CS + and CS-. We validated a new circuit by applying the decoding models trained with its neural activations to multiple external datasets using other paradigms (n = 815 across 7 datasets). Permutation tests were used to assess the significance of the decoding accuracies. Results: Neural signal within the ‘threat network’ successfully decoded conditioned stimuli-induced representations on the discovery dataset (accuracies 62.6%~75.2%) and generalized to the two external datasets (dataset 1: 58.3%~70.8%, dataset 2: 58.7%~82.7%). Importantly, classification performances were substantially improved using activations from distributed networks (discovery dataset: 72.6%-88.6%, external dataset 1: 66.7%-80.2%, external dataset 2: 60.3%-90.6%). The results from the distributed network that significantly contributed to the decoder included somatomotor regions, prefrontal and parietal regions, thalamus, cerebellum, and caudate. All regions that significantly contributed to the classification of the CS + and CS- (including the threat circuit) composed an updated circuit which we refer to as the ‘fear homeostasis circuit’. These regions either consistently coded the CS + /CS- across the experimental phases, or dynamically switched its preference to CS + /CS- depending on the experimental phase. Classifiers trained by neural patterns from the newly defined circuit successfully applied to three visual threat conditioning datasets that used different paradigms (n = 299, 94 and 48, highest accuracy: 91.0%, 91.0%, 87.5%), one auditory threat conditioning dataset (n = 68, highest accuracy: 83.8%), and one dataset examined subjective reported fear (n = 65, highest accuracy: 81.5%). Applying the classifiers to brain activations pertaining to neural representations not related to associative learning and memory (yet still related to emotional stimuli) led to reduced accuracies: picture-evoked negative affect (n = 182, highest accuracy: 75.3%), physical pain (n = 59, highest accuracy: 71.2%), and social rejection (n = 59, highest accuracy: 61.0%). Conclusions: Optimizing classifiers using a large fMRI dataset enabled us to show that conditioned stimuli can be classified using neural activations of the ‘threat network’, and that better performance can be achieved when neural activations from broadly distributed neural networks were used. Based on these results, we updated the ‘fear homeostasis circuit’ by extending the ‘threat circuit’ to include other brain regions that are important to threat and fear processing. Analyses on the external datasets showed robust generalizability of the decoder across different experimental settings, sites, and scanners with different acquisition parameters. And lastly, the sensitivity and specificity of the ‘fear homeostasis circuit’ in representing threat conditioning, fear, and emotionally salient cues were verified. Keywords: Multivariate Pattern Analysis, Fear Conditioning and Extinction, Classification, Generalizability Disclosure: Nothing to disclose. P51. Computational Mechanisms Underlying Cognitive Processing During Threat Exposure Reflect a History of Panic Attacks in Anxiety Patients Adam Gorka, Christian Grillon, Monique Ernst National Institute of Mental Health, Bethesda, Maryland, United States Background: Patients diagnosed with clinical anxiety disorders exhibit heightened sensitivity to threatening information and altered cognitive processing. However, it is currently unclear whether threatening information impacts similar or distinct computational processes in healthy participants and patients with clinical anxiety disorders. Previous research has suggested that experimental models of anxiety (e.g. induced via unpredictable threat of shock), function to facilitate performance during on the Go/No-Go paradigm. We set out to determine how clinical anxiety disorders and a history of panic attacks impact Drift Diffusion model parameters underlying cognitive processing during experimental anxiety. Methods: 39 healthy participants and 42 anxiety patients, 14 of which had a history of panic attacks, performed the Go/No-Go paradigm during periods of safety and periods of induced anxiety. Induced anxiety facilitated performance during on the No-Go trials irrespective of clinical diagnosis. Results: Our computational analysis demonstrated that induced anxiety significantly reduced the “boundary separation” parameter. Within the group of anxiety patients, the impact of induced anxiety on “boundary separation” was significantly weakened by a history of panic attacks. Conclusions: Boundary separation corresponds to the amount of information required in order to trigger a decision and our results may reflect more cautious decision-making during threat exposure in anxiety patients with a history of panic attacks. Collectively, our results suggest that computational models can help us understand the mechanisms through which experimental models of anxiety influence cognitive processes in clinical patients. Keywords: Anxiety, Computational Cognitive Neuroscience, Panic Attacks Disclosure: Nothing to disclose. P52. Visualizing Functional Connectivity in Posttraumatic Stress Disorder: A Novel Use of Multidimensional Scaling Nicholas Petrosino, Amin Zandvakili, Jennifer Barredo, Noelle Marcotullio, Noah Philip Brown University and VA Providence, Providence, Rhode Island, United States Background: Posttraumatic stress disorder (PTSD) is debilitating, difficult to diagnose, and often treatment resistant. Functional imaging has the potential to provide a deeper understanding of PTSD neurobiology. However, such data is inherently complex, and finding a tractable approach is challenging. Here, we have adopted a network visualization algorithm called multidimensional scaling (MDS), which reduces high-dimensional data while preserving pairwise distances, in order to visualize functional connectivity networks and evaluate connectivity changes associated with PTSD severity. Methods: We collected resting-state fMRI on a sample of n = 50 PTSD subjects. Using 100 relevant regions of interest, maps of pairwise functional connectivity were calculated. Participants were divided into two groups: those with high versus low PTSD symptom severity using a median split of total scores on the PTSD Checklist for DSM-5 (PCL-5). The Pearson cross-correlation values for regional pairs comprising the maps were transformed to dissimilarities and a range of data sparsity was generated using a negative exponential transformation. MDS was then performed, embedding the 4,950 regional connectivity pairs in a two-dimensional space. Permutation testing (500 MDS iterations on randomly shuffled data) was used to determine which regions’ connectivity differed significantly between groups. The experiment was repeated for PTSD symptom clusters of intrusion, avoidance, cognition/mood, and arousal based on median splits of the respective PCL-5 subscale scores. Results: The median total PCL-5 score for our sample fell in the moderate severity range (median=46.5; 95% CI 24-71), and PCL-5 total and subscale scores were highly and significantly correlated with each other (Pearson correlation coefficients ranging from 0.38 to 0.81). For total PTSD severity, three regions demonstrated significant group connectivity differences: the left dorsolateral prefrontal cortex (DLPFC) and the right ventrolateral prefrontal cortex (both frontoparietal network nodes) as well as the left anterior paracingulate (salience network node) (p = 0.021, 0.044, and <0.002 respectively). Analyses on PTSD symptom clusters similarly showed significant connectivity differences between high and low severity groups. For the intrusion cluster, 9 regions were significant: the left thalamus, the DLPFC bilaterally, the ventromedial prefrontal cortex bilaterally, the right orbital cortex, the right mid cingulate/paracingulate of the frontoparietal network, as well as the left striatum and the right anterior cingulate/paracingulate in the default mode network (p = 0.009, 0.040 [left], 0.025 [right], 0.005 [left], 0.003 [right], 0.033, 0.035, 0.042, and 0.034 respectively). The avoidance cluster demonstrated differences in the right basolateral amygdala as well as the left orbital cortex and the left medial prefrontal cortex in the default mode network (p = 0.029, 0.041, and 0.035 respectively). Connections to several other relevant network nodes also showed significant differences for the cognition/mood and arousal symptom clusters. Conclusions: We have successfully adopted a network analysis tool for novel application to functional imaging data, revealing associations of PTSD total and cluster severity with connectivity to and from many relevant network nodes, with the most robust findings in the intrusion cluster. This provides new insight into computational and functional modeling of PTSD severity which can help to guide future objective diagnostics and treatments. Keywords: Post Traumatic Stress Disorder, Resting State Functional Connectivity, Computational Psychiatry Disclosure: Nothing to disclose. P53. Computational Measures of Altered Uncertainty Learning in Transdiagnostic Anxiety Vanessa Brown, Timothy Allen, Laura Taglioni, Rebecca Price, Alexandre Dombrovski University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Difficulty learning and responding to uncertain threat is a core impairment in anxiety (Grupe and Nitschke, 2013). Neurocomputational modeling can disambiguate competing hypotheses about sources of uncertainty learning disruptions in anxiety (Brown et al., 2021; Montague et al., 2012; Pulcu and Browning, 2019). Specifically, different computationally-derived forms of uncertainty (from noise versus changepoints in stimulus-outcome contingencies) have different neural substrates and effects on learning (Mackintosh, 1975; Nassar et al., 2018; Pearce and Hall, 1980; Piray and Daw, 2021; Yu and Dayan, 2005). Even with the aid of modeling, these types of uncertainty can be methodologically challenging to measure simultaneously; as a result, how clinically anxious people are impaired in learning about uncertainty is unknown. Whether impaired uncertainty learning is related to somatic and hyperarousal symptoms present in fear-based disorders (Craske et al., 2009; McTeague and Lang, 2012; Watson, 2005), or shared across internalizing distress-based disorders more generally, is also unclear. In the current study, we developed a novel task and computational learning model to measure learning about uncertainty. We assessed relationships between model-derived behavioral indices of uncertainty learning and measures of anxiety and distress to understand how uncertainty learning is disrupted in anxiety. Methods: Data collection is ongoing; preliminary results are reported from 40 participants (age mean [SD]: 32.3 [9.7]; gender # [%]: 29 [73%] female, 10 [25%] male, 1 [2.5%] non-binary; race # [%]: 29 [73%] White, 7 [17.5%] Black, 1 [2.5%] Asian, 3 [7.5%] multiracial) oversampled for high anxiety symptoms. Participants completed modules from the DIAMOND and MINI semi-structured interviews (Lecrubier et al., 1997; Tolin et al., 2018) to assess DSM-5 diagnoses as well as a dimensional self-report measure of depression and anxiety (IDAS-II; (Watson et al., 2012). Uncertainty learning was measured with an aversive learning task where participants chose among options that differed in two types of uncertainty: noise and changepoint probabilities. Participants’ choices were first fit to an established uncertainty learning model (Nassar et al., 2010, 2016), which assumes participants learn near-optimally about uncertainty. This model estimates values for different sources of uncertainty (noise and changepoint probability) for each option on each trial assuming normative learning. Next, a novel version of this model allowing for individual differences in each type of uncertainty learning was developed. Participants’ choices were also fit to this individual differences model to estimate trial-level uncertainty values accounting for individual differences in uncertainty learning. Relationships among trial-by-trial noise and changepoint probability estimates of uncertainty (from the normative and individual differences models) and switching behavior were assessed for relationships with anxiety and distress using multilevel regression models. Results: Participants showed a range of psychiatric symptoms: most (30/40) met criteria for an anxiety, trauma-related, obsessive-compulsive, and/or mood disorder, and of those, most (25/30) met criteria for multiple disorders. In the normative model, trial-by-trial measures of noise decreased, and changepoint probability increased, the likelihood of switching options (main effect of noise: z = -7.39, p < 0.001; changepoint probability: z = 5.02, p < .0.001). A factor measuring anxiety-specific symptoms on the IDAS-II, but not a factor measuring distress symptoms, was related to a reduced effect of noise on switching behavior (anxietynoise interaction: z = 3.08, p = 0.002; distressnoise: z = -1.052, p = .29). The model allowing individual differences in learning showed excellent parameter recovery (correlation between simulated and recovered parameters: rs from 0.89 to 0.99; correlation between recovered parameters: all rs < 0.35), suggesting that the model could independently measure different uncertainty-related changes in learning with low error. The relationships between trial-by-trial measures of true noise and changepoint probability versus estimates from the individual differences model had negative interactions with anxiety symptoms (true noiseanxiety on estimated noise: t = -2.38, p = 0.017; true change point probabilityanxiety on estimated changepoint probability: t = -3.58, p < 0.001), indicating a weaker relationship between true and learned uncertainty measures with greater anxiety. Conclusions: We successfully measured learning from different types of uncertainty in participants with a range of anxiety and distress symptoms. Anxiety symptoms were related to reduced effects of noise, assessed using a normative model, on behavior. Using a model that could represent individual differences in learning, participants’ estimated values of uncertainty (both noise and changepoint probability) diverged more from the true values as anxiety symptoms increased. Some of these relationships were specific to anxiety and not distress symptoms. These findings support difficulties with uncertainty learning in anxiety; specifically, that uncertainty learning differences result in perceptions of uncertainty diverging from true values and in a way that affects choices in anxiety. Keywords: Anxiety, Aversive Learning, Computational Modeling, Uncertainty Disclosure: Aya Technologies: Consultant(Self) P54. Large-Scale Neural Network Computations and Multivariate Representations During Approach-Avoidance Conflict Decision-Making Nicole Moughrabi, Ameera Azar, Kevin Crombie, Joseph Dunsmoor, Zachary Stowe, Joshua Cisler University of Texas at Austin, Austin, Texas, United States Background: Many real-world situations require navigating decisions for both reward and threat. While there has been significant progress in understanding mechanisms of decision-making and mediating neurocircuitry separately for reward and threat, there is limited understanding of situations where reward and threat contingencies compete to create approach-avoidance conflict (AAC). Methods: Here, we leverage computational learning models, independent component analysis (ICA), and multivariate pattern analysis (MVPA) approaches to understand decision-making during a novel task that embeds concurrent reward and threat learning and manipulates congruency between reward and threat probabilities. 30 adult participants recruited from the general community completed the task during fMRI. Results: Computational modeling supported a modified reinforcement learning model where participants integrated reward and threat value into a combined total value according to an individually varying policy parameter, which was highly predictive of decisions to approach reward vs avoid threat during trials where the highest reward option was also the highest threat option (i.e., approach-avoidance conflict). ICA analyses demonstrated unique roles for salience, frontoparietal, medial prefrontal, and inferior frontal networks in differential encoding of reward vs threat prediction error and value signals. The left frontoparietal network uniquely encoded degree of conflict between reward and threat value at the time of choice. MVPA demonstrated that delivery of reward and threat could accurately be decoded within salience and inferior frontal networks, respectively, and that decisions to approach reward vs avoid threat were predicted by the relative degree to which these reward vs threat representations were active at the time of choice. Conclusions: These data shed light on computational and neurocircuitry mechanisms supporting approach-avoidance conflict decision-making. Computational modeling suggested that participants integrate reward and threat according to an individually varying preference for approaching reward vs avoiding threat. Reward, threat, and reward vs threat conflict were separately encoded in unique large-scale neural networks. The MVPA results suggest that navigating AAC decisions involves generating mental representations for possible decision outcomes, and relative activation of these representations may bias subsequent decision-making towards approaching reward or avoiding threat accordingly. Keywords: Approach-Avoidance Conflict, Computational Modeling, Independent Component Analysis, Multivariate Pattern Analysis Disclosure: Nothing to disclose. P55. Prenatal Citalopram Exposure Promotes Resilience in Male Offspring Exposed to Maternal Stress Anne Andrews, Merel Dagher, Sara Erwin, Katie Perrotta, Olena Lukoyanova, Audrey Nashner, Weiye Dai, Julia Brock, Alexandre Bonnin University of California - Los Angeles, Los Angeles, California, United States Background: Mood and anxiety disorders are highly prevalent during pregnancy and can lead to adverse maternal and offspring outcomes. Selective serotonin reuptake inhibitors are the most common medications used to treat mood and anxiety disorders. Both human and animal studies suggest that serotonin signaling plays an important role in the vulnerability to and the manifestation of stress-associated affective disorders. Moreover, the serotonin system is an early orchestrator of brain development. Methods: In this study, timed-pregnant mice underwent chronic, unpredictable stress during the latter half of their pregnancies using ethologically relevant and/or mild stressors. Some of these mice received the antidepressant citalopram concomitantly in their drinking water. After birth, brain tissue serotonin levels at three postnatal developmentally relevant timepoints for serotonin system maturation were assayed in the offspring. A subset of the adult offspring was tested in behavioral assays to assess the long-term effects of in utero exposures. Finally, male adult offspring underwent microdialysis in the ventral hippocampus to investigate long-term neurochemical changes. Results: Offspring of stressed mothers had higher serotonin tissue levels and protein concentrations in the forebrain at postnatal day seven compared to control animals. Male adult offspring displayed greater anxiety-like behavior and stress responsiveness than sex-matched control animals. These effects were rescued in male mice whose mothers were exposed to concomitant citalopram. No changes were observed in basal or stimulated hippocampal serotonin levels during adulthood. However, male adults exposed to in utero stress had increased kappa opioid receptor agonist-induced serotonin release in the presence of serotonin transporter inhibition, which was attenuated by in utero exposure to citalopram. Conclusions: These findings suggest intergenerational benefits of treating maternal depression or anxiety during pregnancy particularly for imparting stress resilience in adult male offspring. Keywords: Maternal Stress, Prenatal Antidepressant Exposure, Ventral Hippocampus, Intergenerational Transmission of Stress, Stress Resilience and Susceptibility Disclosure: Nothing to disclose. P56. Aberrant Brain Function Reconfiguration During Naturalistic Fear Induction in Pediatric Anxiety Disorders: Novel Approach With Implications for Pathophysiological Mechanisms Rany Abend, Julia Linke, Jordan Galbraith, Grace Ringlein, Laura Jett, Song Qi, Anita Harrewijn, Andre Zugman, Anderson Winkler, Daniel Pine Reichman University, Herzliya, Israel Background: Anxiety disorders typically emerge during childhood and adolescence, are prevalent and impairing, and predict negative outcomes. As such, early intervention is of great clinical importance. While anxiety disorders consistently manifest as excessive fear responses in anticipation of threat, understanding of pathophysiological mechanisms is lacking, hindering treatment development for affected youth. Limited mechanistic insight is primarily due to challenges in experimentally evoking fear in children and the distributed nature of brain networks subserving fear responses. Naturalistic paradigms evoking fear states during functional imaging may provide an ecological way to examine fear mechanisms which is particularly suitable for children with anxiety. Graph theory is a computational approach generating metrics that can quantify distributed functional interactions across the brain during naturalistic paradigms. Combining naturalistic fear induction with graph theory approaches could constitute a novel method to identify pathophysiological mechanisms underlying excessive fear in children with anxiety disorders. Methods: 30 youths with anxiety disorders (AD; treatment-seeking, medication-free) and 34 healthy comparisons (HC) (aged 9-17 years, 38 females) completed two functional neuroimaging (fMRI) scans: resting-state scan (baseline) and one with an animated “scary” movie (fear induction). We recorded cognitive (nervousness ratings) and physiological (skin conductance) fear responses, and used a graph-theory approach based on functional connectivity to identify links between brain networks reconfiguration and pathological anxiety. Results: Increase in nervousness in anticipation of the movie, t(60)=3.06, p = 0.003, and decrease after it, t(60)=2.74, p = 0.008, confirmed fear induction. AD reported greater nervousness than HC before the movie, t(62)=4.19, p < 0.001, and demonstrated greater physiological responses during the movie, t(57)=2.25, p = 0.029, indicating successful elicitation of excessive fear responses. Imaging analyses indicated changes in brain network configuration from baseline to movie, p = 0.001. Importantly, a reduction in global between-network connectivity during the movie was noted in HC, V = 122, p = 0.02, but was absent in AD. This effect also manifested dimensionally with anxiety symptom severity, r(62)=-0.39, p = 0.002. Follow-up analyses indicated a reconfigured control-salience-default-mode network that was more strongly connected to attention, visual, and reward networks in AD relative to HC, all pFDRs<0.05. Conclusions: The movie successfully and robustly induced excessive cognitive and physiological fear responses in youth with anxiety disorders. The induced fear state was associated with aberrant reconfiguration of distributed circuitry that related to pathological anxiety, pointing to a putative pathophysiological mechanism. This encourages continued research on pathological mechanisms using naturalistic paradigms and a network perspective that captures aberrant patterns of distributed brain function. Keywords: Anxiety, Functional MRI (fMRI), Developmental Psychopathology, Graph-based Analysis Disclosure: Nothing to disclose. P57. PTSD Independently Associates With Major Adverse Cardiovascular Events: Analysis of a Large Biobank Sample Antonia Seligowski, Hadil Zureigat, Simran Grewal, Shady Abohashem, Michael Osborne, Ahmed Tawakol McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Posttraumatic stress disorder (PTSD) has repeatedly been observed to associate with an increased risk of major adverse cardiovascular events (MACE; e.g., myocardial infarction, stroke, heart failure), as well as MACE risk factors (e.g., elevated blood pressure). However, controversy exists as to whether this association survives adjustment for potential confounders, including socioeconomic and lifestyle factors. A National Institutes of Health-led working group identified numerous gaps in this literature and emphasized a need to evaluate the PTSD-MACE link further in large biobanks, as well as to characterize sex differences. Accordingly, the current study utilized a large healthcare biobank to determine whether PTSD diagnosis was associated with increased risk for MACE while controlling for important covariates. Further, we examined potential sex differences in this association. Methods: Participants included N = 118,995 adults who consented to participate in the Mass General Brigham Biobank. A subset of N = 49,795 completed a health behavior questionnaire. PTSD and MACE diagnoses, as well as covariates, were determined by the presence of corresponding ICD-10 codes. All analyses were conducted in SPSS version 24 using a significance level of p < .05. Covariates included socioeconomic (education, employment, income), lifestyle (exercise, smoking, obesity, alcohol use), and cardiovascular disease risk factors (hypertension, hyperlipidemia, diabetes). Results: PTSD diagnosis significantly associated with MACE (Odds ratio [95% confidence interval] = 2.04 [1.91, 2.18], p < .001, N = 102,395) after adjustment for cardiovascular risk factors. This effect remained significant after also controlling for socioeconomic factors (1.90 [1.68, 2.13], p < .001, N = 50,881), as well as lifestyle factors (1.81 [1.60, 2.04], p < .001, N = 49,795). A univariate ANOVA was used to test the main effects of PTSD and sex on MACE while controlling for cardiovascular risk factors. A significant main effect was observed for PTSD (F[1,102,477] = 23.74, p < .001), such that MACE risk was higher among those with versus without PTSD. A significant main effect was also observed for sex, such that MACE risk was higher in men compared to women (F[1,102,477] = 37.32, p < .001). The PTSD by sex interaction was not significant. Conclusions: Our results from a large healthcare biobank extend those of prior studies by showing that PTSD is a significant risk factor for MACE after adjusting for key confounders, including cardiovascular disease risk factors, lifestyle factors, and socioeconomic factors. We also demonstrated that MACE risk is greatest among men with PTSD, which is consistent with prior studies demonstrating that premenopausal women experience lower MACE risk than men (likely due to higher estradiol levels). Mechanistic studies are critical to determine the pathways by which men versus women with PTSD develop MACE risk and how sex hormones affect these pathways. Keywords: PTSD, Cardiovascular Disorders, Sex and Gender Disclosure: Nothing to disclose. P58. Mapping the Cell Type-Specific Regulome of PTSD in the Human Cortex Matthew Girgenti, Mario Skarica, Jing Zhang, Jiawei Wang, Hongyu Li, Dianne Cruz, Douglas Williamson, Hongyu Zhao, John Krystal Yale School of Medicine, New Haven, Connecticut, United States Background: Post-traumatic stress disorder is a multigenic disorder occurring in the aftermath of severe trauma exposure. Recent studies have begun to detail the molecular biology of the postmortem PTSD brain using bulk-tissue transcriptomic and epigenetic analyses. However, given the array of PTSD-perturbed molecular pathways identified thus far (e.g. glucocorticoid signaling, GABAergic transmission, and inflammatory signaling) it is unlikely that a single cell type is responsible. It is therefore necessary to uncover the individual cell type contributions to the molecular pathology of PTSD. Methods: We isolated ~1M nuclei from human postmortem dorsolateral prefrontal cortex (BA 9/46) from cases and controls for single nucleus RNA sequencing across three diagnostic cohorts: PTSD, MDD (Psychiatric control), and neurotypical controls to identify neuronal and non-neuronal cell type clusters and cell type-specific gene expression changes. We then performed paired sequencing of the same samples for ATAC-sequencing, to measure differential chromatin accessibility. We identified open genomic regions harboring risk alleles for PTSD and through integration of snRNA and snATAC we identified disease specific cis-regulatory elements. We used the most current and largest PTSD and MDD GWAS from the Million Veteran Program to identify cell types enriched for risk variants. We performed spatial transcriptomics on a subset of our cohorts to identify specific cortical lamina where PTSD gene expression changes are aggregating. Finally, we confirmed our findings using a combination of small molecule fluorescent in situ (FISH) and digital quantitative PCR. Results: We identified 67 distinct cell type clusters including neuronal and non-neuronal cell types. We identified over 800 FDR significant differentially expressed genes across many cell types and confirmed expression changes of several genes implicated in PTSD pathophysiology by FISH and digital PCR. We found PTSD specific cis-regulatory elements for several genes including ELFN1, FKBP5, and SGK1. Integration of large GWAS data with our snATAC dataset showed enrichment of variants for PTSD, MDD and other quantitative clinical phenotypes in excitatory and inhibitory neurons and genome-wide spatial transcriptomics confirmed alterations of GABAergic signaling transcripts in specific layers of the DLPFC. Conclusions: Taken together, this work is the first step in the creation of a cell type-specific atlas of stress disorders. Applying functional genomic approaches to characterize risk alleles within specific cell types will help determine which processes are most impacted by stress. These findings provide a global picture of the cell type-specific molecular regulatory mechanisms that govern stress effects on the human cortex and provides a blueprint for integrating single cell type genomic data to characterize the molecular landscape of other brain regions implicated in traumatic stress. Keywords: PTSD, MDD, Genomics, Single Cell Omics Disclosure: Nothing to disclose. P59. An Integrated Multi-Omics Analysis of PTSD Across Brain Regions and Cell Types Vena Martinez, Jiawei Wang, Hongyu Li, Dianne Cruz, Douglas Williamson, Hongyu Zhao, Ronald Duman, John Krystal, Matthew Girgenti Yale University, New Haven, Connecticut, United States Background: The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating many biological processes across distinct cell types. Complex psychiatric disorders such as PTSD result from differences at various levels of regulation (epigenomic, transcriptomic, and proteomic) and converge on specific biological pathways with clinical significance. We are beginning to understand more about the neurobiology of PTSD and the molecular mechanisms associated with it, however, many critical questions still remain. Methods: Here we generated a large multi-omic, postmortem brain dataset of PTSD donors compared to neurotypical controls and a psychiatric control group (major depressive disorder) that includes RNA expression, DNA methylation, and protein expression from the dorsolateral prefrontal cortex, amygdala, and the hippocampus. Single nucleus RNA-seq data was generated to infer cell-type-specific expression. Results: Mutli-omics integration identified pathways, including glucocorticoid signaling, GABAergic transmission, and inflammation as differentially enriched in PTSD and MDD. At the RNA and methylation level there was an aggregation of differential expression in specific PTSD risk loci including CRHR1, ELFN1, and MAD1L1. Conclusions: This approach highlights unique genomic characteristics of both disorders and demonstrates how convergent pathways across molecular modalities contribute to stress-associated etiology. Keywords: PTSD, GWAS, ATAC-seq, RNAseq Disclosure: Nothing to disclose. P60. Epigenome-Wide Markers and Mechanisms of Disease Risk and Psychotherapy Response in Anxiety Disorders Katharina Domschke, Miriam Schiele, Jan Lipovsek, André Pittig, Benjamin Straube, Jan Richter, Ulrike Lüken, Andreas Ströhle, Elisabeth Binder, Tilo Kircher, Alfons Hamm, Volker Arolt, Hans-Ulrich Wittchen, Angelika Erhardt, Anna Köttgen, Jürgen Deckert University of Freiburg, Freiburg, Germany Background: Within the vulnerability-stress pathogenetic model of mental disorders, epigenetic mechanisms such as DNA methylation have been suggested to play a dynamic role at the interface between the genetic and the environmental level. Here, genome-wide DNA methylation patterns were investigated as disease markers of anxiety disorders and as potential predictors and/or mechanisms of response to psychotherapy. Methods: A longitudinal epigenome-wide association study (Illumina MethylationEPIC BeadChip) was performed at baseline (BL), post-treatment (POST) and 6-month follow-up (FU) in the so far largest sample of patients with anxiety disorders (N = 415, f = 238; mean age±SD: 33.29 ± 11.3 years; panic disorder with or without agoraphobia: N = 257; social anxiety disorder: N = 129; multiple specific phobias: N = 29) treated with a standardized exposure-based cognitive behavioral therapy (CBT), and in 317 healthy controls. Results: In the panic disorder subsample as compared to healthy controls, significant epigenome-wide association was discerned for altered DNA methylation at 256 CpGs. In the overall sample of patients with anxiety disorders, suggestive evidence was found for altered DNA methylation at ten and two CpG sites, respectively, to predict treatment response at POST and FU. Suggestive evidence for epigenome-wide DNA methylation changes along with CBT response in anxiety disorders was observed at 13 CpGs from BL to POST and at nine CpGs from BL to FU. Several hits map to genes previously implicated in anxiety-, learning- or plasticity-related processes. Conclusions: The identification of epigenetic biomarkers of anxiety disorders may aid in developing resilience-increasing preventive measures. The definition of epigenetic signatures as predictors or core mechanisms of action of exposure-based interventions is hoped to contribute to the development of more targeted, personalized treatments of anxiety disorders. Keywords: EWAS, Epigenetic biomarkers, DNA Methylation, Psychotherapy, Precision Medicine for Mood and Anxiety Disorders Disclosure: Janssen Inc.: Advisory Board (Self) P61. Changes in Basolateral Amygdala Activity Correlate With Distinct Innate Defensive Behaviors During Predator Odor Exposure in Male and Female Mice Amanda Morgan, Veronika Kondev, Sachin Patel Northwestern University, Chicago, Illinois, United States Background: Life-threatening traumatic experiences can have lasting negative consequences, many of which involve the development of maladaptive pathologies. A complex relationship exists between traumatic stress events and chronic neuropsychiatric conditions, including PTSD, anxiety, and depression. One translational model of life-threatening trauma in rodents uses exposure to a predator odor (2MT) to elicit fear behavior in mice. In both rodents and humans, the neural substrates underlying traumatic experiences are multifaceted, with the basolateral amygdala (BLA) heavily involved in trauma and stress processing. Methods: We examined in vivo BLA neural activity during predator odor exposure by measuring calcium transients with fiber photometry in male and female mice. Results: We have previously shown 2MT increases amygdalar endocannabinoid levels (2-AG), and here we report systemic pharmacological manipulation of endocannabinoid modulates BLA activity during predator odor exposure. Conclusions: Different ways of responding to trauma correlate directly with different patterns of activity in the BLA, and these effects are mediated by the endocannabinoid system. Keywords: Amygdala, Acute Traumatic Stress, Endocannabinoids Disclosure: Nothing to disclose. P62. Using the Genetically Encoded Sensor iSeroSnFR2.0 to Measure Serotonin Release During Fear and Stress Behaviors in Free Moving Mice Emily Wright, Rochelin Dalangin, Sally Ho, Lin Tian University of California, Davis, Davis, California, United States Background: We developed a novel serotonin biosensor, iSeroSnFR2.0, which has improved sensitivity and a lower dissociation constant (Kd) compared to iSeroSnFR1.0, thus granting increased ability to measure serotonin concentrations on a subsecond basis across multiple trials using fiber-photometry. Here we used this powerful tool to record serotonin release dynamics during two anxiogenic conditions. We recorded from bed nucleus of the stria terminalis (BNST) and orbitofrontal cortex (OFC) during fear learning, because these regions are innervated by distinct populations of serotonergic neurons in the dorsal raphe. We also investigate the impact of chronic social defeat on serotonergic transients. By measuring serotonin release across chronic stress conditions as well as acute, we aim to gain a more complete picture of how exposure to adverse stimulus modulates serotonin release. Methods: Experiment 1: All recording was done via fiber photometry using either iSeroSnFR1.0 or iSeroSnFR2.0 expressed in BNST or OFC of adult male mice. Mice then underwent a 3-day fear conditioning paradigm. During the first day of testing mice were exposed to 5 tone trials, followed by 10 tone/shock trials. On the second day of testing mice were exposed to 15 tone/shock trials. On the third day of testing mice were exposed to 5 tone/shock trials, followed by 10 tone trials. n = 7-9 per group. Experiment 2: All recording was done via fiber photometry using iSeroSnFR2.0 expressed in BNST of adult male mice. Mice underwent 10 consecutive days of chronic social defeat (or control handling) with 10 different CD1 stressor mice. 24 hours after the last day of defeat mice were tested in a caged social interaction test. During this test mice spent 3 minutes in an empty neutral arena, 3 minutes with an empty wire cage placed into the arena, and 3 minutes with a caged, novel CD1 aggressor placed into the arena. Two days later mice were tested again, this time in an uncaged social interaction test. During this test mice spent 3 minutes in an empty neutral arena, 3 minutes with a novel same strain male, and 3 minutes with a novel CD1 stressor male. Control n = 2, stressed n = 5. Statistical analysis: Raw photometry data was down sampled 100x. The isosbestic channel was then fitted to a biexponential curve and that curve was subtracted from the signal to correct for bleaching. ΔF/F% was calculated as [100∗(465 signal - fitted signal) / fitted signal)] and those results were then z-scored. Data was then averaged across trials and animals. Area under the curve analyses (AUC) were conducted from the start of tone to immediately prior to the onset of shock. The AUC was calculated as the sum of the area below the mean trace and above y = 0. Parametric t-tests were used to assess for significant differences between AUC values. I also include preliminary data of machine learning tracking of mice during caged and uncaged social interaction testing using deeplabcut. Use of the position estimation data to track animal behavior and correlate with photometry data is in-progress. Results: Experiment 1: AUC analysis shows significantly less response to tone-only trials in trials before shock exposure than in trials comprised of a tone + shock, or in trial-only after previous shock exposure with both iSeroSnFR1.0 BNST (p < 0.01), iSeroSnFR1.0 OFC (p < 0.01), iSeroSnFR2.0 BNST (p < 0.01), and iSeroSnFR2.0 OFC (p < 0.01). In the beginning tone/shock trials during day 1, we found that serotonin is released into BNST/OFC after the mouse has received the foot shock, but as the trials progress the serotonin influx becomes cued by the onset of the tone instead. This pattern is most strongly apparent in mice injected with iSeroSNFR2.0. Experiment 2: For caged social interaction: All test mice had no significant response to time spent in the empty arena or placement of an empty cage. All test mice had increased serotonin release into the BNST in response to the placement of the caged CD1 aggressor into the arena, with no significant difference between groups. For uncaged social interaction: There were no statistical difference in significant response for time spent in the empty arena or at the start of an uncaged interaction with a novel C57 male. Stressed males showed an increase of serotonin into BNST at the start of an uncaged interaction with a novel CD1 stressor male that is not seen in unstressed males (AUC p < 0.05). Conclusions: While the firing patterns of serotonergic neurons have been described, other neuroimaging techniques did not allow for measurement serotonin release from spatially displaced terminals with high degree of temporal specificy. Here my work demonstrates that iSeroSnFR2.0 is able to track trial-specific subsecond changes of endogenous serotonin release. These results indicate that the increased sensitivity to lower volumes of serotonin that is achieved by iSeroSNFR2.0 allows for the collection of a completer and more accurate picture of serotonin release dynamics than is possible with iSeroSNFR1.0. Results from both fear conditioning and chronic social stress show subsecond serotonin release temporarily and rapidly tracks fear state and anticipates punishment. Indicating serotonin may play a critical role in preventing risky behavior or promote rapid withdrawal from an adverse situation. Future directions will include acute vs. chronic SSRI administration prior to fear conditioning or chronic social stress. Keywords: Serotonin, Fiber Photometry, Optical Biosensors, Acute and Chronic Stress Disclosure: Nothing to disclose. P63. Fatty Acid Amide Hydrolase Levels in Social Anxiety Disorder: Preliminary Findings From a PET Study Using [11C]CURB Mashal Ahmed, Christina F. Pereira, Laura M. Best, Rachel F. Tyndale, Dafna Rubin-Kahana, Bernard Le Foll, Isabelle Boileau, Stefan Kloiber* Centre for Addiction and Mental Health, University of Toronto, Toronto, Canada Background: Social anxiety disorder (SAD) is one of the most common psychiatric illnesses in the world. Despite ongoing clinical efforts response rates to conventional pharmacotherapies remain low, constituting a need for the investigation of new neurobiological mechanisms. Preclinical evidence suggests that deficient signalling of the major endocannabinoid neurotransmitter, anandamide, through upregulated activity of its catabolic enzyme, fatty acid amide hydrolase (FAAH), may be associated with the pathophysiology of anxiety-spectrum disorders. However, there are no clinical in-vivo studies investigating brain FAAH status in individuals with SAD. The aim of this study was to determine whether whole brain FAAH levels are elevated in individuals with SAD compared to healthy controls (HC) using positron emission tomography (PET) imaging with the novel FAAH radioligand, [11C]CURB. Methods: Sixteen participants meeting DSM-5 diagnostic criteria for SAD completed PET imaging with arterial blood sampling. [11C]CURB λk3, an index of brain FAAH levels, was investigated in while brain and 9 regions of interest (ROI) using a repeated-measures ANCOVA, controlling for genetic variability known to affect [11C]CURB binding (FAAH rs324420 C > A). SAD symptom severity was assessed using the Liebowitz Social Anxiety Scale (LSAS). Results: Individuals with SAD (n = 16; F/M = 13/3; 26.88 ± 5.78 years; FAAH rs324420: 11 CC, 4 AC, 1 AA) demonstrated 8.78% higher whole brain [11C]CURB λk3 (F(1,59)=5.190, p = 0.026) compared to HC (n = 46; F/M = 24/22; 27.17 ± 8.47 years old; FAAH rs324420: 32 CC, 12 AC, 2 AA). A significant Group[2] x ROIs[9] interaction was also observed (F(5.023,296.36)=2.52; p = 0.029). Post-hoc LSD pairwise comparisons revealed that [11C]CURB λk3 was significantly elevated in 3 of 9 ROIs (p < 0.05), (hippocampus, ventral striatum, dorsal striatum). A partial correlation, controlling for FAAH genotype revealed that whole brain [11C]CURB λk3 was not correlated with total LSAS score or any LSAS sub-scores. Conclusions: In line with our hypothesis and preclinical evidence, our preliminary findings suggest that whole brain FAAH levels are significantly elevated in individuals with SAD compared to HCs, as inferred from [11C]CURB binding. The mechanism behind enzymatic activity of FAAH in SAD is unknown. However, initial results from this pilot study support up-regulated FAAH activity as a potential neurobiological mechanism in SAD and may inform future development and research of FAAH-targeted interventions. Keywords: Social Anxiety Disorder, Endocannabinoid System, PET Imaging, FAAH Disclosure: Empowerpharm: Consultant (Self) P64. 7T Functional Magnetic Resonance Spectroscopy of Glutamate Variations During Working Memory in PTSD Meredith Reid, Sarah Whiteman, Thomas Denney, Abigail Camden, Stephanie Jeffirs, Frank Weathers Auburn University, Auburn, Alabama, United States Background: People with PTSD commonly report difficulties with working memory, yet the neural basis of its dysfunction is not well understood. Previous fMRI studies of PTSD have shown reduced activation in the dorsolateral prefrontal cortex (DLPFC), a crucial brain region for working memory. Converging evidence from animal models and human studies points to glutamatergic dysfunction in key brain regions in PTSD. It is possible that glutamatergic abnormalities could underlie the differential activation patterns observed in the DLPFC; however, this has not been directly tested. Functional magnetic resonance spectroscopy (fMRS) can potentially address this issue by measuring glutamate changes associated with neural activity in response to stimuli. Moreover, there is a lack of MRS studies of the DLPFC in PTSD. Therefore, in this study, we used 7T resting-state MRS as well as fMRS during the N-back working memory task to measure glutamatergic changes in the DLPFC of people with PTSD, trauma-exposed people without PTSD, and people without trauma. We hypothesized that (1) glutamate would be reduced in people with PTSD, (2) glutamate would increase with working memory load, and (3) the glutamate-working memory relationship would be altered in PTSD. Methods: Participants completed the Life Events Checklist (LEC-5 extended version) and the PTSD Checklist for DSM-5 (PCL-5). This study included 26 people with PTSD (age: 28.2 + /- 8.3; sex: 20 F / 6 M), 21 trauma-exposed people without PTSD (TE; age: 29.6 + /- 9.0; sex: 10 F / 11 M), and 24 people without trauma (NT; age: 29.6 + /- 11.2; sex: 15 F / 9 M). Imaging was performed at the Auburn University MRI Research Center on a Siemens MAGNETOM 7T MRI scanner equipped with a 32-channel head coil. Structural images were obtained for voxel placement. Spectra were acquired from the left DLPFC (25 x 25 x 25 mm) using an ultra-short TE STEAM sequence (TR/TE/TM = 10,000/5/45 ms), FASTESTMAP shimming, and VAPOR water suppression. For resting-state MRS, 32 averages with water suppression and 4 averages without water suppression were acquired while participants fixated on a crosshair. During fMRS, participants completed the N-back task. Stimuli were single letters presented one at a time. The task included 9 alternating blocks of 0-back, 1-back, and 2-back conditions (3 blocks of each working memory load). For fMRS, 8 water-suppressed averages were acquired during each block, and the spectra were combined in MATLAB. MRS and fMRS spectra were analyzed in LCModel using a simulated basis set. Spectra were eddy current corrected and quantified using the unsuppressed water signal. MRS levels of glutamate were compared between groups using one-way ANCOVA controlling for age and sex. A linear mixed effects model controlling for age and sex was used to examine the effects of group, condition, and groupcondition interaction on fMRS levels of glutamate. Results: For resting-state MRS, we observed a significant effect of group (F(2,65) = 5.20, p = 0.0008). Specifically, glutamate was significantly lower in PTSD vs. NT (p(Tukey) = 0.023) and in TE vs. NT (p(Tukey) = 0.019). For fMRS, the effect of condition and the groupcondition interaction were not significant. However, we observed a significant effect of group (p < 0.001). Specifically, glutamate was lower across all conditions in PTSD vs. NT (t = 3.79, p < 0.001) and in TE vs. NT (t = 2.95, p = 0.005). The percentage change in glutamate was calculated relative to the MRS passive fixation and was largest for NT (5.0-5.9%) compared to PTSD (2.5-2.7%) and TE (1.5-2.2%). Conclusions: We observed (1) lower glutamate in trauma-exposed people with and without PTSD compared to people without trauma and (2) increased glutamate during the task relative to passive visual fixation. Our findings are consistent with prior studies reporting lower glutamate in the anterior cingulate cortex of people with PTSD and add to the growing evidence for glutamatergic abnormalities associated with traumatic stress. In future work, we will examine the association between glutamate and brain activation from fMRI. Since fMRS and fMRI probe different aspects of neuronal firing and synaptic activity, the combined approach of these techniques could better characterize the neurobiological basis of working memory deficits in PTSD. Keywords: PTSD, Functional Magnetic Resonance Spectroscopy, Glutamate, Working Memory Disclosure: Nothing to disclose. P65. Lesion Network Localization of Neuromodulation Targets for PTSD in Veterans Shan Siddiqi, Noah Philip, Stephan Palm, Heather Bouchard, Jennifer Barredo, Jordan Grafman, Rajendra Morey, Michael Fox Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts, United States Background: The optimal transcranial magnetic stimulation (TMS) target for posttraumatic stress disorder (PTSD) remains unknown, and clinical trials have yielded mixed results. Connectivity of symptom-causing lesions have been shown to predict efficacy of neuromodulation targets for the same symptoms, including depression, motor parkinsonism, cognitive symptoms of Parkinson’s disease, and tics. PTSD risk has been shown to be reduced after amygdala lesions, but the amygdala is too deep to access directly with TMS. Thus, we used brain lesions to map a circuit-based TMS target for PTSD in Veterans. We analyzed three datasets to derive a circuit-based TMS target that is specific to PTSD in military Veterans. Methods: First, in Veterans who sustained penetrating traumatic brain injury (n = 193), we tested whether PTSD risk was reduced after damage to specific brain regions (using voxel lesion symptom mapping) or specific brain circuitry (using lesion network mapping). We used split-half cross-validation to confirm significance. Second, we tested whether our “PTSD circuit” derived from brain lesions was abnormal in Veterans without brain lesions (n = 180) using resting-state functional connectivity (rsFC). Finally, we tested whether connectivity changes in this circuit were associated with improvement in PTSD symptoms following TMS (n = 10 active, 10 sham). We controlled for depression and tested for circuit specificity in all analyses. Results: Lesion locations that reduce PTSD risk map to a common brain circuit that includes the medial prefrontal cortex (mPFC), amygdala, and anterior temporal lobe. Lesion network maps derived from half the patients predicted PTSD status in the other half (p = 0.01). This result survived even when excluding all lesions that overlap with the amygdala (p < 0.05), demonstrating that this is a circuit-level phenomenon. Connectivity within this circuit, but not other circuits, was associated with PTSD diagnosis in patients without brain lesions (p < 0.01) and with PTSD improvement following TMS (p < 0.05). Conclusions: Lesion locations that protect against PTSD map to a common brain circuit. This circuit is abnormal in PTSD patients without brain lesions, associated with TMS-induced improvement in PTSD severity, and may serve as a neuromodulation target for Veterans with PTSD symptoms. Keywords: PTSD, TMS, Circuitry-Based Approach, lesion Disclosure: Magnus Medical: Consultant (Self), Brainsway Inc: Stock / Equity (Self) P66. Effect of Glucocorticoid Treatment During Acute Infectious Illness on Hippocampal Function in Survivors Alice Hill, Huzefa Khalil, Klaudia Laborc, Savannah Kounelis-Wuillaume, Colin Johnston, Isaac Agranoff, Swapnil Gavade, Benjamin Singer, Huda Akil, Joanna Spencer-Segal* University of Michigan, Ann Arbor, Michigan, United States Background: Neuropsychiatric symptoms afflict half of survivors of critical illnesses leading to substantial public health burden. Glucocorticoids are commonly administered to acutely ill patients for their cardiovascular and anti-inflammatory properties, but they also influence brain outcomes. Intriguingly, observational and randomized clinical trials show that glucocorticoid treatment during critical illness decreases the risk of post-traumatic stress disorder (PTSD) in survivors, but the mechanisms are unknown. We hypothesized that glucocorticoid treatment during illness might influence hippocampal-dependent emotional and/or cognitive processes in survivors. Methods: We used cecal ligation and puncture (CLP) to induce systemic infection in male and female mice (N = 80 divided equally among groups) and treated them with corticosterone (16 mg/kg) or vehicle during the acute illness (Days 1-5). Illness severity was measured using locomotion and daily weights. Beginning on Day 14, we studied affective behavior and neuroendocrine function. The Open Field and Elevated Plus Maze were used to measure negative affect, while the Object Recognition test was used to measure memory. Basal and stress-induced (forced swim) ACTH and corticosterone were measured from plasma using an ELISA, and systemic inflammation was measured using splenic cell counts by flow cytometry. Dorsal and ventral hippocampi were dissected from half the mice and RNA was isolated and prepared for paired-end 50 bp RNA Sequencing, which was performed on an Illumina NovaSeq S4. Results: Corticosterone prevented CLP-induced weight loss without significantly affecting sickness-induced locomotor behavior or overall survival. After a 14-day recovery, male and female CLP survivor mice showed persistent negative affect based on decreased exploration of open areas in the Open Field and Elevated Plus Maze, and this was not rescued by corticosterone treatment. On the other hand, corticosterone treatment during illness impaired survivors’ performance on the Object Recognition test. CLP survivor mice showed evidence of enhanced central hypothalamic-pituitary-adrenal (HPA) axis activity based on elevated basal ACTH levels, but corticosterone treatment during illness had no effect on HPA axis function in survivors. There was also evidence of persistent systemic inflammation in CLP survivors based on splenic cell counts that was not rescued by corticosterone treatment. RNA sequencing showed distinct neural signatures of CLP and corticosterone treatment. 103 genes were differentially expressed in CLP survivors and 2076 genes were differentially expressed after corticosterone treatment, with only 6 overlapping genes. CLP survivors showed persistent upregulation of inflammation- and immunity-related genes, which were not altered by corticosterone treatment. Correlation of behavior in the Elevated Plus Maze with gene expression revealed a set of 126 genes in the ventral hippocampus that related to affective behavior after CLP, most of them involved in neuroendocrine signaling rather than inflammatory or immune processes. Conclusions: In summary, murine survivors of CLP-induced acute infectious illness showed persistent negative affect and heightened central HPA axis activation. While persistent systemic and neuro-inflammation was seen in CLP survivors, correlation of gene expression with behavior related altered neuroendocrine signaling in the ventral hippocampus, rather than neuroinflammation, to affect in CLP survivors. Corticosterone treatment during illness did not rescue negative affect in survivors, but it impaired object memory and altered the expression of 10% of the detectable transcripts in the dorsal and ventral hippocampus. These findings provide the first separation of mechanisms related to affective and cognitive impairments after acute illness. The data suggest that neuroendocrine signaling in the ventral hippocampus is important for emotional outcomes while highlighting a role for glucocorticoids in cognitive outcomes. Future work will pursue the resulting hypothesis that glucocorticoids may alter the risk for PTSD by altering hippocampal-dependent memory processes. Keywords: Glucocorticoids, Hippocampus, Infection Disclosure: Camurus AB: Contracted Research (Self), Recordati Rare Diseases: Advisory Board (Self), Chiasma Inc.: Advisory Board (Self) P67. The Prediction of Long-Term PTSD Symptom Development in Military Personnel: Applying Machine Learning to Pre-Deployment Risk Factors Sija van der Wal, Elbert Geuze, Livia Dominicus, Edwin van Dellen, Remko van Lutterveld, Eric Vermetten Ministry of Defence, The Netherlands, Utrecht, Netherlands Background: Active-duty army personnel are frequently exposed to traumatic events during deployments, yet only a minority of them develop mental health disorders such as posttraumatic stress disorder (PTSD). Why some are at increased risk for developing PTSD after deployment is still not fully understood. A large amount of literature has been published on the identification of risk factors for PTSD, but have not yet led to the development of effective pre-deployment screening tools. Machine learning might be a promising approach for developing better prediction models. Methods: The present study utilized a random forest method to predict the development of PTSD symptoms up to ten years after deployment in the PRISMO cohort of Dutch Afghanistan veterans (N = 963). The dataset consisted of both psychological and biological pre-deployment variables. Results: The predictive model had a performance well above chance (AUC = 0.71, sensitivity = 0.63, specificity = 0.69). Among the top five highest-ranked predictive features were self-reported symptoms (depression, anxiety and distrust and personal sensitivity) and lab markers (vasopressin and DEX-sensitivity). A random forest model using a dataset with only psychological predictors performed as well as the random forest model based on both psychological and biological information. Conclusions: The results suggest that a random forest approach can be effective in the identification of important predictive markers to define novel risk mitigation interventions. As the model performance in the present study was modest and no external validation could be performed, more research is needed to increase the usability for pre-deployment screening. Keywords: PTSD, Machine Learning, PTSD, Prediction, Causal Modeling, Trajectory Modeling, Cortisol Disclosure: Nothing to disclose. P68. Evaluating if CRP Contributes to Risk for Developing PTSD-Like Behaviors in Trauma-Exposed Mice Samantha Friend, Sorana Caldwell, Rahul Nachnani, H. Kirk Hammond, Victoria Risbrough VA San Diego Healthcare System, San Diego, California, United States Background: Increasing evidence suggests inflammation plays a role in psychiatric disorders caused by trauma exposure. Studies suggest post-traumatic stress disorder (PTSD) is associated with altered serum C-reactive protein (CRP) and CRP gene mutations. We examined the potential causal role of CRP in mouse models for PTSD, hypothesizing that CRP expression may confer a higher risk for PTSD-like behavior. Methods: Avoidance and conditioned fear processes were tested in CRP null male and female C57BL6J mice. Male wild-type C57BL6J mice received an intra-jugular injection of 10^11 genome copies of AAV8.CRP or AAV8.Null and four weeks later were tested in the predator stress model for PTSD which assesses enduring avoidance behavior and trauma-specific fear responses 1-2 weeks after exposure to a feline predator. Results: CRP null female mice have at baseline enhanced recall fear extinction (FCRP = 1.794, n = 15, p = 0.0053). Despite near three-fold protein level increases (17.63 pg/mL for AAV8.CRP vs. 6.34 pg/mL for AAV8.null), male mice with AAV8.CRP overexpression did not confer a higher risk for PTSD avoidance-like behaviors or alter cued fear extinction in males after predator stress. Conclusions: Loss of CRP signaling supports increased fear extinction in females but not males. Males also do not show CRP overexpression effects, suggesting females may be more susceptible to CRP effects on PTSD-relevant behaviors. Future studies will examine how constitutive CRP expression contributes to fear behaviors after trauma, as well as how CRP overexpression contributes to trauma effects in female mice. Studies are ongoing regarding how CRP alters peripheral and central immune responses after trauma. Keywords: C-Reactive Protein, Fear Conditioning and Extinction, Neuroimmunology, Stress and Anxiety Behavior Disclosure: Nothing to disclose. P69. Gut Feelings: Connectome-Microbiome Dynamics Associated With Anxiety Paul Thomas, Beatriz Penalver, Suzanne Alvernaz, Jun Ma, Olusola Ajilore* University of Illinois at Chicago, Chicago, Illinois, United States Background: There has been an increasing recognition of the role of the gut-brain-axis in internalizing psychopathologies. The present study explored dynamic connectomes by formulating a joint analysis of brain/microbiome networks in participants with depression and obesity to understand post-intervention coupled temporal changes. Methods: Participants were from a study evaluating the effectiveness of specialized behavioral therapy over 2 months. A subset (n = 26, 8:usual care, 18:intervention) with gut microbiome/neuroimaging data at baseline and 2 months were included in our analyses. For each subject, a brain and individual sample microbiome network at both time points were derived and represented as tensors. Joint tensor factorization was used to couple the decomposition of the tensors at the subject mode to identify shared variation in both brain/microbiome networks, and subject factors derived from the analysis were used to relate clinical scales to network changes. Results: One subject factor was significantly correlated with GAD-7 decreases (rho=0.547, p = 0.007, q = 0.045). The associated brain network factor consisted of negative connections between limbic/default mode network regions. For the microbiome network factor, the predominant connectivity was from the Bacteroidaceae/Lachnospiraceae families. Additionally, we found that the coupled factorization provided the best clustering of participants, with 96% accuracy, followed by independent taxa (85%) and brain network (69%) factorizations. Conclusions: Coupled tensor decomposition can be used to identify shared temporal changes in the brain/microbiome networks of participants undergoing treatment for comorbid obesity and depression, suggesting that incorporating heterogeneous data fusion uniquely reveals salient network features. Keywords: Gut Microbiome, Brain Connectome, Depression, Obesity, Anxiety Disclosure: KeyWise AI: Founder (Self), Embodied Labs, Blueprint, Milken Institute, Sage Therapeutics: Advisory Board (Self), P70. A Neurosurgery Case Series on the Role of the Amygdala and Temporal Pole in Development and Maintenance of PTSD Sanne van Rooij, Sean Minton, Timothy Ely, Cecilia Hinojosa, Nathaniel Harnett, Abigail Powers, Tanja Jovanovic, Daniel Drane, Kelly Bijanki, Jon Willie Emory University School of Medicine, Atlanta, Georgia, United States Background: A better understanding of the neurobiology of posttraumatic stress disorder (PTSD) could reveal novel neuromodulation targets and improve treatment outcomes. One such target could be the amygdala, a key region in threat neurocircuitry. An overreactive right (R) amygdala in combination with lower ventromedial prefrontal cortex (vmPFC) control have been related to PTSD. Moreover, brain injury inclusive of the amygdala has been shown to be protective against the development of PTSD; however, due to its invasive nature, neurosurgical interventions have not been studied for PTSD. Methods: This unique case series reports prospective data of 10 epilepsy patients who underwent unilateral ablation for their treatment-refractory epilepsy. The data are presented as a case series because the differential nature of the ablations does not allow for group-level analyses. Before and >6 months after surgery PTSD symptoms were assessed with the Clinician Administered PTSD Scale (CAPS) for DSM-5, and functional MRI scans were collected at both timepoints to assess threat reactivity and functional connectivity (for N = 5). Neuroimaging analyses were performed in native space using Statistical Parametric Mapping 12 and the CONN Toolbox. Post-ablation scans were co-registered to pre-ablation scans of the same modality to assure all scans were in the same native space. Regions of interest (ROIs) were the L amygdala (CITI168 probabilistic atlas) and the vmPFC (WFU PickAtlas, BA25) and first created in standard MNI space. The pre-ablation structural image was normalized to MNI space and the invert normalization parameters were used to convert ROIs to native space. Mean ROIs were extracted using the REX toolbox. The mean difference for the contrast value for fearful vs neutral faces was calculated as a measure for threat reactivity. The CONN toolbox was used to calculate amygdala-vmPFC functional connectivity (FC) during threat reactivity. Results: Five patients met for PTSD before or after ablation. Patients (Pt) 1 and 2 no longer met for PTSD after R amygdala ablation. Pt 2 showed a pre- to post-treatment decrease in left (L) amygdala threat reactivity (-0.27), an increase in vmPFC reactivity (+0.14) and an increase in vmPFC-amygdala FC ( + 0.29). Pt 3 did not recover from PTSD after R amygdala + R temporal pole ablation. Additionally, she developed suicidal ideation post-surgery. She showed a minimal change in threat reactivity (-0.07 for the L amygdala and +0.04 for the vmPFC) and a decrease in vmPFC-amygdala FC (-0.36). Pt 4 was trauma exposed with no PTSD, but developed PTSD after R amygdala + temporal pole ablation. Pt 5 underwent prior surgery sparing her R amygdala. A car accident resulted in recurrence of seizures and development of PTSD. She received R amygdala open resection after which her PTSD ameliorated, but seizures continued. Five non-PTSD epilepsy patients (MRI data for N = 3) who underwent L amygdala ablations were evaluated as controls. Conclusions: This case series support the R amygdala as critical for maintenance of PTSD symptoms, bolstering earlier work on the importance of the amygdala in PTSD treatment non-response, and supporting the R amygdala as a key neuromodulation target. The findings also suggest R temporal pole ablation is related to poor PTSD outcomes, following another case report and findings of lower activation and size in PTSD, suggesting the temporal pole is an important brain region for further study. Keywords: Amygdala Ablation, Post Traumatic Stress Disorder, Epilepsy, Temporal Pole, Threat Reactivity Disclosure: Nothing to disclose. P71. Treating Transdiagnostic Dysphoria Using a Sequence of Virtual Reality and Two Accelerated Transcranial Magnetic Stimulation Protocols F. Andrew Kozel, Megan Senda, Isabelle Taylor, Mariah Jensen, Kevin Johnson Florida State University, Tallahassee, Florida, United States Background: Dysphoria is a transdiagnostic symptom of unease or dissatisfaction experienced across a range of diagnoses, including mood disorders and pain. Dysphoria may have underlying commonalities in mechanisms, and thus treatments targeting dysphoria may have broad impact. Traditionally, studies have focused on narrow diagnosis-specific inclusion criteria, while excluding co-morbid diagnosis. In clinical practice, co-morbid conditions involving dysphoria are quite common (e.g. depression and pain), so a transdiagnostic approach may better reflect clinical reality. The goal of this project was to pilot a sequenced approach for rapidly treating dysphoria. In the first step (“Phase 1”), we sought to determine the feasibility and effectiveness of Virtual Reality (VR) treatment sessions. In subsequent steps as indicated, we sought to determine the effectiveness, tolerability, and feasibility of Accelerated Transcranial Magnetic Stimulation (accel-TMS) for dysphoria starting with sessions stimulating the left dorsolateral prefrontal cortex (“Phase 2A”), sequentially followed by sessions stimulating the dorsomedial cortex (“Phase 2B”). Methods: Participants were age 18 years and above, recruited from the community. Inclusion criteria for dysphoria was depressive (PHQ-9 ≥ 10), anxiety (GAD-7 ≥ 10), PTSD (PCL-5 ≥ 45), or chronic pain (Average Pain Intensity ≥ 4/10 for > 3 months) symptoms. We operationally defined "response" as a 30% improvement on the primary symptom scale, with efficacy separately assessed by clinician-rated scales. Participants were excluded for medical contraindications to TMS and other major psychiatric diagnoses. Phase 1 was a series of VR mindfulness exercises, twice daily for 10 days. Those who did not respond to VR treatments went on to Phase 2A of accel-TMS (five sessions a day with 50 minutes breaks, for 5 days, totaling 25 sessions). Phase 2A involved stimulation of left dorsolateral prefrontal cortex (modified Beam F3), 110% hand motor threshold, 1800 pulses of iTBS. At one-week post treatment, participants with a response continued for 5 more sessions over 5 weeks. Participants advanced to Phase 2B treatments if they failed to respond to Phase 2A or if they remained symptomatic after Phase 2A as defined by inclusion criteria. Phase 2B was accel-TMS (five sessions a day with 50 minutes breaks, for 5 days, totaling 25 sessions), plus 5 more sessions over 5 weeks. Phase 2B involved stimulation of dorsomedial prefrontal cortex (midline, 25% of nasion-inion distance, from nasion), 110% foot motor threshold, 1800 pulses of theta-burst primed 10 Hz (600 pulses of iTBS followed by 1200 pulses of 10 Hz). Results: This interim report is based on 20 participants enrolled, with 1 screen failure and 19 initiated for Phase 1. For Phase 1 (VR mindfulness), 10 participants completed the full 10 days, 3 participants completed 5 days, and 6 participants discontinued before 5 days. Reasons for dropping out of VR were no change in symptoms (n = 9), did not enjoy VR (n = 6), motion sickness (n = 1), and pain precluded continuing coming to lab for VR (n = 1). There were 5 participants that did not continue to Phase 2A (1 responder with improvement in chronic pain and 4 participants chose to not continue). There were 14 participants that continued to Phase 2A (3 responders completed the study and 3 are currently pending to complete Phase 2B). Eight participants went on to Phase 2B with two of eight (25%) demonstrating response. All participants completed full acute courses of Phase 2A and 2B without any major adverse events. Conclusions: Virtual reality treatments were tolerated by most, but many 37% (7/19) felt the experience was negative. Of the 14 who went on to Phase 2A accel-TMS, all completed the full week (25 sessions) and tolerated treatment well. For both Phase 2A accel-TMS and subsequent Phase 2B accel-TMS, there was a modest degree of response (21%, 25%) despite only 1 week of treatment. Our enrolled participants had experienced multiple failed treatment trials in their current episode. This likely factored into perceptions of the VR treatments and modest TMS response rate. Future work is required to optimized progressive sequencing of treatment approaches to provide rapid relief to patients suffering from symptoms of dysphoria. Keywords: Theta Burst Transcranial Magnetic Stimulation, Virtual Reality, Dysphoria Disclosure: Neuronetics: Other Financial or Material Support (Self) P72. Evidence of Between-Session but Limited Within-Session Psychophysiological Habituation in Response to Standardized, Context-Relevant Virtual Reality Exposure for PTSD Mascha van ’t Wout-Frank, Sydney Brigido, Emily Aiken, Noah Philip Alpert Medical School of Brown University, Providence, Rhode Island, United States Background: Virtual reality (VR) exposure therapy has been used for the treatment of posttraumatic stress disorder (PTSD) based on the idea that its immersive nature promotes the emotional engagement necessary for clinical improvement. However, VR, just like non-VR exposure is intense and emotionally demanding. Previously, we demonstrated that non-personalized, standardized VR warzone scenes which capture the context of the traumatic experience distinguishes Veterans with PTSD from those without and reduces PTSD symptom severity with high completion rates. Because clinical response to non-VR exposure is associated with between-session habituation to trauma cues as compared to habituation within a therapy session, we examined between- and within-session psychophysiological habituation to standardized, warzone-related VR exposure to test whether standardized VR specifically captures clinically meaningful between-session emotional learning. Methods: As part of a double-blind, randomized clinical trial (NCT03372460), Veterans with PTSD (n = 28) completed six, 25-minute sessions warzone virtual reality exposure over the course of 2-3 weeks. Within each session, participants completed three times the same virtual reality Iraq or Afghanistan Humvee driving scenario or “run”. Psychophysiological habituation of arousal was quantified through skin conductance reactivity to VR events and measured continuously throughout each session. Results: To test for the presence of between- and within-session habituation as well as their interaction, a repeated measures ANOVA included the variables Session (session 1-6 that occurred over 2-3 weeks reflecting between-session habituation) and Run (runs 1-3 that occurred within each session reflecting within-session habituation). Mauchly’s sphericity test was significant (p < 0.001) and Greenhouse-Geisser values are reported to reflect adjusted degrees of freedom. The interaction SessionRun term was significant (F(4.5,122.2)=2.46, p = 0.04, observed power 0.73) as was the main effect of Session was significant (F(2.9,79.2)=3.48, p = 0.02, observed power 0.75), but not the main effect of Run (F(1.3,35.8)=1.75, p = 0.19, observed power 0.28). These results reflect within-session habituation in the first VR session only, but between-session habituation across all VR sessions. Conclusions: These data show robust between-session habituation to non-personalized, warzone-related VR exposure. However, aside from within-session habituation in the first VR session, no further within-session habituation took place. This suggests that the reduction in arousal to the VR trauma context is due to learning over the course of multiple sessions consistent with traditional, non-VR exposure. The use of standardized, non-personalized VR environments might offer clinical benefit without the need to highly personalize the exposure experience, which in turn could improve treatment adherence and completion. Keywords: Posttraumatic Stress Disorder, Virtual Reality, Safety Learning, Emotional Arousal, Skin Conductance Response Disclosure: Roche: Consultant (Self) P73. Does Protocol Matter for PTSD? A Multisite Analysis of Veterans Receiving Therapeutic Transcranial Magnetic Stimulation Noah Philip, Megan Senda, Yosef Berlow, Stephanie Gee, F. Andrew Kozel, Michelle Madore Brown University and VA Providence, Providence, Rhode Island, United States Background: Repetitive Transcranial magnetic stimulation (rTMS, hereafter TMS) is an evidence-based treatment for pharmacoresistant major depression (MDD). We recently demonstrated that standard parameters used to treat MDD can also effectively reduce symptoms of posttraumatic stress disorder (PTSD) in Veterans, yielding statistically significant and clinically meaningful improvements. However, prior PTSD TMS studies often utilized different device protocols and parameters, which fall under the broad categories of high frequency, left-sided stimulation and lower frequency right-sided stimulation, with more recent inclusion of theta burst stimulation (TBS). In MDD, TBS and 10Hz TMS the left dorsolateral prefrontal cortex (DLPFC) are non-inferior to each other, although from the limited data available higher frequency stimulation in PTSD appears to yield greater effect sizes. To this end, we examined clinical outcomes in a large, multisite registry study of TMS; based on our prior work, we hypothesized that high-frequency stimulation would demonstrate greater effectiveness than other TMS protocols. Methods: VA Palo Alto/Stanford Institutional Review Board approved procedures related to this report. Training was standardized through VA Palo Alto and data gathered using a centralized VA REDCap database. Veterans were eligible for TMS if they met standard inclusion/clearance criteria, and other treatments were recommended to be stable for approximately 6 weeks prior to stimulation and held unchanged during 30 sessions of treatment, generally applied using individual measurements of head anatomy (Beam/F3). Regarding TMS protocols, we compared the magnitude of clinical outcomes in patients treated with FDA-cleared vs. non-cleared (i.e., off-label) TMS parameters. FDA cleared parameters, at the time of this writing, include 10Hz for Magstim, Neuronetics or Magventure systems for 4s, 11-26s intertrain interval (ITI), 3000 pulses per session; Brainsway devices using 18Hz for 2s, 20s ITI, 1980 pulses; and intermittent theta burst delivering 50Hz triplets at 5Hz, 2s stimulation with 8s ITI, 600 pulses). All other utilized parameters were off-label. These included 5Hz TMS (120% MT, 3000 pulses/session), right-sided intermittent TBS (80% of MT, 1800 pulses/session), right-sided 1Hz TMS (120% of MT, up to 3000 pulses/session), and right-sided 1Hz dTMS. Depression and PTSD symptoms were measured using the 9-item patient health questionnaire (PHQ9) and PTSD symptom checklist for DSM5 (PCL-5); outcomes of interest included mean changes and response (>10 point reduction in PCL, > 50% reduction in PHQ9). Outcomes were analyzed using paired-sample t-tests and missing data were excluded listwise. This data includes Veterans that received TMS from October 2017 through February 2022, which represents an expanded cohort from prior reports. Results: The cohort included N = 1,256 Veterans who received care at 35 different VA TMS clinics with usable data. Of these, n = 883 (70.3%) received “on-label” FDA protocols; Representation was greatest for groups receiving 10Hz (n = 736), 5Hz (n = 66), left-sided iTBS (n = 72), and dTMS (n = 75); other protocols were more sparsely represented. There were no statistically significant differences in PTSD outcomes between on- and off-label TMS parameters, and ANOVA did not reveal significant differences between examined protocols (all ps > .1). TMS was associated with a statistically significant and clinically meaningful reduction in PTSD symptoms (Baseline mean (SD) = 45.0 (18.3) and post-treatment mean SD 28.6 (19.7) p < .001, d = 1.04). There were also no statistically significant differences in MDD outcomes between any of the examined protocols (all ps > .1) TMS was also associated with significant and meaningful reduction in MDD symptoms (Baseline score = 18.3 (5.1), post-treatment score = 10.9 (7.0) p < .001, d = 1.21). Sensitivity analyses evaluating outcomes in only those who received an adequate dose (defined as 30 TMS sessions) did not change results. Conclusions: This large, multisite naturalistic data did not provide any clear indication that a single TMS protocol is superior for the treatment of PTSD. Similarly, no protocol outperformed another for MDD. This data shows, at the population level, that decisions regarding clinical parameters may be made based on convenience for the patient or clinic, and that they can be held in equipoise regarding their effectiveness. As previously demonstrated, TMS was able to provide significant and meaningful reductions in PTSD and MDD symptoms. Limitations are those inherent to naturalistic cohort studies and may not have captured multiple changes in protocols that occurred over time. These results represent population-level data without biomarkers, and it is possible that biological data may help whether individually customized parameters or targeting might improve outcomes. Within the off-label group, individual protocols were not evenly represented, limiting statistical power. Accelerated and neuronavigated TMS were not well represented. Ongoing studies, nested within this coordinated VA network, are currently investigating biomarkers (fMRI, EEG) and their relationship with clinical outcomes. In summary, this data did not find any compelling evidence of superiority of any TMS protocol for PTSD or MDD, underscores existing data on the effectiveness of TMS in this patient population, and provides important information on protocol selection for treating physicians. Keywords: Repetitive Transcranial Magnetic Stimulation, Posttraumatic Stress Disorder, Veterans Disclosure: Neurolief, Wave Neuro: Other Financial or Material Support (Self) P74. Paternal Deprivation Increases Social Vigilance in the Absence of Threat in Adult California Mice (Peromyscus Californicus) Erica Glasper, Shakeera Walker, Rita Beyene The Ohio State University, Columbus, Ohio, United States Background: Adverse early-life experiences increase susceptibility to developing mental health disorders, like anxiety disorders, which are often characterized by social avoidance behaviors. In addition to a reduction in time spent engaging with others, social avoidance also includes the inability to initiate interactions. This may be a consequence of less social motivation (i.e., desire to interact) or a result of distress experienced during social interactions. In rodents, social approach is reduced by social stressors (e.g., social defeat, predator stress) and is accompanied by increased social vigilance (i.e., looking at, but not approaching, an unfamiliar individual from a safe distance). In humans, reduced social approach coupled with increased social vigilance may predict the development of social anxiety disorders. Since interventions are successful in reducing social avoidance, determining which early-life adverse experiences are associated with social avoidance, and the trajectory of this behavior, is important for early detection and improving outcomes. To test the exploratory hypothesis that the early adverse experience of father absence leads to maladaptive social behavior, we utilized a unique model of early-life stress in biparental rodents, where the paternal male is permanently removed from their offspring (paternal deprivation [PD]) and examined the impacts of PD on the relationships among sociability, social anxiety, and social vigilance in adult offspring. Methods: Adult male and female control-reared and PD-reared California mice (Peromyscus californicus) were behaviorally assessed in a three-chambered apparatus over the course of two days. Social vigilance and time spent in the chamber with a confined, non-threatening unfamiliar same-sex conspecific was measured during sociability (Day 1) and social anxiety (Day 2) testing. Statistical analyses included two-way analysis of variance (ANOVA) testing (rearing x sex; n = 10-11/group), followed by Sidak’s post-hoc comparisons (when appropriate), and simple linear regressions. Statistical significance was reached when p≤0.05. Results: Paternal deprivation did not significantly influence social vigilance in males or females during sociability testing on Day 1 (two-way ANOVA; rearing x sex: F (1,37) = 2.768, p = 0.10; sex: F (1,37) = 3.962, p = 0.054; rearing: F (1,37) = 1.703, p = 0.199). However, a marginally significant interaction between rearing and sex was observed in social vigilance behavior during social anxiety testing on Day 2 (two-way ANOVA; rearing x sex: F(1,37) = 4.059, p = 0.051). Sidak’s multiple comparison’s test revealed significantly less social vigilance in PD-reared males, compared to PD-reared females (padjusted=0.03). Social vigilance did not differ in control-reared offspring (padjusted=0.90). A simple linear regression was used to predict social vigilance behavior during sociability testing on Day 1. Social vigilance explained a significant amount of variance in time spent interacting with the unfamiliar conspecific in PD-reared males (F(1,6)=9.27, p = 0.022, R2 = .61) and females (F(1,9)=11.81, p = 0.007, R2 = .57). The regression indicated that an increase in social vigilance corresponded with less time in the chamber with the unfamiliar same-sex conspecific. This relationship was absent in control-reared adult offspring on Day 1 sociability testing (p > .05, all comparisons). By Day 2, when mice were again presented with the now-familiar same-sex conspecific (social anxiety testing), social vigilance no longer explained the variance in time spent with the same-sex conspecific in any group (p > .05, all comparisons). Conclusions: These analyses highlight the unanticipated relationship between the adverse early-life experience of paternal deprivation and social vigilance during a low-risk social interaction. Paternal deprivation may augment perceived threat-related mechanisms in both males and females, thus contributing to this maladaptive behavior. Importantly, the negative relationship between social vigilance and social interaction is reduced with repeat exposure to the same conspecific. Future work will focus on identifying pathways and manipulating targets to directly link alterations in social behaviors with the loss of the paternal male in a biparental rodent species. Collectively, this work adds to our understanding of the behavioral consequences of early adverse experiences and may advance therapeutic interventions. Keywords: Early Life Stress, Social and Behavioral Deficits, Social Anxiety Disclosure: Nothing to disclose. P75. ENX-102, a GABA-A Alpha2,3,5 PAM That Blocks Alpha1, for the Treatment of Generalized Anxiety Disorder: A Phase 1 Single Ascending Dose Clinical Study Kimberly Vanover, Eve Taylor, Stephanie Parks, Jordi Serrats, Stephen Cunningham Engrail Therapeutics, Inc., San Diego, California, United States Background: Generalized anxiety disorder (GAD) can manifest as excessive worry and/or tension associated with a variety of somatic symptoms and substantially impairs daily life. Nonselective gamma-aminobutyric acid type A (GABA-A) positive allosteric modulators (PAMs) such as benzodiazepines, are proven treatments for GAD, but their use is generally limited to short-term therapy due to the risk of drug dependence and significant side effects such as sedation as well as cognitive and motor impairment, consequences largely mediated by activation of alpha1 subunit–containing GABA-A receptors. ENX-102 is a GABA-A PAM that activates neurotransmission via GABA-A receptors containing alpha2, alpha3, or alpha5 subunits, while blocking 1. ENX-102 is an investigational new drug in development for the treatment of GAD. A Phase 1 clinical study was designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of ENX-102 in healthy volunteers. Methods: ENX-102 was evaluated in a randomized, double-blind, placebo-controlled, single ascending dose study in healthy male and female volunteers. The primary objective was to evaluate the safety and tolerability of ENX-102 after single oral administration of increasing doses in comparison to placebo. The secondary objective was to evaluate the PK of ENX-102 after single dose administration. After signing written informed consent, subjects were screened up to 28 days prior to Study Day 1 (day of dosing). Eligible subjects were admitted to a clinical unit one day before dosing and remained confined for at least 4 days after dosing to monitor safety and PK and an outpatient follow-up visit occurred approximately 7 days after discharge. Eight (8) subjects were planned per dose cohort and were randomized to receive ENX-102 (N = 6/cohort) or placebo (N = 2/cohort). Within each cohort, 2 sentinel subjects were dosed and followed for at least 24 hours before the remainder of the cohort was dosed. A Dose Escalation Committee reviewed safety, tolerability, and PK data before escalating to the next higher dose. Safety was measured by adverse events (AEs), vital signs, 12-lead electrocardiograms (ECGs), clinical laboratory tests (hematology, serum chemistry, coagulation, urinalysis), physical exams, and the Modified Observer’s Assessment of Alertness/Sedation (MOAA/S) scale. Safety measures were analyzed using descriptive statistics. Blood samples for PK analysis were collected pre-dose and then 15, 30, 45, 60, 90, and 120 minutes and 4, 6, 8, and 12 hours after dosing. Plasma concentration-time data for ENX-102 were analyzed using non-compartmental methods. PK parameters included maximum plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration-time curve (AUC), and half-life (t1/2). Results: Of the fifty-six (56) healthy volunteers who participated, 30 (53.6%) were male and 26 (46.4%) were female, and 53 (94.6%) were White, 1 (2.4%) was Asian, 1 (2.4%) was Black, and 1 (2.4%) reported mixed races; none reported being Hispanic/Latino, though 19.6% declined to report ethnicity. Participants were randomized to receive ENX-102 or placebo (6 active:2 placebo per cohort) across 7 cohorts that evaluated doses of 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 and 10.0 mg. All 14 subjects randomized to placebo completed the study and 41/42 (97.6%) subjects randomized to ENX-102 completed the study; one subject randomized to 10.0 mg ENX-102 withdrew consent after the inpatient portion of the study was completed and declined the follow-up visit. There were no deaths or other serious adverse events in the study. There were no discontinuations due to adverse events. Treatment-emergent adverse events (TEAEs) were reported in 16/42 (38.1%) subjects randomized to ENX-102 and in 1/14 (7.1%) subjects randomized to placebo. Consistent with a central GABAergic mechanism of action, the most frequently reported adverse events in subjects receiving ENX-102 were nervous system disorders and included mild dizziness in 8/42 (19.0%), mild fatigue in 4/42 (9.5%), and mild to moderate somnolence in 3/42 (7.1%). Postural dizziness occurred in similar rates in subjects receiving ENX-102 (3/42; 7.1%) and placebo (1/14; 7.1%); instances of postural dizziness were mild to moderate in ENX-102-treated subjects and was moderate in the placebo-treated subject. Other TEAEs occurred in one subject each. None of the TEAEs were severe. There were no notable, dose-related changes in physical exams, vital signs, ECGs, or clinical laboratory values. On the MOAA/S scale, no frank sedation was observed. Pharmacokinetic analyses indicated increases in ENX-102 exposure with dose. Mean (standard deviation) Cmax values ranged from 7.013 ng/mL (1.2663) associated with the lowest dose of 0.5 mg to 95.633 ng/mL (11.7959) associated with the highest dose of 10 mg, and AUC to the last nonzero time point ranging from 244.296 hng/mL (55.1034) to 4400.502 hng/mL (1710.469). Tmax was reached between 1 and 4 hours. Mean t1/2 was approximately 60 hours. Conclusions: ENX-102 was considered safe and well-tolerated in healthy volunteers. A maximally tolerated dose was not defined. ENX-102 exhibited predictable dose-related exposure with a long half-life consistent with once daily oral administration. The data support continued development of ENX-102 for the treatment of GAD. ENX-102 has advanced into a Phase 1b multiple ascending dose study. Keywords: Phase 1, GABA-A, Positive Allosteric Modulators, Generalized Anxiety Disorder Disclosures: Engrail Therapeutics: Employee(Self), Intra-Cellular Therapies: Stock / Equity(Self), Evolution Research Group: Consultant (Self) P76. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Infection and “Long COVID” in Adolescents With Anxiety Disorders: A Prospective Longitudinal Study Jeffrey Strawn, Jeffrey Mills, Heidi Schroeder, Zoe Neptune, Ashley Specht, Susana Keeshin University of Cincinnati, Cincinnati, Ohio, United States Background: The impact of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on youth with anxiety disorders has not been prospectively examined. Further, there are limited prospective data on post-acute sequelae COVID-19 (PASC), including symptoms that constitute the long COVID neuropsychiatric syndrome. With this in mind, we examined the longitudinal course of anxiety and long COVID symptoms following SARS-CoV-2 infection in affected adolescents. Methods: In December 2019, we began an IRB-approved, longitudinal study of adolescents aged 12-17 with DSM-5 primary anxiety disorders treated with either duloxetine or escitalopram. At each follow-up visit, affective and anxiety symptoms during the prior interval, week-by-week were assessed using the Longitudinal Interval Follow-up Examination (LIFE). Each follow-up interview included all items from the Generalized Anxiety Disorder-7 (GAD-7) and Quick Inventory of Depressive Symptoms (QIDS) scales each week and a weekly psychiatrist-rated Clinical Global Impression-Severity (CGI-S) scale. To examine pre- and post-SARS-CoV-2 infection differences in anxiety symptoms, the distribution of the difference in means for GAD-7/QIDS items and CGI-S were computed using Monte Carlo simulation from a Student-t distribution for the mean pre- and post-COVID symptom scores. Results: Twenty-six patients contributed 1,308 weeks of data (1019 patient weeks from patients who did not contract COVID-19, 289 patient weeks from patients with COVID-19 infection, and 139 patient weeks post-COVID-19). Patients were, on average, 14.3 ± 1.64 years (14.8% male) and 3.8% Black and were followed for 50.3 ± 30.4 weeks (range: 7 to 96 weeks, median: 55.5 weeks). All patients met criteria for generalized anxiety disorder, 19% had separation anxiety disorder, 78% had social anxiety disorder, 30% had panic disorder, 26% had ADHD, and 37% had a history of a depressive disorder. All patients with COVID-19 were unvaccinated and experienced mild symptoms; none were hospitalized or received monoclonal antibodies. Post-COVID-19, anxiety symptoms significantly worsened for all GAD-7 subscales (all p < 0.001), and patients had significant syndromic worsening, reflected by CGI-S scores (p < 0.001). SARS-CoV-2 infection was also associated with within-patient worsening in long COVID symptoms (anergia, β = 0.37, p < 0.001; amotivation, β = 0.57, p < 0.001; concentration, β = 0.35, p < 0.001, and irritability, β = 0.52, p < 0.001). Conclusions: This prospective study of the longitudinal impact of COVID-19 in pediatric anxiety disorders reveals that COVID-19 is associated with worsening anxiety symptoms and a disquieting 33% worsening in syndromic severity. Further, these data raise the possibility that, in anxious youth, COVID-19 is associated with a surfeit of neuropsychiatric symptoms, representing a long COVID neuropsychiatric syndrome like that described in adults. Keywords: Adolescent Anxiety, COVID-19, Antidepressant Treatment Practice Disclosures: Myriad Genetics: Other Financial or Material Support (Self), AbbVie: Contracted Research (Self), MedScape, Neuroscience Educational Institute: Speakers Bureau (Self), Yung Family Foundation: Grant(Self), Springer Publishing: Royalties (Self), Cerevel: Consultant(Self) P77. Preclinical Evaluation of ENX-102, a GABAA α2,3,5 PAM That Blocks α1, in the Elevated Plus Maze and Pharmaco-EEG Jordi Serrats, Krishna Vadodaria, Kimberly Vanover, Eve Taylor, Stephen Cunningham Engrail Therapeutics, San Diego, California, United States Background: Nonselective gamma-aminobutyric acid type A (GABAA) positive allosteric modulators (PAMs) such as benzodiazepines, are proven anxiolytics, but their clinical use is limited due to significant side effects, largely mediated by activation of α1 subunit–containing GABAA receptors. There have been several efforts to generate subtype selective (α2, α3, or α5 subunits) GABAA modulators driving the beneficial effects of GABA modulation without the undesired effects that result from the activation of α1 subunit-containing receptors. ENX-102 is a GABAA PAM that activates neurotransmission via GABAA receptors containing α2, α3, and α5 subunits, while blocking α1. We evaluated the potential for anxiolysis of ENX-102 in rodent anxiety models such as the elevated plus maze (EPM) after acute and chronic dosing, and we characterized the sleep-wake electroencephalography (EEG) profile in rats induced by increasing doses of ENX-102. Methods: The in vitro pharmacology of ENX-102 was assessed with the SyncroPatch platform in PAM mode on the human GABAA receptors α1/β3/γ2 α2/β2/γ2, α2/β3/γ2, α3/β3/γ2, and α5/β3/γ2. Adult male SD rats (N = 130; N = 10 per condition) were used in the in vivo pharmacology studies. ENX-102 was evaluated in the EPM test which assesses anxiety. The following measures were automatically recorded: distance travelled, time spent in each arm, and entries into each arm. ENX-102 was orally dosed at 0.1, 0.3 and 1 mg/kg to target receptor occupancies of approximately 30%, 50% and 80% at 1 hr post-dosing, respectively. Acutely dosed rats were also tested 24 hrs post-dose. A different cohort was dosed chronically, once daily for fourteen days and tested 1 hr following the last dose to assess the anxiolytic properties of ENX-102 after continuous target engagement. Chlordiazepoxide was used as a positive control. Data were analyzed by ANOVA followed by post hoc analyses where appropriate. Adult male SD rats (N = 12 in a cross-over design) were used in the pharmaco-EEG studies. Sleep-stage specific pharmaco-EEG signature of ENX-102 was assessed after orally dosing ENX-102 (0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg and 1 mg/kg) to target a broad range of receptor occupancies. Lorazepam (1 mg/kg) was used as a positive control. Rats were implanted with telemetry devices. Recordings started 2 hrs prior to compound administration and recorded continuously for 48 hrs after administration. Spectral analysis included quantifying the changes in spectral power for the traditional EEG bands (Delta-0.5-3.9 Hz, Theta-4-7.9 Hz, Alpha-8-11.9 Hz, Beta 12-29.9 Hz, Low Gamma 30-49.9 Hz, and High Gamma 50-100 Hz). Sleep analysis was also conducted for 2 hrs prior to ENX-102 administration and for 12 hours after ENX-102, as well as 23-25 hrs and 46-48 hrs after ENX-102 was administered. Several variables were analyzed: sleep architecture as percent time in active awake, quiet wake, NREM, REM and latency to sleep. ANOVA with Dunnett’s multiple comparisons was applied independently to each 15-minute bin to assess the drug effects on the power of different EEG spectral bands as well as across sleep stages. All experimental protocols in animal studies were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Results: The functional in vitro activity of ENX-102 on GABAA receptors showed that ENX-102 is a subtype-specific GABAA PAM. ENX-102 did not drive Cl- flux on cloned human GABAA subtype α1/β3/γ2. ENX-102 displayed EC50 of 0.80 nM, 0.82 nM, 2.47 nM and 0.20 nM at GABAA subtypes α2/β2/γ2, α2/β3/γ2, α3/β3/γ2, and α5/β3/γ2, respectively; and Emax of 50%, 89%, 113% and 96% at GABAA subtypes α2/β2/γ2, α2/β3/γ2, α3/β3/γ2, and α5/β3/γ2, respectively. In vivo characterization of ENX-102 in the EPM assay showed that ENX-102 increased distance traveled in the open arms demonstrating an anxiolytic-like profile. The effect was equivalent to what was demonstrated by the reference anxiolytic compound, chlordiazepoxide. Chronic dosing of ENX-102 showed a similar magnitude of anxiolytic effect. These data demonstrate strong anxiolytic activity in rodents that is maintained with chronic dosing. The EEG profile induced in rats by dosing ENX-102 was also studied. ENX-102 induced EEG changes indicative of an anxiolytic profile and that mimic the spectral EEG signature that has been associated to other subtype-selective GABA modulators (see Christian et al., 2014). This spectral EEG signature can be used as an indication of central pathway engagement in rodents and as a translational bridge into clinical studies. Conclusions: ENX-102 is anxiolytic-like in rodents after acute and sub-chronic (14 days) dosing without any loss of effect despite engaging the target for the entire duration of the study. Sleep-wake pattern and EEG profile analysis suggest a non-sedative anxiolytic profile of ENX-102. These data support the clinical study of ENX-102 in anxiety-related indications. Keywords: Anxiety, GABA-A, Positive Allosteric Modulators, Elevated Plus Maze, EEG Biomarkers Disclosure: Engrail Therapeutics: Employee (Self) P78. The Role of Matrix Metalloproteinases 2 and 9 in the Nucleus Accumbens After an Exposure to an Acute Stress and a Stress-Conditioned Cue Ritchy Hodebourg, Constanza Garcia-Keller, Peter Kalivas Medical University of South Carolina, Charleston, South Carolina, United States Background: There is a strong comorbidity between post-traumatic stress disorder (PSTD) and substance use disorders. For example, a person with PTSD is 2 to 4 times more likely to have a substance use disorder than a person without PTSD. Preclinical studies have shown that both addictive drug use and acute stress exposure produce long-lasting neuroadaptations at glutamatergic synapses in the nucleus accumbens core (NAcore) including an increase in the AMPA/NMDA ratio and dendritic spine density, and a downregulation of the glutamate transporter. Furthermore, like drug associated cues, a stress-conditioned stimulus (CS) is able to reinstate drug seeking behavior. Knowing that cue-induced drug seeking requires activation of NAcore matrix metalloproteinases (MMPs), we previously found that a stress-CS also increased MMP-2,9 activity in NAcore, and induced burying behavior in a defensive burying task (DBT). However, it is unknown whether the increase of MMPs is mediated by MMP-2, MMP-9 or both. In this study, we firstly sought to determine which MMP is involved in the stress-CS induced burying behavior. Then we sought to quantify the MMPs activity in a cell-specific manner in NAcore. We hypothesized that the burying behavior is mediated by MMP-9, an MMP known to be involved in cue-induced drug seeking. Methods: To quantify the effect of an acute stress on MMP-2,9 activity, Long Evans rats were microinjected with an MMP-9 inhibitor followed by FITC-quenched gelatin into the NAcore immediately before 30 min of acute restraint stress or 30 min in the home cage. Then, to evaluate the role of a stress-CS on the behavior and MMP-2,9 activity, rats were restraint stressed for 2h and simultaneously exposed to an odor that became the stress-CS. Control rats were exposed to the same odor in the home cage. 3 weeks after the stress, rats were pretreated with MMP-2 or -9 inhibitors, then FITC-quenched gelatin was injected into the NAcore and the effect of the CS or a novel stimulus (NS) was tested in a DBT for 15 min. The burying and immobility behaviors, as well as MMP-2,9 activity were quantified. Finally, the MMP-2,9 activity was quantified in a cell specific manner. To this end, an AAV cre-dependent mCherry virus was used to transfect medium spiny neurons (MSN) in D1 and D2 cre-dependent rats. Results: We found that an acute stress increased the MMP-2,9 activity in the NAcore. This increase is prevented by an MMP-9 inhibitor. Both MMP-2 and -9 inhibitors prevented the stress-CS induced burying behavior, without affecting the immobility. However, only the MMP-2 inhibitor significantly reduced MMP activity in NAcore. Finally, we found that the stress-CS significantly increased MMP-2,9 specifically around D1 but not D2 MSNs. Conclusions: These findings contribute to a growing literature suggesting that PTSD and SUDs share common neural substrates and offer new targets for treating both disorders. Keywords: Matrix Metalloproteinases, Post Traumatic Stress Disorder, Nucleus Accumbens Disclosure: Nothing to disclose. P79. Simultaneous Targeting of NMDARs and 5-HT4Rs Exerts Additive Effects in Preventing Stress-Induced Hyponeophagia Briana Chen, Holly Hunsberger, Indira Mendez-David, Denis David, Alain Gardier, Christine Ann Denny Columbia University, New York, New York, United States Background: Serotonin (5-HT) receptors and N-methyl-D-aspartate receptors (NMDARs) have both been implicated in the pathophysiology of affective and anxiety disorders. In particular, 5-HT type 4 receptor (5-HT4R) agonists and NMDAR antagonists have been suggested to act as rapid-acting antidepressants and resilience-enhancing prophylactic drugs. Here, we evaluated whether targeting both receptors through combined dosing of (R,S)-ketamine and prucalopride, a 5-HT4R agonist would have additive effects, resulting in reductions in stress-induced fear, behavioral despair, and hyponeophagia. Methods: A single injection of saline, (R,S)-ketamine, prucalopride, or a combined dose of (R,S)-ketamine + prucalopride was administered before or after contextual fear conditioning (CFC) stress in male and female 129S6/SvEv mice. Drug efficacy was assayed using a variety of behavioral tests, including the forced swim test (FST), elevated plus maze (EPM), open field (OF), marble burying (MB), novelty-suppressed feeding (NSF), and contextual fear discrimination (CFD). All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the New York Psychiatric Institute (NYSPI). c-fos and parvalbumin (PV) expression in the hippocampus was assayed using immunohistochemistry. Generally, the effect of Drug was analyzed using an analysis of variance (ANOVA), using repeated measures where appropriate. Post-hoc Dunnett, Sidak, or Tukey tests were used where appropriate. Results: A single dose combination of prophylactic (R,S)-ketamine + prucalopride (10 + 3 mg/kg) attenuated learned fear in male mice (n = 6-10 male mice per group; ANOVA; p = 0.0035) and decreased behavioral despair in both sexes (n = 6-10 male mice per group; ANOVA; p < 0.0001; n = 6-12 female mice per group; ANOVA, p < 0.0001). Combined administration of (R,S)-ketamine + prucalopride exerted an additive effect in preventing stress-induced hyponeophagia in both male and female mice, but not when administered separately (n = 6-10 male mice per group; ANOVA; p = 0.0004; n = 6-12 female mice per group; ANOVA, p = 0.0467). Prophylactic (R,S)-ketamine + prucalopride also exerted an additive effect of enhancing and facilitating contextual fear discrimination in male mice (n = 6-7 mice per group; RMANOVA; p < 0.0001). Combined, but not separate, administration of (R,S)-ketamine + prucalopride significantly increased neural activity and PV expression, as well as overlap, in ventral CA3 of the hippocampus (n = 7-8 mice per group; ANOVAs; p = 0.0001, p = 0.0197, p = 0.0178, respectively). Conclusions: Our results indicate that simultaneously targeting NMDARs and 5-HT4Rs using a drug combination of (R,S)-ketamine + prucalopride exerts additional and distinct neural and behavioral effects in reducing a wide variety of stress-induced fear, behavioral despair, and hyponeophagia behaviors in both male and female mouse models of stress. Simultaneously targeting NMDARs and 5-HT4Rs is sufficient to enhance both excitatory and inhibitory signaling in specific subregions of the HPC, a brain region critically involved in stress processing and psychiatric disorders. Together, our findings demonstrate the potential of leveraging combinatorial pharmacological treatment to advance targeted therapies for stress-induced psychiatric disorders. Keywords: (R,S)-ketamine, Prucalopride, NMDA Receptor, 5-HT4 receptor, Depression and Anxiety Disclosure: Patent: Patent (Self) P80. Orally Administered Heteroaromatic Salvinorin A Analogue (AK-1402) Elicits Antinociceptive and Anxiolytic-Like Effects in Mice Caroline Moreira, SA Filho Alberto, Adam Keasling, Jordan Zjawiony, Elson Costa, James Fajemiroye Universidade Federal de Goias, Goiania, Brazil Background: Despite the attribution of the analgesic and anxiolytic properties of Salvia divinorum to neoclerodane diterpene called Salvinorin A (SA), the limiting pharmacological profile and dearth of clinical applications of SA support the preliminary screening of its C(22)-fused-heteroaromatic analogue [2-O-salvinorin B benzo[b]thiophene-2-carboxylate (AK-1402)] in mice nociception and anxiety models while assessing potential mechanism of action. Methods: Acute oral administration of AK-1402 (1, 3, or 10 mg/kg), morphine (MOR 5 mg/kg), diazepam (DZP 1 mg/kg), or vehicle 10 ml/kg in male and female Swiss mice [5-week-old with weight = 25 ± 3 g; n = 8 (4 mice per sex) randomly distributed into the treatment groups to the acetic acid-induced abdominal writhing, formalin-induced hind paw licking, the thermal reaction in the hotplate as well as aversive response in the elevated plus-maze (EPM), and light-dark box (LDB). Additionally, mice were pretreated intraperitoneally with naloxone (NAL 3 mg/kg; non-selective opioid receptor antagonist), naloxonazine (Nalzine10 mg/kg; selective μ-OR antagonist), and nor-binaltorphimine (nor-BNI 10 mg/kg; selective ĸ-OR antagonist), or vehicle before the oral administration of AK-1402 10 mg/kg, MOR 5 mg/kg, DZP 1 mg/kg or vehicle 10 ml/kg and behavioral evaluations. The NIH Guidelines for the Care and Use of Laboratory Animals as approved by the Ethics Committee of the Federal University of Goiás (protocol number 104/08) were adhered to in all experimental procedures. Data were subjected to ANOVA followed by Dunnett’s s or Bonferroni’s post hoc tests and expressed as mean ± SEM (p < 0.05 was considered statistically significant). Results: The oral administration of AK-1402 (3 and 10 mg/kg) and MOR 5 mg/kg reduced the abdominal writhing activity [F (4, 39) = 61.6], licking behavior at the first phase of formalin [F (4, 25) = 19.8, p < 0.05], and increased latency to thermal stimulus response on hot plate [F(4,35)= 8.6, p < 0.05], thereby showing antinociceptive property. Like DZP 1 mg/kg, an increase in the time spent and entry into the open arm of EPM [F (4, 35) = 12.5 and F (4, 35) = 8.3, respectively; p < 0.05] as well as the transition and time spent at the light area of LDB [F (4, 35) = 10.1 and F (4, 35) = 9.3, respectively; p < 0.05] suggest the attenuation of aversive-like response by AK-1402 at the dose of 10 mg/kg. The blockade of the antinociceptive and anxiolytic-like properties following NAL, nor-BNI, and/or FLU pretreatments suggests opioid receptors and benzodiazepine site involvement in the activities of AK-1402. Conclusions: The antinociceptive and anxiolytic-like effects of AK-1402 are promising and demand additional pharmacological characterizations. Keywords: Salvinorin A, AK-1402, Behavioral Model, Opioid System, Gabaergic System Disclosure: Nothing to disclose. P81. Association of Early Life Stress and Dietary Factors With Mitochondrial DNA Deletions in Healthy Young Adults Teresa Daniels, William Lewis-de los Angeles, Brooke Hjelm, Emily Zitkovsky, Audrey Omidsalar, Marquis Vawter, Audrey Tyrka Brown University, Bradley Hospital, Riverside, Rhode Island, United States Background: Increasing evidence indicates that mitochondria respond to psychosocial and environmental stressors. In a recent study, elevated mitochondrial DNA (mtDNA) deletions were found in the postmortem brains of individuals with psychiatric disorders, but no prior work has examined the impact of early life stress (ELS) and health behaviors in adulthood on mtDNA deletions. This study assessed mtDNA deletions, diet, and cardiometabolic risk in peripheral blood mononuclear cells (PBMCs) in healthy young adults, with and without ELS. Methods: Participants (N = 181; 69% female) ages 18-40 were recruited from community. Participants with ELS (n = 108) experienced childhood maltreatment and parental loss. Controls (n = 73) had no maltreatment, loss, or psychiatric disorders. Standardized interviews and self-reports assessed demographics, adversity, psychiatric history, cardiometabolic risk factors, and dietary intake using the Healthy Eating Index. PBMCs were isolated for sequencing of mtDNA deletions. Splice-Break pipeline with long-range PCR and next-generation sequencing was utilized to quantify mtDNA deletions, with statistical control for coverage depth. mtDNA deletions were log transformed for analysis due to skew. Results: In a linear model adjusted for age, sex, and coverage depth, participants with ELS demonstrated significantly more unique mtDNA deletions (F(4,176)=19.2, p < .001) than participants without ELS. Current psychiatric disorders, adult stressors, and current perceived stress were each associated with deletions (p’s < .05) but did not account for ELS associations with unique mtDNA deletions. A healthier diet was associated with fewer unique mtDNA deletions (p = 0.046) in a linear model adjusting for age, sex and coverage depth, though diet did not account for the association of childhood adversity and mtDNA deletions (p < .001). There was no association between a composite metabolic risk score and mtDNA deletion burden (p = 0.50). Conclusions: In this sample, participants with ELS demonstrated higher numbers of unique deletions, regardless of psychiatric status. Additionally, diet, but not cardiometabolic risk, predicted mtDNA deletions in healthy young adults. These preliminary findings add to recent evidence indicating that mtDNA is impacted by childhood adversity, psychiatric disorders, and health behaviors. Next steps include additional assessments of relationships with mitochondrial structure and function, metabolic hormones, and type and timing of adversity. Keywords: Mitochondrial DNA, Early Life Stress (ELS), Diet Disclosure: Nothing to disclose. P82. Fear Potentiated Startle to an Unconditioned Aversive Stimulus Predicts Individual Differences in Physiological Conditioned Fear and Self-Reported Fearful Traits in a Posttraumatic Stress Sample Michael Lewis, Eylul Akman, Scott Rauch, Isabelle Rosso McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Posttraumatic stress symptoms (PTSS) are characterized, in part, by pathological fear, anxiety, and stress reactivity (FASR). Distinct aspects of FASR are indexed using physiological measures of acute conditioned fear responding and self-report measures of dispositional fearful reactivity. Elevated physiological or self-reported FASR has been observed in some, but not all, PTSS samples and may contribute to variability in clinical presentation and treatment response. Unconditioned physiological responding (UCR) to a naturally aversive stimulus may contribute to individual differences in FASR. Studies in rodents, healthy humans, and humans with fear- and-anxiety-related disorders have found that UCR predicts physiological responding to a conditioned fear cue during fear acquisition. In a recent fear-potentiated startle (FPS) study of trauma-exposed individuals with low PTSS (Kreutzmann et al., 2021), UCR predicted FPS to conditioned fear and safety cues during acquisition. Some prior studies have found that physiological stress responding is related to self-reported FASR. In a transdiagnostic study of fear and anxiety disorders (Marin et al., 2020), UCR predicted a composite score of self-reported dispositional reactivity to stress. Here, we extend prior work by testing the association of UCR with both physiological and self-reported fearful reactivity in a trauma-exposed sample with a continuum of PTSS severity. We hypothesized that higher UCR would be positively correlated with FPS to conditioned fear cues during acquisition and extinction, with FPS to conditioned safety cues during acquisition and extinction, and with self-reported dispositional FASR. We also explored associations of UCR with PTSS severity. Methods: Participants were 79 trauma-exposed adults (61 female) with a continuum of PTSS severity. PTSS severity was quantified as total score on the Clinician Administered PTSD Scale (CAPS) for DSM-5. A composite score of self-reported Fear Anxiety and Stress Reactivity (FASR) was derived by averaging z-scores from four self-report measures that index distinct aspects of dispositional FASR and previously have been associated with PTSS and fear conditioning: Fear Survey Schedule-II, Anxiety Sensitivity Index-3, State-Trait Anxiety Inventory–Trait Anxiety Scale, and 10-item Connor-Davidson Resilience Scale. FPS was recorded during fear Acquisition and Extinction. The auditory startle probe was a burst of white noise. The unconditioned stimulus (US) was an airblast to the larynx and the conditioned stimuli were two colored shapes. The conditioned fear cue (CS+) was paired with the US during Acquisition and the conditioned safety cue (CS-) was never paired with the US. Acquisition consisted of 12 trials of each CS, with the US following each CS + trial. Extinction consisted of 16 trials of each CS type. Mean FPS for UCR was calculated using all 12 US presentations during Acquisition. Mean FPS for CS + and CS- during Acquisition and Extinction were calculated after omitting the first trial of each CS type for each phase. Six separate linear regressions tested the correlation of UCR with FPS to CS + during Acquisition, FPS to CS + during Extinction, FPS to CS- during Acquisition, FPS to CS- during Extinction, FASR score, and total CAPS. All analyses were performed both with and without biological sex as a covariate. Results: UCR was positively correlated with CS + during Acquisition (R2 = 0.24; p < 0.0001), CS + during Extinction (R2 = 0.13; p = 0.0028), CS- during Acquisition (R2 = 0.14; p = 0.0015), CS- during Extinction (R2 = 0.21; p < 0.0001), and FASR (R2 = 0.15; p = 0.0004). UCR was not correlated with total CAPS (R2 = 0.03; p = 0.1352). All significant correlations survived Bonferroni correction and remained Bonferroni significant when controlling for sex. Female sex was associated with greater total CAPS scores (B = 0.66, p = 0.0184), but this association did not survive Bonferroni correction. Conclusions: Higher UCR was associated with greater conditioned FPS to four distinct conditions (CS + Acquisition, CS + Extinction, CS- Acquisition, CS- Extinction) and with a composite measure of self-reported dispositional FASR in this trauma-exposed sample with a continuum of PTSS severity. Thus, in trauma-exposed individuals, elevated UCR to naturally aversive stimuli may lead to elevated physiological fear reactivity to a broad array of conditioned stimuli and to an increased tendency to feel fearful and anxious in response to stress. Conditioned fear extinction is the basis of prolonged exposure therapy for PTSD and inability to inhibit fear in the presence of conditioned safety cues is theorized to represent a distinct phenotype of PTSD with regard to treatment responsivity or resistance. Thus, future studies are needed to determine whether elevated UCR predicts differences in treatment response in PTSD. Keywords: Post Traumatic Stress Disorder, Fear-Potentiated Startle, Fear Conditioning and Extinction, Unconditioned Responses, Anxiety and Stress Disclosure: Nothing to disclose. P83. Neighborhood Poverty is Associated With Reduced Acoustic Startle in Individuals With Fewer PTSD Symptoms Meghna Ravi, Sriya Karra, Tanja Jovanovic, Jennifer Stevens, Abigail Powers, Vasiliki Michopoulos Emory University School of Medicine, Atlanta, Georgia, United States Background: Neighborhood poverty is associated with exposure to chronic stressors including environmental stressors like noise and pollution and traumatic events like police violence and other violent crimes. Recent work from our group has shown that neighborhood poverty is associated with worse posttraumatic stress disorder (PTSD) symptoms, both in chronically trauma-exposed Black women and in acutely traumatized men and women of varied race. PTSD is also characterized by dysregulation of autonomic function. For example, individuals with PTSD take longer to habituate to a startle tone than those without PTSD in the aftermath of a traumatic event. Furthermore, several studies demonstrate increased startle magnitude in individuals with PTSD as compared to individuals without PTSD. Despite the relation between neighborhood poverty and PTSD symptoms, little is known about how neighborhood poverty relates to psychophysiological functions that are dysregulated in PTSD. Thus, in the current study we determined associations between neighborhood poverty and acoustic startle in a fear potentiated startle paradigm. We hypothesized that individuals living in areas with high rates of poverty with high PTSD symptoms would show the highest startle magnitude. We also explored the interactions of PTSD symptom subclusters with rates of neighborhood poverty on startle magnitude. Methods: Participants (N = 64, 70% female) were enrolled between 2013 and 2014 in the waiting rooms of a large publicly funded hospital in Atlanta, GA. Participants were recruited out of medical clinics, including primary care or obstetric and gynecology waiting rooms. The sample was 93.8% Black, 3.1% white, 1.6% Latinx, and 1.6% mixed. Basic sociodemographic information including zip code where the participant lived was collected. PTSD symptoms were assessed with the modified PTSD Symptom Scale (PSS). Neighborhood poverty was assessed using the 2014 5-year American Community Survey, an annual survey that determines socio-demographic information (such as poverty) for a given area within the US. Neighborhood poverty was defined as the percentage of individuals living below the federally determined poverty line in a participant’s zip code. Lastly, startle magnitude was determined using an acoustic startle paradigm. Startle magnitude was defined as the average eye blink electromyogram (EMG) response to 106 decibel white noise probes across 12 trials of the task. Moderation analyses were used to determine the effect of neighborhood poverty on startle magnitude and whether there was a moderating role of PTSD symptoms. Results: The model with total PTSD symptoms as the moderator accounted for 13.45% of the variance in startle magnitude, F(3,60)=3.11, MSE = 1080.49, p = 0.03. Neighborhood poverty significantly predicted startle magnitude, where living in areas with more poverty predicted lower startle magnitude (B = -132.27, SE = 54.56, p = .02). There was also an interaction between neighborhood poverty and PTSD symptoms on startle magnitude (B = 6.19, SE = 3.15, ΔR2 = .06, p = .05). Follow up analyses were run to determine conditional effects of PTSD symptoms. Results showed that the effect of neighborhood poverty on startle magnitude was only significant at the 16th percentile of PTSD symptoms (B = -136.08, SE = 52.39, p = .02), but not at the 50th (B = -70.42, SE = 39.49, p = .08) or 84th percentiles of PTSD symptoms (B = 13.70, SE = 53.09, p = .80). For symptom subcluster analyses, the model with hyperarousal symptoms as the moderator accounted for 11.88% of the variance in startle magnitude, F(3,60)=2.70, MSE = 1100.11, p = .05. In this model, neighborhood poverty significantly predicted startle magnitude (B = -124.33, SE = 56.70, p = .03), though the interaction between hyperarousal symptoms and neighborhood poverty was not significant (B = 14.88, SE = 8.69, ΔR2 = .04, p = .09). For intrusive symptoms, the model accounted for 7.86% of the variance in startle magnitude and was not significant, F(3,60)=1.7, MSE = 1150.34, p = .18. Finally, for avoidance and numbing symptoms, the model predicted 12.54% of the variance in startle magnitude, F(3,60)=2.87, MSE = 1091.84, p = .04. Neighborhood poverty significantly predicted startle magnitude, (B = -132.77, SE = 53.11, p = .02) and the interaction between avoidance and numbing symptoms and neighborhood poverty was significant (B = 14.04, SE = 6.80, ΔR2 = .06, p = .04). Follow up analyses revealed that neighborhood poverty predicted startle magnitude at the 16th (B = -132.77, SE = 53.11, p = .02) and 50th (B = -83.63, SE = 40.97, p = .05) percentiles of avoidance and numbing symptoms, but not at the 84th percentile of avoidance and numbing symptoms (B = 2.02, SE = 48.97, p = .97). Conclusions: Our results indicate that neighborhood poverty is associated with blunted startle magnitude only for those with few PTSD symptoms. This effect seems to be driven by the avoidance and numbing symptom cluster of PTSD. It is possible that individuals living in areas with high rates of poverty are more habituated to loud noises, potentially due to greater exposure to loud noise in their everyday environments, resulting in decreased responses to acoustic startle probes. This habituation may no longer be present when an individual has greater PTSD symptoms. Overall, results imply that neighborhood poverty can impact basic autonomic functioning. Future studies should further explore the role of different types of PTSD symptoms and their interaction with neighborhood poverty in impacting psychophysiology. Keywords: Acoustic Startle, PTSD, Neighborhood Poverty Disclosure: Nothing to disclose. P84. HIV Interacts With PTSD to Impact Fear Extinction and Estrogen Receptor Expression in Trauma-Exposed Black Women Vasiliki Michopoulos, Susie Turkson, Paul Howell, Igho Ofotokun, Tanja Jovanovic, Gretchen Neigh Emory University School of Medicine, Atlanta, Georgia, United States Background: People living with HIV experience high rates of trauma exposure over their lifetime, which increases individual risk for the development of posttraumatic stress disorder (PTSD) and psychophysiological hyperarousal, including deficits in fear extinction. Additionally, under-resourced and minoritized Black women living in urban environments tend to be exposed to high levels of trauma, suffer from chronic PTSD, and are at increased risk for HIV infection. Because evidence-based PTSD treatments, such as prolonged exposure, rely on fear extinction, it is important to understand how HIV serostatus interacts with PTSD to impact fear extinction. We hypothesized that individuals with both HIV and PTSD would show greater impairments in fear extinction. Additionally, it is important to understand the biological mechanisms by which HIV and PTSD may impact fear extinction processes. Estradiol (E2) and its receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) affect neurobiological processes underlaying fear extinction. More specifically, preclinical and translational studies in women indicate that limbic regions of the brain critical for fear extinction and learning process are densely populated with ERα and ERβ and show that E2 impacts fear extinction processes. Thus, in the current study we also assessed the impacts of HIV and PTSD on the ratio of peripheral gene expression of ERα to peripheral gene expression of ERβ. We hypothesized that both HIV and PTSD would be associated with differences in ERα to ERβ gene expression ratios. Methods: Women (18-65 yrs) recruited from the Women’s Interagency HIV Study (WIHS) in Atlanta, GA (n = 70, 26 without HIV, 44 with HIV) provided informed consent and underwent a semi-structured interview to assess adult (Traumatic Events Inventory; TEI) and childhood trauma (Childhood Trauma Questionnaire; CTQ) exposure, as well as PTSD using the PTSD Checklist for DSM-5 (PCL-5). Participants also underwent a fear-potentiated startle (FPS) paradigm to assess fear extinction. Blood samples were collected within two weeks of the FPS paradigm to assess peripheral gene expression of ERα and ERβ. A ratio of ERα to ERβ gene expression was calculated. ANCOVAs assessing the effects of HIV, PTSD, and their interaction on fear extinction and ERα/ERβ expression controlled for age, income, education, and adult trauma exposure were conducted. Results: PTSD diagnosis (p = 0.021; η2 = 0.088) and HIV status (p = 0.046; η2 = 0.067) were both associated with deficits in the percent decrease of fear extinction. There was also a significant interaction of HIV serostatus and PTSD diagnosis (p = 0.023; η2 = 0.082) on fear extinction, as HIV was associated with impaired fear extinction only in women with a PTSD diagnosis (p = 0.026; partial eta2 = 0.078). Although there were no main effects of PTSD or HIV status on ERα/ERβ expression, there was a significant interaction between PTSD and HIV (p = 0.006; η2 = 0.142). ERα/ERβ expression was significantly lower in women without PTSD and with HIV (p = 0.007), and women with HIV and without PTSD (p < 0.001) compared to women without either HIV or PTSD. Conclusions: Our findings suggest HIV impacts fear psychophysiology in women with a PTSD diagnosis, demonstrating deficits in fear extinction. Given the use of prolonged exposure as a prominent evidence-based therapy for PTSD, these findings are clinically relevant, suggesting that HIV may impair the efficacy of such treatment in women living with HIV. Our findings also suggest that HIV and PTSD impact ERα/ERβ expression in women, but not in a synergistic manner. Future studies are necessary to determine how alterations in expression of ERα/ERβ may contribute to deficits in fear extinction in women living with HIV, including inflammatory pathways implicated in psychophysiological hyperarousal in trauma-exposed individuals. Keywords: PTSD, HIV, Fear Extinction, Estrogen Receptor, Women’s Mental Health Disclosure: Nothing to disclose. P85. Maternal-Infant Emotional Connection is Related to Reduced Physiological Stress Response in Mothers and Their Babies: Preliminary Results From a Trauma-Exposed Sample of Primarily Black Mother-Infant Dyads Abigail Powers, Catherine Abrams, Elizabeth McAfee, Shimarith Wallace, David O’Banion, Rebecca Hinrichs, Vasiliki Michopoulos, Jennifer Stevens Emory University School of Medicine, Atlanta, Georgia, United States Background: Trauma and posttraumatic stress disorder (PTSD) related risks are transmitted across generations, yet little is known about early pathways of risk or resilience in the context of maternal PTSD. Infancy is a critical time for socioemotional development. Emotional connection (EC) between mother and infant is particularly important for an infant’s secure attachment and ability to regulate stress. Infant cardiac reactivity and low vagal tone, measured with heart-rate (HR) and respiratory sinus arrhythmia (RSA), are significantly impacted by parenting behaviors and may be an important early biomarker of risk for later vulnerability to trauma-related psychopathology. Additionally, heightened sympathetic arousal is a biomarker of PTSD, and maternal emotional reactivity may impact a mother’s ability to sensitively respond to their infant’s needs. However, no prior studies have examined how mother-infant EC relates to maternal and infant stress response or its relation to maternal PTSD. Thus, examining physiological reactivity patterns to stress in newborns and their mothers in a sample of women with high levels of PTSD symptoms and trauma exposure will aid in our understanding of potential pathways through which maternal PTSD impacts early emotional and stress regulation development in children at risk for negative outcomes. Methods: The current pilot study measured maternal PTSD symptoms, mother-infant emotional connection (EC), and maternal and infant stress response to the Double Still Face Paradigm (DSFP, 4m) in 20 mother-infant dyads (mean maternal age = 27; infant age = 4 months; 91% Black or African American) recruited from an urban safety net public hospital with high levels of trauma exposure and PTSD (45% met for probable PTSD diagnosis). DSFP is a laboratory stressor task used to assess infant response to maternal disengagement. Dyads completed a behavioral task assessing baseline EC and post-stressor EC using the Welch Emotional Connection Scale, which includes measurement of attraction, vocal, facial, and sensitivity scales and an overall EC score. PTSD symptoms were assessed using the PTSD Checklist for DSM-5. Infant respiratory sinus arrhythmia (RSA) and heart rate (HR) was measured during DSFP. Maternal skin conductance response (SCR) during DSFP was also obtained on a subsample of participants (n = 8). Bivariate correlations between maternal PTSD, baseline mother-infant EC, and physiological response to stress were analyzed. For infant stress response, baseline, initiation of first recovery phase, and final recovery phase time points were used. Bivariate correlations between PTSD symptoms and post-stressor EC were also run. Results: Bivariate correlation analyses indicated higher mother-infant EC was significantly associated with higher infant RSA response during initiation of the first recovery phase of DSFP (r = .48, p = .034), but not baseline (r = -.15, p = .53) or final recovery (r = -.33, p = .19). There were no significant associations between mother-infant EC and HR. Additionally, mother-infant EC was inversely related to levels of maximum maternal skin conductance response during the DSFP (r = -.85, p = .008, n = 8). No significant correlational associations between PTSD symptoms and baseline mother-infant EC or infant RSA or HR were found. However, higher levels of PTSD symptoms were related to lower post-stressor mother-infant EC for facial and sensitivity subscales (r = -.54, p = .031 and r = -.52, p = .041, respectively; n = 16). Conclusions: Results suggest that EC in mother-infant dyads relates to how both mothers and their infants respond to stress and point to an important potential pathway through which intergenerational trauma and PTSD-related risk or resilience may occur. Keywords: Posttraumatic Stress Disorder, Intergenerational Transmission of Trauma, Psychophysiology Disclosure: Nothing to disclose. P86. Quality of Sleep Moderates Cardiovagal Reactivity to the Trier Social Stress in Black Women With PTSD Ida Fonkoue, Katherinne Fox, Sayra Medina Banuelos, Abigail Powers, H. Drew Dixon, Rachel Gluck, Guillermo Umpierrez, Tanja Jovanovic, Thaddeus Pace, Kathryn Cullen, Vasiliki Michopoulos, Charles Gillespie University of Minnesota Medical School, Shoreview, Minnesota, United States Background: Increased rates of trauma exposure are strongly associated with poverty. Women exposed to trauma develop post-traumatic stress disorder PTSD at twice the rate observed in than men. Previous research suggests that presence of PTSD is associated with poor sleep and greater risk of cardiometabolic diseases. However, given the possible link between poor sleep and cardiometabolic diseases, it remains unclear whether sleep disturbance is the mechanism through which PTSD leads to greater cardiovascular disease risk. In this study, we examined the impact of PTSD diagnosis and symptom severity on cardiovascular and autonomic responses to an acute psychological stress. In addition, we examined the effect of sleep quality and comorbid depression on these responses. We hypothesized that women with a diagnosis of PTSD will have exaggerated blood pressure, heart rate and sympathetic reactivity to the Trier Social Stress Test (TSST), and that these responses will be moderated by sleep quality. Methods: To test this hypothesis, we recruited 74 black women (age between 31 and 63 years old) with a history of trauma exposure and type 2 diabetes from a large county hospital in the southeastern United States. We measured cardiovascular and sympathovagal response during the TSST in women with (n = 45) and without (n = 29) a current diagnosis of PTSD. Categorical diagnosis of PTSD and PTSD symptom severity were determined using the Clinician Administered PTSD Scale (CAPS), depressive symptom severity was assessed with the Beck Depression Inventory (BDI) and quality of sleep was assessed with the Pittsburgh Sleep Quality Index (PSQI). Baseline systolic and diastolic blood pressure (BP) was measured via automated SphygmoCor. Participants underwent the 20-minute Tier Social Stress Test (TSST) and were seated in a quiet room before and after the standardized psychosocial stressor test. Heart rate (HR) and Low-Frequency to High Frequency Ratio (LF/HF) were obtained via 3-lead ECG measured before, during and after TSST. Pearson correlation was used to probe for the relationship between CAPS, BDI and PSQI scores. BP, HR and LF/HF reactivity was analyzed using repeated measures ANOVA with time as within factor and PTSD diagnostic as between factor. PSQI and BDI were covariates in the analysis. Results: We found significant correlation between PSQI and CAPS severity (r = 0.47, p = <0.001) and between PSQI and BDI (r = 0.58, p = <0.001. Heart rate response to TSST was significantly higher in the PTSD group vs. the non-PTSD group (time, p = <0.001; group, p = 0.02). LF/HF response was also significantly higher in those with PTSD vs. those without (time, p = 0.46; group, p = 0.05). However, systolic blood pressure response was not different between groups (time, p = <0.001; group, p = 0.22). Given the strong correlation between PSQI and BDI, we chose to control for one of the two variables in the repeated measures ANOVA. Sleep was selected because of the literature supporting the link between sleep deprivation and exaggerated sympathetic response to acute stress in women. When controlling for sleep, the difference in heart rate (group, p = 0.05) and LF/HF (group, p = 0.044) reactivity between the group was affected. Conclusions: Our results show that PTSD status is associated with heart rate over-reactivity and autonomic imbalance during a standardized psychosocial stressor in this sample of traumatized black women with type 2 diabetes. Furthermore, we observed that sleep quality moderates that response. This suggests that sleep disturbances are a hallmark of PTSD and might be the factor driving the reported sympathetic overactivity to acute stress described in PTSD. Therefore, improvement of sleep quality may serve as a target for interventions aiming at reducing cardiovascular risk associated with PTSD. Funding: Supported by R01MH099211 (CFG). Keywords: PTSD, Women, Sleep, Heart Rate Variability, Heart Rate Disclosure: Nothing to disclose. P87. Plasticity in Dissociable Neural Circuits Supports the Divergent Consequences of Severe Stress Zachary Pennington, Alexa LaBanca, Zhe Dong, Patlapa Sompolpong, Denise Cai Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Acute severe stress is able to produce an array of changes in fear and anxiety-like behavior, including defensive responses to stress-associated stimuli, heightened reactions to novel aversive events, and increases in anxiety-like behavior. Whether these diverse consequences of severe stress arise from plasticity in a common circuit, or if distinct circuits support the various consequences of severe stress, remains unknown. Addressing this question is fundamental to understanding how the pathological consequences of stress might be mitigated. Here, utilizing a combination of region-specific protein synthesis inhibition, chemogenetics, and activity-dependent cell tagging, we show that stress-induced plasticity within the ventral hippocampus (vHC) and basolateral amygdala (BLA) support dissociable consequences of severe stress. Methods: Experiment 1 examined the role of stress-induced protein synthesis in the vHC and BLA on subsequent behavior. Mice were exposed to a an acute severe stressor in which they received 10 foot-shocks, or not, and protein synthesis in either structure was blocked just after. A week later, associative fear of the stressor was examined by placing the animals back in the stressor environment and measuring freezing, anxiety-like behavior was assessed in a light-dark test, and learning about novel aversive events was assessed by exposing them to a loud auditory stressor in a distinct conditioning chamber. Experiment 2 then assessed how the BLA and vHC support the expression of post-stress behavioral changes. An identical behavior procedure to that of Experiment 1 was used, but chemogenetics were utilized to inhibit the vHC or BLA during the expression of post-stress behavioral phenotypes. Lastly, Experiment 3 utilized a transgenic activity-dependent tagging approach to permanently label stress-reactive cells and then looked at their reactivation in response to subsequent events utilizing of immunohistochemistry. n = 8-12/group. Male mice were used for these experiments. Results: In Experiment 1, blockade of stress-induced protein synthesis in the BLA profoundly reduced subsequent associative freezing in the stressor context and mitigated heightened responses to a novel stressor, but had no impact on the anxiety-like behavior of the same animals. Conversely, blockade of stress-induced protein synthesis in the vHC attenuated changes in anxiety-like behavior but had no impact on responses to novel stressors and had minimal impact on associative freezing. In Experiment 2, using chemogenetics, we demonstrated that neural activity in BLA and vHC have similar dissociable contributions to the expression of these post-stress phenotypes. Lastly, in Experiment 3, we found that stress reactive ensembles in the BLA (but not the vHC) are more likely to be reactivated by novel stressors, suggesting that stress-induced plasticity specifically within this cell population supports the heightened response to novel stressors Conclusions: Collectively, these results indicate that plasticity in separate BLA and vHC circuits support distinct impacts of stress on behavior, and ongoing research aims to elaborate on the unique role of stress-reactive ensembles. Critically, these results suggest that the impacts of severe stress are biologically divergent. If true, this might indicate that interventions geared towards one these post-stress phenotypes (e.g., associative memories) in conditions like PTSD may not affect other post-stress phenotypes (e.g., anxiety). Moreover, by targeting the specific cells within these networks that are activated by stress, we might be able to mitigate post-stress phenotypes. Keywords: Fear, Anxiety, Amygdala, Hippocampus Disclosure: Nothing to disclose. P88. Examining the Role of BNST CRF in Models of Aversive Learning Thomas Kash, Olivia Hon University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: Anxiety disorders affect roughly one third of the population and a core symptom of anxiety and trauma-related disorders is excessive and generalized fear. While identifying and responding to threats that predict danger is essential for survival, persistent apprehension about future threats can become maladaptive. Recent work suggests that the predictability of if and when danger will occur plays a critical role in development of pathological anxiety. Recent studies in rodents have shown that unpredictable, but not predictable threat engages the bed nucleus of the stria terminalis (BNST), a part of the extended amygdala that has long been studied with respect to stress, anxiety and drugs of abuse. Methods: We used multiple approaches to probe this question. To determine if CRF in the BNST regulates fear and antecedent behaviors, we used viral deletion of CRF coupled to behavior in the mouse brain. To determine activity profiles of neurons and modulators, we used fiber photometry and miniscope recordings in mice. Results: First we found that partially (PRF), but not fully reinforced fear (FRF) conditioning led to exaggerated acoustic startle response in males but not females. Next, we used multi-region fiber photometry to characterize BNST cell body and terminal dynamics in the periaqueductal gray, a midbrain structure that regulates adaptive motor responses. We found that during fear conditioning, BNST cell body and terminal activity did not differ between fear groups, but that differences emerged during fear recall 24 hours later. Interestingly, the size of BNST responses during conditioning positively correlated with recall freezing behavior in PRF but not FRF mice. We next were curious about the modulatory signaling that drove these behaviors. Extensive work from Mike Davis and others over the years have suggested that the neuropeptide Corticotropin Releasing Factor (CRF) played an important role in this behavior. Thus, we aimed to determine the role of BNST CRF neurons in partially reinforced fear in male and female mice. Using a transgenic floxed CRF line, we found that knockdown of CRF in the BNST selectively enhanced PRF in females but not males. Next, we implanted GRIN lenses and used miniature head-mounted microscopy to record individual BNSTCRF cells during behavior. We found that in both sexes, BNSTCRF activity was higher after conditioning in partially reinforced compared to fully reinforced fear mice, and that cells responded differently during fear recall across groups and sexes. Finally, using fiber photometry coupled to a novel CRF biosensor, we identified differences in CRF release in the PAG but not BNST during fear recall across groups. Conclusions: Overall, we find sex-specific effects of BNSTCRF signaling in partially and fully reinforced fear, suggesting potential sex-dependent treatment strategies based on CRF modulation. Notably we find that deletion of BNST CRF leads to enhanced post fear startle in male mice exposed to reliable fear, suggesting fear evoked release of CRF constrains arousal. Keywords: Amygdala, Neuropeptides, Acoustic Startle Response Disclosure: Nothing to disclose. P89. Adolescent Chronic Social Defeat Produces Robust Social Avoidance and Changes in Neural Activation in the Adult Mouse Erin Hisey, Emily Newman, Kerry Ressler Harvard Medical School, McLean Hospital, Belmont, Massachusetts, United States Background: Adolescence is a time of monumental cognitive and neurobiological changes. Trauma during this time period can in turn alter normal brain development and result in lifelong psychiatric disorders. While models of early life neglect in rodents do exist, models of early life trauma are nearly absent. In order to better understand the neurobiological underpinnings of early life trauma on the adult brain, we have developed a model of adolescent chronic social defeat in male mice. Methods: Adolescent chronic social defeat stress (aCSDS): On the first day of aCSDS, an experimental C57BL/6J (C57) male mouse (P27 + /-2 days) is placed directly into an adult (>8 weeks) CFW male resident’s territory for a 3-minute aggressive encounter (ended after 30 bites). After the aggressive encounter, the experimental C57 mouse is housed adjacent to that resident for 24 hours until the aggressive encounter on the next day. This procedure of defeat and housing is repeated with a novel resident each day for 10 days. Non-defeated control C57 mice are housed in divided home cages identical to those used for defeat next to an unfamiliar control C57 mouse. On the tenth day of aCSDS, defeated and control C57 mice are housed side-by-side in divided cages that allow both olfactory and visual contact until adulthood. Open Field Social Interaction with CFW male (OFSI): After 1 hour of habituation to the testing room, C57 male mice (P50 for late adolescent testing, P70 for adult testing) are placed opposite an inverted black wire cup in a large (44 x 44 cm) arena. After 150 seconds of exploration of the arena and empty cup, the C57 is placed back in the arena with the cup now containing a novel nonaggressive CFW male inside and allowed to explore for 150 seconds. All interactions are captured and scored with Ethovision. Social interaction ratio is calculated as time spent with the empty cup divided by time spent with the cup with a CFW inside. Home Cage Social Interaction with ovariectomized CFW female (HCSI): Adolescent (~P50) C57 male mice remain in their home cage and an ovariectomized CFW female is placed inside the home cage for 90 seconds. If any biting is observed from either male or female, interaction is immediately ended, and the female is replaced with another female. The interaction is recorded at 30 fps and later analyzed for specific behaviors with DeepLabCut. Whole Brain c-fos imaging: After OFSI, mice are placed in a clean empty cage in a quiet room for 1 hour and then perfused with 1x PBS followed by 4% PFA. Brains are removed and placed in PFA overnight then stored in 0.02% sodium azide PBS. Only brains without any visible redness or damage were used for tissue clearing followed by whole brain c-fos labeling and imaging (LifeCanvas Technologies). Results: After chronic defeat exposure in early adolescence, mice display robust social avoidance that is maintained throughout late adolescence and adulthood (t-test for OFSI social interaction ratio with nonaggressive male CFW; n = 40 defeated, 38 control male mice; in late adolescence (~P50), p < 0.0001; in adulthood (>P70), p < 0.0001). This social avoidance behavior also generalizes to female mice of the same strain as those used as aggressors during defeat (HCSI with female CFW at ~P50 characterized with DeepLabCut, n = 14 defeated, 14 control; t-test for reductions in following (p < 0.001) and contact (p < 0.01), increases in defensive posturing (p < 0.001) and freezing (p < 0.05) in defeated mice compared to controls). Re-exposure to a nonaggressive male mouse during adulthood drives dramatic increases in c-fos activity, with changes in network connectivity, in the brains of male mice defeated as adolescents, as revealed by whole brain c-fos imaging. Significant increases in c-fos expression in comparison to control mice were found in hippocampus (CA3), medial and lateral entorhinal cortex and anterior insula (n = 6 defeated brains, 6 control brains, t-test for comparison of defeated and control brains, p < 0.05). Conclusions: Our aCSDS model produces robust and long-lasting deficits in social interaction which allows us to examine the neural circuits underlying avoidant behavior, a hallmark of neuropsychiatric disorders such as generalized anxiety and PTSD, in the aftermath of adolescent social defeat stress. One of the strongest areas of activation after adult social interaction in mice defeated as adolescents is CA3, a region implicated in pattern completion. Intriguingly, changes in hippocampal volume and connectivity are among the most robust findings in human imaging studies of adults that experienced trauma during childhood and adolescence. We are currently using 10x RNA sequencing to compare differential gene expression in CA3 of adult mice defeated as juveniles to that of CA3 in adult humans with a history of early life trauma. By using this reverse translational approach, we hope to better understand how circuits are altered after early life trauma, with the hopes of developing more effective treatments for psychiatric disorders resulting from early life trauma. Keywords: Adolescent Stress, Chronic Social Defeat Stress, Early Trauma Disclosure: Nothing to disclose. P90. Norepinephrine Dynamics Represent Threat Prediction Errors Under Uncertainty Aakash Basu, Abigail Yu, Jen-Hau Yang, Samira Glaeser-Khan, Yulong Li, Alfred Kaye Yale University, New Haven, Connecticut, United States Background: Posttraumatic stress disorder involves an internal state of hypervigilance to reminders of prior traumatic experiences. Norepinephrine (NE) has been proposed as a mediator of this process, since excessive NE release after stress induces anxiety in animals and exacerbates PTSD symptoms in humans. Understanding the moment to moment processing of traumatic memories and how they alter threat computation via internal states thus requires direct measurement of NE over the course of traumatic memory expression. Methods: We expressed the fluorescent norepinephrine sensor GRAB-NE2h in the mouse medial prefrontal cortex (mPFC) using AAV9. We measured NE release via fiber photometry during fear conditioning while varying the delay between cues and outcomes (in groups of n = 10-13 animals). NE dynamics were measured over the course of 20 training trials and 20 extinction trials. Reinforcement learning models were fit to cue- and footshock-induced NE release over the course of learning and extinction. Models included 1) temporal difference learning, 2) TD learning with belief states (Starkweather et al 2017), and 3) TD learning with state uncertainty (Mikhael et al 2022). Results: NE is released during footshock, and NE release during threat-predictive cues increases with repeated cue/shock associations (p = 0.0026, t = 3.882, forward 1st training vs 11th training cue). Cue-evoked NE persists in the absence of cue-shock pairings (p < 0.0001, t = 7.606, forward 1st training vs 1st extinction cue)). Trace fear conditioning also increases cue-evoked NE release in 5 second-trace conditioning(p = 0.0011, t = 4.385, 1st training vs 1st testing cue) but not 15s (p = 0.0806, t = 1.968, 1st training vs 1st testing cue) or 30s trace conditioning (p = 0.4501, t = 0.7896, 1st training vs 1st testing cue), and testing cue evoked NE decreases with increased trace length (Trace: F(2,30)=7.136, p = 0.0029). When modeling trace conditioning using TD learning under state uncertainty, the prediction error term recapitulated several important features of NE release, such as sustained cue-evoked release, cue-offset evoked release, and release suppression during trace periods. Conclusions: Our results suggest that prefrontal norepinephrine represents a more precise threat prediction error signal than has previously been described. Reminders of a traumatic event induce NE release that scales with the intensity of the threat memory. Reinforcement learning models of threat required modification to incorporate constant feedback from the environment and temporal uncertainty to reproduce observed patterns of NE release. Thus, NE appears to multiplex threat and uncertainty during retrieval of fear memories, offering a computationally rich signal for navigating dangerous environments. Keywords: Norepinephrine, Anxiety Circuitry, Reinforcement Learning, Optical Biosensors, Uncertainty Disclosures: Transcend Therapeutics, Freedom Biosciences: Contracted Research (Self) P91. Cell-Type-Specific Dopamine Signaling in Ventral Hippocampus Tracks Anxiety-Related Behaviors Arthur Godino, Marine Salery, Angelica Minier-Toribio, Vishwendra Patel, John Fullard, Eric Parise, Carole Morel, Sarah Montgomery, Scott Russo, Panos Roussos, Robert Blitzer, Eric Nestler Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Despite accumulating evidence for both the ventral hippocampus (vHipp) and the mesocorticolimbic dopamine system in encoding anxiety-relevant information, surprisingly little is known about how dopamine signaling selectively affects vHipp representations of emotionally-salient stimuli to guide approach/avoidance behaviors. To address these shortcomings, we here study dopaminoceptive neurons in mouse vHipp – which can be segregated based on their expression of either the dopamine D1 or D2 receptor – to delineate a model of dopamine action in vHipp. Methods: We capitalized on D1-Cre and D2-Cre mouse lines to visualize, record and manipulate vHipp D1 and D2 cells. The reporters lines were used for anatomy, snRNA-sequencing, slice electrophysiology, and cell-type-specific in vivo calcium imaging, chemogenetics and optogenetics. Fluorescent dopamine sensors were used to record dopamine release in vHipp. Results: At the histological level, D1- and D2-expressing cells exhibit a precise topographical organization across vHipp subfields, which we further dissected using RNA-sequencing of single, sorted nuclei from D1 and D2 cells. We also confirmed dopamine’s pharmacological effects on the electrophysiological properties of these cell types. Functionally, we found that anxiogenic environments and approach/avoidance conflicts trigger distinct patterns of calcium activity in D1 vs. D2 vHipp neurons in concert with dopamine release in in this region. Bidirectional chemogenetic and optogenetic manipulation of D1 or D2 vHipp neuron activity causally demonstrated their opposite roles in mediating anxiety and approach/avoidance behaviors. Intriguingly, vHipp dopaminoceptive mechanisms also contribute to cocaine-related behaviors, suggesting drug-induced plasticity in this circuit as well. Conclusions: Together, we propose that dopamine dynamics in vHipp operate as a feedback loop that bidirectionally tracks anxiety levels to gate exploratory behaviors through differential recruitment of vHipp D1 and D2 neurons, which in turn mediate opposite approach/avoidance and anxiety-like responses. This work paves the way for further studies of dopamine signal processing in limbic regions, and underscores the complexity of the circuit mechanisms that govern affective states. Keywords: Anxiety, Dopamine, In vivo Imaging Disclosure: Nothing to disclose. P92. Early Life Adversity in Mice Leads to Heightened Threat Reactivity and Sex-Differences in Activity of Corticotrophin Releasing Hormone Neurons in the Central Amygdala Camila Demaestri, Margaux Pisciotta, Naira Altunkeser, Madalyn Critz, Dayshalis Ofray, Kevin G. Bath Columbia University, New York, New York, United States Background: Experiencing early life adversity (ELA), such as resource scarcity, is associated with an increased risk for anxiety-related disorders. Females are twice as likely to develop anxiety-related disorders compared to males. A core feature of these disorders is enhanced reactivity to real and perceived threat. Sex biases in risk and symptom presentation may be related to changes in corticotropin-releasing hormone (Crh), a neuropeptide that is released in response to stress and is important for driving hyperarousal associated with threat reactivity. In males, ELA has been associated with changes in Crh levels in the amygdala, however, the effects on females and the impact on threat reactivity is still largely unexplored. Methods: We used the limited bedding and nesting (LBN) model of ELA in mice to test its effects on fear-potentiated startle, a translationally relevant behavioral phenotype used to assess threat reactivity, and the activity of Crh+ cells in the amygdala. Mice underwent LBN rearing from postnatal day 4 (PD4) to PD11 and were tested on the fear-potentiated startle task as adults ~PD70. The magnitude of startle was measured during both moderate- and high- threat anticipation states, in which mice were presented with the startling 50ms white noise either randomly (moderate anticipation) or preceded by a tone previously associated with a foot shock (high anticipation). The activity of Crh+ neurons in the central amygdala was measured using two methods. First, the number of active Crh+ neurons was quantified post-testing using cFos as a marker of neuronal activity. In a separate cohort of mice, in-vivo Ca2+ of Crh+ neurons was measured during fear-potentiated startle using fiber photometry. Two-way ANOVAs were used to test for main effects of rearing (control x LBN), sex (male x female) and an interaction on the startle response to the random and cued white noise (n = 15-17/group), on the percent of Crh+ neurons co-expressing cFos (n = 6-7/group), and on fiber photometry z-score and AUC calculations (n = 5-7/group). Results: We found that LBN enhanced acoustic startle both when the startling white noise was preceded by the conditioned tone (rearing: F(1,57)=7.37, p = 0.008, eta2partial = 0.11) and when presented randomly (rearing: F(1,57)=9.61, p = 0.003, eta2partial = 0.14) indicating heightened threat reactivity across both high and moderate anticipatory states. This effect was more robustly observed in LBN females, who show greater change in startle compared to control females. LBN female mice also had increased co-labeling of cFos+ and Crh+ in the central amygdala (interaction: F(1,23)=4.29, p = 0.04), eta2partial = 0.15) and sustained Ca2+ activity of Crh+ neurons during the conditioned tone (interaction: F(1,23)=4.63, p = 0.04), eta2partial = 0.16), suggesting a role of Crh+ central amygdala neurons in threat anticipation. Conclusions: The current work identifies a potential sex-specific mechanism for enhanced threat responding as a result of ELA. Ongoing studies will lay the groundwork for understanding sex differences in pathology and will contribute to the improvement of individualized treatment and interventions informed by the prior experience and sex of the individual. Keywords: Early-Life Adversity, Fear-Potentiated Startle, Corticotropin-Releasing Hormone, Sex-Differences Disclosure: Nothing to disclose. P93. CRF1 Receptor-Expressing Central Amygdala Projections to Lateral Hypothalamus Mediate Stress-Induced Escalation of Alcohol Self-Administration and Anxiety-Like Behavior Marcus Weera, Jason Middleton, Nicholas Gilpin LSU Health New Orleans, New Orleans, Louisiana, United States Background: In humans, stressful events may elicit avoidance coping (i.e., persistent avoidance of stress-associated stimuli) in some individuals, which is associated with negative consequences such as increased alcohol drinking and increased anxiety. Using rats, our lab has shown that predator odor (i.e., bobcat urine) stress produces persistent avoidance of a stress-paired context in a subset of subjects, termed ‘Avoiders’. Interestingly, Avoider rats display long-lasting increases in alcohol self-administration (SA) following stress exposure, a phenomenon that is absent in stress-exposed Non-Avoiders, and that recapitulates findings in humans. Our published work show that antagonism of CRF1 receptors in the central amygdala (CeA) rescues stress-induced escalation of alcohol SA and blunts avoidance of a stress-paired context in Avoider rats. In addition, we have demonstrated that avoidance of a stress-paired context is mediated by CeA neurons that project to the lateral hypothalamus (LH), a brain region that is known to modulate motivated behaviors. The main goal of this study is to test the hypothesis that stress-induced escalation of alcohol SA and avoidance behavior is supported by activation of CRF1 receptor-expressing CeA-LH neurons. Methods: All experiments were performed in male and female CRF1-Cre-tdTomato rats that were generated in our lab. In Experiment 1, we used a combination of retrograde tracing and c-Fos immunohistochemistry to test the hypothesis that stress produces more c-Fos+ CRF1 CeA-LH neurons in Avoiders compared to Non-Avoiders (N = 11-14 rats/group). In Experiment 2, we used ex vivo slice electrophysiology to characterize the synaptic transmission and intrinsic properties of CRF1 + CeA-LH neurons in stressed Avoiders and Non-Avoiders, and unstressed Controls (N = 38-42 cells/group). In Experiment 3, we used chemogenetics to test the hypothesis that in vivo inhibition of CRF1 CeA-LH neurons rescues stress-induced escalation of alcohol SA, avoidance of a stress-paired context, and anxiety-like behavior as measured using the elevated plus maze (N = 7-11 rats/group). In Experiment 4, to inform future hypotheses, we used a Cre-dependent viral-mediated anterograde tracing approach to identify additional brain areas that receive projections from CRF1 + CeA neurons (N = 4 rats). Data were analyzed using multifactorial ANOVAs and Tukey’s post-hoc tests where appropriate. Results: In Experiment 1, stress increased the number of c-Fos+ CRF1 CeA-LH neurons in Avoider rats (p < 0.01). In Experiment 2, we found that stress blunts sIPSC frequencies in CRF1 CeA-LH neurons (p < 0.05), which may contribute to increased excitability of these neurons. In Experiment 3, chemogenetic inhibition of CRF1 CeA-LH neurons rescued stress-induced escalation of alcohol SA (p < 0.05), anxiety-like behavior, but not avoidance of a stress-paired context. In Experiment 4, Cre-mediated anterograde tracing showed that CRF1 CeA neurons project to a variety of brain areas (in addition to the LH), including the nucleus accumbens, insula, lateral septum, bed nucleus of stria terminalis, lateral preoptic area, ventromedial hypothalamus, medial amygdala, lateral habenula, paraventricular nucleus of the thalamus, zona inserta, piriform cortex, and periaqueductal gray. In all experiments, no sex differences were detected. Conclusions: These results suggest that increased excitability of CRF1-expressing CeA-LH neurons supports escalated alcohol consumption and anxiety-like behavior in Avoider rats following stress exposure. Current work is focused on elucidating the extended neurocircuitry that contributes to this phenomenon. Future work focused on identifying the molecular mechanisms that modulate the activity CRF1 CeA-LH neurons and related neurocircuitry may unveil novel targets for the management of alcohol misuse and anxiety related to stress. Keywords: Anxiety and Stress, Alcohol, Central Amygdala Disclosure: Roche In Vitro Diagnostics: Consultant (Spouse) P94. Suppressing Fear in the Presence of a Safety Cue Requires Infralimbic Cortical Signaling to Central Amygdala Ka Ng, Michael Pollock, Abraham Escobedo, Brent Bachman, Susan Sangha Indiana University, Indianapolis, Indiana, United States Background: Stressful events can have lasting and impactful effects on behavior, especially by disrupting normal regulation of fear and reward processing. Post-traumatic stress disorder (PTSD) has become a growing public health concern, with a lifetime prevalence of ~6.4% of the US population. Increasing evidence suggests PTSD may alter the ability to discriminate among different stimuli, in particular where PTSD disrupts the ability to use signals that indicate safety, and overgeneralize the impact of fear signals. Our lab uses a unique rodent behavioral task that allows us to examine stimulus discrimination alongside regulation of fear and reward processing behaviors. Male Long Evans rats show significant discrimination among cues representing reward, fear and safety. Specifically, they spend more time reward seeking during a reward cue predicting sucrose compared to a fear or safety cue, and more time freezing during a fear cue predicting footshock compared to a reward or safety cue. Critically, they also show suppressed freezing levels when the fear cue is presented concurrently with the safety cue but without footshock (fear+safety cue). Prior stress exposure reduces this fear suppression effect. Our prior work has shown areas of the prefrontal cortex and amygdala contribute to this learning. However, the precise connections amongst these subregions in the ability to express learned safety have not been previously tested. Methods: Adult male Long Evans rats (n = 25-30) were used in the present study since we have previously shown that female rats do not learn to suppress fear in the presence of a safety cue. Using a dual virus approach, we expressed mCherry-tagged hM4D(Gi) receptors in the infralimbic cortex (IL) targeting either basolateral amygdala (BLA) projecting or central amygdala (CeA) projecting neurons for CNO-induced (3mg/kg, i.p.) neuronal inhibition. All rats underwent stimulus discrimination training where a reward cue was paired with sucrose, a fear cue paired with footshock, a safety cue paired with neither sucrose or footshock, and a compound fear+safety cue without footshock. All rats received vehicle i.p. injections across the first 3 discrimination sessions. Then, using a within subjects design, rats received either vehicle or CNO during the 4th and 5th discrimination sessions. Freezing and reward seeking behaviors were quantified to each cue and collapsed across the 4th and 5th discrimination sessions followed with 2-way repeated measures ANOVAs comparing drug and cue. Results: We show that the infralimbic projection to the central amygdala is necessary for suppressing freezing in the presence of the safety cue, while the projection to the basolateral amygdala is not. Specifically, only CNO inhibition of IL-CeA neurons resulted in a failure to suppress freezing during the fear+safety cue compared to the fear cue (p > 0.05). In contrast, freezing was significantly lower to the fear+safety cue than the fear cue in the same rats (n = 5) under vehicle conditions (p < 0.05), and for rats (n = 6) with unconfirmed mCherry expression injected with either CNO (p < 0.01) or vehicle (p < 0.05). Conclusions: The loss of discriminative fear regulation seen specifically during IL-CeA inhibition is similar to the disruption seen in PTSD individuals that fail to regulate fear in the presence of a safety cue. Keywords: Safety Learning, Fear Regulation, Amygdala, Medial Prefrontal Cortex Disclosure: Nothing to disclose. P95. Visualizing the Longitudinal Development of Stress-Induced Anhedonia From Representations of Valence in the PFC Austin Coley, Jeremy Delahanty, Assaf Ramot, Rachelle Pamintuan, Lexe Linderhof, Vivian Liu, Caroline Jia, Harini Advikoluna, Sama Shathaya, Kanha Batra, Deryn LeDuke, Felix Taschbach, Romy Wichmann, Hao Li, Kyle Fischer, Marcus Benna, Takaki Komiyama, Kay Tye The Salk Institute for Biological Studies, La Jolla, California, United States Background: A critical issue within the mental health field is the lack of granularity in diagnostic practices. For example, a patient that is sleeping and eating too much may be prescribed the same antidepressant as a patient who is sleeping and eating too little. This may explain the low rates of efficacy for first line antidepressants and begs further dissection beyond the DSM. Anhedonia, described as the inability to experience pleasure, is a core feature expressed in both major depressive disorder and schizophrenia. Anhedonia is linked to a dysregulation within the brain reward pathways that includes the medial prefrontal cortex (mPFC), which is highly involved in emotional and valence processing, critical for encoding hedonic values. Dopamine (DA) transmission tightly regulates mPFC cortical activity and associated behavior and is implicated in anhedonia. However, it remains unknown how DA modulates mPFC valence-specific neuronal population activity during stress-induced anhedonia. Methods: We implemented learned helplessness (LH) and chronic mild stress (CMS) protocols to induce anhedonia in mice. We designed an anhedonic phenotype score for individual mice. To investigate the effects of DA modulation within mPFC valence-specific neuronal populations during anhedonia, we performed longitudinal in vivo 2-photon calcium imaging with optogenetics techniques to photostimulate DA ventral tegmental area (VTA) inputs in the mPFC while measuring mPFC population activity and dynamics during a Pavlovian conditioning task in mice exposed to LH and CMS. Both males and females were used in this study. Results: Our preliminary findings showed a significant reduction in reward consumption and sociability in LH mice (Pearson Correlation, r = -0.69, p = 0.03), but not CMS mice (Pearson Correlation, r = -0.16, p = 0.58), suggesting a difference in behavioral phenotypes depending on the stress. Following initial exposure to CMS, we observed a significant decrease in mPFC activity during aversive trials in susceptible mice compared to resilient and control groups (One-way ANOVA (F(2,398)=13.66, p < 0.001; Tukey-Kramer post hoc Susceptible/Resilient, p = 0.004, Susceptible/Control, p < 0.001, Resilient/Control, p = 0.18). Interestingly, susceptible mice revealed a significant increase in activity during reward trials compared to resilient groups (One-way ANOVA (F(2,398)=7.26, p = 0.0008; Tukey-Kramer post hoc Susceptible/Resilient, p = 0.002, Susceptible/Control, p = 0.999, Resilient/Control, p = 0.003), but no change in control mice. Conclusions: These results indicate that anhedonic states influence mPFC valence encoding properties, and that acute, severe stressors can lead to distinct etiologies from chronic, mild stress. Altogether, these experiments point to the need for increased granularity in the measurement of both behavior and neural activity, as these factors can decode the induction conditions of stress-induced anhedonia, as well as the underlying biological pathology, propelling us towards a future of individualized medicine. Keywords: Valence, Anxiety and Depression, Medial Prefrontal Cortex Disclosure: Nothing to disclose. P96. The Hippocampal Spatial Code is Dynamically Altered During Context Fear Discrimination Robert Rozeske, Leonie Runtz, Aaron Sossin, Alexandra Keinath, Mark Brandon University of Toronto, Scarborough, Toronto, Canada Background: Assessing a situation as either threatening or safe is critical for our survival. However, this process can be impaired in individuals with post-traumatic stress disorder (PTSD). Consequently, neutral environments may not be discriminated from a traumatic memory and lead to re-experiencing the psychological distress of the original trauma. One theory for the pathophysiology of PTSD is impaired context processing. Prior work indicates that contexts are represented in the hippocampus (HPC) by ensembles of ‘place cells’ that collectively construct a spatial map. Although the HPC is essential for episodic memory, the dynamics of the HPC spatial representation during the formation of an aversive memory, and the retrieval of a threatening or neutral memory, are not fully understood. Methods: To investigate context fear discrimination we used one-photon microendoscope calcium imaging in freely behaving male C57Bl/J6 mice (n = 8-11) during a novel context memory retrieval task. Mice received surgical AAV injection to express the calcium indicator GCaMP6f in CaMKII+ cells of HPC CA1; a GRIN lens was later implanted for imaging. A customized fear conditioning apparatus composed of audio speakers and a cylindrical LED screen (90 cm dia.) presented two different contexts, A and B, while mice remained in the same physical space. On Days 1 and 2, mice were placed in neutral context B in the AM and fear conditioned with footshock in context A in the PM. On Day 3 mice were placed in the apparatus for 12 minutes and context A and B presentations were repeatedly alternated at 2-minute intervals. Throughout the task HPC activity and freezing behaviour were recorded. To investigate spatial encoding, spike likelihoods were calculated from neuronal calcium transients and place cell rate maps were generated. For each mouse, neuronal activity was correlated with a population vector of the mouse’s place cell ensemble activity. This population vector correlation was used to assess the strength of the HPC spatial map at a given moment. A separate group of non-conditioned control mice (n = 4) underwent similar behavioural and CA1 recording procedures. Data analysis was performed with Matlab and statistical significance was calculated using Goodness-of-fit tests, non-parametric tests, Pearson correlation, and within-subject comparisons to shuffled controls. Results: We report three primary findings in our fear conditioning task: (1) On Day 1 fear conditioned mice significantly increased freezing (p < 0.01 Friedman) and were different from non-conditioned controls (p < 0.01 Kruskal-Wallis). Conditioned and non-conditioned mice had significantly different freezing on Day 2 conditioning (p < 0.01 Kruskal-Wallis). On Day 1 the HPC spatial map during fear conditioned context A was significantly remapped compared to neutral context B (p < 0.01 Kolmogorov-Smirnov; p < 0.05 Wilcoxon signed-rank for context A). (2) During Day 3 context “teleportation” freezing behaviour significantly changed during test (p < 0.01 Friedman) and was different between conditioned and control mice (p < 0.01 Kruskal-Wallis). The HPC spatial maps of contexts A and B were significantly altered during context transitions (p < 0.05 Wilcoxon signed-rank for conditioned mice). (3) During the time surrounding context transitions, stronger context A spatial maps were correlated with greater freezing and stronger context B maps were associated with less freezing (Pearson correlation p < 0.05). Conclusions: These results reveal the temporal relationship between the HPC spatial map and memory formation/retrieval processes. Despite an unchanged context, formation of an aversive memory destabilized and altered the spatial map. Moreover, context alteration in the “teleportation” studies illustrated the dynamic relationship between spatial map strength and fear memory expression. These studies bridge the field of spatial navigation with emotional behaviour and introduce a task to identify circuits of safe and dangerous context representations that may guide the development of targeted therapeutics for PTSD. Keywords: Contextual Fear, Fear Generalization, Hippocampus, PTSD, Spatial Memory Disclosure: Nothing to disclose. P97. Circuit-Reorganization and Transformation for Observational Contextual Fear Memory Takashi Kitamura, Joseph Terranova, Sachie Ogawa The University of Texas Southwestern Medical Center, Dallas, Texas, United States Background: The formation and maintenance of long-term memories for episodes are important for daily life. A feature of episodic memory is autobiographical; requires own experience accompanied with representation of the self in an episode and preserves at first-person viewpoint, which allow to distinguish own episode from other’s episode. While it has been speculated that the self-reference effect may be closely related to the transformation process from episodic memory to semantic memory in human, it remains unknown about the neural circuit mechanisms for the time-limited self-reference effect on memory recall. Methods: To direct examine the self-reference effect on fear memory recall in mice, we have established observational contextual fear conditioning (observational CFC) paradigm and examined the neural circuit mechanisms for recent (1 day) and remote memory (28 days) recall for observational CFC in the observer side and demonstrator side. Because, the observer mice showed the freezing behavior in the observer side but not in demonstrator side at recent time point, while the observer showed the freezing behavior in both at remote time point. Importantly, the enhanced freezing response in the demonstrator side is not due to the fear generalization. This behavioral model is suitable to evaluate the time-limited self-reference effect on observational fear memory recall. 10-15 weeks C57BL6J mice were used in the observer and the demonstrator. Experimenters were blinded with respect to the genotype of the mice and the experimental designs were counterbalanced. Equal numbers of animals from each experimental group (i.e. genotype, sex, age) were assigned. Sample size determination: Using data from our previous reports (Terranova et al., 2022, Neuron), we performed a power analysis with effect size at 0.9 and alpha value of 0.05 yielding a group size of 10 mice. Based on our previous studies (Kitamura et al., 2017, Science, Terranova et al., 2022, Neuron), we determined 6 mice per a group for histology. Statistical analysis: Comparisons between two-group were analyzed by unpaired t test or paired t test, if normally distributed, otherwise by Wilcoxon sum rank or signed-rank test. Multiple group comparisons were assessed using a one-way, two-way, or repeated-measures analysis of variance (ANOVA), followed by the post-hoc test when significant main effects were detected. The null hypothesis was rejected at the P < 0.05 level. Results: We found that dorsal hippocampal activity is crucial for the recent memory recall of observational CFC in the observer side (U = 13, P = 0.004, Mann-Whitney U test), while medial prefrontal cortical (mPFC) activity is essential for the remote memory recall of observational CFC in both observer and demonstrator sides (t19 = 2.42, P = 0.03, unpaired t-test). Consistently, the recent memory recall of observational CFC in the observer side increased Arc expression in dCA1 (t8 = 2.69, P = 0.03, unpaired t-test). but not mPFC (t8 = 0.74, P = 0.48, unpaired t-test), while the remote memory recall of observational CFC increased Arc expression in mPFC in both observer (t8 = 3.13, P = 0.01, unpaired t-test) and demonstrator side (t10 = 3.32, P = 0.01, unpaired t-test), but not dCA1. Optogenetic terminal inhibition further demonstrated that mPFC-BLA pathway is crucial for remote memory recall of observational CFC. Conclusions: Observational CFC memory is long-lastingly maintained. While observational CFC requires the dorsal HPC (dHPC) at recent time point, observational CFC requires the mPFC-basolateral amygdala (BLA) pathway at remote time point. Furthermore, we revealed that recent observational CFC memory can be only egocentrically retrieved, while remote observational CFC memory can be retrieved as both egocentric and allocentric recall depending on the animal’s context. Finally, we demonstrated that mPFC neural activity during observational CFC is necessary for the successful recall of both egocentric and allocentric observational CFC memory at the remote time point. Keywords: Dorsal Hippocampus, Medial Prefrontal Cortex, Observational Learning, Fear Conditioning, Mouse Models Disclosure: Nothing to disclose. P98. Cholinergic Interneuron Control of Cue-Dependent Salience and Aversive Learning Tanner Francis, Andrew Belilos, Christie Sanders, Ayesha Sengupta, Geoffrey Schoenbaum University of South Carolina, Columbia, South Carolina, United States Background: Surviving harmful environmental stimuli requires encoding for appropriate response to future threat. The Nucleus Accumbens (NAc) is a hub for processing motivational information, including aversive events, and dictates response to these salient events. Highly salient, aversive events promote phasic changes in striatal cholinergic interneuron (ChI) firing, which may contribute to learning. In addition, substance P is known to enhance ChI activity over the timescale of seconds to minutes, an increase that is observed following a pause in activity in response to aversive stimuli. Subsequent downstream signaling by enhanced ChI activity through substance P activation promotes plasticity on NAc medium spiny neurons (MSNs) via muscarinic 1 receptors, which may provide a synaptic substrate for learning about these salient, aversive events. Therefore, we aimed to test the hypothesis that enhanced acetylcholine release by substance P predicts and supports associative learning of salient aversive events. Methods: A Pavlovian fear conditioning paradigm was used to study aversive learning in mice and freezing in response to the associative cue was used as the primary measure of learning. To determine selective release dynamics of acetylcholine, mice were injected with an adeno-associated virus expressing the genetically encoded acetylcholine sensor GRAB-ACh3.0 in the NAc core and in vivo signals were measured using fiber photometry in wild-type C57Bl/6J mice during various phases of fear conditioning and recall. All GRAB-ACh signals were normalized by z-scoring and changes were quantified using the area under the curve. To determine acetylcholine responses to salient environmental features, mice received foot shocks at varying intensities (0.1-1.0 mA) at random intervals. To further test the role of acetylcholine in salience, a subset of mice underwent latent inhibition procedures to diminish salience of aversive cues. To assess the role of substance P in mediating acetylcholine signals and aversive learning, mice were injected with L-733,060, an antagonist of the primary receptor for substance P, the neurokinin 1 receptor (NK1R). In addition, a ChI-Cre transgenic mice were injected with a NK1R guide RNA in the presence of Cas9 for ChI-selective, conditional knockdown of the NK1R. Lastly, to determine the necessity of acetylcholine signaling in cue-dependent learning, the NAc of mice were directly infused with pirenzepine, a muscarinic 1 receptor-like antagonist via a NAc-implanted cannula. Results: Cue-dependent fear conditioning produced a multi-phasic response including activity in response to a novel cue, a decrease in release following a foot shock (pause), and a lasting increase in activity (1-2 sec) following the pause. Shock alone produced an identical pause followed by an increase in release as was observed during conditioning, which indicates salient stimuli in the absence of cues produces these phasic changes in acetylcholine. Increasing shock intensity caused increased acetylcholine release (F(3,28) = 3.420, p < 0.05) and the response diminished over subsequent cue exposures (F(1,28) = 6.793, p < 0.05), suggesting this enhanced acetylcholine release signals salience. NK1R antagonist treatment blocked the increase in activity at 0.6 mA (t(9) = 2.017, p < 0.05), indicating substance P signaling may be responsible for encoding salience of the aversive stimulus. Cue recall was significantly diminished by NK1R treatment (t(40) = 3.801, p < 0.001), NK1R CRISPR knockout (t(26)=3.332, p < 0.01), and latent inhibition (t(10) = 2.170, p < 0.05). In all treatment cases, motor responses did not explain the change in freezing. In addition, latent inhibition (t(10) = 3.886, p < 0.01) and NK1R antagonist treatment (t(13) = 2.140, p < 0.05) suppressed rebound cholinergic activity without affecting the pause during cue recall. Lastly, pirenzepine diminished cue recall when administered directly in the NAc prior to conditioning (t(17) = 3.253, p < 0.01), but not prior to cue recall (t(11) = 1.220, p > 0.05). Conclusions: These results provide evidence for a NAc core microcircuit that requires substance P and acetylcholine release for plasticity underlying cue-dependent aversive learning. Additionally, this work implicates substance P signaling, and not necessarily intrinsic mechanisms, in the ChI firing rebound during aversive events and cue learning, which signals salient environmental features. This work reveals a mechanism for a peptide-acetylcholine microcircuit that shapes response to aversive events by dictating salience of stimuli. These results could have broader implications for processing stressful events driving anxiety and depression. Keywords: Salience, Acetylcholine, Neuropeptides, Aversive Learning, Ventral Striatum Disclosure: Nothing to disclose. P99. Decoding Stress Susceptibility From Activity in Amygdala-Ventral Hippocampal Network Frances Xia, Nina Vishwakarma, Valeria Fascianelli, Frances Ghinger, Stefano Fusi, Mazen Kheirbek University of California - San Francisco, San Francisco, California, United States Background: Major depressive disorder is characterized by network-level functional alterations, with the amygdala (AMY)-ventral hippocampus (vHPC) pathway as well as their connected regions being central components. However, how changes in emotional state are represented in the activity patterns of single neurons and populations in the AMY-vHPC network remains unclear. Methods: Here, using high-density Neuropixels probes, we recorded single unit activity across the AMY-vHPC network, including AMY and vHPC subregions, as well as medial prefrontal cortex (mPFC), and thalamus. This allowed us to investigate how stress modulates representations of emotional information in the AMY-vHPC circuit and the effects on reward-seeking behavior. Mice (males and females, total n = 60) were first subjected to chronic social defeat stress, a well-validated rodent paradigm that stratifies stressed mice into those that exhibit a depression-like behavior (susceptible) vs. those that do not (resilient). After defeat, we recorded single unit activity across a AMY-vHPC-mPFC-thalamic network as mice performed a head-fixed sucrose preference test where they freely choose between consuming water vs. sucrose rewards, a classic test of anhedonia-related behavior. Statistical significance was assessed using two-way ANOVA followed by post-hoc tests where appropriate. If data were significantly non-normal or variances were significantly unequal, non-parametric tests including Kruskal-Wallis, Mann-Whitney, and Wilcoxon signed-rank tests were used. Results: Our results show that sucrose preference after defeat was correlated with social interaction ratio (P = 0.0002), a standard measure of stress susceptibility. At the single neuron level, we found that spiking activity was greatly elevated during reward consumption in control (non-stressed) mice across the AMY-vHPC -mPFC-thalamic network, but stressed mice showed region-specific changes in firing patterns, including decreased activity in AMY and vHPC neurons (P < 0.0001), and elevated firing rates in mPFC neurons (P < 0.0001). Interestingly, despite reduced sucrose preference behaviorally in the stressed mice, neurons in AMY and vHPC showed increased reward selectivity. To probe this phenomenon further at the population level, we used support vector machine classifiers to decode reward representations. We found that in stressed mice, future reward choice (sucrose or water) could be decoded seconds before the choice was made in the AMY, mPFC, and vHPC neurons (P < 0.0001), suggesting a rigidity in future reward representations. Furthermore, in susceptible mice, neurons in these regions showed smaller change in decoding accuracy from pre- to post-reward (P < 0.0001), suggesting reduced reward sensitivity in comparison to controls and resilient mice. We next investigated how stress alters interaction between brain regions. We found that coordinated activity between AMY and vHPC neurons is correlated with individual animal’s sucrose preference (P = 0.003), suggesting that reduced AMY-vHPC interaction may underlie anhedonia. In line with this, chemogenetic activation of AMY-vHPC pathway rescued anhedonic behavior in susceptible mice (P = 0.01). Conclusions: Together, our results show that chronic stress alters single neuron firing patterns and population representations of reward in the AMY-vHPC network, and these changes may be responsible for ultimately driving anhedonic behavior following stress. Keywords: Mood Disorders, Chronic Social Defeat Stress, Neural Circuit and Animal Behavior, Neural Decoding, Reward Representation Disclosure: Nothing to disclose. P100. A Thalamo-Ventral Hippocampal Circuit That Supports Anxiety-Related Behavior Mark Gergues, Joshua Bratsch-Prince, Shazreh Hassan, Mona Li, Arsine Kolanjian, Victoria Turner, Mazen Kheirbek University of California, San Francisco, San Francisco, California, United States Background: Recent work has revealed the hippocampus (HPC), which has classically been implicated in learning, for its role in encoding emotionally charged environments and generating appropriate behavioral responses. More specifically, emerging evidence from our lab has identified HPC neurons in the ventral pole that project to the hypothalamus encode anxiogenic environments and bidirectionally modulate avoidance behavior. Yet how and which inputs are involved in the generation of anxiety-related representations in the ventral HPC remain unknown. Using a whole-brain cell-type specific anatomical screen, we identified the anterior portion of the paraventricular nucleus of the thalamus (PVT), an area implicated in the processing of salient emotional stimuli, as a putative source of anxiety-related information to the HPC. In this study, we dissect the anatomical organization of the PVT-vHPC circuit and its contribution to anxiety-related behavior. Methods: We used a Cre-dependent strategy to label vHPC-projecting cells of the paraventricular thalamus (PVT) with the inhibitory DREADD receptor hM4Di. We injected retroAAV-Cre in vHPC and a Cre-dependent chemogenetic inhibitor, hM4Di, into PVT, both bilaterally. Four weeks later we injected animals with CNO (3mg/kg) 15 minutes before running the elevated plus maze and open field test. To assess the collateral projections of vHPC-projecting cells in PVT, we used anatomical tracers (a Cre-dependent expression of Synaptophysin-mRuby in PVT and retroAAV-Cre in vHPC) and viral barcoding approaches. Four weeks later, animals were sacrificed, brains extracted, followed by whole-brain histology to identify axon fibers to other brain regions. We then sought to identify how PVT input differs from other inputs to vHPC during behavior with dual fiber photometry. We injected a retrograde jGcAMP8m in vHPC then implanted optical fibers over both the PVT and Basal-lateral amygdala (BLA) to record BLA-vHPC and PVT-vHPC neurons simultaneously. Four weeks later we recorded calcium signals in PVT and BLA while animals explored the elevated plus maze and open field. Results: Inhibition of PVT-vHPC pathway resulted in reduced time in the open and center of the elevated plus maze (p < 0.05) and reduced time in the center of the open field test (p < 0.05). In our synaptophysin experiment we identified axon fibers to the nucleus accumbens, central amygdala, bed nucleus of the stria terminalis, and zona incerta. Dual fiber photometry recordings revealed differential responses of BLA-vHPC vs aPVT-vHPC in the elevated plus maze and open field test, indicating these two areas convey different information to vCA1 during exploration of anxiogenic environments. Conclusions: Here, we identify a role for PVT-vHPC projections in modulating anxiety-related behavior. These PVT-vHPC projections collateralize to the amygdala and nucleus accumbens suggesting that broadcasting PVT-vHPC neurons may play an important role in orchestrating behavioral responses to threatening stimuli. Responses in PVT-vHPC neurons to anxiogenic compartments of the elevated plus maze differed from that of BLA-vHPC, which showed consistent increases in activity when animals explored anxiogenic areas. This indicates differential, specialized processing of anxiogenic information PVT-vHPC neurons when compared to the well-studied BLA-vHPC pathway. Keywords: Ventral Hippocampus, Paraventricular Nucleus of the Thalamus, Anxiety Circuitry Disclosure: Nothing to disclose. P101. Dopamine Release at the Time of a Predicted Aversive Outcome Causally Controls the Trajectory and Expression of Conditioned Behavior Munir Kutlu, Jennifer Tat, Jennifer Zachry, Erin Calipari Vanderbilt University School of Medicine, Nashville, Tennessee, United States Background: The execution of adaptive behavior depends on the ability of organisms to predict potential threats in their environment. To this end, animals learn to predict when aversive stimuli will occur and learn what actions are necessary to avoid contexts and situations with potential negative outcomes. However, equally important, is the ability to learn when aversive stimuli are not likely to be presented. This balance allows animals to avoid potential negative outcomes while still exploring their environment when it is safe and unlikely to result in harm. Dopamine release in the nucleus accumbens (NAc) has been causally linked to adaptive aversive learning and its dysregulation is a core phenotypic characteristic of anxiety and stress disorders. Thus, understanding the role of dopamine in aversive learning is important for both understanding neuromodulatory signaling in the brain as well as how its dysregulation is important in psychiatric disease states. Methods: We employed an aversive conditioned inhibition design combined with fiber photometry and optogenetics to test the role of NAc dopamine in aversive learning. In this task, a target cue was presented in isolation (to predict the presentation of a footshock) or in combination with a secondary cue (which predicted that footshock would be omitted on the current trial). We recorded dopamine response to the cues that predict the presence and absence of aversive footshocks using a fluorescent dopamine sensor (dLight1.1). Then, we optogenetically manipulated the dopamine responses during the absent footshocks to show causal relationship between NAc dopamine and conditioned inhibition learning. Finally, we used a theory-driven computational model of associative learning (Kutlu-Calipari-Schmajuk, KCS, model) in order to explain our results and derive novel predictions regarding the role of dopamine in aversive learning. Results: Using optical approaches to directly record and manipulate dopamine release in the NAc in awake and behaving animals, we show that dopamine responses evoked at the time of omitted aversive outcomes are causal to the expression of conditioned responses. The magnitude of the dopamine response at the time of an omitted footshock scaled positively with the prediction of the aversive outcome on that trial; however, this effect was only apparent at the time of the omitted outcome, but not in response to the predictive cue itself. Importantly, we show, via optogenetic manipulation of this signal, that dopamine in this context is not transmitting an error-based signal, as enhancing this signal disrupted learning rather than enhancing it. Finally, using the KCS model, we showed that these effects can be explained by dopamine signaling the perceived saliency of predicted aversive events, but not prediction errors. Conclusions: Together, we show that NAc core dopamine responses to expected but omitted aversive stimuli causally determine associative learning for aversive stimuli in mice. These results add to the growing literature supporting the dopaminergic encoding of perceived saliency by dopamine in the NAc. Our conclusions regarding the dopaminergic information encoding in associative learning also have important clinical implications for anxiety and stress disorders. In sum, these results have far-reaching implications for the theory of learning and memory, the understanding of the mesolimbic neurocircuitry, and the psychopathology of anxiety and stress disorders. Keywords: Dopamine, Safety Learning, Aversive Learning Disclosure: Nothing to disclose. P102. Ultrasonic Vocalization Patterns Reveal a Sex-Dependent Dimension of Fear Conditioning Mikaela Laine, Julia Mitchell, Johanna Rhyner, Leena Ziane, Emmett Bergeron, Rose Clark, Akshara Kannan, Jack Keith, MaryClare Pikus, Rebecca Shansky Northeastern University, Boston, Massachusetts, United States Background: Pavlovian fear conditioning is a widely used paradigm for investigating the neural mechanisms that mediate aversive learning. However, the behavioral outcome metric has historically been the singular "freezing" response, despite the complex nature of the task and multi-dimensional fear responses animals can exhibit. Particularly in light of increased consideration of sex as a biological variable, expanding the repertoire of our behavioral observations in this classic task will be critical to a more nuanced understanding of learning and memory processes. Methods: Male (n = 100) and female (n = 100) Sprague Dawley rats underwent auditory cued fear conditioning in standard chambers, with either 1.0mA, 0.5mA, 0.3mA, or no unconditioned stimulus. Some animals also underwent extinction learning and recall procedures on consecutive days. Locomotor behavior was recorded using overhead cameras and tracked using Ethovision software. Vocalizations were recorded and analyzed using DeepSqueak software. Some animals were euthanized 90 minutes after fear conditioning concluded, and areas of the prefrontal cortex, periaqueductal gray (PAG), and spinal cord were immunostained for c-fos expression. Results: We observed significant main effects of sex and shock intensity in the number of alarm calls elicited during fear conditioning. Males exhibited significantly more alarm calls than females, and these calls also persisted for longer across the training session in males compared to females. The propensity to make alarm calls tracked shock intensity and corresponded to freezing behavior in males but not females. Alarm calls emerged later when CS-US presentations were explicitly unpaired than when paired, suggesting that alarm calls may reflect associative learning. However, alarm calls could be observed during ITIs as well as during CS presentation. We also observed sex differences in correlations between c-fos activity and both conditioned and unconditioned responses. Conclusions: This work provides novel evidence that males and females process a Pavlovian fear conditioning session differently, both in several dimensions of behavior, as well as in the activity of key brain regions. Our data hold important implications for both basic scientists who use Pavlovian fear conditioning to study learning and memory processes, as well as for translational neuroscience hoping to use rodent models to understand aversive learning-related disorders like PTSD. Keywords: Fear Conditioning, Sex Differences, Ultrasonic Vocalization, Periaqueductal Grey (PAG), Prefrontal Cortex Disclosure: Nothing to disclose. P103. Investigating the Role of Nucleus of the Solitary Tract Projections to the Central Amygdala in Mediating Hyperarousal Symptoms After Traumatic Experience Claire Stelly, Alyssa Hall, Kasey Anderson, Anh Duong, Naseem Azadi, Jeffrey Tasker, Jonathan Fadok* Tulane University, New Orleans, Louisiana, United States Background: Persistent hyperarousal symptoms, including exaggerated startle responses and increased sympathetic tone, are a hallmark of post-traumatic stress disorder. We hypothesize that interoception of sympathetic activity directly regulates threat responses to promote and maintain hyperarousal. Noradrenergic neurons in the nucleus of the solitary tract (NTS) receive ascending input from major organ systems and send noradrenergic/glutamatergic projections to the central amygdala (CeA), a key region for orchestrating responses to threats. We propose that traumatic experience alters NTS activity to promote exaggerated threat responses. Methods: Hyperarousal symptoms were induced with the Traumatic Experience with Reminders of Stress (TERS) paradigm, consisting of a single exposure to a traumatic footshock (10 s, 2 mA) followed by six brief, intermittent exposures to a contextual reminder of the traumatic experience over 24 days. Male and female C57Bl6/J mice were randomly assigned to Control (n = 8) or TERS (n = 22) groups. Acoustic startle responses, resting heart rate, and sociability were measured before and after the trauma paradigm. Population activity of NTS noradrenergic neurons was examined by injection of AAV5-CAG-FLEX-GCaMP6f in the NTS of TH-Cre mice (n = 2) and implantation of an optical fiber for photometry. Chemogenetic activation of NTS noradrenergic input to the CeA was achieved by injection of AAV5-hSyn-DIO-hM3dGq in the NTS of TH-Cre mice (n = 2) and local application of DREADD agonist deschloroclozapine/vehicle via bilateral cannulae in the CeA. Results: Mice that underwent the TERS paradigm had enhanced acoustic startle (p = .006, Welch’s t-test) and elevated heart rate (p = .009, Mann-Whitney test) compared to unshocked controls. Based on the acoustic startle response, the traumatic exposure group can be subdivided into a susceptible group with significantly elevated startle responses, and a resilient group with startle responses like control. Sociability did not differ between groups (social interaction events p = .56; social interaction time p = .86, Welch’s t-tests). No sex differences were observed in any measure. Preliminary fiber photometry data suggest that startling and aversive stimuli elicit calcium responses in NTS noradrenergic neurons. Additionally, chemogenetic activation of NTS noradrenergic axon terminals in the CeA potentiates the acoustic startle response in naive mice (p = .05, paired t-test). Conclusions: Traumatic experience, paired with situational reminders, elicits hyperarousal symptoms in male and female mice. Noradrenergic neurons within the NTS are likely activated during salient or aversive stimuli, and activation of noradrenergic NTS projections in the CeA potentiates startle. These findings suggest that the NTS may regulate threat responses by its projections to the central amygdala. Keywords: Hyperarousal, Central Nucleus of the Amygdala, Noradrenergic System, Nucleus Tractus Solitarii Disclosure: Nothing to disclose. P104. Offensive Attack Initiation Triggered by an Amygdala CRH + Cell Activity Signature Emily Newman, Erin Hisey, Hector Bermudez, Nicholas Ressler-Craig, Kerry Ressler Harvard Medical School McLean Hospital, Somerville, Massachusetts, United States Background: Stress-induced psychopathologies like posttraumatic stress disorder can severely disrupt emotion regulation and increase reactivity – in some patients, this manifests as an increase in aggression. The central amygdala (CeA) is a point of intersection for neural circuits that modulate both threat responding and aggression. In our present work, we employed mouse models of naturalistic agonistic behavior to examine the neural bases of offensive (i.e., territorial) and self-defensive attacks. We used single-cell calcium imaging and chemogenetics to evaluate CeA neurons that express the well-characterized stress-signaling neuropeptide, corticotropin releasing hormone (Crh). Methods: Male CRH-ires-Cre mice received intra-CeA AAV5-EF1a-DIO-GCaMP6s and were implanted with a gradient index lens to record calcium-dependent activity in Crh+ CeA cells. Cell activity (n = 16-32 cells/mouse) was video-recorded using a miniature microscope during 5-minute home-cage offensive aggressive encounters with a submissive intruder mouse or during 1-minute self-defensive interactions with an aggressive conspecific. Crh+ CeA cell ensembles were identified based on their activity (3 z-score peak event threshold) relative to offensive attack initiation. Additional CRH-ires-Cre males received intra-CeA AAV5-hSyn-DIO-hM4D(Gi)-mCherry to drive inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) or control virus in Crh+ CeA neurons. Five minutes before offensive or self-defensive aggression trials, mice were injected systemically with vehicle or deschloroclozapine (0.02 mg/kg). Behavioral videos were collected from each trial and analyzed using a supervised machine learning pipeline. Specifically, animal tracking was conducted in multi-animal deeplabcut (maDLC; Lauer et al. 2022). Pose estimations generated by maDLC served as the input for SimBA (simple behavioral analysis; Nilsson et al. bioRxiv) which employed random forest classification to identify social approach, social contact and attacks. Sequences of classified behaviors were identified to quantify behavioral transition states, e.g., the probability of transitioning from social approach to social contact to attack. Results: Crh+ CeA cells (n = 85) were classified into three ensembles – 41% were active in the 10 seconds before offensive attack initiation, 32% were active during attacks, and 27% were inactive during aggression. Chemogenetic inhibition of Crh+ CeA cells selectively prevented offensive attacks without affecting social approach, contact, or adaptive self-defensive bites toward an attacking intruder. Conclusions: CeA Crh+ cell activity is necessary for the transition from social contact to the initiation of offensive attacks. CeA Crh+ cell activity was not necessary for self-defensive bites, suggesting distinct neural circuitry underlying offensive vs. self-defensive aggression. All-optical closed-loop approaches are presently being used to block attack initiation using Crh+ CeA cell activity to remotely trigger real-time inhibitory optogenetics. Keywords: Aggression, Amygdala, Corticotropin-Releasing Factor (CRF), in Vivo Calcium Imaging, Chemogenetics Disclosure: Nothing to disclose. P105. Basolateral Amygdala Reactivity and Functional Connectivity Over Time Following Trauma: Evidence for a Dynamic Model Alyssa Roeckner, Rebecca HInrichs, Timothy Ely, Sanne van Rooij, Nathaniel Harnett, Lauren LeBois, Vishnu Murty, Tanja Jovanovic, Stacey House, Samuel McLean, Karestan Koenen, Ron Kessler, Kerry Ressler, Jennifer Stevens Emory University School of Medicine, Atlanta, Georgia, United States Background: Amygdala hyperreactivity early post-trauma is a demonstrable physiological correlate of future posttraumatic stress disorder (PTSD). Two areas of the amygdala are particularly known to play a part in the threat response and PTSD – the basolateral amygdala (BLA) which is involved in threat processing and memory, and the central amygdala (CeA) which is involved in autonomic threat responses. However, there is little evidence regarding how the dynamics of amygdala reactivity over time relate to longitudinal PTSD symptomology. Two opposing hypotheses are that (1) trait differences in amygdala threat response predict PTSD and persist over time, or (2) dynamic changes in threat response over time impact symptoms. Exploring how reactivity and functional connectivity in the BLA and CeA persists or changes over time following trauma may allow for better understanding of the physiology of PTSD and future treatment options. Methods: As part of a larger multisite study, “AURORA”, participants were enrolled in the emergency department (ED) within 72 hours of a traumatic event. Participants experienced an event that involved actual or threatened serious injury, sexual violence, or death, either by direct experience, witnessing it, or learning about it. Participants were between the ages of 18-75 and English speaking. PTSD symptoms were assessed using the PTSD Symptom Checklist for the DSM-5 (PCL-5) at 2 weeks and 6 months post-trauma. Left and right basolateral amygdala (BLA) and central amygdala (CeA) responses to threat (i.e., fearful>neutral faces) during fMRI were collected 2 weeks (N = 305, 199 female) and 6 months post-trauma (N = 100, 69 female). Contrast values for fearful > neutral faces for the four regions of interest (ROIs) were tested for correlations with 2-week and 6-month PTSD symptoms. Follow-up task-based functional connectivity (FC; fearful>neutral faces) was analyzed for regions showing significant associations in the ROI analyses using Conn Toolbox with a voxel threshold of 0.005 to allow for a final FWE-corrected cluster threshold of 0.05. Results: Left BLA response at 6 months post-trauma negatively correlated with both 2-week (r = -.21, p = .04) and 6-month PTSD symptom severity (r = -.27, p = .006). Similarly, a decrease in left BLA reactivity from 2 weeks to 6 months predicted 6-month PTSD symptom severity (r = -.20, p = .04). There were no correlations between PCL-5 scores and CeA or right BLA reactivity at either 2 weeks or 6 months. Follow-up FC analyses were then performed with the left BLA as a seed region. At 2 weeks, current PTSD symptoms were associated with more positive FC with the cerebellum and more negative FC with the anterior and posterior cingulate, the left middle frontal gyrus, and the left supramarginal gyrus. At 6 months, current PTSD symptoms were associated with more positive FC with the posterior cingulate gyrus, the left inferior temporal gyrus, and the left cerebellum, and more negative FC with the right temporal pole and the right superior frontal gyrus. Conclusions: Findings suggest that PTSD symptoms persist in individuals who show a strong decrease in left BLA reactivity to threat over the months following trauma, supporting a dynamic model of threat response changes post-trauma in PTSD. The left BLA may experience a form of exhaustion in those with chronic PTSD, potentially due to the downregulation of norepinephrine or glutamate receptors in response to chronic stress. FC results suggest positive left BLA-cerebellum connectivity is consistently linked with higher PTSD severity across time points, while connectivity associated with higher PTSD severity between the left BLA and areas involved in the default mode network (posterior cingulate gyrus; anterior cingulate) starts as negative at 2 weeks and changes to positive by 6 months. This suggests the default mode network may play a major role in left BLA reactivity changes for those with higher PTSD severity. Findings support the importance of affective dynamics in PTSD risk and the need for further longitudinal neuroimaging studies. Keywords: PTSD, fMRI, Basolateral Amygdala, Central Amygdala, Longitudinal MRI Disclosure: Nothing to disclose. P106. Serotonergic Effects of Viloxazine Measured Using 5-HT2 Agonist Radioligand, [11C]CIMBI-36, in Cynomolgus Monkeys: A PET Imaging Approach Jennie Garcia-Olivares, Brittney Yegla, David Zweibaum, Chungping Yu Supernus Pharmaceuticals Inc, Rockville, Maryland, United States Background: Viloxazine ER (viloxazine extended-release capsules; Qelbree®), is a novel, non-stimulant FDA-approved treatment for attention-deficit/hyperactivity disorder (ADHD) in children (≥6 years) and adults. Viloxazine therapeutic effects in ADHD have been ascribed to norepinephrine reuptake inhibition; however, its moderate potency (Ki=0.630 µM) compared to traditional NRIs (e.g. atomoxetine Ki=0.005 µM) for binding in cells expressing human norepinephrine transporters (NET) suggests alternate mechanisms of action may be involved. Emerging in vitro data show viloxazine has moderate affinity for human isoforms of serotonin (5HT)- receptors [5-HT2C (Ki=1.5 µM), 5-HT7 (Ki=1.9 µM) and 5-HT2B (Ki=3.4 µM)], but no expected clinically relevant effect at 5-HT reuptake transporters SERT (Ki =17.3 µM). Moreover, newly completed in vivo microdialysis experiments in rats are showing viloxazine significantly increases 5-HT levels in the rat prefrontal cortex at interstitial fluid (ISF) concentrations associated with clinically relevant plasma concentrations, an effect that cannot be explained by the NET inhibition alone. To build upon these newly emerging data and further elucidate viloxazine’s effects on serotonergic neurotransmission in vivo, we conducted a positron emission tomography (PET) imaging study in cynomolgus monkeys. For this study, the radioligand agonist (for 5-HT2A and 5-HT2C), [11C]CIMBI-36, was used to assess the ability of viloxazine to induce changes in extracellular serotonin levels in cortical regions and to occupy 5-HT2C receptors in the choroid plexus Methods: Four anesthetized cynomolgus monkeys were administered bolus infusions of viloxazine (3, 6, and 12 mg/kg), with a 6-week resting period between scans. PET scans were performed at baseline and 30 minutes after infusion (1-2 scans per dose, 123 minutes per scan). [11C]CIMBI‐36 was administered via bolus infusion immediately prior to initiation of the scanning period. During image acquisition, arterial blood samples were drawn at eight timepoints 2-90 minutes post-scan initiation for quantification of [11C]CIMBI-36 metabolite. Plasma concentrations of viloxazine were measured in blood samples collected at four timepoints 15-120 minutes post-administration of viloxazine. PET images were analyzed using spatial processing to enable automated definition of regions of interest (ROIs). ROIs analyzed included frontal cortex, choroid plexus, and cerebellum. The choroid plexus was manually delineated. To estimate total volume of distribution (VT) or the ratio of [11C] CIMBI-36 in a target region, time-activity curves were estimated from the data using the established multilinear analysis (MA1) modeling method. From the calculated VT values, non-displaceable binding potential (BPND) was computed for each ROI using the cerebellum as a reference region. The effect of viloxazine on the displacement of the radioligand at 5-HT receptors is evaluated based on the change in BPND between baseline and post viloxazine treatment. Results: Administration of 3 mg/kg (n = 1), 6 mg/kg (n = 2), and 12 mg/kg (n = 2) of viloxazine produced respective reductions in BPND in the choroid plexus of 60%, 76% and 100% (the latter representing complete blockade of [11C]CIMBI-36 binding). These same doses induced changes in BPND of 25%, 15-32%, and 38%-42%, respectively in the cortical regions. At 3 mg/kg, the unbound viloxazine plasma concentration (Cmax) was 3.9 µM, which is close to the range of 2.2-3.3 µM unbound viloxazine plasma concentration (Cmax) observed at the therapeutic dose of 400 mg/day viloxazine ER in children between 6 to 17 years old. Conclusions: Our results establish a dose-dependent binding effect for viloxazine at the 5-HT2C receptor in the choroid plexus. The 60% displacement of the radioligand by viloxazine at a clinically relevant dose (3 mg/kg) may be attributable to direct occupancy of the 5-HT2C receptor by viloxazine. In the cortical regions, a dose-dependent displacement of [11C]CIMBI-36 by viloxazine was also seen. The effect of viloxazine in the cortical regions (rich in 5-HT2A receptors) may be attributable to either direct occupancy of 5-HT2A or due to increased release of synaptic 5-HT. An estimated EC50 value of viloxazine for the changes in BPND at the 5-H2A receptor is significantly higher than the unbound plasma concentrations of viloxazine at all doses tested. This suggests that the observed effect on radioligand binding may result from a viloxazine-mediated increase in endogenous 5-HT release rather than direct binding to the 5-HT2A receptor in the cortex. Overall, our experiments suggest that, at clinically relevant doses, viloxazine increases serotonergic neurotransmission in the PFC and acts on the 5-HT2C receptor, which could play a role in its efficacy in the treatment of ADHD. Keywords: ADHD, Serotonin, PET imaging, PFC, Viloxazine Disclosure: Supernus Pharmaceuticals Inc.: Employee (Self) P107. Pubertal Stress Alters Future Maternal Behavior in Mice Through Lasting Disruptions to Chromatin and Transcriptional Landscapes in the Hypothalamus Gretchen Pifer, Karissa Gautier, Samantha Higley, Briana Karem, Kathleen Morrison West Virginia University, Morgantown, West Virginia, United States Background: Adverse childhood experiences, specifically during the pubertal transition, are one of the greatest predictors for affective dysfunction in women. As puberty is marked by dynamic hormonal changes and ensuing reorganization of the brain, it represents a window of sex-specific vulnerability to adverse experiences. We have previously shown that stress during puberty alters the hypothalamic-pituitary-adrenal (HPA) stress axis response in adult female mice only during pregnancy and postpartum. In humans, pubertal adversity led to a blunted HPA response to a maternal separation stressor, which was associated with increased postnatal depression score. We previously examined puberty-stress reprogramming in the paraventricular nucleus (PVN) of the hypothalamus, which initiates the HPA axis response. We found that pubertal stress led to an altered chromatin landscape and transcriptome phenotype in the PVN. Further, we found that the pregnancy-associated hormone allopregnanolone is necessary and sufficient to produce the blunted stress response in pubertally stressed females. It is possible that pubertal stress creates a disorganization of maternal responsiveness and vulnerability to affective dysfunction. To extend our understanding of the risks posed by pubertal stress to alter future behavior, we have begun to examine various aspects of forming and performing maternal behavior. Here, we examined pup-directed behavior both before (pre-maternal) and after pregnancy, as well as further investigated the lasting programming of the stress response by pubertal stress. We hypothesized that pubertal stress would lead to both deficits in the ability to form and display maternal behavior and alterations in the chromatin and transcriptional landscapes of maternal- and stress-related brain regions. Methods: Mice were exposed to chronic variable stress (CVS) from postnatal day 21–34 using our previously established paradigm. CVS consisted of two stressors (tactile, olfactory, or auditory) per day. In adulthood, virgin females were either left undisturbed (no exposure) or were exposed to novel pups for 2h per day for 4 days (maternal exposure). All females were tested for pup retrieval in a maze 24h following the last maternal exposure, after which they were left undisturbed for two weeks. The pup retrieval task was repeated, and brains were collected. Pup-directed behavior was analzyed both during maternal exposures and during the pup retrieval task (n = 34-35/group). To examine molecular consequences associated with maternal behavior, the medial preoptic area (mPOA) of the hypothalamus was extracted from the brains, RNA was isolated, and quantitative PCR was used to measure gene expression (n = 9-10/group). In another set of mice, males and females were exposed to CVS as above and were left undisturbed until adulthood when pharmacological treatment and brain collection occurred. Mice were given either allopregnanolone (100ng/200nl per side) or vehicle (25% w/v HP-β-CD) via intra-PVN cannulae 2h before brain collection. RNA from PVNs was isolated and gene expression was measured using quantitative real-time PCR (n = 2-6/group). Data were analyzed by ANOVA with Tukey post-hoc tests when necessary or Chi square. Results: Adult, pubertally stressed females were more likely to show aggression towards pups throughout exposures and behavioral testing than were control females (X2(1,69) = 6.031, p = 0.014). In the mPOA, there was a significant interaction between pubertal stress and maternal exposure on expression of Crebbp (p = 0.0034). Specifically, there was upregulation of Crebbp in the mPOA of pubertally stressed females who had no maternal exposure compared to control females who had no exposure (p = 0.0225). This upregulation was associated with poor performance on the memory task, indicating that pubertal stress disrupted how Crebbp canonically influences memory. In the PVN, preliminary findings from intra-PVN allopregnanolone administration recapitulated prior findings showing that pubertally stressed mice respond differently to allopregnanolone than do control mice at the level of gene expression. Conclusions: These results provide novel insight into the impact of adversity during puberty on lifelong risk for altered behavior. We found that stress during puberty led to disrupted pup-directed behavior and altered transcription in hypothalamic brain regions that regulate maternal and stress responses. Multiple areas of the hypothalamus are sensitive to pubertal stress, suggesting potentially extensive and lasting consequences of adversity during this developmental stage. Previous and ongoing work implicates disrupted histone acetylation as a potential mechanism of this lasting effect. This translationally-relevant mouse model provides the opportunity to understand the molecular underpinnings of risk for behavioral and hormonal stress dysregulation following pubertal stress. Keywords: Epigenetics, Adolescent Stress, Risk and Resilience Disclosure: Nothing to disclose. P108. Sexual Dimorphisms in Neuronal Structure, Function, and Behavior in a Model System of Autism Spectrum Disorders Olivia Williams, Madeleine Coppolino, Cecilia Micelli, Melissa Perreault* University of Guelph, Guelph, Canada Background: Autism Spectrum Disorders (ASD) are showing increasing prevalence in North America. Individuals are commonly given a standard diagnosis of ASD, despite symptom presentation and severity varying upon the spectrum, and sexual dimorphisms in age of onset, prevalence, etiology, and presentation are also observed. Animal models are often used to study specific aspects of ASD, however females have been historically underutilized, leading to a dearth of knowledge of the possible sex-dependent mechanisms that may underlie the disorders. In this study, we therefore aimed to characterize sexual dimorphisms in neuronal structure and function in the valproic acid model, a model often used for the study of idiopathic ASD. Methods: Pregnant Sprague Dawley rats were injected with VPA (500 mg/kg) or saline i.p. on gestational day 12.5. To assess alterations in neuronal structure and function in vitro, cortical and hippocampal (HIP) tissues were dissected from postnatal day 0-1 male and female pups. On DIV 21, neurons were stained with microtubule associated protein-2 (MAP-2) for Sholl analysis, or neuronal systems activity evaluated on a multi-electrode array (MEA) (Axion Biosystems) for 30 min. To evaluate sexual dimorphisms in behavior, adolescent male and female rats (PND 31-38) underwent behavioral testing in the three-chamber social test, novel object recognition (NOR), object location (OL), and elevated plus maze (EPM). Following testing, rats were bilaterally implanted with stainless steel electrodes into the medial prefrontal cortex (PFC) and dorsal HIP. Once recovered, local field potential recordings were performed in awake, freely moving animals, for 30 min (N = 10 rats/group). Chronux software for MATLAB was used to evaluate the spectral power at each frequency band within each region. Results: : Sholl analysis revealed sex-dependent VPA-induced alterations in the mean number of intersections in both the cortical and HIP neurons that were dependent upon the distance from the soma. Only female VPA cortical neurons exhibited reduced length compared to sex-matched controls (p = 0.029). When neuronal activity was evaluated, sex-specific alterations in systems function were evident in both the VPA group and control neurons. Control male-derived cortical neurons displayed elevated mean firing and bursting compared to female-derived control neurons (p < 0.001), although this was reversed in the VPA neurons (p < 0.001). VPA neurons from either sex also displayed elevated firing and bursting compared to their sex-matched controls (p < 0.001). Cortical neurons derived from males had a significantly greater synchrony index compared to those from females (p < 0.001), although this effect was lost between sexes in the VPA group. Overall, VPA neurons displayed significantly greater synchrony compared to controls [F(1,62)=58.06, p < 0.001]. In the HIP neurons, the control firing rate of male-derived neurons was greater than those from females (p < 0.001), an effect lost with VPA exposure. Only female-derived VPA cells displayed increased firing compared to their sex-matched controls (p < 0.001). Unlike the cortex, bursting activity was decreased in male-derived VPA HIP neurons (p < 0.001) and increased for the female VPA group (p < 0.001) compared to sex-matched controls. No significant group differences were observed for synchrony index. In vivo, behavioural testing in adolescence revealed sex-specific ASD-like characteristics in the VPA group. VPA-exposed female rats displayed greater anxiety in the EPM compared to sex-matched controls (p = 0.005), showed deficits in the NOR task (p < 0.001), and displayed a deficit in social index scores (p = 0.007) although they were more sociable than VPA-treated male rats (p = 0.014). VPA males, in contrast, showed deficits in both the OL task (p = 0.012) and social index score (p = 0.036) compared to male controls. Following behavioural testing neuronal oscillatory activity was examined. Female VPA rats displayed significantly greater PFC delta power (p < 0.001) and reduced theta power (p < 0.001) compared to female controls and had higher delta (p < 0.001) and reduced theta (p < 0.001) power compared to VPA males. In high frequency bands, VPA females displayed lower beta, low gamma, and high gamma (p < 0.001, p = 0.012, and p < 0.001 respectively) compared to VPA-exposed males, effects that were not evident between the sexes in the controls. In the HIP, differences were observed selectively in the low frequency bands, delta, and theta, in which VPA females displayed significantly greater delta power (p = 0.037) compared to VPA males and had lower theta power (p = 0.008) to control females. When coherence was examined between the PFC and dHIP, VPA females had lower delta coherence (p = 0.029), while VPA males exhibited no change compared to sex-matched controls. Conclusions: These preclinical findings identify key sex differences in neuronal structure and function in vitro, and in PFC and HIP systems function and behavior in vivo, in a model commonly used to study ASD. Notably, the impact of VPA exposure in the PFC and HIP was more substantial females exposed to VPA. Overall, these findings demonstrate clear sexual dimorphisms in the VPA model that may have relevance to the sexual dimorphisms observed in ASD. This work also highlights the critical need for employing both sexes when utilizing animal models in the study of neuropsychiatric disorders. Keywords: Autism, Neuronal Oscillations, Dendritic Arborization, Behavior, Systems Neuroscience Disclosure: Nothing to disclose. P109. The Paraventricular Nucleus of the Thalamus Contributes to Early-Life Adversity-Induced Disruptions in Reward-Related Behaviors Cassandra Kooiker, Matthew Birnie, Yuncai Chen, Tallie Z. Baram University of California - Irvine, Irvine, California, United States Background: Early-life adversity (ELA) is associated with poor cognitive and emotional health, including an increased risk for a variety of affective disorders, such as depression and substance use disorders. Many of these disorders are characterized by impairments in reward-related behaviors, and we find that these same deficits are provoked by rodent models of ELA. However, the brain regions and processes underlying these long-term consequences of ELA remain largely unknown. The paraventricular nucleus of the thalamus (PVT) is an important node of the reward circuit that encodes remote emotionally salient experiences to influence future motivated behaviors. We hypothesize that the PVT encodes adverse experiences as remote as the early postnatal period in mice, and that ELA-engaged PVT neurons subsequently contribute to alterations in reward-related behaviors in adults. Methods: We employ TRAP2 mice, which we exposed to a week of simulated ELA in a limited-resource cage between postnatal days 2-9. We induced the TRAP2 system using tamoxifen on P6, triggering Cre-dependent recombination in neurons activated during P6-P8. This leads to permanent labeling of neurons activated during this time frame. We validated our findings using routine cFos immunohistochemistry in WT mice. We then chemogenetically inhibited these ELA-engaged neurons during an adult reward-seeking task with the goal of ameliorating ELA-induced changes in reward-seeking behaviors. Results: ELA robustly and selectively activates significantly more PVT neurons than typical rearing conditions (p = 0.0154, unpaired t-test; N = 24), and a large proportion of these ELA-engaged PVT neurons express CRFR1 (40% vs 20% in controls, p < 0.001, unpaired t-test; N = 12). Silencing ELA-engaged PVT neurons during reward-related tasks in adult female mice ameliorates the observed ELA-induced changes in reward-seeking behaviors (N = 16). Conclusions: The PVT is robustly and almost uniquely activated in response to emotionally salient events in neonatal mice, and inhibition of these ELA-engaged neurons ameliorates ELA-induced changes in reward-seeking. The PVT is thus poised as a potential contributor to deficits in reward-related behaviors following ELA. Keywords: Paraventricular Nucleus of the Thalamus, Early-Life Adversity, Affective Disorders, Reward Circuitry, Motivated Behaviors Disclosure: Nothing to disclose. P110. Adolescent Social Isolation Disrupts Developmental Profiles of GABAergic and Glutamatergic Gene Expression in the Reward Circuitry of Males but Not Females Natasha Fowler, Allison Milian, Bendersky Cari, Mason Andrus, Deena Walker Oregon Health and Science University, Portland, Oregon, United States Background: Adolescence is a period of reward development and social experience in adolescence has robust effects on reward behaviors. Reduced social connections increases the risk for substance use disorder. Our lab and others have shown that adolescent social isolation (SI) alters cocaine-related behaviors and transcription in adulthood, but little is known regarding how SI disrupts development of the reward circuitry to influence behavior. Methods: We hypothesized that SI disrupts developmental profiles of GABAergic and glutamatergic systems in 3 brain regions (prefrontal cortex - PFC, basolateral amygdala - BLA and ventral hippocampus – vHIP) to influence adult behavior. Animals were isolated from ~postnatal (P)22 – P42 and then group housed. Male and female mice were euthanized on the day of isolation (P22), mid-isolation (P32), rehousing (P42), and on P72. Punches of PFC, BLA and vHIP were collected for RT-qPCR (n = 6-8 animals per group; Total: ~150 animals) of 24 GABAergic and glutamatergic genes. Reward-related behaviors including palatable food consumption and cocaine conditioned place preference were also assessed in a subset of adult males and females. Results: Preliminary analysis of behavior suggests an increased preference for palatable food and cocaine in males. Three-way ANOVA for sex, SI and age revealed main effects or interactions (p < 0.05) for glutamatergic receptor subunits (Gria1 and 2; Grin1, 2a, 2b and 2c) and the glutamate transporter (Slc17a6) in all 3 brain regions. Although region specific effects were observed, post hoc analysis revealed that expression of many glutamatergic genes was enhanced by SI in males on P32. However, GABAergic genes were only affected in the PFC. Three-way ANOVA revealed a significant Sex X Age X SI interaction for GABAA receptor subunits (Gabra1, Gabrg1; p < 0.05), GABA transporter (Slc32a1; p = 0.006) and Gad2 (p = 0.001). Post hoc analysis revealed a significant decrease in expression on P32 in males. Conclusions: These data suggest that SI results in a sex-specific enhancement of the excitatory system across the reward circuitry in males and a concomitant suppression of the GABAergic system (PFC only). These finding provide a sex-specific mechanism by which SI may reprogram the reward circuitry to influence behavior in adults Keywords: Mesolimbic Reward Circuitry, Gene Expression, Stress in Adolescence, GABA-A Receptors, NMDA Glutamate Receptors Disclosure: Nothing to disclose. P111. Physical and Functional Convergence of the Autism Risk Genes SCN2A and ANK2 in Neocortical Pyramidal Cell Dendrites Andrew Nelson, Amanda Catalfio, Julie Philippe, Lia Min, Rene Caballero-Floran, Kendall Dean, Carina Elvira, KImberly Derderian, Henry Kyoung, Atehsa Sahagun, Stephan Sanders, Kevin Bender, Paul Jenkins* University of Michigan Medical School, Ann Arbor, Michigan, United States Background: Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel NaV1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how NaV1.2 is anchored within dendritic regions is unknown. Methods: Here, we paired cellular and molecular biology with electrophysiology and two-photon imaging to demonstrate that the protein products of these two ASD risk genes, SCN2A and ANK2, interact in neocortical pyramidal cell dendrites to mutually regulate dendritic excitability. Results: Using a novel epitope-tagged NaV1.2, we found that NaV1.2 co-localizes with ankyrin-B in the dendrites of mature neocortical neurons. Removal of ankyrin-B eliminated NaV1.2 dendritic localization. Furthermore, dendritic ankyrin-B loss was not compensated for by other ankyrin family members, indicating that ankyrin-B has a unique scaffolding role in this neuronal compartment. Ex vivo studies revealed that Ank2 haploinsufficiency results in intrinsic and synaptic dendritic deficits that closely phenocopy those observed in Scn2a heterozygous neurons. Conclusions: Thus, these findings suggest that deficits in dendritic excitability may be a common point of convergence in ASD, with direct convergence between two high-risk genes SCN2A and ANK2. Keywords: SCN2A, Dendrites, Excitatory Synapses, Autism Spectrum Disorders Disclosure: Nothing to disclose. P112. The Synaptic Proteome of Autism Spectrum Disorder Across Postnatal Development in Human Visual Cortex Shelby Ruiz, Lambertus Klei, Bernie Devlin, Matthew MacDonald* University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Despite diversity in symptoms and severity in autism spectrum disorders (ASD), spine alterations are observed in many genetic (ASD) models and subject tissue. Genetic studies of ASD have identified rare variants and common loci that implicate the synaptic protein networks crucial for spine formation and stabilization. Additionally, transcriptomic studies of cortical regions in ASD have found altered synaptic gene expression. However, transcriptomic changes are not always well correlated with synaptic protein levels and a survey of the synaptic proteome in ASD subject tissue has not yet been performed. Thus, the extent to which transcriptomic alterations manifest at the synapse in ASD subjects and contribute to spine alterations is currently unknown. Here, in a preliminary study, we assayed synaptic protein levels in primary visual cortex tissue from postnatal ASD and matched neurotypical subjects. Methods: Postmortem primary visual cortex tissue from 31 pairs of ASD and neurotypical subjects (ages 4-33) matched for sex, age and postmortem interval were obtained from the University of Maryland branch of the NIH NeuroBioBank. Synaptosome enrichments were prepared with SynPER. Synaptosomes were digested with trypsin, TMT-labeled, fractionated, and analyzed on an Orbitrap Eclipse with SPS and real time search. Peptide and protein identification and quantification were performed in Proteome Discoverer 2.5. Results: 7,577 proteins were identified in synaptosome enrichments across all subjects. Of these, 4,601 (including 115 ASD risk gene products, SFARI) were quantified with > 50% present call and included for statistical comparison with age and diagnosis. Synaptosome levels of 1,462 proteins were significantly associated with age (q > 0.05); with 860 decreasing during postnatal development and 539 increasing. Interestingly, the 860 proteins that decreased with age were significantly enriched for the GO term synapse in SYNGO (q = 5.82e-15, relative to the 7,577 proteins quantified) while the proteins that increased with age were not. Similarly, the proteins that decreased with age included 40 ASD risk genes while increasing proteins included only 4 ASD risk genes. Of the ASD risk genes assayed, only one, Receptor-type tyrosine-protein phosphatase F differed significantly between ASD and neurotypical subjects (FC = 1.2, q > 0.05). We are currently assaying protein expression and phosphorylation in this cohort. Conclusions: Synaptic protein levels are robustly altered across postnatal development in the human primary visual cortex. The decreased synaptosome levels of canonical synaptic proteins and ASD risk genes likely reflects the rearrangement and loss of synaptic proteins that accompanies synaptic pruning in postnatal development and the importance of ASD genes in prenatal synaptic development. Our findings further suggest a role for Receptor-type tyrosine-protein phosphatase F, which may be involved in postsynaptic organization and regulation of tyrosine kinases, in postnatal synaptic alterations in ASD. Keywords: Autism, Proteomics, Postmortem Brain Tissue Disclosure: Nothing to disclose. P113. Hippocampal Mossy Cells Are Regionally Reduced in Adolescent and Adult 22q11.2 Deletion Syndrome Model Mice Alan Lewis, James Bauer, Leann Seanez, Catherine Cerroni, Yuval Guetta, Sam Kwon, Alex Steiner Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: Localized hyperactivity of the anterior hippocampal formation is a reproducible finding in patients with early psychosis. Identifying causes and consequences of regional hippocampal pathology at molecular and cellular levels of resolution is challenging in living humans, supporting the use of animal models of aberrant neurodevelopment predisposing to psychotic disorders, such as 22q11.2 deletion syndrome. Using a mouse model of 22q11.2 deletion syndrome, we tested whether mossy cells (MCs), a glutamatergic neuron in the dentate gyrus (DG) that is vulnerable to loss in other disorders of hippocampal hyperexcitability such as temporal lobe epilepsy, are reduced in number in adolescent and adult mice. We then performed RNA-sequencing to explore molecular pathways with potential relevance to localized MC changes. Methods: Df(h22q11)/+ and D2-Cre mice were purchased from Taconic Biosciences and UC Davis, respectively, and bred in the Lewis Lab at Vanderbilt. Male and female D2-Cre;22q/+ or D2-Cre;+/+ were sacrificed at P28 (adolescent) or at >12 weeks of age (adult) followed by immunohistochemistry for the MC marker GluR2/3, confocal microscopy, and manual cell counting, performed blind to genotype. Statistical analysis of cell counts was performed using t test (2-sided or 1-sided if testing for predicted reduction) or ANOVA with Sidak’s multiple comparisons test. RNA-sequencing was performed by the Vanderbilt VANTAGE genomics core using RNA extracted from 12-week-old Df(h22q11)/+ and + /+ littermate ventral hippocampal tissue. Library preparation was performed utilizing a ribo-depletion total RNA library preparation kit and sequencing performed at Paired-End 150 bp on the Illumina NovaSeq 6000 followed by differential gene expression analysis and p value correction for multiple comparisons. Results: Mossy cell number was reduced in the ventral but not dorsal DG of adult D2-Cre;22q/+ mice compared to D2-Cre;+/+ littermates (region x genotype interaction: F(1,19) = 9.087, p = 0.007; post-tests between genotypes: dorsal region: p = 0.87; ventral region: p = 0.001; N = 10-11/group). However, in this group of mice there was no reduction in DG granule cell density (p = 0.82) or DG parvalbumin+ interneuron number (p = 0.73). We next explored whether this reduction was also present during adolescent development, and found ventral MCs were reduced in 28-day-old D2-Cre;22q/+ mice versus + /+ littermates (p = 0.03, N = 6/group, 1-sided t test). RNA-sequencing from adult ventral hippocampal tissue (N = 3 Df(h22q11)/+, 3 + /+ mice) showed a significant reduction in 18 of 27 genes within the Df(h22q11)/+ deletion. This analysis also identified significant reduction in Fos and JunB, two members of the activator protein-1 (AP-1) family of transcription factors not encoded within the Df(h22q11)/+ deletion region. Immunohistochemistry revealed that both Fos and JunB were expressed in ventral DG MCs in wildtype mice. Conclusions: These studies show that MCs are reduced in number in mice with a genetic deletion orthologous to the human 22q11.2 deletion syndrome. This deficit occurs at least as early as P28, arguing that the loss is not due to cell death in later development. Several questions arise from this study: 1. Is the reduction in ventral MCs due to a problem with progenitor populations and/or cell-autonomous developmental dysfunction? 2. Do abnormalities in DG networks early in development, such as excitation-inhibition imbalance, result in cell non-autonomous deficits in MC maturation or stability? 3. Are AP-1 transcriptional networks actually perturbed in MCs as opposed to other cell types? If so, is this cell autonomous or non-autonomous? Taken together, regardless of whether the loss of ventral MCs per se contributes to cognitive or other aspects of 22q11.2 deletion syndrome, exploring the mechanisms underlying MC regional loss may yield a more general understanding of hippocampal molecular and cellular pathology in conditions predisposing to psychosis. Keywords: Hippocampus, 22q11 Deletion Syndrome, Dentate Gyrus, RNA-Sequencing, Psychosis Disclosure: Nothing to disclose. P114. Understanding Primate Amygdala Development: A View From Microglia Dennisha King, Judy Cameron, Ania Majewska, Julie Fudge University of Rochester, Rochester, New York, United States Background: The amygdala of nonhuman primates is remarkably similar to that of humans, developing over a lengthy postnatal period. This long development means that environmental events may influence neuronal growth and connectivity during early life. Our group and others–including those working with human postmortem tissue–have shown that throughout life, the primate amygdala has a repository of immature post-mitotic neurons (the paralaminar nucleus, PL) that surrounds and interdigitates with the main basal nucleus (Bpc). While most PL neurons are immature at birth, by adolescence a proportion have developed mature profiles suggesting ongoing differentiation. The Bpc in contrast is largely populated by mature neurons based on immunocytochemical markers. While we are beginning to understand normal neural maturation in the PL, the role of microglia, the brain’s immune cells, in PL development is unknown. However, microglia are critical for differentiation of precursor cells to neurons, clearing excess neuroblasts and pruning synaptic contacts. Each of these functions is associated with different morphological and molecular signatures. Microglia phagocytose unneeded neural precursors (large microglia soma, short thick processes, clustered), prune neural synapses (small microglia soma, ramified processes, relatively dispersed), and release growth factors that assist in neural differentiation. Methods: As a first step in characterizing the role of microglia in the developing PL, we assessed microglia in 3-month-old (infant) and 4-year-old (adolescent) macaques (n = 4/group) in both the PL and adjacent Bpc. We immunostained 1:12 sections through the amygdala for Iba1 (ionized calcium-binding adaptor molecule 1), which is a marker for microglia. The region of the PL and Bpc were identified using adjacent sections stained for mature and immature neurons. High-power photomicrographs were then taken at similar levels across cases, in a blinded fashion. We then analyzed morphologic features (microglia density, ’clustering’, and soma size/shape) using FIJI/Image J. Studies of microglia branching (Sholl) and immunostaining for chemical markers for synaptogenesis are ongoing. Results: The average density of microglia in the PL was 37% greater in adolescents (390 microglia/mm2) compared to infants (267 microglia/mm2; p = 0.0143), with no differences in density across medial, central, and lateral PL. The Bpc microglia density was also greater in adolescents (341 microglia/mm2) compared to infants (290 microglia/mm2), although by a lesser percentage: 16% (p = 0.0143). The clustering (spacing) index, which measures microglial distribution while accounting for density (calculated as: (average nearest neighbor distance)2 microglia density) was similar between the infant and adolescent groups in the PL (p = 0.395). In contrast, in adolescent Bpc the spacing index (0.473) was 10% less relative to infants (0.530), indicating closer spacing (p = 0.0277). Conclusions: These preliminary data show an increase in microglia numbers from infancy to adolescence in both the PL and Bpc. There is also increased clustering of Bpc microglia in adolescence, suggesting potential increased microglia interactions with neurons. Ongoing data analyses will determine whether differences in increasing microglia densities in each region are associated evidence of regional synaptogenesis. Keywords: Paralaminar Nucleus, Immune Markers, Cytokines, Synapses, Schizophrenia, Autism, Basal Nucleus Disclosure: Nothing to disclose. P115. Preliminary Observations From an Open Phase II Trial of Cannabidiol in Children With Autism Spectrum Disorder Francisco Castellanos, Paige Cervantes, Rebecca Shalev, Greta Conlon, Yuliya Yoncheva, Lauren Robinson, Glenn Hirsch, Andrea Troxel, Orrin Devinsky NYU Grossman School of Medicine, New York, New York, United States Background: Autism spectrum disorder (ASD) is common (~2.3% of children) and lacks specific pharmacologic treatments. Anecdotal reports and one placebo-controlled trial (of a 20:1 ratio of cannabidiol (CBD) and THC; Aran et al., Molecular Autism, 2021) suggest CBD may be helpful for some autistic children. CBD has been approved by the US FDA to treat refractory seizure syndromes in children and was well tolerated. We undertook an ongoing open trial of pharmacologic grade FDA-approved CBD without THC in children and adolescents without seizures and at least average intellectual ability as a basis for hypothesis generation and selection of dose and outcomes in future controlled trials. CBD is being provided at no cost by JAZZ Pharmaceuticals, which has had no had a role in designing the study, nor in analyzing or reporting results. Methods: Nineteen youth, ages 7-17 (M = 11.3 ± 2.9) with ASD completed an ongoing, 6-week Phase 2 open trial of 98% CBD (100 mg/mL) at 3, 6 or 9 mg/kg/d; target N = 30 [NCT03900923]. Dose was determined per a Bayesian optimal interval design starting at the 6 mg/kg/d dose with an initial expectation of 60% response rate. Inclusion criteria were a confirmed ASD diagnosis, verbal fluency, IQ ≥ 80, Social Responsiveness Scale (SRS-2) Total T-score ≥ 66, and Clinical Global Impression Scale–Severity (CGI-S) score ≥ 4 on an individualized target symptom domain identified at baseline by clinician consensus from informant report, rating scales, and clinical observation. Response was defined as CGI–Improvement (CGI-I) score ≤ 2 in the target domain. Adverse events (AEs) were assessed at weeks 2, 4 and 6 by clinician administered UKU Side Effects Rating Scale – Patient Version-5 to dyads, and at weeks 1, 3, 5 by coordinator phone calls to parents. Clinician consensus determined relatedness of AEs to treatment. In response to COVID-19, all screening procedures and assessments (excluding physical exams and blood testing to monitor safety) were conducted via secure virtual teleconferencing. Surveys were administered securely via REDCap software. Plasma CBD levels and clinical labs were obtained at the last session. Results: Demographics: We enrolled 18 males and 1 female. Full-scale IQ scores ranged from 81 to 136 (M = 104 ± 17). Seventeen had co-occurring psychiatric diagnoses: ADHD–Combined (n = 12), ADHD–Inattentive (n = 4), OCD (n = 4), Anxiety (n = 3), and Other–Specified Disruptive and Impulse Control Disorder (n = 2). Participants’ racial and ethnic identities included white (n = 12), white/Hispanic (n = 5), Asian (n = 2), Black/African American (n = 1), multiracial (n = 2), unknown race/Hispanic (n = 1), and unknown race/ethnicity (n = 1). All 19 enrolled participants completed the trial; overall, eight were classified as responders (42%); response rate was related to dose, ranging from 17% at 3 mg/kg/d (n = 6), to 44% on 6 mg/kg/d (n = 9) and 75% on 9 mg/kg/d (n = 4; this phase is ongoing). Dose correlated with CGI-I (r(18)=-0.43, p = .05). CGI–S scores improved significantly from pre-(M = 4.68 ± 0.48) to post-treatment (M = 3.95 ± 0.78), paired t(18)=3.98, p = <.001; Cohen’s d = 0.92; 95% CI [0.37, 1.44]. In a post-hoc analysis, we noted that none of the five participants taking stimulants for ADHD responded to CBD, while 8 of 11 (73%) who have ADHD but were not being treated with a stimulant responded (Fisher exact p = 0.013). A total of 83 AEs were reported; 79 were considered mild, 3 mild-moderate, and 1 moderate. Of 30 AEs judged by clinician consensus to be related to CBD, none were both related and unexpected. There were 17 unexpected AEs, all deemed unrelated. Liver function lab tests and complete blood counts were unaffected by CBD. Plasma CBD levels after 6-week treatment ranged widely from 2.4 ng/ml to 430.6 ng/ml and were related to dose (r(18)=0.46, p = 0.05) but not to CGI-I (r(18)=-0.19). Conclusions: CBD is well tolerated and seems to be beneficial for autistic youth although with a lower overall response rate than expected. We speculate placebo effects may have diminished based on informal tests conducted by families, facilitated by CBD legalization, although mean CBD doses in the community are 2 to 3 mg/kg/day (DiLiberto et al., 2022). Our experience suggests that higher doses are more likely to be beneficial and that benefits, when they emerge, do so over several weeks. Unexpectedly, we observed a high response rate (73%) in youth with comorbid ADHD not being treated with stimulants. These results resonate with Seeman’ 2016 observation (in Translational Psychiatry) that CBD has partial agonist properties at striatal D2 receptors, comparable to aripiprazole, one of two atypical neuroleptics approved to treat irritability in pediatric autism. We plan to continue enrollment until our IRB-approved accrual of 30 is reached, with a focus on ASD + ADHD comorbidity in the remaining participants. Keywords: Delta9-Tetrahydrocannabinol, Cannabidiol, Autism Spectrum Disorder and Related Syndromes, Phase 2, Attention Deficit Hyperactivity Disorder Disclosures: BOL Pharma: Advisory Board (Self), Frontiers Media: Honoraria (Self) P116. Pharmacological Interventions for the Treatment of Tourette’s Syndrome in Youth: A Systematic Review and Network Meta-Analysis Luis Farhat, Emily Behling, Angeli Landeros-Weisenberger, Jessica Levine, Pedro Macul Ferreira de Barros, Ziyu Wang, Michael Bloch Faculdade de Medicina FMUSP, Universidade de São Paulo, São Paulo, Brazil Background: Tourette’s syndrome (TS) is a neurodevelopmental disorder characterized by several motor, and at least one phonic, tics which onset during childhood or adolescence and last longer than 1 year. Practice guidelines from the American Academy of Neurology (AAN), the American Academy of Child and Adolescent Psychiatry (AACAP), and the European Society for the Study of Tourette Syndrome (ESTSS) agree that medications should be offered for individuals whose TS symptoms are moderate to severe, persistent, and associated with impairment. However, there is no agreement regarding what medications should be used as first-, second- or third-line treatments. The inconsistencies in treatment recommendations arise from the fact that pharmacological randomized double-blinded controlled trials (RDBCTs) in TS are scarce and based on small sample sizes, which creates difficulties when trying to determinate the head-to-head effects of different medications. Network meta-analyses facilitate the estimation of the comparative efficacy of two or more interventions, even when they have not been investigated in head-to-head RDBCTs and are required to inform treatment guidelines as the highest level of evidence. To fill this gap, we conducted a systematic review and network meta-analysis of pharmacological treatments evaluated in RDBCTs for the treatment of youth with TS. Methods: We searched PubMed, the Cochrane Central Register of Controlled Trials, Web of Science, Embase, PsycINFO, and the WHO International Trials Registry Platform, including ClinicalTrials.gov, from the date of database inception to November 19, 2021. We also hand-searched the US Food and Drug Administration, European Medicines Agency, and relevant drug manufacturer’s websites, as well as references of previous systematic reviews and guidelines, to look for additional studies. We included randomized, double-blinded controlled trials (RDBCT) that enrolled children and adolescents (aged ≥ 4 and ≤ 17 years), adults (≥ 18 years), or both with a primary diagnosis of TS according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-III/III-R/IV/IV-TR/5), the International Classification of Diseases (ICD-9/10/11) or the Chinese Classification of Mental Disorders 3rd edition (CCMD-3). We did not restrict eligibility based on medications provided they were administered as monotherapy. For the outcome, we considered efficacy, measured as change in tic symptom severity; tolerability, measured as the proportions of participants who discontinued the study due to adverse events; and acceptability, measured as the proportions of participants who discontinued due to any reason. Pairs of researchers independently reviewed the records to select the studies and extracted data. We performed random-effects network meta-analysis in a frequentist framework. We assumed a common heterogeneity variance τ2 across the various treatment comparisons. We quantified heterogeneity as low, moderate or high by comparing τ2 with its empirical distribution. We evaluated inconsistency globally (design-by-treatment interaction) and locally (separate indirect from direct evidence, SIDE). All analyses were done in R with packages meta or netmeta. We assessed risk of bias of RDBCTs with the Cochrane tool. We plotted comparison-adjusted funnel plots to investigate publication bias. We assessed certainty of findings from the network meta-analyses with Confidence in Network Meta-Analysis (CINeMA) framework. Results: Of the 12,088 records screened, 39 RDBCTs involving 4,578 participants (mean age [SD] 11.83 [4.55]; 80% male) were included in the network meta-analyses. Assessments of heterogeneity, inconsistency, and publication bias were incorporated in the ratings of quality of evidence. In terms of efficacy, aripiprazole (SMD -0.60; 95% CI -0.83, -0.38), haloperidol (SMD -0.51; 95% CI -0.88, -0.14), olanzapine (SMD – 0.83; 95% CI -1.49, -0.18), pimozide (SMD -0.48; 95% CI -0.84, -0.12), risperidone (SMD -0.66; 95% CI -0.98), clonidine (SMD -0.20; 95% CI -0.37, -0.02) and ecopipam (SMD – 0.34; -0.63, -0.06) led to larger changes in tic symptom severity than placebo (moderate certainty of evidence). There were no significant differences between antipsychotic medications in head-to-head comparisons (low to very low certainty of evidence), however aripiprazole (SMD -040; 95% CI -0.69, -0.12) and risperidone (SMD -0.46; 95% CI -0.82, -0.11) were more efficacious than clonidine (moderate certainty of evidence). In terms of tolerability and acceptability, there were no relevant findings for any of the efficacious medications against each other or placebo (low to very low certainty of evidence). Conclusions: Overall, our results support that antipsychotics are the most efficacious medications in the management of TS. Because there were no differences in efficacy between any pairs of antipsychotics based on the currently available data, the choice of antipsychotic should be guided mainly by their tolerability. Nevertheless, whether antipsychotics should be used as first-line pharmacological interventions in the treatments of TS is debatable given they are potentially toxic and may lead to somatic and serious adverse events, particularly in vulnerable populations such as children and adolescents. Although alfa2-agonists are less efficacious, their benefit-to-risk ratio may be higher because of less worrisome adverse events. Keywords: Tourette Syndrome, Psychopharmacotherapy, Meta-Analysis Disclosure: Nothing to disclose. P117. An Open-Label, Tolerability and Efficacy Study of ZYN002 (Cannabidiol) Administered as a Transdermal Gel to Children and Adolescents With 22q11.2 Deletion Syndrome (INSPIRE) Helen Heusslar, Jonathan Cohen, Caroline Buchanan, Carol O’Neill, Stephen O’Quinn, Terri Sebree, Steven Siegel Keck School of Medicine, USC, Los Angeles, California, United States Background: 22q11.2 deletion syndrome (22q) is caused by a microdeletion of region 11.2 on the long arm of chromosome 22 and is the most common recurrent contiguous gene deletion syndrome, estimated to occur in about 1 in 4000 live births. 22q is associated with a range of developmental anomalies including congenital heart defects, palate and pharyngeal defects and immunodeficiency. Behavioral problems, autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), and mood disorders occur frequently in children with 22q. ZYN002 is a pharmaceutically produced cannabidiol transdermal gel in development for treatment of the behavioral symptoms in 22q, Fragile X syndrome and ASD. INSPIRE was an open-label, phase 2 trial to evaluate the safety/tolerability and efficacy of ZYN002, in children and adolescents ages 4 to <18 years, in the treatment of behavioral and anxiety-related symptoms in 22q. Methods: Males and females with 22q confirmed by genetic testing, with or without autistic features, a Clinical Global Impression-Severity (CGI-S) score ≥4 and a Pediatric Anxiety Rating Score-Revised (PARS-R) score ≥10 were enrolled. Patients weighing ≤35 kg received 250 mg/day and those weighing >35 kg received 500 mg/day of ZYN002 in divided doses every 12 hours added to current stable therapy for 14 weeks. Patients with <25% improvement from baseline in the Aberrant Behavior Checklist-Community (ABC-C) Irritability subscale at week 6 could have their dose increased to either 500 mg/day or 750 mg/day based upon weight. Safety assessments included adverse events, vital signs, laboratories, and electrocardiograms (ECGs). Efficacy assessments included change from baseline on the Anxiety, Depression and Mood Scale (ADAMS), ABC-C, PARS-R, CGI-Improvement (CGI-I), and the Children’s Sleep Habits Questionnaire (CSHQ). A qualitative caregiver reported behavioral problems survey was also collected. Results: Twenty patients, 60% males, with a mean age of 9.9 years (5 to 15 years) were enrolled. Seventeen patients completed and 3 withdrew during the treatment period. Sixteen patients had evaluable efficacy assessments at week 14 and 13 patients entered a six-month extension period. Statistically significant improvements were seen in the ADAMS, ABC-C and PARS-R scales. Mean change and mean/median percent improvement from baseline on the ADAMS were as follows: Total Score -18.4, 45.3%/43.0%, p = 0.0005; General Anxiety -5.4, 43.6%/48.8%, p = 0.0005; Depressed Mood -4.3, 50.3%/52.8%, p = 0.0033; Social Avoidance -4.4, 41.3%/50.0%, p = 0.0084; Obsessive/Compulsive Behavior -1.9, 64%/66.7%, p = 0.0037; Manic/Hyperactive Behavior -3.1, 38.2%/27.4%, p = 0.0032. Mean change and mean/median percent improvement from baseline on the ABC-C were as follows: Social Withdrawal -6.4, 27.6%/46.4%, p = 0.011; Inappropriate Speech -1.8, 18.3%/50.0%, p = 0.0166; Stereotypic Behavior -2.3, 52.1%/58.3%, p = 0.0155; Irritability -8.4, 36.3%/39.6%, p = 0.0055; Hyperactivity -7.6, 16.5%/38.1%, p = 0.0091. Mean change and mean/median percent improvement from baseline on the PARS-R was: Total Score -6.2, 40.6%/40.0%, p = 0.0005. Twelve of 16 patients (75%) were rated as “improved”, “much improved” or “very much improved” at week 14, with 62.5% being “much improved” or “very much improved” on the CGI-I. Three patients reported treatment related adverse events which were all mild application site adverse events which were transient and resolved with continued dosing. One patient discontinued treatment due to adverse events not related to ZYN002. No serious adverse events or clinically significant changes in vital signs, ECGs or laboratories were reported. Conclusions: INSPIRE provides initial evidence suggesting a positive risk–benefit profile for ZYN002 in improving behavioral and anxiety-related symptoms in children and adolescents with 22q when added on top of stable standard of care. Further studies are warranted. Keywords: 22q11 deletion syndrome, Adolescence, neurodevelopmental disorders, cannabidiol, CBDV, translational models, clinical trials Disclosure: Zynerba: Consultant (Self) P118. Molecular Variation in Fragile X Syndrome: Impact on Drug Target Engagement Craig Erickson, Lauren Schmitt, Lisa DeStefano, Rui Lui, Lauren Ethridge, Kelli Dominick, Rebecca Shaffer, Meredith Will, Elizabeth Smith, John Sweeney, Ernest Pedapati Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States Background: Fragile X syndrome (FXS) is the most common single gene cause of autism and the most inherited form of developmental disability. Despite the available of fragile X knockout (KO) animal models, the translation of preclinical treatment success has not yet resulted in successful approved treatments in humans with FXS. We have discovered molecular variation in fragile X messenger ribonucleoprotein (FMRP) expression in males with FXS described with clinical genetic testing as having a fully methylated full mutation (CGG expansion) in the FMR1 gene. We have added peripheral blood FMRP assay to recent target engagement drug trials in FXS as a means to characterize patient-specific molecular pathology. We describe initial experience with this approach in placebo controlled trial of single dose baclofen (GABA B agonist) and two weeks daily dosing of BAER-101 (selective GABA A alpha 2,3 agonist) in humans with FXS. Methods: Utilizing a Luminex-based system we obtained peripheral blood FMRP levels in subjects with FXS enrolling in two Phase Ib target engagement studies in adolescents and adults with FXS. We then analyzed the outcome results of each trial using molecular FMRP level as a quantitative means to subgroup patients. The first trial was a double-blind, placebo-controlled crossover single dose study of acamprosate, lovastatin, minocycline, and baclofen in adolescents and adults with FXS. The second trial of a double-blind, placebo-controlled, crossover, two-week treatment period study of the selective GABA A alpha 2,3 agonist BAER-101 in adults with FXS. Both trials included performance-based outcome measures such as eye tracking, memory testing, and computer-base testing. Each project additionally focused on EEG-specific outcomes employing high density EEG study pre- and post-treatment in both studies. Results: In our single-dose challenge study, acamprosate, minocycline, and lovastatin dosing was not associated with any consistent changes in EEG signal. In 17 adolescents and adults (11 males and 6 females; mean age 26.3 years; range 16-43 years), baclofen (single 30mg dose) was associated with significant reduction in frequency gamma band EEG activity at rest. Increased gamma band activity is a known pathological EEG feature associated with FXS. In a FMRP-driven subgroup analysis, males who were Southern Blot and PCR diagnosed with a fully methylated full FMR1 mutation but who expressed trace levels of FMRP in blood showed a significant reduction in gamma band activity that correlated with improvement in social gaze on an eye tracking measure. This positive association between EEG and clinical change was not noted in females or males expressing zero FMRP in blood. Overall, a significant association was noted between FMRP level and gamma band EEG change with lower FMRP levels associated with greater reduction (rescue) of gamma band activity. In our study of BAER-101 in 13 adults with FXS, low dose (5mg BID) drug was associated improvement in memory as measured by the Repeatable Battery of Neuropsychiatric Status (RBANS) in males only. No significant BAER-101 associated improvement in EEG measures were noted across all patients. Our FMRP-driven subgroup analysis noted that males expressing zero FMRP in blood had low-dose BAER-101 associated improvement in social withdrawal as measured by the Aberrant Behavior Checklist (ABC), an opposite directional change compared to males expressing some FMRP. Additionally, this zero FMRP male subgroup showed a relevant correlation between gamma band power change and memory improvement with gamma band reduction being associated with more improvement in memory as measured by the RBANS. Conclusions: We have noted early evidence that FMRP status in fragile X syndrome (FXS) may represent a disease/disorder continuous molecular measure that impacts small molecule treatment response. The ability to effectively identify molecular patient subgroups based on peripheral FMRP analysis holds promise for use in larger Phase II and III studies to match patient groups to particular drugs under study. We believe FMRP levels will support future small molecule studies in FXS and potentially enable personalized medicine approaches in this field. We need to gather data from larger patient groups to potentially confirm our initial results that baclofen use is associated with a strongest positive signal in males expressing trace levels of FMRP and BAER-101 use is associated with a positive signal in males expressing zero FMRP. Keywords: Fragile X Syndrome, Clinical Trial Methodology, EEG Disclosures: Forge Therapeutics, Impel, Scioto Bioscience, Stalicla: Consultant (Self) P119. Stress-Induced Plasticity of a Novel CRH GABA Projection Disrupts Reward Behaviors Matthew Birnie, Annabel K. Short, Gregory de Carvalho, Benjamin G. Gunn, Aidan L. Pham, Christy A. Itoga, Xiangmin Xu, Lulu Y. Chen, Stephen V. Mahler, Yuncai Chen, Tallie Z. Baram University of California-Irvine, Irvine, California, United States Background: Disrupted operation of reward circuits is thought to underlie several emotional disorders including depression and drug abuse, disorders commonly arising after early-life stress. Yet, how early-life adversities (ELA) impact the functional maturation of reward circuitries to promote disease remains unclear. The nucleus accumbens (NAc) is a major component of the reward circuit and key structure mediating pleasure, motivation, and emotional processes. Multiple inputs converge onto the NAc to modulate reward behaviors, including the basolateral amygdala (BLA). The BLA mediates associative learning for aversive and appetitive stimuli, and stimulation of glutamatergic projections from the BLA to NAc promotes appetitive behaviors. Here, we identified a novel projection that expresses the stress neuropeptide corticotropin-releasing hormone (CRH) to connect the basolateral amygdala (BLA) and nucleus accumbens (NAc). In the NAc, CRH + axon terminals modulate reward and motivational behaviors. Here, we identify the role of this CRH + BLA-NAc projection during reward in naïve and ELA mice. Methods: Pairing viral-genetic approaches with CRH-IRES-Cre mice and Cre-dependent viruses, we identified CRH + BLA projections to the NAc. To determine the function of this novel CRH + BLA-NAc projection we used chemo-, optogenetic and electrophysiology strategies in control (CTL) and ELA mice. In these mice, excitatory or inhibitory Cre-dependent DREADDs and optogenetic viruses were injected into BLA, followed by medial NAc shell targeted microinjections of CNO or light activation. In behavior, we tested the function of this pathway using reward, and non-reward tasks. Results: Male ELA mice have reduced preference for sucrose, palatable food, and a sex-cue, compared with CTLs. Viral-genetic tracing combined with electrophysiology identified a novel GABAergic projection that co-expresses the stress neuropeptide CRH from the BLA to the medial NAc shell. In freely behaving mice, exciting this projection using chemo- and optogenetic techniques reduced preference for sucrose, palatable food, and a sex-cue, but did not alter non-reward-mediating tasks. In adult ELA mice, chemogenetic inhibition of the GABAergic CRH + BLA-NAc projection rescued these reward behaviors. Conclusions: Here, we identify a novel GABAergic CRH + BLA-NAc projection and establish its role in mediating the effects of stress on reward behavior. These discoveries provide potential selective targets for prevention and intervention in the disruption of such behavior that accompanies several psychopathologies. Keywords: Basolateral Amygdala, Nucleus Accumbens, CRH, Early Life Stress, GABA Disclosure: Nothing to disclose. P120. Quantitative Sensorimotor Traits as Familial Endophenotypes Associated With Autism Spectrum Disorder (ASD) Matthew Mosconi, Erin K. Bojanek, Shannon Kelly, Lauren Schmitt, Stormi P. White, John Sweeney University of Kansas, Lawrence, Kansas, United States Background: Sensorimotor impairments, including reduced feedforward and feedback control of motor behaviors, are common in autism spectrum disorder (ASD) and have been documented in unaffected first-degree relatives. These findings suggest sensorimotor traits may serve as endophenotypes useful for understanding heritable risk pathways associated with ASD. To determine the extent to which sensorimotor traits were familial in ASD, sensorimotor behaviors were examined across a cohort of family trios that included individuals with ASD (probands) and each of their biological parents. We also tested multiple separate motor behaviors (rapid and sustained) across multiple effector systems (hand and eye). Methods: Fifty-eight individuals with ASD (probands; ages 5-17 years), 109 parents (ASD parents; ages 29-54 years), and 89 neurotypical (NT) control participants matched on age, sex, and nonverbal IQ completed manual and oculomotor tests. To assess feedforward control processes, participants completed tests of rapid precision gripping (manual motor) and visually guided saccades (VGS; oculomotor). During the rapid gripping test, participants viewed a static red/green target bar and were instructed to press the load cells as quickly as possible when the red target bar turned green so that a white force bar reached the level of the green target bar set to 15, 45, or 75% of their maximum force. The accuracy of initial force output was examined. During the VGS test, participants fixated a central cross-hair and made reactive saccades to suddenly appearing peripheral targets at + 12 or 24 deg. The accuracy of saccades was examined. To assess sensory feedback control of motor behavior, participants completed sustained precision gripping (manual) and smooth pursuit eye movement tests (oculomotor). The sustained gripping test was similar to the rapid grip test, but participants were instructed to maintain a constant force at the level of the target bar for 8 sec. The variability of sustained grip force was examined. During the smooth pursuit test, participants tracked targets moving from 0 to + 15 deg at varying velocities (2-30 deg/s). Smooth pursuit gain, or the ratio of pursuit velocity relative to target velocity, was examined. We also conducted subgroup analyses comparing families with at least one parent showing broader autism phenotypic traits (BAP+) and those with two parents without BAP traits (BAP-). Results: During the rapid precision grip and VGS tests, probands showed reduced accuracy relative to NT controls. These differences were specific to probands with BAP- parents. BAP- parents also showed reduced saccade accuracy relative to BAP + parents and NT controls. Saccade accuracy was inter-related among family trios. During the sustained precision grip and smooth pursuit eye movement tests, probands showed performance deficits relative to NT controls, including increased variability during gripping and reduced gain during smooth pursuit. Increased force variability was more severe in probands with BAP + parents than those with BAP- parents. BAP- parents showed increased precision grip force variability relative to BAP + parents and controls. Conclusions: Findings that rapid sensorimotor behaviors are selectively impacted in BAP- probands and parents suggest they may reflect polygenic liabilities independent of familial autistic traits. Sustained sensorimotor behaviors were affected in BAP + probands and BAP- parents implicating overlapping or additive polygenic risk with core autism traits. These findings are consistent with prior research indicating sensorimotor impairments may serve as useful, quantitative endophenotypes reflecting inherited polygenic risk for ASD. Results also indicate that separate sensorimotor behaviors associated with distinct neurophysiological processes may represent unique pathways of familial risk for ASD. Keywords: Autism, Sensorimotor, Endophenotypes Disclosure: Nothing to disclose. P121. Identifying Cerebral Cortex Indices That Predict Autistic Symptom Severity Yi-Ling Chien, Susan Shur-Fen Gau National Taiwan University Hospital, Taipei, Taiwan (Republic of China) Background: Abnormal cortical anatomy is among the significant features of the neuropathology in autism spectrum disorders (ASD). Several cortical regions have been identified to be altered in individuals with ASD. However, which cortical index can be correlated with clinical severity of autistic symptoms is inconclusive. This study aims to identify the cortical components that can better explain the overall severity of ASD. Methods: We recruited 122 patients with ASD and 118 typically-developing controls (TDC). All the participants underwent brain MRI assessment. Cortical thickness was analyzed by using FreeSurfer software with 74 automatic parcellations. Autistic symptom severity was measured by Social Responsiveness Scale (SRS), Social Communication Questionnaire (SCQ), and Autism Spectrum Quotient (AQ). We adopted principal component analysis on the cortical thickness and cortical volumes of cerebral cortex to identify the main components of the cortical indices, and examined their correlations with autistic symptom severity. Results: In principal component analysis, there were 9 components for white matter volume, 3 for cortical volume, and 6 for cortical thickness. To correlate these factors with autistic traits, we found that none of the 9 white matter components were significantly correlated with AQ or SRS total scores. For cortical volume components, we found that the second component was correlated with AQ and SRS total scores. The correlation coefficients were 0.27 for AQ total scores, 0.37 for SRS total scores. Both are low-level correlations. As for cortical thickness component, the first three components were correlated with AQ total scores. The correlation coefficients were ranging from 0.28 to .45, with the highest correlation on factor 3. For SRS, factor 2, 3, 4 were correlated with total scores, correlation coefficients ranged from 0.24 to 0.34, the highest one on factor 3, too. The top one is the third component, to the level of medium correlation. We further examined other cortical thickness parameters to see whether that can better correlate with ASD severity. We adopted the other two cortical parcellation instead of FreeSurfer. Using 7 network built based on the intrinsic functional connectivity proposed by Yeo, the factor 1 and 3 were correlated with AQ and SRS, correlation coefficients were as high as 0.39, a bit lower than those in DKT40 cortical thickness components that reaches 0.45. Finally, regarding the BA principal components, the first two factors were correlated with both AQ and SRS. The correlations between factor 1 and AQ or SRS can be as high as 0.7. Conclusions: This study found a major component of cortical thickness that was significantly correlated with autistic symptom severity. Autistic severity is associated with the principal components of cortical thickness and volume, but not with those of white matter volumes. Autistic severity is also associated with the principal components based on function connectivity. The highest correlations were among the sensory and motor area components based on BA area. Our findings warrant further validation. Keywords: Autism, Cerebral Cortex, ASD Core Symptoms Disclosure: Nothing to disclose. P122. Differential Symptom Response to 12-Week Mixed Amphetamine Salts in ADHD Youth With Versus Without Familial Risk for Bipolar I Disorder: Associations With Polyunsatutared Fatty Acid Biostatus Robert McNamara, Alexis Brown, Maxwell Tallman, Thomas Blom, Jeffrey Welge, Jenni Farrow, L. Rodrigo Patino, Melissa DelBello* University of Cincinnati, Cincinnati, Ohio, United States Background: The initial onset of bipolar I disorder (BD) frequently occurs during the peripubertal period, and is commonly preceded by attention deficit/hyperactivity disorder (ADHD). Prospective studies have shown that ADHD increases risk for developing mood disorders including BD, and is associated with an earlier age at onset of mood symptoms. However, vulnerability factors associated with the risk of developing BD in youth with ADHD remain poorly understood. Having a first-degree relative with BD robustly increases risk for BD in offspring, and youth with a first-degree BD relative exhibit higher rates of ADHD and more severe ADHD symptoms. Additionally, retrospective studies suggest that antecedent stimulant exposure may precipitate or exacerbate manic symptoms and accelerate the onset of mania in a subset of individuals. Lastly, BD youth exhibit deficits in omega-3 polyunsaturated fatty acids (n-3 PUFA) including docosahexaenoic acid (DHA), and developmental DHA insufficiency in rodents alters dopamine neurotransmission and response to psychostimulants. To investigate these potential risk factors, this study compared the effects of 12-week psychostimulant treatment in ADHD youth with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD. It was hypothesized that HR youth would exhibit greater increases in symptoms previously found to precede and predict the initial onset of BD, including manic symptoms and parent-reported ratings of dysregulation, compared with LR youth, and that higher n-3 PUFA biostatus would be negatively correlated with these changes. Methods: ADHD youth (ages 10-18 years) with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD were enrolled. LR youth received 12-week open-label mixed amphetamine salts-extended release (MAS-XR), and HR youth were randomized to MAS-XR or placebo (PBO). All ADHD patients met DSM-5 criteria for ADHD (any type), had no exposure to psychostimulants for at least 3 months prior to enrollment, and had no comorbid mood, conduct, eating, or psychotic disorders. Clinician ratings of ADHD (ADHD-rating scale, ADHD-RS), mania (Young Mania Rating Scale, YMRS), depression (Children’s Depression Rating Scale-Revised, CDRS-R), global functioning (Children’s Global Assessment Scale, CGAS), and global symptom severity (Clinical Global Impression-Severity Scale, CGI-S) were performed, and parents completed the Child Behavior Checklist (CBCL), at baseline and week 12. Baseline red blood cell (RBC) membrane PUFA (DHA, eicosapentaenoic acid [EPA] + DHA, arachidonic acid, AA) levels were determined by gas chromatography. Group x time interactions in baseline-endpoint change scores were calculated, and correlations between symptom change scores with baseline fatty acid levels were performed. Results: A total of n = 96 (HR: n = 47; LR: n = 49) ADHD youth (mean age: 13.9 ± 2.5 years) were enrolled (LR-MAS: n = 49; HR-MAS: n = 31; HR-PBO: n = 16). There were no significant baseline group differences in age, sex, race, pubertal status, body mass index, or prior exposure to psychostimulants. The HR-MAS group had lower baseline RBC levels of DHA (p = 0.01) and EPA + DHA (p = 0.007), but not AA (p = 0.91), compared with LR-MAS but not HR-PBO. Following 12-week MAS-XR treatment (median MAS-XR dose: LR: 17.7 ± 5.2 vs HR: 15.8 ± 5.7, p = 0.12), the LR-MAS group exhibited significantly greater reductions in ADHD-RS total scores (p = 0.015) and inattentive subscale scores (p = 0.0001) compared with HR-MAS. A greater percentage of LR-MAS achieved remission (ADHD-RS endpoint score <18) compared with HR-MAS (92% vs. 61%, p = 0.0001). LR-MAS youth also exhibited significantly greater improvements in overall illness severity (CGI-S, p = 0.0001) and global functioning (CGAS, p = 0.0008) compared with HR-MAS, and there was a trend for greater reductions in CBCL-dysregulation profile subscale score (p = 0.08). For all ratings, there were no significant differences in baseline-endpoint change in HR-MAS versus HR-PBO groups, and no significant group differences were observed for changes in YMRS and CDRS total scores. Among all ADHD subjects (n = 96), higher baseline RBC DHA levels were associated with greater improvements in global functioning scores (p = 0.04), and higher AA levels were associated with greater reductions in depression (p = 0.02), CBCL total score (p = 0.02) and CBCL externalization (p = 0.03), internalization (p = 0.04), and dysregulation (p = 0.01) subscale scores. In HR-MAS and LR-MAS groups, AA levels were differentially associated with changes in CGI-S (group interaction, p = 0.028), ADHD-RS total score (p = 0.040), and ADHD-RS hyperactivity/impulsivity subscale score (p = 0.02). Conclusions: Following 12-week MAS-XR treatment, ADHD youth without a BD family history exhibit greater reductions in inattention symptoms, and greater improvements in overall illness severity and global functioning, compared with ADHD youth with a BD family history. ADHD youth with a BD family history exhibited lower DHA biostatus compared with ADHD youth without a BD family history, and both DHA and AA were associated with different symptom changes. These findings suggest that ADHD youth with a BD family history may require alternative or adjunctive treatments for inattention and global functioning, and PUFA associations with symptom changes warrant further investigation. Keywords: Bipolar Disorder, ADHD, Psychostimulants, Prodrome Disclosures: Myriad, Medscape: Advisory Board(Self), Alkermes, Janssen: Consultant (Self), Alkermes, Lundbeck, Janssen, Allergan, Shire: Contracted Research (Self), P123. Community Sample Data-Derived Communication Profiles and Early Autistic Phenotypes Angela Tseng, Amy Yang, Bobbi Rohwer, Jason Wolff, Jed Elison, Suma Jacob* University of Minnesota, Minneapolis, Minnesota, United States Background: Delays in language development are one of the earliest features associated with Autism Spectrum Disorder (ASD) (Tager-Flusberg, 2016); often, observations of atypical communication abilities serve as a tocsin for parents to seek neurodevelopmental evaluations for their child (Richards et al., 2016). Autistic children pronounce their first words and phrases later than their peers (Roemer et al., 2019, Charman et al., 2003) and delays in non-verbal communication (e.g., gestures) associate with impaired verbal communication - even before language development (Colgan et al., 2006, Luyster et al., 2008, Mitchell et al., 2006). Further, receptive and expressive language skills associate negatively with restricted and repetitive behaviors in both autistic (Ray-Subramanian and Ellis Weismer, 2012) and neurotypical (NT) children (Larkin et al., 2017) with improvements in language skills predicting symptom reduction. Yet, little is known about the role of early verbal and non-verbal language development on sensory-motor and communication trajectories in ASD and NT samples. In the present analysis, we first used the MacArthur Bates-Communication Development Inventory (MB-CDI): Words and Gestures (Infant Form) (Fenson et al., 1994) to delineate data-driven, early communication profiles in a community sample. The MB-CDI infant form is a widely-used and validated measure designed and normed for use with 8- to 18-month old and older, developmentally-delayed children; caregivers are asked to rate their child’s abilities on several components of language development, yielding raw scores and percentile rankings for expressive (Words Produced; WP) and receptive (Words Understood; WU, Phrases Understood; PhU) language, as well as a child’s use of action and gestures (Total Gestures; TG). In an effort to explore the informative potential of these novel profiles, we then examined associations between these groupings with data collected concurrently using the Video-Referenced Rating of Reciprocal Social Behavior (vrRSB; 18-30 month version), a parent-report measure that has demonstrated strong reliability quantifying autistic traits in community samples (Marrus et al., 2018, Marrus et al., 2015, Marrus et al., 2020) and the Repetitive Behavior Scales – Early Childhood Supplement (RBS-EC) (Wolff et al., 2016, Sifre et al., 2021), a parent-report measure of restrictive and repetitive behaviors for children (8 months - 8 years). Methods: Survey measures (MB-CDI: WG, vrRSB, RBS-EC, demographics) were collected online from parents of N = 904 (47.9% Female), community-ascertained children (Mean Age = 18.08 ± 0.53 months) during the baseline phase of a larger, descriptive longitudinal study. Applying data-driven, two-step cluster (TSC) analysis with MB-CDI percentile rankings (WP, WU, PhU, TG), we employed a log-likelihood distance measure, Akaike’s Information Criteria (AIC) clustering criterion, and a maximum of 15 clusters. Silhouette coefficients of cohesion and separation, along with membership variables of each cluster solution, were used to derive parsimonious profile TSC groupings. Multiple general linear models (GLMs) were used to examine associations between MB-CDI data-derived TSC groups, sex, age, vrRSB, and RBS-EC scores. Results: TSC analysis with percentile rankings for TG/WP variables yielded a four-cluster solution with a good silhouette measure of cohesion and separation coefficient (0.62): 1. LowWP/LowTG (N = 390); 2. HighWP/LowTG (N = 139); 3. LowWP/HighTG (N = 243); 4. HighWP/HighTG (N = 132). A similar four-cluster solution was found for WU/WP in the good cluster quality range (coefficient = 0.56): 1. LowWU/LowWP (N = 354); 2. LowWU/HighWP (N = 233); 3. HighWU/LowWP (N = 170); 4. HighWU/HighWP (N = 147). TSC analysis with PhU alone yielded a three-cluster solution (coefficient = 0.74): 1. LowPhU (N = 385); 2. MidPhU (N = 362); HighPhu (N = 157). Covarying for age, GLM analyses demonstrated discriminant grouping by profile cluster membership and sex for ASD characteristics on vrRSB subscale and composite scores (p < 0.01) as well as mean frequency and interference subscale and composite scores on the RBS-EC (p < 0.05). Broadly, clusters with lower MB-CDI percentile rankings on all included variables (e.g., LowWP/LowTG, LowWU/LowWP) associated with higher vrRSB and RBS-EC scores (i.e., increased risk for ASD). However, clustering also suggested distinct profiles for individuals showing asymmetrical development of verbal and non-verbal communication skills (e.g., LowWP/HighTG, HighWU/LowWP) that may provide further insight into the clinical heterogeneity of ASD. Conclusions: Emergent ASD features. Of particular interest, infant siblings of autistic children at heightened risk for an ASD diagnosis (18.7% recurrence rate) demonstrate substantial variability in their language and gesture development (Ozonoff et al., 2011, Iverson et al., 2018) suggesting that associations between expressive and receptive verbal abilities and gestures should be examined in the broader population to capture heterogeneity in communicative development. Our community sample data will inform efforts to characterize early markers of ASD and focus early therapeutic targets for more tailored interventions. Keywords: Autism, Communication, Language Delay, Cluster Analysis, Community Detection Disclosure: Nothing to disclose. P124. Local Network Architecture and Attention Problems in Children With Concussion Sonja Stojanovski, Guido Guberman, Eman Nishat, Jean-Christophe Houde, Maxime Descoteaux, Anne Wheeler* SickKids Research Institute, Toronto, Canada Background: Attention problems are common after concussion in children. Short superficial white matter (SWM) fibers in the brain are particularly vulnerable to concussion in children due to their protracted myelination and location at the grey-white matter interface beneath the cortex. The objectives of this study are to describe alterations in the SWM in children with concussion, and their impact on network community structure and attention. Methods: Male and female children age 9-10 years with concussion (N = 339) were matched to children without concussion (N = 339) from the Adolescent Brain Cognitive Development Study. Particle filtering tractography was applied to multishell diffusion MRI data to generate matrices weighted by four measures from the tensor model (FA, MD, AD, RD) and two from fiber orientation distribution functions obtained from Constrained Spherical Deconvolution (AFD, NuFO). SWM was derived via length thresholding (<85mm), while measures of local community (modularity, mean clustering coefficient) were calculated from the complete matrices. Attention was assessed with the CBCL attention problem scale. Differences between the concussion and control group and relationship between measures were assessed with linear mixed effects models. Results: Children with concussion had more clinically significant attention problem scores (p = 4.9x10-6). Concussed children had elevated FA, MD, RD, AFD and NuFO (ps<2.0x10-8), lower AD (p = 2.0x10-16) in SWM as well as lower network modularity (p = 2.0x10-16) and mean clustering coefficient (p = 2.0x10-16) compared to controls. Interactions between SWM measures and age showed that in younger children with concussion altered SWM measures were associated with more attention problems. Conclusions: Maturation of SWM fibers and local network structure may be altered by concussion and impair attention. Keywords: Concussion, Attention, Diffusion MRI, Brain Networks, White Matter Microstructure Disclosure: Nothing to disclose. P125. Predicting Longitudinal Trajectories of Child Psychopathology With Explainable Neural Network Ensembles: An Adolescent Brain and Cognitive Development Study Analysis Jason Smucny, Linhao Chen, Hiroshi Sakakibara, Soumil Shekdar, Ruyi Yang, Ian Davidson, Cameron Carter University of California - Davis, Sacramento, California, United States Background: Early identification of children with behavioral and psychopathological problems is of critical importance to clinicians and families alike. Although predictive modeling methods such as machine learning (ML) have been used to identify predictive factors, to date most of this work has been cross-sectional, i.e., using baseline predictors to predict baseline symptoms. Here we use an explainable, multimodal, ensemble ML approach using a large dataset (the Adolescent Brain and Cognitive Development (ABCD) Study), in which we use baseline features to predict the emergence of “Persistent” (vs. “Absent/Remitting”) Child Behavior Checklist (CBCL) scale scores (Anxious/Depressed, Withdrawn/Depressed, Somatic Complaints, Social Problems, Thought Problems, Attention Problems, Rule-Breaking Behavior, Aggressive Behavior, Internalizing, Externalizing, Total Problems) a two-year follow-up period. Methods: Demographic, environmental, neurocognitive, and brain imaging data were trained in separate neural network-based models and combined in an ensemble to predict Persistent CBCL scores (defined as a CBCL t-score of ≥ 65 on at least two timepoints (baseline/one-year/two-year follow-ups) vs. Absent/Remitting (defined as a CBCL t-score of ≥ 64 on at least two timepoints). Neuroimaging features were reduced by principal components analysis (PCA) prior to inclusion, with separate PCAs performed for each modality (volumetric structural MRI, diffusion tensor imaging, resting state fMRI, task fMRI for each contrast of interest). Site was also included as a predictor. Because the sample was highly imbalanced (~95% were Absent/Remitting for all CBCL scales), the minority class (Persistent) was upsampled and loss function weights were modified such that incorrectly predicting the minority class resulted in greater loss. Learner performance was validated by k-fold cross validation. The explainable artificial intelligence (XAI) integrated gradient method was used to identify the most salient predictors. Results: Out of a sample size of 7902 with complete data, 378 were defined as Persistent and 7524 as Absent/Remitting on average across CBCL scores. Mean model accuracy across all scales was 79% (95% C.I. 78%-80%), including 83% accuracy (95% C.I. 82%-84%) for Total Problems, 83% accuracy for Externalizing broadband score (95% CI 81%-85%), and 74% accuracy for Internalizing broadband score (95% C.I. 73%-75%). Predictive accuracies for 2-year trajectories of specific syndromes ranged from 70% (for Somatic Complaints) to 90% (for Rule Breaking Behavior). XAI suggested Reasoning scores on the WISC-V battery, School Risk and Protective Factors Questionnaire School Involvement and Environment Scores, family income, and sleep problems were the five most salient predictors of syndrome trajectories. Conclusions: These results suggest that machine learning using baseline data can predict 2-year trajectories of CBCL scores in children. Cognitive reasoning, school problems, family income levels, and issues with sleep may be particularly effective predictors of clinically significant trajectories. Keywords: Developmental Psychopathology, Machine Learning, explainable-AI, ABCD Study, Sleep Disturbances Disclosure: Nothing to disclose. P126. Efficiency of Evidence Accumulation as a Potential Mediator of the Relation Between Neural Response to Cognitive Demand and ADHD Symptoms in Youth Alexander Weigard, Mike Angstadt, Aman Taxali, Zvi Shapiro, Mary Heitzeg, Chandra Sripada University of Michigan Medical Center, Ann Arbor, Michigan, United States Background: Efficiency of evidence accumulation (EEA), a cognitive mechanism formally defined in computational models that drives people’s cognitive performance across many contexts, has recently been found to be impaired in several psychopathologies, and most clearly in childhood Attention-Deficit/Hyperactivity Disorder (ADHD). Although emerging findings suggest that EEA may be supported by the frontoparietal network and related neural systems that respond to external task demands, there is currently a dearth of research testing whether EEA plausibly mediates relations between the functioning of these neural systems and psychopathology. In the Adolescent Brain Cognitive Development (ABCD) study sample, we investigated relations between individual differences in neural responses to cognitive demand on the n-back task, which is known to engage frontoparietal and other task-positive networks while suppressing networks associated with off-task processing (e.g., default mode network), and individuals’ EEA. We then evaluate whether EEA is a potential mediator of the relationship between neural responses to cognitive demand and ADHD symptoms measured one year later by both parent- and teacher-report. Methods: We focused on a subsample of youth from ABCD Release 4.0 (n = 2347) who 1) had n-back neuroimaging data from the baseline timepoint that passed quality control checks, and 2) had valid parent and teacher reports of ADHD symptoms at the year-1 follow-up visit. EEA was measured by fitting the diffusion decision model (DDM) to data from the n-back task and data from a separate task completed in the neuroimaging session, the stop-signal paradigm (“go” trials only), using Bayesian estimation within the Dynamic Models of Choice R suite. We created multivariate “expression scores” for individuals’ neural responses to cognitive demand by taking voxel-wise parameter estimates for the group-level 2-back versus 0-back contrast – which robustly activates task-positive networks and deactivates task-negative networks – and multiplying these estimates by each individual’s voxel-wise parameters for the same contrast. Hence, higher values of such scores indicate that an individual displays a greater expression of the normative pattern of neural responses to cognitive demand. ADHD symptoms were measured dimensionally by fitting a bifactor model to parent- and teacher-report data from the 1-year follow-up and estimating scores for the general factor that represents shared variance across the reporters and across home and school contexts. We first tested bivariate relations between the neural expression scores, EEA, and ADHD symptoms. We then tested formal mediation models in which EEA mediates the relation between the neural expression scores and future ADHD symptoms. All inferential models were estimated in MPlus with clustered bootstrapping used to estimate standard errors while accounting for the nesting of participants within families and ABCD study sites. Results: Expression scores for neural responses to cognitive demand on the n-back task showed a strong positive relation with EEA on the same task (standardized Β = 0.58, p < .001) as well as a smaller relation with EEA on the stop-signal task (Β = 0.19, p < .001). Expression scores were negatively related to ADHD symptoms (Β = -0.16, p < .001). EEA was related to future ADHD symptoms regardless of whether it was measured on the n-back task (Β = -0.17, p < .001) or stop-signal task (Β = -0.16, p < .001). The mediation model involving EEA from the n-back displayed an effect of the expression score on EEA (Β = 0.58, p < .001), an effect of EEA on future ADHD symptoms (Β = -0.12, p < .001), and a significant total indirect effect involving mediation by EEA (Β = -0.07, p < .001). A remaining direct effect of the expression score on ADHD symptoms was also detected (Β = -0.09, p = .001). Despite the smaller bivariate relation between the neural expression scores and EEA measured on the stop-signal task, all of the above relations remained significant in a second mediation analysis involving EEA on the stop-signal rather than the n-back, suggesting that this mediation effect is not specific to n-back performance. Conclusions: In a subsample from the ABCD study, we found that neural responses to cognitive demand on the n-back – which reflect activation of frontoparietal and other task-positive networks and deactivation of the default mode network – show strong relations to EEA on this task as well as more general relations to EEA measured on a separate stop-signal task. Furthermore, we find that EEA partially mediates the relation between neural responses to cognitive demand and future ADHD symptoms measured across parent and teacher reports and that this mediation effect is robust to whether EEA is measured on the same task as the neural responses. These findings both establish neural responses to cognitive demand as a key neural correlate of individual differences in EEA in childhood and provide the first formal test of whether EEA plausibly mediates the relationship between neural functioning and ADHD symptoms. Keywords: Attention Deficit Hyperactivity Disorder, Computational Cognitive Neuroscience, Computational Psychiatry, Frontoparietal Network Disclosure: Nothing to disclose. P127. Combined Effects of Gestational Flame Retardants and Immune Activation on Behavior in Mice Victoria Rodriguez, Hayley He, Alexa Soares, Ariana Navar, Joseph Slama, Dionisio Amodeo, M. Margarita Behrens, Susan Powell* University of California, San Diego, La Jolla, California, United States Background: Evidence suggesting a role for environmental factors contributing to the origin of several neurodevelopmental disorders has accumulated in recent years, and increasingly points to a possible interaction between genetic vulnerability and the environment in the generation of autism and schizophrenia. Indeed, epidemiological studies have consistently found that maternal infection and chemicals in the environment, on their own or in conjunction with genetic factors, increase the risk for ASD. Animal models of maternal infection have determined that the maternal immune activation (MIA) is a key factor in the neurodevelopmental alterations observed in offspring. In addition to exposure to infectious agents, there is increasing evidence that exposure to environmental toxins may contribute to ASD risk. Polychlorinated biphenyls (PCBs, banned since 1976) and polybrominated diphenyl ethers (PBDEs, used as fire retardants) have been accumulating in the environment for decades. Mouse models have demonstrated that exposure to these compounds produce alterations in neurodevelopment leading to changes in synaptic plasticity, and behavioral alterations. Because of the adverse effects observed in humans and the toxicity observed in animal models, it has become an increasing concern that PBDE chemicals contribute adversely to neurodevelopment. Our previous studies showed that prenatal poly(I:C) exposure impaired probabilistic reversal learning, decreased social approach in offspring and produced long term effects on the genes involved in glutamine neurotransmission, mTOR signaling and potassium ion channel activity. This study tested if gestational and early prenatal exposure to PBDE chemicals found in flame retardants will exacerbate the effects of maternal immune activation on behavioral phenotypes linked to neurodevelopmental disorders. Methods: After completing a dose response of BDE-47 in a previous experiment, we concluded that a 0.03 mg/kg/day dose of BDE-47 was well tolerated in dams. Female C57BL6/J mice (8 weeks of age; Jackson Lab, Bar Harbor, ME) were exposed to 0.03 mg/kg BDE-47 in corn oil via a cornflake for 4 weeks prior to mating and then during gestation through weaning (~10 weeks total). A separate group of female mice received a cornflake with corn oil only. After timed-matings pregnant females were exposed to either Poly(I:C) (20 mg/kg) or saline on E12.5. Offspring were tested in a behavioral battery with relevance to neurodevelopmental disorders. Results: Overall, litter survival was low in the experiment. The effects was more pronounced in BDE-exposed litters, with litter survival being ~ 35%, compared to Vehicle-exposed litters (~58%). BDE-exposed female mice showed increased locomotor activity compared to vehicle-exposed mice [F(1,46)=5.98, p < 0.05] with the effects slightly more pronounced in the combined BDE-PolyI:C group. There were no differences in locomotor activity in male mice as a result of BDE or PolyI:C exposure; however, male BDE-exposed mice showed increased Rearing behavior [main effect of BDE; p < 0.05]. When mice were tested in the social approach task at 7 weeks old, no differences in social behavior were observed. Because our previous studies showed that male mice are more sensitive to social approach deficits in the MIA model, we re-tested male mice in the social approach task at 6-7 months old. BDE had no effect on social approach; however, male offspring of dams treated with PolyI:C on E12.5 did not spend siginificantly more time investigating the novel mouse vs an empty cup (NS). This resulted in a trend for Vehicle- PolyI:C mice to show decreased percent time with the stranger mouse. Mice were then tested for sensitivity to the dopamine agonist, amphetamine. Female mice exposed to PolyI:C during gestation showed decreased sensitivity to the locomotor-stimulating effects of amphetamine [main effect of PolyI:C; F(1,37)=4.66, p < 0.05] with the effect primarily being driven by the Vehicle-PolyI:C group [block x gestational exposure x PolyI:C interaction; F(8,296)=2.30, p < 0.05]. A different pattern of effects was observed in male mice, with BDE-exposed male mice showing decreased sensitivity to amphetamine-induced locomotor activity [p < 0.05]. Conclusions: Overall, BDE-exposed mice showed alterations in locomotor activity, investigatory behavior, and sensitivity to amphetamine in a sex-specific manner. Whereas, gestational exposure to PolyI:C produced a mild social approach deficit in male mice and altered response to amphetamine in female mice. Interestingly, there were no significant interactive effects between gestational exposure to BDE-47 and PolyI:C. One limitations of the study is the overall low litter survival. Keywords: Maternal Immune Activation, Flame Retardant, Neurodevelopmental Disorders Disclosure: Nothing to disclose. P128. Functional Analysis and Pharmacological Screening in Zebrafish Mutants of ASD Risk Genes Hellen Weinschutz Mendes, Tianying Chen, Yunqing Liu, Weimiao Wu, Uma Neelakantan, Ningshan Li, Sumedha Chowdhury, Andrea Gorodezky, Jeffrey Eilbott, Brent Vander Wyk, Zuoheng Wang, Ellen Hoffman Yale University, New Haven, Connecticut, United States Background: Autism spectrum disorders (ASDs) are a group of complex neurodevelopmental disorders that affect social behavior and communication, characterized by the presence of repetitive, restrictive behaviors. Pharmacological interventions that target the core deficits in ASD are still lacking, mainly due to its complex biology and clinical heterogeneity. Whole-exome sequencing has revealed “high confidence” risk genes associated with ASD (hcASD), which are advancing our understanding of the biology of ASDs. In this context, the zebrafish has become an optimal vertebrate model for genetic studies due to its fast development, transparency of its embryos, availability of efficient methods for generating mutant lines, and tractability for high-throughput analyses. Methods: Using CRISPR/Cas9 technology, we have generated zebrafish mutant lines of ten hcASD genes. To investigate how gene disruption alters basic sensory processing and rest-waking activity, we have established a novel behavioral high-throughput in vivo system, which was used across the different hcASD zebrafish mutant lines. Zebrafish mutant larvae at 5-7 days-post-fertilization are placed in a 96-well plate where responses to lights on and off stimuli and rest-wake behavior are tracked. Results: These analyses generated a unique behavior fingerprint for each mutant. In parallel, this system was used to screen wild-type zebrafish larvae treated with over 750 FDA-approved drugs to identify their effects on these behaviors, generating a drug-behavior fingerprint. By comparing the effect of the drug-behavior profile to mutant behavioral fingerprints, we were able to identify several dysregulated pathways in mutants and potential suppressors of hcASD behavioral phenotypes. More specifically, using linear mixed model (LMM) analysis, we identified the most significant anti-correlating pharmacological compounds for each zebrafish mutant line. These drugs include compounds representing a range of mechanisms, including estrogenic compounds, anti-inflammatory, and anti-cancer drugs. Next, we exposed mutants to select anti-correlating compounds to identify suppressors of mutant behavioral phenotypes. Conclusions: The discovery of pharmacological suppressors of these phenotypes has the potential to identify drug candidates for further investigation in ASDs. Keywords: Autism, Zebrafish, Drug Discovery - New Approaches, High Throughput Screening Disclosure: Nothing to disclose. P129. Adverse Childhood Experience and Grey Matter Volume: Results From a Pilot Sample of Puerto Rican Children in New York State Tamara Sussman, Albert Wakhloo, Jonathan Posner, Cristiane S. Duarte Columbia University Medical Center, New York, New York, United States Background: Increased exposure to adverse childhood experiences (ACEs) is a well-established risk factor for a wide range of negative mental health outcomes (Dube et al, 2003; Dube et al, 2006; Campbell et al, 2016; Shin et al, 2018; Hughes et al, 2017; Leza et al, 2021). ACEs have also been found to relate to changes in brain structure and function (e.g. Teicher et al, 2016; Philips et al, 2014), leading some to theorize that ACE-related mental health risks could be driven by ACE-related neural alterations (Puetz et al, 2015; Edalati et al, 2015). In line with this perspective, some authors have suggested that low or moderate stress early in life can prepare organisms for future stressful experiences, shaping the brain and behavior to adapt to the environment (Oshri et al, 2020). Indeed, studies in rodents and primates have found that exposure to stress early in life is associated with greater resilience to later novel stressors (Gapp et al, 2014; Parker et al, 2004; Parker et al 2004). Investigations into ACE-related neurodevelopmental alterations have yielded results consistent with this viewpoint. For example, studies have found lower grey matter volumes (GMV) in visual brain regions in adults who witnessed violence as children, and increased GMV in adults who experienced parental verbal abuse (Teicher et al, 2016; Tomoda et al, 2011; Tomoda et al, 2012). The current pilot results are an early look from a study examining the impact of ACEs on neurodevelopment in children aged 10-14 whose families have participated in the Boricua Youth Study (BYS) in New York, New York. The BYS is an intergenerational, representatively sampled cohort of Puerto Rican families, originally recruited from the South Bronx, New York, and San Juan, Puerto Rico. BYS families have been followed for 20+ years, with a focus on mental health outcomes. Here, in preliminary data, we examined the relationship between ACE-exposure and GMV in children of the original BYS cohort using non-negative matrix factorization (NMF), a constrained dimension reduction technique designed to yield sparse components. In this way, we limit the number of statistical comparisons without sacrificing the interpretability of our results. Methods: Sixteen children (ages 10 to 14; 9 female) participated in a study at the New York State Psychiatric Institute, New York, NY. ACEs were measured using the CDC Kaiser scale (Fellitti et al, 1998). All participants underwent MR imaging during which T1-weighted images were acquired. Cortical GMVs were extracted from these data using the FreeSurfer pipeline, version 7.2.0. NMF was applied to the resulting cortical GMV data matrix. When applied to brain morphometry data, matrix factorization techniques decompose the dataset into a set of components, or clusters of brain regions, with a set of subject-specific weights for each component. Each subject’s data can then be reconstructed by taking a weighted sum of the components. Therefore, the subject-specific weights serve as a low-dimensional representation of the original dataset, reducing the number of subsequent statistical tests. NMF enforces a non-negativity constraint on both the weights and the components. This leads to sparse components, each concentrated on only a few brain regions. Thus, relationships between subject-level component weights and exposures of interest can be more easily interpreted. Prior to dimension reduction, all volumes were divided by their standard deviation to promote interpretability of the resulting components. The NMF algorithm was initialized using non-negative double singular value decomposition in order to encourage sparsity and was run with coordinate descent under a Frobenius norm loss using the scikit-learn package until the default numerical tolerance was reached. Sixteen components were used for this decomposition allowing for near perfect data reconstruction while significantly reducing the dimensionality of the data. Following NMF, the relationship between GMV data and ACE exposure was examined through a series of linear models. Each model used subject-level ACEs to predict the reduced dataset (i.e., the weights). These models were fit using the python statsmodels package, version 0.13.2. Results: In this preliminary sample, ACEs ranged from 0 – 2 (mean total ACEs = 1.06, SD = 0.77). A component primarily concentrated about the left supramarginal gyrus, right lateral occipital cortex, and bilateral precentral cortex showed a significant, positive, relationship with ACEs (beta = 1.01, p = 0.043), indicating a positive correlation between greater ACE-exposure and increased GMV in these regions. Conclusions: In these pilot analyses of preliminary data, we found exposure to ACEs was in the low range (0-2 ACEs). Within this lower range of ACEs, without correction for multiple comparisons, greater exposure to ACEs was associated with increased GMV in brain regions including the left supramarginal gyrus, right lateral occipital cortex, and bilateral precentral cortex. Given that increased GMV in left supramarginal gyrus in early childhood has been associated with improvements in cognitive control (Berger, 2022), it is possible that ACE-related increases in GMV in this region are a sign of neurocognitive adaptations to environmental stressors. Future analyses in a larger sample can further test these findings, as well as associations between GMV and task performance, to determine if ACE-related structural brain changes relate to cognitive task performance. This research was funded by NIDA: K08DA049913 Keywords: Adverse Childhood Experiences (ACE), Grey Matter Morphometry, Neurodevelopment Disclosure: Nothing to disclose. P130. Functional and Physiological Neurobiomarkers of Vicarious Threat Extinction Learning in Parent-Child Dyads Sara Hess, Grace George, Justin Russell, Ryan Herringa University of Wisconsin - Madison, Madison, Wisconsin, United States Background: A critical aspect of socioemotional development during childhood is the learning and unlearning of fear associations from one’s environment, much of which occurs while observing a parent or caregiver. While mechanisms of vicarious threat extinction have been explored in animal models and adults, the neurobiological substrates of this formative learning process during childhood remains largely unexplored. Methods: In this study, we investigated whole-brain activation during day vicarious threat extinction using 27 parent-child dyads (youth ages 10-14). To characterize how previously learned threat associations can be modified or extinguished through parent observation, dyads completed a threat extinction paradigm where acquisition consisted of viewing visual stimuli with (CS+) and without (CS-) partial reinforcement of electrodermal stimulation (ES). During vicarious extinction, youth observed a video of their parent undergoing direct extinction, during which exposure to the CS- and CS + was not paired with ES (CS + vic). The parent completed the behavioral paradigm during simultaneous recording of skin conductance response, while youth completed all phases while undergoing functional magnetic resonance imaging and skin conductance recording. To estimate stimulus specific patterns in whole-brain activation, scans during vicarious extinction training were preprocessed using FMRIPREP and results comparing activation during the CS + vic and CS- presentation following cluster-size thresholding are presented (voxelwise threshold p < 0.0001). Finally, parent-child physiological synchrony was estimated using cross-recurrence quantification analysis (CRQA). Results: During vicarious extinction training, youth exhibited significantly decreased activation in the bilateral posterior insular cortex (pIC) while watching their parent view the CS + vic, as compared to watching their parent view the CS- (left, k = 423, pcorr<0.01; right, k = 447, pcorr<0.01). Differential activation in the right pIC during the CS + vic was further positively correlated with parent-child autonomic synchrony during the task (r = 0.502, p = 0.034). Interestingly, parent-child synchrony measures were not predictive of pIC activation while observing the CS- (r = -0.222, p = 0.374). Conclusions: These results first demonstrate novel biomarkers of the transmission of threat and safety learning in parent-child dyads during a critical period of adolescence and suggests a potential mechanism of this transmission via parent-child autonomic synchrony. Differential activation in the bilateral pIC while viewing a parents’ exposure to previously learned aversive stimuli provides additional support for the critical role of the pIC in extinction memory consolidation and may further be involved in decoding parent modeling of threat responses to aversive stimuli. Keywords: Functional MRI (fMRI), Parent - Child Dyads, Extinction Learning Disclosure: Nothing to disclose. P131. Developmental Patterns and Cognitive Relevance of Intrinsic Neural Timescales in Humans Kenneth Wengler, Andrew Goldberg, Seonjoo Lee, Guillermo Horga Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Many neuropsychiatric disorders are thought to have a developmental origin given their typical early onset and substantial genetic risk. As such, it is imperative to describe the normative developmental trajectories of neuroimaging phenotypes to contextualize neurodevelopmental alterations in individuals who develop a neuropsychiatric disorder. One such neuroimaging phenotype is the resting-state functional MRI measure of intrinsic neural timescale (INT). INT reflects the time window of neural integration and is thought to reflect the strength of recurrent excitation in cortical microcircuits—an index of excitation-inhibition balance (E/I) critical for cognition. We, and others, have recently reported reduced INT in patients with schizophrenia, but it is not clear how these reductions relate to neurodevelopment. Importantly, E/I is affected in a range of neuropsychiatric disorders and recurrent excitation plays a critical role in cognitive functions such as working memory. Persistent activity in higher-order cortical circuits, including dorsolateral prefrontal cortex, has long been thought to be a key substrate for maintaining information over delay periods in working-memory tasks. Synthetic biophysical models of canonical microcircuits, even in their simplest form, can themselves perform working-memory tasks, recapitulating primate behavior and electrophysiology. Also consistent with these models is the observation that single-neuron INT positively correlates with working-memory performance, although this has yet to be tested with non-invasive INT measures in humans. Here, we used two large-scale neuroimaging studies from the Human Connectome Project (Development [HCP-D] and Young Adult [HCP-YA]) to characterize age-related developmental patterns of INT throughout the brain and to test the hypothesized link between resting-state INT and working-memory performance. Methods: Resting-state fMRI data were collected from two publicly available datasets (n = 1601; 55% female): 1) HCP-D (n = 591, 6–21 years old); 2) HCP-YA (n = 1010, 22–37 years old). INT maps were estimated as previously described (Wengler et al., eLife, 2020). Briefly, the autocorrelation function of the fMRI signal at each grayordinate was estimated and the sum of the autocorrelation coefficients during the initial positive period was calculated. INT maps were parcellated using the HCP-MMP-v1.0 atlas and averaged across hemispheres for a total of 188 parcels. INT maps were harmonized using ComBat. Developmental curves were calculated for each parcel using general linear models: INT = B0 + B1age + B2age^2 + B3sex + B4motion. Inflection points were calculated for each parcel and hierarchical effects evaluated by correlation; hierarchical rank was determined by ranking INT values from the HCP-YA group-averaged INT map. The relationship with cognition was evaluated in the HCP-YA sample (n = 781, excluding subjects with worse performance on 0-back than 2-back) by partial correlation between N-back performance and the average INT in parcels significantly activated by the N-back task—2nd-level results for the HCP-YA sample determined 47 a priori parcels (contrast: 2-back>0-back)—controlling for motion (mean framewise displacement). Permutation tests were used to determine statistical significance and correct for multiple comparisons. Results: Nearly all parcels showed significant age effects characterized by an inverted-U shape with an increase in INT across early childhood/adolescence and a decrease in INT in adulthood (age: 180/188 parcels positive effect, P < 0.05, P_FWE,permutation = 0.001; age2: 182/188 parcels negative effect, P < 0.05, P_FWE,permutation = 0.001). Inflection points (INT peaks of fitted quadratic functions) ranged from ~21–36 years of age and occurred later in higher-order brain regions (correlation between inflection point and hierarchical rank: r = 0.75, P_permutation = 0.001). A sex-stratified analysis demonstrated that females had later inflection points than males (mean difference = 4.20 years, t = 18.6, P_permutation = 0.001). Results were robust to controls for motion, including in a motion-matched subgroup (n = 1343) and a subgroup with more stringent motion exclusion-criteria (n = 690). Subjects with better cognitive performance (as indexed by 2-back minus 0-back accuracy on the N-back task) had longer INT in brain regions which are activated by the task (r_partial = -0.09, P_permutation = 0.012; i.e., less difference between 2-back and 0-back accuracy). This was driven by subjects with longer INT having better performance during the 2-back condition requiring working memory (r_partial = 0.12, P_permutation = 0.001). Conclusions: Our results describe the age-related developmental patterns of INT across pre- and post-pubertal development, showing that almost all brain regions exhibit an inverted-U-shaped pattern across the studied age range (5–37 years). Higher-order brain regions (e.g., association cortex) reach their developmental peak (longest INT value) later in life than lower-order brain regions (e.g., sensory cortex). In addition, we showed that subjects who have longer INT—measured at rest—performed better on an N-back task. These results confirm the theoretical link between temporal integration windows at rest (i.e., INT) and a subject’s ability to maintain information in working memory, supporting the potential value of INT for studying developmental cognitive dysfunction in neuropsychiatric illness. Keywords: Intrinsic Neural Timescale, Brain Development, Cognition, Working Memory, Magnetic Resonance Imaging Disclosure: Nothing to disclose. P132. Sex-Dependent Effect of Adolescent Stress on Adult Affective Dysregulation and PV/SST Content in the pLPFC Daniela Uliana, Anthony Grace University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Stress is a socio-environmental risk factor for the development of psychiatric disorders, with the age of exposure potentially determining the later outcome. Early life adversity, such as during adolescence, has a significant impact on the development of neuropathological states in which females respond differently to stressors compared to males. Adolescence is a critical period for maturation of the medial prefrontal cortex (mPFC) and it is also when sex differences in PFC structure begin to emerge. Dysfunction of GABAergic network within the prelimbic portion of PFC (plPFC) during development increases vulnerability to adult affective dysregulation. However, the precise neurobiological mechanisms/circuits that contribute to differential sex responsivity to adolescent stress are understudied. Here we investigated the impact of stress exposure during adolescence on adult susceptibility to anhedonia, helplessness and dopaminergic activity in both sexes. Methods: Male Sprague-Dawley rats were subjected to a combination of footshock/restraint stress during adolescence (postnatal day 31-40). The rats were tested for sucrose preference (PD70), learned helplessness (PD72-73; Day 1, inescapable footshock; Day 2 escape session) and single-unit extracellular electrophysiology recording of VTA DA neurons (>PD78; four days after LH day 2). Independent group of animals were submitted or not to stress and perfused at PD31, PD41, PD51, and PD75 for posterior immunohistochemistry analysis of PV/SST content in the plPFC. All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Results: Adolescent stress decreased the sucrose preference (n = 4 each group, t6 = 4.41, p < 0.05, t-test) and increased the % of helplessness behavior only in males (Naïve 35.71%, 9 nonhelplessness, 5 helplessness; Stress 75%, 4 nonhelplessness, 12 helplessness, Chi-square=30.79, p < 0.05). No significant effect was found regarding sucrose preference or helplessness behavior in all female groups (Sucrose preference, n = 4 naïve, n = 5 stress, t7 = 0.21, p > 0.05, t-test; Helplessness; Naïve 46.15%, 7 nonhelplessness, 6 helplessness, Stress 43.75%, 9 nonhelplessness, 7 helplessness, Chi-square=0.0022, p > 0.05). Moreover, a decreased number of spontaneously active DA neurons was observed in rats showing helpless behavior across all groups exposed to LH. However, adolescent stress increased the number of DA neurons when the rats were not exposed to LH (Behavior, F5,37 = 24.29, ANOVA, p < 0.05). This is similar to data showing that chronic mild stress attenuates the hyperdopaminergic state in MAM rats. Adolescent stress increased the PV (n = 4 each group, Age x Condition F2,18 = 3.85, Two-way ANOVA, p < 0.05) and SST (n = 4 each group, Age x Condition F2,18 = 4.49; Two-way ANOVA, p < 0.05) expression in the plPFC at PD51 only in males. Conclusions: Our findings indicate that stress during adolescence increases negative affect behavior, such as decreased sucrose preference and increased helplessness, along with downregulation of dopaminergic activity in the VTA only in males. Males have an increased expression of GABAergic markers PV and SST at PD51. Females are resilient to adolescent stress at all behavioral and physiological markers. Therefore, a sex-dependent predisposition of early life adverse events that impair plPFC activity may enhance susceptibility to affective disturbance in adulthood. Keywords: Adolescence, Sex Difference, Early Life Stress, Dopamine, Affective Behavior Disclosure: Nothing to disclose. P133. Maturation of Nucleus Accumbens Synaptic Transmission Signals a Sensitive Period for the Rescue of Social Deficits in a Mouse Model of Autism Spectrum Disorders Melina Matthiesen, Carl Junior Steininger, Abdessattar Khlaifia, Maryam Dadabhoy, Unza Mumtaz, Maithe Arruda-Carvalho University of Toronto - Scarborough, Toronto, Canada Background: Social behaviour emerges early in development, a time marked by the onset of neurodevelopmental disorders featuring social deficits, including autism spectrum disorders (ASD). Although deficits in social interaction and communication are at the core of the clinical diagnosis of ASD, very little is known about their neural correlates at the time of clinical onset of the disorders. The nucleus accumbens (NAc), a brain region extensively implicated in social behaviour, undergoes synaptic, cellular and molecular alterations in early life, and is particularly affected in ASD mouse models. Methods: Male and female C57BL/6J and BTBR T + Itpr3tf/J mice were bred in house and tested in a three-chamber social interaction or social memory tests at postnatal day (P)30 (n = 27, 32). Slice electrophysiology of nucleus accumbens medium spiny neurons was conducted as in our previous work (Arruda-Carvalho et al., J Neuroscience 2017) at P4, P6, P8, P15, P21 and P30 (n = 10-22 cells/group) examining spontaneous excitatory and inhibitory transmission. Rapamycin (0.5mg/kg) intraperitoneal injections took place daily from either P4-P8 (n = 12, 14) or P60-P64 (n = 9,10) in BTBR mice, which were tested for social interaction at P30 and P86, respectively. Results: To explore a link between the maturation of the NAc and neurodevelopmental deficits in social behavior, we compared age-dependent changes in spontaneous synaptic transmission in NAc shell medium spiny neurons between the highly social C57BL/6J mouse strain and the idiopathic ASD mouse model BTBR T + Itpr3tf/J, which show deficits in social interaction (Two-way RM ANOVA, significant effect of chamber F1.536, 90.61 = 38.79, p < 0.0001, and chamber x strain interaction F2, 118 = 16.71, p < 0.0001) at postnatal day (P) 4, P6, P8, P15, P21 and P30. We found changes in spontaneous excitation and inhibition started as early as P4, highlighting a potential critical period in the maturation of NAc which could modulate the efficacy of interventions aimed at NAc-dependent behavior. To test this possibility, we injected BTBR mice in either early life (P4-P8) or adulthood (P60-P64) with the mTOR1 antagonist rapamycin, a well-established rescue intervention for ASD-like behavior. We found that rapamycin treatment rescued social interaction deficits in BTBR mice when injected in infancy (Two-way RM ANOVA, significant effect of chamber F2, 48 = 22.42, p < 0.0001, and chamber x treatment interaction F2, 48 = 9.436, p < 0.0003), but not in adulthood (Two-way RM ANOVA, significant effect of chamber F2, 34 = 7.904, p = 0.0015 only). Conclusions: In this study, we found age-dependent changes in the maturation of spontaneous excitatory and inhibitory transmission within and between strains, with BTBR mice showing overall increased spontaneous inhibition and decreased excitation at timepoints as early as P4. Given the timeline of these changes, we hypothesized that early developmental timepoints might encompass a critical period for the efficacy of rescue manipulations correlating with improved outcomes. Consistent with this, rapamycin treatment in infancy, but not adulthood, reversed the social interaction deficits in BTBR mice. Studying brain regions involved in the pathophysiology of neurodevelopmental disorders at clinically-relevant timepoints may offer novel insight into the timing and targets of therapeutic interventions to maximize positive outcomes. Keywords: Social and Behavioral Deficits, Nucleus Accumbens, Brain Development, Autism Spectrum Disorder Disclosure: Nothing to disclose. P134. Single-Nucleus Transcriptomic Analysis of the Prefrontal Cortex During Development and Early-Life Stress Edenia Menezes, Fabiula Abreu, Melissa Alldred, Catia Teixeira* Nathan S Kline Institute, New York University School of Medicine, Orangeburg, New York, United States Background: The brain contains interconnected circuits which are neither completed at birth or invariant across life. This neuronal plasticity is essential for life-long adaptive features like continuous learning and memory. However, this plasticity, especially when associated with severe adverse factors during early-life, can lead to the derailment of normative brain development and contribute to the etiology of behavioral deficits and psychiatric disorders. One of the most influential environmental factors during early-life is parental/caregiver care. Childhood adversity has been estimated to account for a significant percentage of adult-onset mental health disorders. In extreme cases of childhood adversity, institutional rearing where the infants were deprived of caregiver contact, cognitive deficits and dysregulated prefrontal cortex (PFC) function were found. In this study we hypothesize that early-life adversity, in the form of maternal separation, leads to long-lasting changes in the transcriptome of specific PFC cell populations leading to a dysregulation of PFC function. Methods: Here, we use a mouse model of maternal separation. The brains of pups and adults of normal reared or maternal separated (MS) animals were collected. In one set, we performed single-nucleus RNAseq with hashing to contrast the PFC transcriptome of these groups. In a separate cohort, we recorded whole slice voltage dye (VSD) responses to explore how changes in receptors identified by snRNAseq altered PFC responses. Results: Using snRNAseq, as expected, we observed that the changes between ages were much more accentuated than changes between treatments. We also observed that the proportion of oligodendrocytes in adult-MS animals was similar to infant animals. Furthermore, we found that most of the differential gene expression between MS and standard reared animals was in interneurons, affecting pathways related to GABAergic, glutamatergic, and serotonergic functions. Using VSD we observed that adult-MS responses to GABAergic and serotonergic agonists were similar to the responses of more immature normal-reared animals. Conclusions: This study suggests that MS leads to an immature PFC that may be linked to the behavioral deficits observed in animals and humans exposed to poor early-life care. Keywords: RNAseq, Prefrontal Cortex, Early-life Stress Disclosure: Nothing to disclose. P135. Effects of Juvenile Ketamine and/or Psychological Stress Exposure on Spatial Memory Performance in Adult Mice Sergio Iniguez, Israel Garcia-Carachure The University of Texas at El Paso, El Paso, Texas, United States Background: Ketamine is currently being used for the management of treatment resistant depression in adolescent patients. However, the possible long-term effects of ketamine exposure during adolescence have not been thoroughly assessed. Thus, we examined whether repeated exposure to concomitant ketamine and/or psychological stress, during the adolescent stage of development, results in long-lasting spatial memory alterations in male and female C57BL/6 mice (N = 36; 9 per group, N = 44; 11 per group, respectively). Methods: Male and female postnatal day (PD)-35 mice underwent 10 days of vicarious defeat stress (VDS; a form of psychological stress) with or without ketamine exposure (20 mg/kg; PD35-44). Once mice reached adulthood (PD70) separate groups were assessed for spatial memory performance adopting a water maze task. Results: We found that singular pre-exposure to ketamine or VDS increased the latency (sec) to locate the escape platform in adult male, but not female, mice – revealing that ketamine, like psychological stress, induces an enduring spatial memory impairment in males only. However, history of concomitant ketamine and VDS prevented spatial memory impairment in adulthood. Conclusions: Together, our findings suggest that ketamine, as a prophylactic treatment for adolescent psychological stress-induced illnesses, does not lead to long-term changes in spatial memory. However, juvenile recreational ketamine-use, like psychological stress history, results in an enduring spatial memory deficit in a male-specific manner. Keywords: (R,S)-Ketamine, Adolescent, Spatial Memory Disclosure: Nothing to disclose. P136. Prepregnancy High BMI is Associated With Sex-Specific Alteration in Maternal and Fetal Heart Rate Parameters and Differential Placental DNA Methylation With Later Effects on Mother-Reported Infant Temperament Sameera Abuaish, Seonjoo Lee, Benjamin Tycko, Frances Champagne, Catherine Monk Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia Background: Increased rates of obesity in general world-wide is also paralleled by increased BMI during pregnancy, which is known to impact pregnancy and birth outcomes. Recent research also indicates that early in-utero factors associated with maternal obesity could affect the long-term health trajectories of individuals including metabolic and neurobehavioral outcomes that are believed to be mediated by an underlying molecular fingerprint set during these early developmental periods by epigenetic mechanisms. High BMI is also associated with altered stress and autonomic nervous system physiology in individuals, which could contribute to these outcomes. Studies indicate that the developing fetus could present early markers of programming effects of altered autonomic nervous system to different maternal environmental exposures through heart rate (HR) measurements and a marker of future child neurobehavioral functioning. The placenta is an integral organ and a key mediator of environmental exposures affecting the developmental programming of the fetus leading to long lasting health impacts. Sex-specific effects of maternal obesity have been reported in the literature; however, no studies have examined its effects sex-specific on fetal neurobehavioral and infant temperament outcomes and the underlying differential placental DNA methylation. In this study we aim to examine the impact of maternal pre-pregnancy BMI on stress related psychological and physiological variables in pregnant women and fetal neurodevelopment by measuring fetal heart rate and later infant temperament. In addition, we investigate the epigenetic modification of genes in the placenta in response to high pre-pregnancy BMI. Methods: Pregnant women (n = 176) answered the Perceived Stress Scale (PSS) questionnaire and gave saliva samples at gestation weeks (GA) 12-22, 23-28, and 34-36, and attended fetal laboratory assessment sessions in GA 23-28, and 34-36 to obtain maternal and fetal HR at baseline and in response to maternal cognitive challenge. Placentae near the fetal surface were collected at birth and DNA methylation was assessed using Illumina array (n = 147). At a 4-month postpartum visit (n = 75), mothers completed the Infant Behavioral Questionnaire to assess infant temperament. Results: We found no association between BMI and diurnal salivary cortisol or PSS. However, only women with high BMI had higher HR, lower HR variability in both temporal and frequency domains. These effects were only exhibited in women pregnant with female and not male fetuses. Moreover, we found similar sex-specific differential effect of high BMI on fetal HR across gestation, with female fetuses of high BMI women exhibiting significantly lower HRV in the 3rd trimester. Interestingly, these sex-specific effects continued into infancy with female infants of high BMI women reported to have lower scores on orienting/regulation dimension of temperament, including soothability. Preliminary differential DNA methylation analysis revealed 4 differentially methylated probes, three of which were on the chromosome 12 and one on chromosome 10. Only the probe on the 10th chromosome was associated with an annotated gene, H2AFY2, and located in the 5’UTR. This probe was hypomethylated. DMR analysis also revealed a region in chromosome 10 in H2AFY2 gene, which codes for macroH2A2, a core histone from the H2A. macroH2A2 is known as an epigenetic repressor associated with X chromosome inactivation and transcriptionally supressed regions genome wide. DNA methylation analysis stratified by sex did not reveal any significant association. Conclusions: Our results indicate that maternal pre-pregnancy high BMI impacts on maternal HR parameters are only observed in women pregnant with females and these effects are mirrored in their female fetuses. These sex-specific effects continued into infancy and were associated with neurobehavioral outcomes related to infant temperament. Maternal pre-pregnancy high BMI left a molecular signature on placental DNA, with reduced methylation in the promoter of a gene coding a histone variant that is associated with transcription repression, which could impact the transcriptional landscape of the placenta and fetal development. Keywords: Obesity, Sex-specific Effects, Infant Behavior, DNA Methylation, Fetal Neurobehavior Disclosure: Nothing to disclose. P137. Relationships Between Adrenarche-Associated Hormone Concentrations and Reward-Related Neural Processing in Prepubertal Children Shau-Ming Wei, Madeleine Goldberg, Katherine Cole, Pedro Martinez, Michael Gregory, J. Shane Kippenhan, Zachary Trevorrow, Oriana Myers, Christina Recto, Philip Kohn, Lynnette Nieman, Jack Yanovski, Peter Schmidt, Karen Berman National Institute of Mental Health, Bethesda, Maryland, United States Background: The pubertal transition involves significant physical, social, emotional, and cognitive changes that are linked to greater propensity to engage in risky behaviors, and is timed with the development of several neuropsychiatric disorders. These puberty-related behavioral changes, such as increased impulsivity and risky choices, have been attributed, at least in part, to the effects of emerging pubertal hormones (adrenal, gonadal, and growth hormones) on reward-related brain regions, including the prefrontal cortex, ventral striatum and subcortical limbic regions. However, it has been challenging for experimental approaches to disambiguate the neurotrophic effects of adrenal androgens from those of gonadal steroids. Adrenarche, marked by increased secretion of adrenal androgens including dehydroepiandrosterone sulfate (DHEAS), is the earliest stage of pubertal development, usually starting between 7 and 8 years of age, and precedes gonadarche (i.e., surges in the gonadal hormones estradiol and testosterone). Here, using a reward paradigm with fMRI, we examined the relationships between DHEAS serum concentrations and reward-related brain activation in healthy, typically-developing boys and girls who were carefully documented to be prepubertal and, thus, prior to increases in gonadal hormones (i.e., gonadarche). Methods: Ninety typically developing, prepubertal children (as ascertained by clinician physical examination; 37 girls, mean age=8.6 + -0.3y; 53 boys, mean age=8.6 + -0.3) completed three runs of a modified monetary incentive delay task in a 3T MRI scanner. Data were derived from the first, baseline prepubertal visit of participants in a larger longitudinal study. The reward paradigm involved trials of low and high reward probability with anticipated levels of difficulty, along with control trials without monetary gain. Serum samples of DHEAS and testosterone were assayed using liquid chromatography-tandem mass-spectrometry, and DHEAS concentrations were log transformed for analysis. Serum estradiol was assayed using a chemiluminescence assay. Two separate whole-brain, voxel-wise multivariate model analyses using AFNI’s 3dMVM were run to test for correlations between DHEAS and activation during anticipation of reward and activation during receipt of reward (reward gain). Log-transformed DHEAS was and included as a continuous variable while controlling for sex. Post-hoc Spearman’s correlations were run in R to determine correlation directionality. Results: Both plasma estradiol and testosterone were at pre-pubertal concentrations, and no significant sex differences were observed in age or serum DHEAS (p’s > 0.6). Among participants, DHEAS ranged from 0.15 mcg/mL to 1.63 mcg/mL (the DHEAS biochemical threshold for onset of adrenarche is reported to be ≥ 0.4 mcg/mL.) During reward gain, log-transformed DHEAS concentrations were positively correlated with left hippocampal and right ventromedial prefrontal cortex (vmPFC) activation (pFDR<0.05). In contrast, negative correlations were observed during reward anticipation between log-transformed DHEAS and activations in bilateral dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex, orbitofrontal cortex, and caudate (pFDR<0.05). Conclusions: These neurofunctional/DHEAS relationships were found in prepubertal children prior to increases of the gonadarche-related hormones estradiol and testosterone, suggesting that adrenal androgens are associated with and may influence the function of key neural systems related to reward processing. Future directions will involve longitudinal analyses to better understand the maturation of these reward-related brain systems, beginning with this prepubertal baseline sample and following through various pubertal stages to disentangle the effects of the relevant endocrinological events (e.g. gonadarche) on brain development. Keywords: Adrenarche, Puberty, Children and Adolescents, Brain Development Disclosure: Nothing to disclose. P138. Prenatal Exposure to Early Life Adversity and Neonatal Brain Connectivity on Early Executive Function at Age 2 Years Rachel Lean, Christopher Smyser, Joan Luby, Emily Gerstein, Ashley Nielsen, Deanna Barch, Barbara Warner, Cynthia Rogers Washington University in St. Louis, St. Louis, Missouri, United States Background: Childhood exposure to early life adversity (poverty and psychosocial stressors) alters brain structural and functional development in frontal, parietal, and temporal regions involved in the neural networks of executive function (EF). Aberrant brain development and related problems in EF increases risks for psychiatric disorders including attention deficit hyperactivity disorder and anxiety disorders. However, whether associations between early life adversity and brain development begin prenatally to alter emerging EF is unknown, which may inform the timing of early preventative interventions. Maternal attunement to infant mental states (Maternal Mind-Mindedness, MMM) is one aspect of the early caregiving environment that supports EF and brain connectivity in older children. The extent that MMM buffers the adverse effects of early life adversity to shape the neural underpinnings of emerging EF is unclear. Methods: This longitudinal study focuses on 399 infant-mother dyads oversampled for poverty exposure, identified from the March of Dimes Research Center at Washington University in St. Louis. During pregnancy, mothers completed background measures analyzed with confirmatory factor analysis which resulted in two latent factors: Social Disadvantage (education level, health insurance status, income-to-needs ratio [INR], area deprivation index, nutritional intake) and Psychosocial Stress (depression symptoms, perceived stress, racial discrimination, stressful/traumatic life events). At birth, neonates (56% male, 44% female) underwent resting state functional (n = 319) and diffusion (n = 303) MRI scans on a Prisma 3T scanner. Key resting state networks included cingulo-opercular (CO), frontoparietal (FPN), ventral (VAN) and dorsal attention (DAN), and default mode (DMN) networks. Key white matter tracts included the cingulum, uncinate, and inferior fronto-occipital fasciculus (IFOF). At the 1-year follow-up, MMM was observed during a parent-child interaction task (n = 232 coded). At age 2 years, the Minnesota Executive Function Scale (MEFS) task was used to assess EF outcomes (n = 125 assessed to date) and INR was collected again as a measure of postnatal disadvantage. Prenatal Social Disadvantage and Psychosocial Stress factors were related to neonatal brain connectivity measures using multiple regression adjusted for covariates (age at scan, preterm birth [<37 weeks gestation], sex). Adversity variables, brain connectivity measures, and MMM ratings were fitted to MEFS scores using multiple regression and moderation analyses adjusted for covariates. Results: Prenatal exposure to Social Disadvantage was independently related to aberrant neonatal CO (β = -.15) and VAN-FPN (β = .13) network connectivity and right dorsal cingulum (β = -.17), bilateral inferior cingulum (β = -.20) and left IFOF (β = -.17) microstructure (all p ≤ .03). Psychosocial Stress was independently related to CO-DMN connectivity (β = .16, p = .007) and left uncinate microstructure (β = .14, p = .01). Social Disadvantage and aberrant FPN-CO, IFOF, and cingulum connectivity predicted poorer EF at age 2 years (all p ≤ .03). Maternal Psychosocial Stress was not independently related to EF. Infants born preterm had poorer EF outcomes at age 2 years than term-born infants (t = 1.98, p = .05) but there was no difference in EF by sex (t = .0.12, p = .90). At follow-up, year 1 MMM was positively associated with EF at age 2 years (β = .24, p = .02). Moderation analysis suggested that prenatal INR was associated with worse EF outcomes in the lower MMM exposure group whereas this association was reduced in the higher MMM exposure group (interaction term B = -.06, p = .03, total model R2 = .19). Although pre- and postnatal INR were highly correlated (r = .92, p < .001), there was no interaction between postnatal INR and MMM on EF (p = .07). Moderation analysis also showed that infants exposed to higher MMM had stronger neonatal cingulum connectivity-EF outcome relationships than infants exposed to lower MMM (interaction term B = -.4.56, p = .03, total model R2 = .28). There was no interaction between MMM and either FPN-CO or IFOF connectivity on EF (all p > .05). Conclusions: Integrating multi-modal neuroimaging, observational, and behavioral data elucidates the pathways by which EF delays emerge in early childhood. Prenatal exposure to poverty and altered neonatal brain connectivity increase risks of poorer EF outcomes. MMM may buffer the effects of poverty exposure and enhance brain-EF relationships, making MMM an important intervention target to support brain-EF development. Keywords: Prenatal Programming, Resting State fMRI, Diffusion MRI, Executive Function Disclosure: Nothing to disclose. P139. Maternal Inflammation During Pregnancy is Associated With ADHD in Children at Age 10: Results From the COPSYCH Study Julie Rosenberg, Jens Richard Jepsen, Parisa Mohammadzadeh, Astrid Sevelsted, Mikkel Sørensen, Rebecca Vinding, Klaus Bønnelykke, Hans Bisgaard, Bjørn Ebdrup* Center for Neuropsychiatric Schizophrenia Research, Mental Health Center Glostrup, University of Copenhagen, Glostrup, Denmark Background: Increasing evidence support that maternal inflammation during pregnancy affects early neurodevelopment, however the evidence for long-lasting risks for aberrant neurodevelopment are scarce and based on preclinical data and human observational register studies. In the current prospective birth cohort, COPSYCH, we investigated associations between early life inflammation in terms of maternal inflammation during pregnancy and psychopathological clinical outcomes in the children at age 10. We aim to repeat the analyses using the inflammatory status from the children at 6 months of age as the independent variable. Moreover, will corroborate clinical findings using diffusion weighted imaging parameters. Methods: COPSYCH is a translational research alliance between CNSR (Center for Neuropsychiatric Schizophrenia Research) and COPSAC (Copenhagen Studies of Asthma in Childhood) (ClinicalTrials.gov: NCT00798226 and NCT00856947). COPSYCH is established on the population based COPSAC2010 cohort consisting of 700 unselected mother-child pairs, who have been followed prospectively since pregnancy week 24. At 10-years of age the children have completed a 2-day visit comprising of a thorough examination of neurodevelopment including categorical and dimensional psychopathological examinations (Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS) and various validated rating scales) and neurocognitive tests as well as a 3 Tesla cerebral MRI scan including diffusion tensor imaging (DTI) sequences. Longitudinally measures of the inflammatory marker high sensitivity CRP (HS-CRP) as well as comprehensive data on potential confounders were obtained from the COPSAC2010-database. Logistic regression and multiple linear regression were used to estimate odds ratios (OR) for psychopathological outcomes. Statistical analyses were conducted using multiple linear regression for continuous outcomes (symptoms scores and ratings scales). The independent variable, HS-CRP, was log 2-transformed to ease interpretation. Logistic regression was used for binary outcomes (ADHD yes/no). Sample size: The COPSYCH2010 cohort at baseline, N = 700. Follow up rate at the current 10 years follow-up was N = 592 (86 %). Complete datasets after adjusting: N = 567. The results were checked for interaction of gender and if present the analyzes were stratified by sex. There was no interaction of sex on diagnosis level, but it was significant for symptom scores and rating scales, indicating that the effect of HS-CRP was driven by the boys (likely due to statistical power). HS-CRP vs K-SADS symptom score ADHD: estimate 0.40, interaction of sex, p = 0.01. Boys: estimate 0.60, p < 0.001. Girls: estimate: 0.16, p = 0.07. ADHD-RS and CBCL DSM 5 ADHD scale showing similar results. Statistical significance was set at p < 0.05. Statistical analyzes were calculated using R-software. Results: Out of the 700 children in the birth cohort, 592 children underwent the COPSYCH visit at age 10 (84.6%). Sixty-five, (10.9% fulfilled a research diagnosis for ADHD (16 girls (25%) and 49 (75%) boys). Maternal HS-CRP in pregnancy week 24 (mean 11.4 mg/l) was significantly associated with K-SADS diagnosis of ADHD, OR 1.48 (1.22-1.88), p < 0.001. The association was preserved at symptom level as well as across validated questionnaires (ADHD-RS and CBCL-DSM5 ADHD scale). Exploratory analyses revealed that the association was driven by the inattentive type of ADHD (i.e. ADD), OR 1.65 (1.24-2.07), p < 0.001 over the conventional ADHD symptomatology including hyperactivity, impulsivity, and inattention OR 1.23 (0.96-1.59), p = 0.1. No significant associations with HS-CRP were revealed for the other neurodevelopmental disorders including autism spectrum Disorder, Tourette’s syndrome, or tic disorder. Conclusions: The results add clinical data to the growing evidence of the importance of prenatal early life exposures including maternal inflammation. Identifying inflammation as an important marker will provide a potential target for future increased awareness and prevention during pregnancy, ultimately improving neurodevelopmental trajectories in children. Keywords: Maternal Inflammation, ADHD, Birth Cohort Disclosures: Boehringer Ingelheim: Contracted Research (Self), Lundbeck: Honoraria (Self) P140. Cortical and Subcortical Structural Differences in ADHD Youth With and Without a Family History of Bipolar I Disorder: A Cross-Sectional Morphometric Comparison Ziyu Zhu, Du Lei, Kun Qin, Maxwell Tallman, L. Rodrigo Patino, John A. Sweeney, Melissa DelBello, Robert McNamara* University of Cincinnati College of Medicine, Cincinnati, Ohio, United States Background: Attention deficit/hyperactivity disorder (ADHD) commonly precedes the onset of bipolar I disorder (BD), and prospective studies have found that prodromal ADHD significantly increases risk for developing mood disorders including BD. Moreover, the prevalence rates of ADHD in youth with BD are substantially higher than the general population, particularly in pre-pubescent children. Additionally, having a first-degree relative with BD robustly increases the risk of developing BD, and youth with a first-degree BD relative exhibit higher rates of ADHD and more severe ADHD symptoms. While these findings suggest that ADHD in conjunction with familial risk for BD may represent a different and more severe illness that confers greater risk for developing BD, associated neuropathogenic mechanisms remain poorly understood. The initial onset of BD frequently occurs during the peripubertal period which is associated with linear increases in frontal cortex gray matter expansion and connectivity with subcortical regions implicated in emotional regulation. Previous structural MRI studies have observed cortical and subcortical gray matter structural abnormalities in youth with BD or ADHD compared with healthy developing youth. However, the majority of these studies did not control for BD familial risk, ADHD comorbidity, and/or psychostimulant exposure. In the present cross-sectional study, we investigated regional cortical and subcortical morphometrics in psychostimulant-free ADHD youth with and without a first-degree relative with BD and a typically developing control group. We additionally evaluated relationships between morphometrics and relevant symptom measures. Methods: ADHD youth (ages 10-18 years) with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD and typically developing healthy controls (HC) with no personal or family history of a DSM-5 Axis I psychiatric disorder were enrolled. All ADHD youth met DSM-5 criteria for ADHD (any type), had no exposure to psychostimulants for at least 3 months prior to enrollment, and had no comorbid mood, conduct, eating, or psychotic disorders. Clinician ratings of ADHD (ADHD-rating scale, ADHD-RS), mania (Young Mania Rating Scale, YMRS), depression (Children’s Depression Rating Scale-Revised, CDRS-R), global functioning (Children’s Global Assessment Scale, CGAS), and global symptom severity (Clinical Global Impression-Severity Scale, CGI-S) were performed, and parents completed the Child Behavior Checklist (CBCL). High-resolution 3D T1-weighted images were acquired using a Philips 3.0 T MR scanner. FreeSurfer image analysis was used to obtain measures of cortical thickness, surface area, as well as the volumes of 14 subcortical structures. Group differences were evaluated using a general linear model with the age and sex as covariates. Post hoc permutation tests evaluated pair-wise group differences (FDR corrected p < 0.05). Partial correlations were computed for symptom severity measures and all significant structural differences in LR and HR groups, and interaction effects were tested by using linear mixed effects models with age and sex as covariates (FDR corrected p < 0.05). Results: A total of n = 142 adolescents (mean age: 14.16 ± 2.54 years, 35.9% female) were included in the analysis (HC, n = 48; low-risk, n = 49; high-risk, n = 45). No significant group differences were observed for age, sex, handedness, or prior psychostimulant exposure in the ADHD groups. Compared with the LR group, the HR group had higher ADHD-RS hyperactivity/impulsivity subscale scores (p = 0.03), YMRS (p = 0.004), and CGI-S (p = 0.01) total scores, and higher CBCL total score (p = 0.002) and internalization (p = 0.01), externalization (p = 0.002), and dysregulation (p = 0.047) subscale scores. For subcortical volumes, no significant differences were found between LR and HC groups. The HR group exhibited smaller bilateral thalamus, caudate nucleus, and hippocampus volumes compared with the LR group as well as the HC group with the exception of the right caudate. For cortical surface area, the LR group exhibited a larger right parsopercularis compared with the HC group. In contrast, the HR group exhibited smaller left postcentral, bilateral inferior parietal, right precuneus, left temporal pole, right fusiform, and left rostro anterior cingulate cortices compared with the HC group, and smaller bilateral lateral orbitofrontal, left superior frontal, left postcentral, bilateral inferior parietal, right precuneus, left temporal pole, right fusiform, and right parahippocampal cortices compared with the LR group. Among LR and HR ADHD subjects (n = 94), ADHD-RS inattention subscale scores were positively correlated with left lateral orbitofrontal surface area (p < 0.0001), and YMRS total scores were inversely correlated with left inferior parietal surface area (p < 0.0001) and right precuneus surface area (p < 0.0001). Conclusions: Psychostimulant-free ADHD youth with a BD family history exhibit a more severe clinical profile, including greater mania and dysregulation symptoms, and more extensive cortical and subcortical volume abnormalities compared with ADHD youth without a BD family history and healthy youth. These findings suggest that BD family history in conjunction with ADHD is associated with a more pervasive disruption of peripubertal neurodevelopmental trajectories that distinguish it from ADHD alone. Keywords: Bipolar Disorder, ADHD, Brain Imaging, Prodrome Disclosure: Nothing to disclose. P141. Family History of Alcohol and/or Substance Use Problems and Frontal Cortical Thickness From 9-10 to 11-12 Years of Age: A Longitudinal Analysis of the Adolescent Brain Cognitive Development (ABCD) Study Priscila Goncalves, Silvia Martins, Neo Gebru, Stacy Ryan-Pettes, Nicholas Allgaier, Alexandra Potter, Hugh Garavan, Matthew Albaugh, Ardesheer Talati Columbia University, New York, New York, United States Background: Individuals with a family history of alcohol or substance use problems (FH+) are themselves at risk for increased use, misuse, and eventual development of alcohol and/or substance use disorders (AUD/SUD). Having a FH + has also been associated with impaired development of prefrontal brain regions, particularly those related to executive functioning (EF) and impulsivity. Lower EF and high impulsivity are also unique contributors to alcohol/substance-related problems and AUD/SUD.6–12 Prior cross-sectional studies showed that FH + adolescents exhibited thinner frontal cortices including aspects of the inferior frontal gyrus (e.g., pars triangularis), as well as lateral and medial orbital frontal cortices. Children with FH + (ages 9-10) had lower whole-brain cortical thickness, including thinner cortices in the left precentral and paracentral lobules, and greater surface area in the right precentral lobule compared to children with FH-. Little is known about changes in cortical thickness as a function of FH + during early adolescence – a critical period for brain maturation. Most studies to date have been cross-sectional and/or include broad age ranges that do not allow sufficient focus on the beginning of adolescence. Our aim was to examine frontal cerebral cortical thickness trajectories among 9–12-year-old preadolescents with a positive (FH+), versus negative (FH-), family history of alcohol/substance use problems. Methods: Data from the ABCD Data Release 4.0 including structural MRI measures at two time points (baseline [n = 11,878] and 2-year follow-up [n = 6,571]). FH + (n = 2,861) was defined as having ≥1 biological parents and/or ≥2 biological grandparents with a history of alcohol/substance use-related problems (e.g., alcohol/substance use-related separation/divorce, being laid off/fired related to alcohol/substance use problems; arrests/DUIs; alcohol/substance harmed health; in an alcohol/substance treatment program; caused arguments or were drunk/intoxicated a lot). Individuals with no parents or grandparents with a history of alcohol/substance use problems were classified as FH- (n = 7,017). Individuals with only one grandparent with a FH of alcohol/substance problems (n = 1,595) or with missing data on all parents and grandparents were excluded (n = 405). Our primary outcome was cortical thickness within 11 frontal regions (caudal middle frontal, frontal pole, lateral orbital and medial orbital frontal, paracentral, pars orbitalis, pars opercularis, pars triangularis, precentral, superior frontal, and rostral middle frontal), based on the Desikan-Killiany cortical parcellation atlas. Statistical Analyses A linear mixed-effects model was fit to test associations between FH + and frontal cortical regions addressing the 11 frontal cortical regions in both hemispheres, while adjusting for the fixed effects of age, sex, race/ethnicity, parental marital status, household income, prenatal exposure to alcohol, drugs, and tobacco, and intracranial total volume, and the random effects of family relationships (e.g., siblings), MRI scanner and participant ID as random effects. To examine the extent to which age-related change in cortical thickness was qualified by FH, we also added an interaction term for FH, timepoint (i.e., baseline and 2-year follow-up) and frontal cortical regions. We also ran pairwise comparisons examining differences in specific frontal cortical regions between FH + and FH- at each timepoint. Analyses were conducted using the ‘lme4’and ‘emmeans’ packages in R. Results: In this sample, 24.9% of the preadolescents had a FH + , and 61.2% a FH- (13.9% with only 1 grandparent with a FH of alcohol/substance use were not included). Having a FH + was associated with more pronounced reductions in frontal thickness in the 2-year follow-up compared to baseline (FH + , β -0.10, p < 0.001; FH- β -0.08, p < 0.001; overall FH x time interaction, p = 0.003). Given the significant overall associations, we next examined thickness measures in each frontal region separately. At baseline, FH + (vs. FH-) was associated with reduced paracentral (β-left -0.11 p = 0.001) and precentral (β-left -0.08 p = 0.001), and thicker medial orbital frontal cortices (β-left 0.07 p = 0.004; β-right 0.05 p = 0.04). At the two year follow-up, FH + continued to be associated with thinner paracentral (β-left: -0.14 p < 0.001, β-right: -0.08 p = 0.008), precentral (β-left -0.11 p < 0.001, β-right -0.12 p < 0.001), superior frontal (β-left -0.08 p = 0.01) and rostral middle frontal cortices (β-left -0.07 p = 0.02), and thicker frontal pole (β-right 0.06 p = 0.04). Conclusions: Whereas previous studies have documented cross-sectional associations between brain structure and FH of alcohol or substance use problems, preliminary results from the present longitudinal study indicate that FH + is associated with altered cortical thickness development in frontal areas (particularly within pre- and paracentral regions). Specifically, when comparing FH + and FH- youths, having FH + exhibited differences in frontal cortical thickness at 9-10 years of age. Further, these differences in cortical structure increased in magnitude during early adolescence (ages 11-12), at which time structural alterations in other regions (including areas implicated in emotion regulation, such as the superior and middle frontal cortices) are also detected. Future research may examine any dose-dependent (i.e., number of relatives) and specific substance effects of FH + on adolescents’ brain development. Keywords: Adolescent Brain Cognitive Development Study, Cortical Development, Cortical Thickness Disclosure: Nothing to disclose. P142. Altered EEG Coherence in Adolescents With Recent Suicidal Behaviors Deniz Doruk Camsari, Molly McVoy, Can Ozger, Farhad Kaffashi, Ken Loparo, Farren Briggs, Charles Lewis, Ayse Irem Sonmez, Parmis Fatih, Deniz Yuruk, Julia Shekunov, Jennifer Vande Voort, Paul Croarkin Mayo Clinic, Rochester, Minnesota, United States Background: Several studies have attempted to characterize suicidality using Quantitative EEG measures. There is limited prior work focused on EEG power and coherence. Most of these studies were examined adults. No prior study has investigated EEG coherence in adolescents with suicidal ideations and behaviors. Methods: Thirty adolescents aged 13-18 years admitted to an inpatient psychiatry unit with suicidal ideations and suicidal behaviors (assessed with the Columbia-Suicide Severity Rating Scale (C-SSRS)) and thirty with no prior psychiatric diagnoses were recruited. All participants underwent diagnostic assessments and resting EEG recordings. Three patients dropped out before completing EEG recordings. The inpatient group was further divided into 2 sub-groups, those with suicidal ideations (n = 9) or suicidal behaviors (n = 18) based on the C-SSRS assessment. ANCOVA models were used to test if intra-hemispheric coherence differed amongst the groups controlled for age and gender. Bonferroni correction was used for post-hoc comparisons. Results: Patients with suicidal behaviors (SB) had higher delta (p = 0.024), alpha(p = 0.024) and beta coherence (p = 0.006) over right PFC (Fp2-F4) compared to patients with suicidal ideations (SI) only. The SB group also had higher delta power (p = 0.027) over left parieto-occipital cortex (P7-01) compared to the SI only group. Over right temporo-parietal (T8-P8) and centroparietal cortex (C4-P4), the SB group had lower theta (p = 0.036), alpha (p = 0.045) coherence compared to healthy controls. Depression severity (assessed with the Children’s Depression Rating Scale Revised) did not differ between SB and SI only groups (p > 0.05). Conclusions: Patients with SB had higher intra-hemispheric coherence within the right prefrontal cortex for both lower and higher frequencies except theta coherence but had decreased coherence in temporo-parietal centroparietal cortex. These results may suggest altered default mode network and inhibitory mechanisms in patients with recent suicidal behaviors as compared to patients with suicidal ideations. Given the paucity of research in this area, further research is needed to replicate the results in larger sample sizes. Keywords: EEG, Adolescent, Suicide, Suicidal Behavior, Suicidal Ideation Disclosures: Neuronetics, Magventure: Other Financial or Material Support (Self), Engrail Therapeutics, Sunovion, Myriad Neuroscience, Procter and Gamble Company: Advisory Board (Self) P143. The Interrelationship Between Myelination, Chronotype, and Stress in Adolescents at High Risk for Psychiatric Disorders Michael McCarthy, Connor McCabe, Alejandro Meruelo* University of California, San Diego, San Diego, California, United States Background: Chronotype characterizes a tendency towards morningness or eveningness (e.g., larks versus night-owls). The relevance of chronotype to the development of depression, diabetes, alcohol use disorder, cannabis use disorder, and obesity has been increasingly established through associational studies. Chronotype is developmentally regulated, and shows the greatest amount of change in adolescence, precisely the time when many psychiatric disorders are first identified and important changes in brain myelination occur. However, whether chronotype or eveningness plays a role in the development of some of these disorders has been much less explored and investigated. A number of studies have investigated the relationship between trauma and chronotype, and found an association between childhood trauma and a chronotype preference towards eveningness suggestive of mediation by emotional dysregulation. However, the relationship between stressful life events and chronotype has not been explored in as much depth. Some genome-wide studies have most prominently related chronotype to the anterior cingulate as a potential neuroanatomical phenotype that could be related to chronotype or eveningness. Increased activation of the anterior cingulate has been observed during suppression of negative emotions when contrasted with maintaining negative emotions. To properly function, the brain needs myelin, an electrically insulating structure that surrounds axonal fibers. It has been shown based on fractional anisotropy imaging that poor sleep quality or short sleep duration are associated with lower myelin content in the human neo-cortex. Intracortical myelin in the anterior orbitofrontal cortex mediates the association between poor sleep and negative emotionality, which can mechanistically explain how poor sleep influences affective behavior. Diffusion tensor imaging, such as fractional anisotropy is sensitive to degree of myelination of anatomical structures like the anterior cingulate. Methods: The Adolescent Brain Cognitive Development (ABCD) study offers a large sample of approximately 11,000 at high risk adolescents followed over a 10-year period with data on chronotype, neuroimaging, and stressful life events. This study tracks biological and behavioral development from adolescence into young adulthood. We used a mediation model to examine the relationship between chronotype, fractional anisotropy of the anterior cingulate, and stressful life events using the Munich Chronotype Questionnaire (MCTQ) and the Life Events Scale. In our comprehensive final model, we included race, sex, and socioeconomic status as covariates to control for. All analyses were conducted in R using lavaan. Results: We found that cross-sectional data suggest a potential relationship between chronotype and myelination of the anterior cingulate mediated by stressful life events. Lesser myelination of the anterior cingulate was associated with more eveningness. Conclusions: Our work provides a new look into the interrelationship between chronotype, myelination, and stressful life events in the ABCD study. Understanding the role of myelination in stress in those predisposed towards eveningness is useful because it may improve our understanding of the biological mechanisms that contribute to stress, and lead to better prevention and treatment efforts using interventions such as increased lifestyle regularity and daytime light exposure. Keywords: Chronotype, Myelination, Adolescence, ABCD, Stress Disclosure: Nothing to disclose. P144. Examining the Impact of Food Insecurity on White Matter in Children and Adolescents: An ABCD Study Amanda Lyall, Shreyas Fadnavis, Suheyla Cetin-Karayumak, Fan Zhang, Ofer Pasternak, Lauren J. O’Donnell, Yogesh Rathi Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Adolescence is a critical phase of life between late childhood and adulthood that is characterized as the second greatest and most rapid period of development after infancy. As a result, adolescence has been recognized as a critical period of increased vulnerability, during which exposure to environmental and social stressors, in addition to the impact of pubertal hormones or other life events, can have long-standing functional and structural impacts. Food insecurity is a growing public health concern in the United States and around the globe. Food insecurity is defined as a lack of means to achieve reliable access to a sufficient quantity of food necessary to maintain a healthy diet. In the US, it is estimated that approximately 14.8% of households suffer from food insecurity, with around 7% of those affected with low or very low food security being children (6.1 million children). While food insecurity does not necessarily equate to extreme states of hunger, starvation, or malnourishment, it can lead to inconsistent or limited access to healthy foods and nutrients. Previous reports show that very young children are often protected from the effects of food insecurity, however, adolescents are often at a greater risk. This lack of access to sufficient resources and diet-derived nutrients may be detrimental during this critical period of brain development, particularly for the brain’s white matter, which is actively maturing during adolescence. White matter is comprised of a lipid-rich substance, called myelin, that surrounds neuronal axons to provide insulation and increase the rate at which electrical impulses are carried along the axon. The creation and maintenance of myelin is governed by oligodendrocytes, which require an array of resources, such as iron, fatty acids, and assorted vitamins such as Vitamin D and Vitamin K, the concentrations of which are principally dependent on food intake. As a result, developing white matter may be particularly susceptible to the potential lack of resources incurred by the inconsistent or limited access to nutritious foods which occur in food insecure homes. However, to date there have been no neuroimaging studies which aim to investigate whether food insecurity influences or impacts white matter development in children and adolescents. In this study, we will utilize extant data from the Adolescent Brain and Cognitive Development (ABCD) study to evaluate the extent to which food insecurity can impact overall white matter health in children and adolescents aged 9 to 15 years. More importantly, given the complex interplay between food insecurity and other known factors that have an impact on brain development (i.e., socioeconomic status, stress, childhood trauma, etc.), we plan to utilize a machine learning approach to determine whether there is a link between food insecurity and alterations in white matter health, independent of the impacts of other environmental, financial, or social stressors. Methods: Of the 11,877 individuals included in the ABCD baseline sample, 954 parents (8%) responded to having had an instance of food insecurity in the last twelve months. We will employ propensity score matching using K-nearest neighbors to construct comparable groups of children/adolescents with food insecurity (FI) matched with respect to age, sex, race, ethnicity, intelligence quotient, and, if feasible, years of parental education and/or socioeconomic status, to children/adolescents without food insecurity (noFI). For the imaging data for those subjects included in our identified groups, we will utilize the diffusion MRI imaging data that has already been pre-processed and harmonized across the 21 imaging sites as part of an on-going NIMH-funded effort led by Dr. Rathi. Whole-brain two tensor tractography has been performed and quality controlled. An unsupervised fiber clustering atlas (whitematteranalysis) has been applied and will be utilized to extract whole brain average fractional anisotropy (FA) for each participant in this sample. Results: The statistical analyses in this study are presently on-going. We will train a machine learning model to determine whether the matched FI cohort exhibits differences in whole brain average FA from the non-FI cohort. To understand the covariance structure of the variables used to train the model, we will employ an interpretability approach which will enable mediation analysis and ranking of the variables based on the learnt weights. This will enable us to understand the relative importance of food insecurity on white matter health in children/adolescents, independent of the effects of other confounds, such as socioeconomic status. Conclusions: To our knowledge, this will be the first study to determine whether there is an association between food insecurity and white matter microstructure in children and adolescents. This study will serve as a first step toward furthering our understanding of the role access to healthy and nutritious foods may play on brain development. Keywords: White Matter Development, ABCD, Food Insecurity Disclosure: Nothing to disclose. P145. Family History of Substance Use Disorder and Parental Impulsivity are Differentially Associated With Neural Responses During Risky Decision-Making Joseph Aloi, Kathleen Crum, John Nurnberger, Leslie Hulvershorn Indiana University School of Medicine, Indianapolis, Indiana, United States Background: Risky decision-making is associated with the development of substance use behaviors during adolescence. Although prior work has investigated risky decision-making in adolescents at familial high risk for developing substance use disorders (SUDs), little research has controlled for the presence of co-morbid externalizing disorders (EDs), another SUD risk factor. Additionally, few studies have investigated the role of parental impulsivity in offspring neurobiology associated with risky decision-making. The purpose of this study was to address these gaps in the literature by (i) investigating differences in neural functioning during risky decision-making in children with externalizing disorders with and without family histories of SUD; and (ii) investigate the relationship between parental impulsivity and child neural functioning during risky decision-making. Based on prior work in decision-making and neurobiology of children at risk for SUDs, we hypothesized that children with co-morbid familial histories of SUD and EDs would show greater responsiveness to negative outcomes of risky decisions within anterior insula cortex (aIC), rostral anterior cingulate cortex (ACC) and lateral orbitofrontal cortex (OFC) compared to both psychiatric controls and healthy controls, and that these differences would be related to parental impulsivity. Methods: One-hundred twenty-five children (28 healthy controls, 47 psychiatric controls (PC) with EDs without a familial history of SUD, and 50 high-risk (HR) children with co-morbid EDs with a familial history of SUD) participated in the Balloon Analogue Risk Task (BART) while undergoing functional magnetic resonance imaging. Impulsivity for parents and children was measured using the UPPS-P Impulsive Behavior Scale. AIC, rostral ACC, and lateral OFC were defined as structural regions of interest (ROIs) using the Freesurfer parcellations from the Desikan-Killiany atlas. A 3 (Group: HC, PC, HR)-by-2 (Laterality: Left, Right)-by-3 (Region: lOFC, rACC, aIC)-by-2 (BART Choice: Inflate, Win) repeated measures ANCOVA was conducted on the BOLD response data modulated by probability of balloon explosion from these ROIs. The covariate of interest in this ANCOVA was parental impulsivity; this analysis was also repeated with child impulsivity included as a covariate of no interest. Results: We found that individuals in the psychiatric control group showed greater activation, as chances of balloon explosion increased, while making choices, relative to the healthy control and high-risk groups in the rostral ACC and lateral OFC. We also found a positive association between greater activation and parental impulsivity in these regions. However, within rostral ACC, this relationship was moderated by group, such that there was a positive relationship between activation and parental impulsivity in the HC group, but an inverse relationship in the HR group. These relationships remained significant when controlling for child impulsivity. Conclusions: These findings suggest that there are key differences in the neurobiology underlying risky decision-making in individuals with EDs with and without a familial history of SUD. These data also indicate that the association between parental impulsivity and rostral ACC activity during risky decision-making may be a marker of future risk for SUDs. We propose that development of biomarkers of SUD risk (based on these findings) would provide further insight into determining level of risk for SUDs across different populations. However, future work regarding the roles of how altered rostral ACC and lateral OFC activity may confer risk for SUDs and following high-risk samples longitudinally is needed. Keywords: Addiction Comorbidity, Alcohol Use Disorder and Drug Addiction, Impulsivity Disclosure: Nothing to disclose. P146. Neural Network Functional Interactions Influence White Matter-Emotional Behavior Relationships in Infants Layla Banihashemi, Vanessa Schmithorst, Michele Bertocci, Alyssa Samolyk, Joao P Lima Santos, Amelia Versace, Megan Taylor, Gabrielle English, Jessie B. Northrup, Vincent K. Lee, Richelle Stiffler, Haris Aslam, Ashok Panigrahy, Alison Hipwell, Mary Phillips University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Low levels of positive emotionality (PE) and high levels of negative emotionality (NE) in childhood signal risk for subsequent behavioral and emotional problems. Elucidating the neural basis of these temperamental features in infancy can identify objective biomarkers of risk that reflect underlying pathophysiological processes. Our goal was to determine how functional interactions among large-scale neural networks supporting emotional regulation influence white matter microstructural-emotional behavior relationships in 3-month-old infants. We hypothesized that microstructural-emotional behavior relationships will be differentially mediated or suppressed by underlying resting-state functional connectivity, particularly between Central Executive Network (CEN) and Default Mode Network (DMN) structures. Methods: This sample comprised caregiver-infant dyads [52 infants (42% female, mean age at scan=15.10 weeks) and their primary caregiver (98% biological mother)], with neuroimaging of the infant and assessments of infant emotional behavior at 3 months. Infant white matter and functional connectivity were assessed by diffusion-weighted imaging/tractography and resting-state magnetic resonance imaging (MRI) during natural, non-sedated sleep. The Infant Behavior Questionnaire-R provided measures of infant PE and NE. Following significant white matter-emotional behavior relationships, multimodal analyses were performed using whole-brain voxelwise mediation. Results: Results revealed that greater cingulum bundle tract volume was significantly associated with lower infant PE (ß = -0.263, p = 0.031); however, a pattern of lower functional connectivity between CEN and DMN structures suppressed (i.e., reduced) this otherwise negative relationship. Greater uncinate fasciculus tract volume was significantly associated with lower infant NE (ß = -0.296, p = 0.022); however, lower orbitofrontal cortex (OFC)-amygdala functional connectivity (supporting emotional regulation), suppressed this otherwise negative relationship, while greater OFC-CEN functional connectivity (supporting executive control over emotion) mediated (i.e., facilitated) this relationship. Conclusions: Functional interactions among neural networks have an important influence on white matter microstructural-emotional behavior relationships in infancy. These relationships can elucidate neural mechanisms contributing to future behavioral and emotional problems in childhood. Keywords: Infant Brain, Infant Emotionality, Multimodal Neuroimaging, Diffusion Weighted Imaging, Resting State Functional Connectivity Disclosure: Nothing to disclose. P147. Prenatal Neighborhood Crime Exposure and Neonatal Frontolimbic Connectivity are Associated With Socioemotional Functioning at 12 Months Rebecca Brady, Cynthia Rogers, Trinidi Prochaska, Tara Smyser, Barbara Warner, Deanna Barch, Joan Luby, Christopher Smyser Washington University School of Medicine, St. Louis, Missouri, United States Background: Growing up in a neighborhood with high crime rates is associated with sleep disturbances, mental health problems, and altered brain function. Our previous study found that prenatal exposure to neighborhood crime was associated with reduced frontolimbic connectivity in the neonatal brain, partially mediated by maternal psychosocial stress (Brady et al. 2022). It is unknown whether prenatal crime exposure and subsequent neonatal brain changes lead to altered behavior in infancy or beyond. It is plausible that prenatal crime exposure relates to later behavior, as studies in middle childhood and adolescence demonstrate a link between living in a high-crime neighborhood and mental health problems. We hypothesize that prenatal crime exposure will be related to poorer socioemotional outcomes, mediated by both maternal psychosocial stress and neonatal frontolimbic connectivity. Methods: The study utilized a longitudinal cohort of 399 women recruited during pregnancy as part of the eLABE study at Washington University. Prenatal addresses were collected for each mother and coded by census block group. Crime data for each block group was obtained from Applied Geographic Solution’s CrimeRisk Database. Violent crimes (e.g., murder, rape, aggravated assault, robbery) and property crimes (e.g., burglary, larceny, vehicle theft, arson) were examined. In the neonatal period (mean postmenstrual age at scan=41 weeks), 319 non-sedated infants (male=55%, mean gestational age=38 weeks) were scanned using a resting-state functional MRI sequence (TR = 800 ms, TE = 37 ms, Voxel size=2.0×2.0×2.0 mm3, MB = 8) on a Prisma 3T scanner and had at least 10 minutes of high-quality resting-state data after pre- and post-processing, including stringent motion correction. At one year of age, the mothers of 293 infants completed the Infant-Toddler Socioemotional Assessment (ITSEA), which contains information about four domains of behavior: externalizing, internalizing, dysregulation, and competence. Pearson correlations were used to examine the relationship of block-tract group crime to the ITSEA t-scores. Linear models probed the relationship between the significant frontolimbic connections in Brady et al. 2022 (i.e. Thalamus-Default Mode Network (DMN), Thalamus-anterior Frontoparietal Network (aFPN), Amygdala-Hippocampus, and Amygdala-DMN) and relevant ITSEA t-scores, controlled for gestational age at birth and scan. Results were corrected for multiple comparisons using an FDR procedure. Structural equation models (SEMs) were created to test whether maternal psychosocial stress and neonatal neuroimaging mediated the relationship between prenatal crime exposure and ITSEA t-scores. To assess the specificity of exposure to crime compared to other forms of adversity, a composite measure of disadvantage experienced during pregnancy (income-to-needs, Area Deprivation Index, education, insurance status, and Healthy Eating Index) was included. Results: The results of this study demonstrated that prenatal exposure to high levels of neighborhood violent crime was related to higher externalizing (q < .001), internalizing (q = .03) and dysregulation (q < .001) t-scores, but not competence t-scores (q = .31) at age 1 year. Living in an area with high property crime during pregnancy was also related to higher externalizing (q = .001) and dysregulation (q < .001) t-scores, but not internalizing (q = .06) or competence t-scores (q = .81). All of these relationships continued to be significant after the addition of disadvantage to the models, except violent crime’s relationship with internalizing symptoms. Frontolimbic connections in the neonatal brain, specifically the Thalamus-DMN (q = .05; q = .005), Thalamus-aFPN (q = .03; q = .004), and Amygdala-DMN (q = .02; q = .005), were related to externalizing t-scores and dysregulation t-scores, respectively. Amygdala-Hippocampus connectivity was not related to externalizing or dysregulation t-scores. In SEM models predicting externalizing symptoms from violent crime, the direct relationship with violent crime remained significant, but mediation was non-significant. In SEM models predicting dysregulation symptoms, the indirect path through maternal psychosocial stress and Thalamus-aFPN mediated the relationship between adversity and dysregulation symptoms. Additionally, there were direct relationships between Thalamus-DMN and Amygdala-DMN and dysregulation scores. For property crime, SEM models showed that the direct relationships with crime and disadvantage both independently predicted externalizing and dysregulation symptoms. Conclusions: These findings provide evidence that exposure to a high crime environment in utero is associated with poorer socioemotional processing in the externalizing and dysregulation domains at age 1 year. Crime seemed to be the most important predictor for externalizing symptoms whereas frontolimbic connectivity seemed to be most important for dysregulation symptoms. A limitation of this analysis is that it does not account for crime exposure during the first year, but this is a planned future direction of the study. Nevertheless, the current findings suggest that in utero exposure to crime and neonatal brain may affect socioemotional functioning. Given the effect of prenatal crime exposure, this study reiterates the need to reduce neighborhood rates of crime, as well as promote the development of frontolimbic connectivity. Keywords: Pregnancy, Neighborhood Crime, Functional MRI (fMRI), Stress and Adversity, Infant Behavior Disclosure: Nothing to disclose. P148. High-Throughput Functional Analysis of Autism Genes in Zebrafish Identifies Convergent Pathways Hellen Weinschutz Mendes, Uma Neelakantan, Yunqing Liu, Sarah Fitzpatrick, Tianying Chen, Weimiao Wu, April Pruitt, David Jin, Kristen Enriquez, Cheryl Lacadie, Ningshan Li, Dejian Zhao, Marina Carlson, Sundas Ijaz, Catalina Sakai, Christina Szi, Brendan Rooney, Marcus Ghosh, Ijeoma Nawabudike, Jeffrey Eilbott, Brent Vander Wyk, Jason Rihel, Xenophon Papademetris, Zuoheng Wang, Ellen Hoffman* Yale University, New Haven, Connecticut, United States Background: Whole-exome sequencing studies in autism spectrum disorders (ASDs) have led to the identification of a rapidly growing list of high confidence (hcASD) risk genes. These genes converge on biologically relevant mechanisms, including mid-fetal glutamatergic projection neurons, chromatin modification, and synaptic function, providing new insights into the biology underlying ASDs. At the same time, understanding how loss of function of hcASD genes affects specific cell types and the establishment of neural circuits in a developing vertebrate brain remains a central challenge. Methods: Here, we leverage the unique advantages of zebrafish as a high-throughput, in vivo system to analyze the function of 10 hcASD genes in parallel at the behavioral, structural, and circuit levels. First, we generated mutants of the zebrafish orthologs of 10 hcASD genes using CRISPR/Cas9. These genes span a range of biological functions, including gene expression regulation (CHD8, CUL3, KDM5B, POGZ, TBR1), neuronal communication (CNTNAP2, SCN1A/SCN2A, GRIN2B), and the cytoskeleton (DYRK1A, KATNAL2). Second, to determine how gene loss of function affects basic behaviors, we performed high-throughput automated assays of visual-startle and sleep-wake behavior. Third, we developed a novel computational pipeline to analyze whole-brain structure and activity across all mutant lines in parallel. Fourth, to identify overlapping molecular pathways, we performed whole-brain RNA-seq in three mutants with the most robust behavioral and brain activity phenotypes. Results: We establish a novel high-throughput pipeline for the parallel functional analysis of 10 hcASD genes in zebrafish and identify multiple points of convergence and divergence across risk genes at the behavioral, structural, circuit, and molecular levels. First, we identify unique behavioral fingerprints affecting arousal and sensory responsiveness across hcASD gene mutants. Second, we identify the forebrain as a significant contributor to brain size differences across mutants. Third, using whole-brain activity mapping, we find that regions involved in sensory-motor control show altered baseline brain activity and identify 22 out of 149 regions in the zebrafish brain with significant differences in baseline brain activity in more than half of mutants. Fourth, RNA-seq reveals the conservation of ASD gene-associated pathways, including neurogenesis and FMRP targets, and implicates novel cellular mechanisms in a subset of mutants. Conclusions: High-throughput parallel in vivo analysis of 10 ASD risk genes in zebrafish identifies convergent phenotypes affecting brain structure and baseline activity, and uncovers shared molecular and cellular pathways in a subset of mutants. Keywords: Autism Spectrum Disorder, Genetics, Zebrafish, Neurodevelopment Disclosure: Nothing to disclose. P149. Multidimensional Neuropsychiatric Phenotypes in Children With Noonan Syndrome Jennifer Bruno, Paige Naylor, Tamar Green* Stanford University, Stanford, California, United States Background: The Ras-mitogen-activated protein kinase (Ras-MAPK) pathway is central to neurodevelopment. Noonan syndrome (NS), the most common disorder associated with Ras-MAPK pathogenic variants, is associated with highly variable neuropsychiatric outcomes. We address limitations in previous research with a focus on prepubertal children, comparison to typically developing (TD) children, comprehensive neuropsychiatric evaluation and controlling for overall cognitive abilities Methods: Forty-five children with NS (8.48 + /-2.13 years; 29 female) and 40 TD children (8.75 + /-1.96 years; 22 female) were evaluated with objective parent report and psychiatric interview measures Results: Children with NS demonstrated higher levels of attention-deficit/hyperactivity disorder (ADHD) (attention, hyperactivity, and inhibition), autism spectrum disorder (ASD) (maintaining social relationships, behavioral rigidity, and sensory sensitivity) relative to TD children, all p’s<0.05 and η2 effect sizes were in the large range (>0.14). Children with NS also demonstrated higher levels of oppositional defiant disorder (ODD) (aggression) symptoms relative to TD children (all p’s<0.05, η2 effect sizes were in the medium range). Forty- eight percent of children with NS met the criteria for ADHD, 19% for ODD, and 25% demonstrated clinically significant symptoms seen in children with ASD. Conclusions: Children with NS are at increased risk for a spectrum of ADHD, ASD, and ODD associated symptoms. Over 50% of children fulfilled criteria for at least one of the tested clinical diagnoses. In contrast to the currently accepted categorical diagnostic approach, a dimensional approach reveals significant ASD symptoms in NS. Keywords: ADHD, Neurodevelopment, Noonan Syndrome, ASD Disclosure: Nothing to disclose. P150. Using an Optimized Fragile Messenger Ribonucleoprotein (FMRP) Assay to Predict Disease Severity in Fragile X Syndrome Lauren Schmitt, Christina Gross, Kelli Dominick, Ernest Pedapati, Craig Erickson Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States Background: The fragile X messenger ribonucleoprotein 1 (FMR1) gene-specific protein product, FMRP, is critical for brain development. In fragile X syndrome (FXS), FMRP levels are markedly reduced, if not absent. Previous studies have demonstrated the relationship between FMRP levels and general cognitive functioning; however, due to limitations of available assays, FMRP was not able to be reliably and reproducibly detected at extremely low values, preventing the examination of clinical relationships across the entire spectrum of individuals with FXS. Identifying these relationships may be critical to planning clinical care and understanding development over time. Methods: A sample of 65 individuals with FXS ( > 200 CGG repeats; 67% male), aged 8-45 years, completed a battery of psychosocial, neurocognitive, and electrophysiological measures. Participants whole blood samples were obtained via venipuncture and analyzed using our validated Luminex-based immunoassay to determine FMRP levels. Pearson correlations were conducted to examine the relationship between FMRP levels and clinical phenotypes. Results: In both males and females with FXS, higher FMRP levels were associated with higher IQ scores (m: r2 = .20, p-.01; f: r2 = .24, p = .02), consistent with prior findings. Of note, full mutation, fully methylated males who produce little to no FMRP showed a leftward shifted (mean=25, standard deviation=15) but otherwise normal distribution of IQ scores (skewness = .15, kurtosis = -.82). In males only, higher FMRP levels were associated with less severe stereotyped behavior (r2 = .12, p = .04) and irritability (r2 = .10, p = .07). In females only, higher FMRP levels unexpectedly were associated with more severe social avoidance (r2 = .40, p = .04) and depressive symptoms (r2 = .58, p = .01). FMRP levels were not significant related to any executive function variables in males or females with FXS. However, lower levels of FMRP were associated with increased relative gamma power, a neurophysiological biomarker of neural hyperexcitability, across FXS patients (r2 = .14, p = .04). Conclusions: Together, our optimized FMRP assay provides new evidence of the link between FMRP expression and clinical and neurophysiological phenotypes in FXS, and these relationships, importantly, may differ by biological sex. In addition, our findings also offer a paradigm-shifting role of FMRP in cognitive function in full mutation males: FMRP drives the leftward shift in IQ, but does not account for the remaining variance in cognition in full mutation males with FXS. This is in contrast to ours and others’ findings indicating FMRP has a dose-dependent effect on IQ in mosaic males and full mutation females who express higher levels of protein. There is much future work to better understand the mechanism by which deficient or absent FMRP leads to clinical phenotype. Still our novel work is a critical step towards establishing molecular markers of disease severity in FXS. Ultimately, this foundational work is paving the pathway towards the development of individualized “precision medicine”. Keywords: Fragile X Syndrome, FMRP, Biomarker Disclosure: Nothing to disclose. P151. Genomic Architecture and Genetic Risk for Autism Spectrum Disorder in Individuals of Latin American Ancestry Marina Natividad Avila, GALA Consortium, Joseph Buxbaum Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Autism spectrum disorder (ASD) is characterized by deficits in social communication and the presence of restricted interests and/or repetitive behaviors. Risk for ASD is complex but largely genetic, with both common and rare genetic variation contributing to risk. While common variation accounts for most genetic liability for ASD, rare variation, often de novo, accounts for substantial individual liability. In anticipation of both the tremendous impact and consequential challenges that high-throughput sequencing would have on ASD genomics, the Autism Sequencing Consortium (ASC) was formed in 2010 and recently completed an analysis of 65,000 exomes (Fu et al. 2022, in press), which included samples from ASC sites as well as SPARK data (SPARK Consortium 2018). In Fu et al, the ASC reported 71 genes associated with ASD at a false discovery rate (FDR) ≤ 0.001; a threshold that approximates exome-wide significance (p < 2.5e-6), and 185 genes at FDR ≤ 0.05. Analysis of the SPARK dataset identified 60 largely overlapping genes with exome-wide significance (Zhou et al. 2022, in press). Despite the success of these large-scale genetic analyses, the question of whether the genetic architecture of ASD differs across ancestral populations remains. Notably, there has been no systematic effort to investigate the genetic architecture of ASD in Latina/Latino populations (hereafter Latinx), the largest minority in the United States. We have established the Genomics of Autism in Latinx Ancestries (GALA) Consortium to develop a large prospective cohort to examine the role of genetic and environmental factors in ASD across ancestral backgrounds and collect genetic materials for large-scale analyses. Our first aim was to explore whether the rare damaging burden signal is similar across ancestral backgrounds. In parallel, we are analyzing existing and emerging GALA and other Latinx samples for ASD gene discovery. Methods: The GALA Consortium has collected ca. 1,000 ASD Latinx participants, largely as trios from South and Central America and/or the USA. Collection of an additional 1,600 Latinx ASD trios is ongoing. All sites will use a similar strategy to screen and phenotype probands. All samples will be subject to genotyping and whole exome sequencing (WES). Single Nucleotide Polymorphism (SNP) data will be used to estimate SNP-based heritability at various levels of ancestry. Rare de novo and inherited single nucleotide variation (SNV), and insertion deletion variation (indel) will be identified from WES. Chromosomal microarray and WES data will identify potentially damaging copy number variation (CNV). With these data, we will first test whether the genomic architecture of ASD, as reflected by (a) the exonic de novo rate, (b) mutation burden in highly conserved genes (pLI ≥ 0.995), and (c) polygenic risk, differs between Latinx and non-Latinx populations. Accounting for ancestry, we will then use the TADA (Transmitted and de novo Association) framework to identify Latinx ASD genes impacted by rare variation, integrating de novo and inherited variation from trios and case-control data, incorporating SNV, indel, and CNV. SNP data from GALA will be contributed to the PGC-ASD analyses to facilitate greater power for cross-ancestry GWAS. Results: We first determined whether the genomic architecture and ASD genes impacted by rare deleterious variation will differ across ancestries. We hypothesize that they will not. To examine this, we took all de novo mutations in Fu et al. and examined rates as a function of ancestry. We looked at highly conserved genes and all remaining genes. We observed that the signal is overwhelmingly found in the most conserved genes across all ancestries. Thus, when separating the samples by genetically defined ancestry, results were similar across groups. We are now analyzing WES data from the existing and emerging GALA samples, together with Latinx samples from SPARK. Power for gene discovery can be estimated based on these results and it is reasonable to estimate at least 25 ASD genes in the full GALA Latinx samples at a strict FDR < 0.05 and more at more lenient thresholds. Genetic findings will be compared with those in European samples and if, as we predict, the genes overlap, we will carry out joint analysis across multiple ancestries. First results from GALA are already implicating known ASD genes, consistent with shared molecular risk architecture. Analyses are ongoing and results from all analyses will be presented. Conclusions: Genetic variation that harbors strong impact on neurodevelopment will overwhelmingly be found in highly conserved genes; those depauperate of deleterious variation in very large, heterogeneous databases. Hence, we hypothesized that the rare mutational burden in conserved genes will not differ across ancestries and that there will be extensive overlap between ASD genes identified. Our analyses are consistent with this hypothesis and support continued ASD gene discovery in Latinx and other populations. Biology, here reflected in genes that are intolerant to deleterious variation due to critical function and dosage sensitivity, is universal and not impacted by “local” genetic processes. Going forward, we can carry out genotype-phenotype correlations in Latinx samples and explore the role of access to care on phenotype and genomic architecture. As we transition from studying rare deleterious variants to standing, inherited variation, improved methods for integrating data across populations will be needed. Keywords: ASD, Rare Genetic Variation, Multi-Ancestry, Copy Number Variation, Gene Discovery Disclosure: Nothing to disclose. P152. The Role of Fragile X Messenger Ribonucleoprotein (FMRP) in Neural Response To and Discrimination of Speech Sounds in Children With Fragile X Syndrome Kelli Dominick, Elizabeth Smith, Lauren Schmitt, Craig Erickson Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States Background: Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability (ID), resulting from the silencing and inactivation of the FMR1 gene with resultant loss of fragile X messenger ribonucleoprotein (FMRP) expression leading to widespread changes in brain structure and function. While delays in language are almost universal in those with FXS, there is limited research examining auditory and language processing, particularly in early life and in males. And few have examined the relationship between FMRP and functional brain response to language in FXS. Here we examine the relationship between FMRP levels and neural activation to speech and nonspeech sounds using fNIRS. Methods: Participants. Current sample includes 38 full-mutation FXS (diagnosis determined via genetic testing) and 15 typically developing control subjects. 18 of 38 participants with FXS also had blood drawn to assess FMRP levels. fNIRS. All fNIRS data were collected using a custom NIRx NIRScout (NIRx Medical Technologies, Brooklyn, NY) machine with lasers at wavelengths of 685 and 830nm and a 7.8125 Hz sampling frequency with an auditory 2x2x4 montage designed to cover temporal lobe areas bilaterally including the primary auditory cortex. Data was collected using NIRStar acquisition software with auditory stimuli programmed and presented using NIRStim software. Auditory Task. The experimental design was a passive listening task lasting approximately 20 minutes (variable due to jitter, actual time was 1,208 seconds plus or minus up to 68 seconds of jitter). The auditory task consisted of a modified version of the Stories paradigm created by Cincinnati MR Imaging of Neurodevelopment (C-MIND) (Schmithorst, Holland, and Plante, 2006; Sroka et al., 2015). Clips of 5 narrative stories for young children read by female voice, interspersed with control nonspeech tones frequency-matched to human speech (200-4000Hz center frequencies, 0.5-2.0Hz duration broadband sweep). FMRP. FMRP was measured using an optimized luminex-based assay (Boggs et al. 2022) which is able to detect a wide range of FMRP levels, including very low protein levels. Blood processing has been completed on 11 of 18 FXS subjects to date, with additional subjects to be added for final analysis. Data Analysis. fNIRS data were then processed using the Homer3 toolbox (https://github.com/BUNPC/Homer3). Activation patterns to speech and nonspeech stimuli were examined in relation FMRP levels in participants with FXS. Results: Children with FXS showed activation for speech and nonspeech sounds in language regions. Controls showed activation for speech and neural differentiation between the speech and nonspeech stimuli. FMRP demonstrated a non-significant relationship to neural activation patterns in early analyses. Additional analyses will be completed on the complete cohort including examination of the relationship between FMRP and language lateralization. Descriptive statistics will be used to characterize males versus females with FXS, however small sample size will limit further subgroup analysis. Conclusions: Effective biomarkers for language impairment and development in FXS will require understanding the mechanisms that lead to impairment, which will, in turn, require functional neuroimaging methods like fNIRS. Optimally, it will include linking neural changes to both phenotypical presentation and molecular etiology. This work provides early examination of the relationship between peripheral FMRP and neural activation patterns in FXS. Keywords: Language Delay, Fragile X Syndrome, fNIRS Disclosure: Nothing to disclose. P153. A Multimodal Approach to Investigating Theta Burst Stimulation and Auditory/Language Processing: Piloting an Autism Spectrum Disorder Study Sunday Francis, Lindsay Oberman, Megan Hynd, Lysianne Beynel, Zeynab Rezaee, Paul Rohde, Jeff Stout, Joelle Sarlls, Jan Willem van der Veen, Audrey Thurm, Sarah Lisanby National Institute of Mental Health, Bethesda, Maryland, United States Background: Challenges in language processing are a central characteristic of Autism Spectrum Disorder (ASD), a complex heterogeneous neurodevelopmental disorder that also includes restricted and repetitive behaviors and deficits in social interaction. Previous literature suggests dysfunctional γ-aminobutyric acid (GABA) transmission may underlie auditory and language processing deficits in individuals with ASD. With current advancements in neuroimaging and neurophysiology technologies, researchers can undertake the challenge of clinical investigations studying GABAergic and other neurochemical transmission, as well as structural and functional connectivity associated with the pathophysiology of auditory and language processing deficits. The pilot phase of this study, presented herein, serves multiple purposes: (1) to confirm feasibility and tolerability of study procedures, (2) to develop and test the performance of novel processing and analysis pipelines, (3) to establish the relationship between baseline local neurochemical concentrations in Wernicke’s area, structural and functional network connectivity, and magnetoencephalography (MEG) indices of auditory and language processing, and (4) to investigate the impact of single session, individually targeted, continuous theta burst stimulation (cTBS) on the abovementioned outcomes in 20 adult non-clinical volunteers. Individualized stimulation targets are determined by task-related peak activation within the left posterior superior temporal cortex (pSTC) during the Auditory Description Decision Task (ADDT). We utilize magnetic resonance spectroscopy (MRS) to quantify neurochemical concentrations (including GABA, glutamate, glutamine, n-acetyl aspartate (NAA), and creatine) within the targeted region. Diffusion-weighted imaging (DWI) is collected to investigate structural connectivity. Functional MRI (fMRI) is acquired to evaluate activation and functional connectivity at rest and during the language task and MEG is conducted to understand the temporal aspects of auditory processing. These measures together provide a comprehensive, overarching picture of brain mechanisms underlying language and auditory processing. Methods: Twenty non-clinical participants (5 males, 23.15 ± 0.97 years) completed MEG, MRI, and MRS at baseline, thereafter two cTBS visits, a minimum of a week apart, were conducted. During cTBS visits MEG (MEG + TMS visit), MRI, and MRS (MRI + MRS + TMS visit) data were collected prior to and post-stimulation. MEG data was collected using a CTF 275 whole head MEG (1200Hz sampling rate), while participants listened to validated auditory stimuli designed to probe evoked power and inter-trial coherence in the gamma frequency band, as well as local and long-range network functioning (as measured by phase-amplitude coupling between the alpha and gamma frequency bands and the latency of evoked fields such as the M100, and MMF). All MRI and MRS scans were acquired using a General Electric (GE) 3T MRI scanner. A 15.6cm3 voxel centered on the individually defined target was used for spectroscopy. A single cTBS session comprised of 200 3-pulse 50Hz bursts with an inter-burst interval of 200ms, totaling 40s. Stimulation was applied at 80% of active motor threshold. Results: As of submission, all 20 participants have been enrolled. No serious adverse events have been reported; indeed, all volunteers have tolerated all procedures. The first objective, to confirm feasibility and tolerability of study procedures has been met. Secondly, processing and analysis pipelines are being developed and tested. The language processing task used for TMS targeting reliably activated (p ≤ 0.05) a defined region within pSTC for each participant. An acute effect of the cTBS on the MEG measures has been noted as well. Data analysis is ongoing. Results of the inferential statistics will be presented at the annual conference. Conclusions: We report both baseline and pre-post cTBS Phase I data investigating the relationship between local neurochemical transmission, structural and functional network connectivity, and MEG indices associated with auditory and language processing networks in 20 adult non-clinical volunteers. In addition to observing the impact of cTBS on auditory and language processing, this phase serves as a proof of feasibility and tolerability of study procedures and enables the development of multimodal imaging analysis pipelines for the main phase of the study, which will include 86 adolescents (age 14-17) with ASD. Keywords: Human Neuroimaging, Multimodal Neuroimaging, Repetitive Transcranial Magnetic Stimulation (rTMS), Autism Spectrum Disorder, Auditory Processing Disclosure: Nothing to disclose. P154. Development of a Novel qEEG Based Diagnostic Classifier for Adolescent Depression Molly McVoy, Deniz Doruk Camsari, Farren Briggs, Farhad Kaffashi, Kenneth Loparo, Paul Croarkin Case Western Reserve University, Cleveland, Ohio, United States Background: The early recognition, accurate diagnosis and prognosis determination in youth with Major Depressive Disorder (MDD) is challenging. Imprecise nosology and overlapping presentations are particularly difficult in childhood (Mendelson and Tandon, 2016). As a result, there is an unmet need for evidence-based biomarkers in childhood psychiatric diagnoses. We previously reported in a pilot biomarker study that quantitative electroencephalogram (qEEG) showed a difference in coherence (one measure of resting connectivity) between an outpatient sample of youth with Major Depressive Disorder (MDD) and healthy controls (HC). Adolescents with MDD showed a pattern of decreased coherence when compared with HC. We have developed predictive model, or diagnostic classifier, in a pilot sample of adolescents with MDD, using EEG measures of coherence, power and cross correlation. We report on a new inpatient sample of youth with MDD and suicidality and measures of connectivity as measured by qEEG compared to HC. In addition, we report on new findings of the use of this predictive model in this inpatient sample of youth with MDD and suicidal ideation. Methods: The initial diagnostic classifier was developed in an outpatient sample of youth with MDD. 21 youth, age 14-17 (F = 17 M = 4) with MDD and 14 healthy controls, age 14-17 (F = 10 M = 4) received a one-time resting EEG. The Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID), the Children’s Depression Rating Scale (CDRS), the Snaith–Hamilton Pleasure Scale (SHAPS), and the Pediatric Anxiety Rating Scale (PARS), were given. QEEG in four frequency bands (alpha, beta, theta, and delta) was collected, and coherence, cross-correlation, and power data streams obtained. A two-stage analytical framework was then used to develop the final logistic regression model which was then evaluated via a receiver-operating characteristic curve (ROC) analysis. The validation sample consisted of a group of 27 MDD youth, age 13-18 (F = 21 M = 6) admitted to an inpatient child psychiatric unit for acute suicidality and 29 HC, age 13-18 (F = 16 M = 13). This cohort were given the MINI-KID, the CDRS and the PARS. This cohort of youth had resting EEG data collected in addition to task based data. The diagnostic classifier was validated in the resting EEG data collected in this sample. Coherence, cross-correlation and power data streams were obtained in the same four frequency bands. Results: Within the initial training data set of outpatient youth with MDD, 6 qEEG dyads (all coherence) had significant predictive values. The top 10% of predictive markers used to develop the classifier included coherence at the following dyads:F3-C3 (alpha frequency(fr)), P3-O1 (theta fr), F3-C3 (theta fr), F3-C3 (beta fr), CZ-PZ (beta fr), Fp2-F4 (theta fr), F3-C3 (delta fr), T8-P8 (beta fr), P3-O1 (alpha fr), Fp2-F4 (alpha fr), and power at the following dyads: C3-P3 (alpha fr), T7-P7 (theta fr), F7-T7 (theta fr), Fp1-F7 (alpha fr), F8-T8 (theta fr), P8-O2 (theta fr), F3-C3 (alpha fr), C4-P4 (delta fr), C4-P4 (theta fr), P7-O1 (theta fr), P4-O2 (beta fr), T8-P8 (theta fr). Within the final biomarkers, just 4 predictors, including F3-C3 (R frontal) alpha coherence, P3-O1 (R parietal) theta coherence, CZ-PZ (central) beta coherence, and P8-O2 (L parietal occipital) theta power were used in the final model, which yielded an ROC area of 0.8226. In the validation sample set of youth with MDD and suicidality, the model that included the top 10% of classifiers yielded an ROC of 0.8122. The final model from the training data set included 4 variables, one of which, CZ-PZ, was not available in the validation sample. This model with only the 3 variables common to both data sets, yielded an ROC 0.5668. In the validation sample of youth with MDD and suicidality, 7 qEEG dyads were statistically significantly different in MDD youth than in HC (p < 0.05): T8-P8 coherence (beta fr), T8-P8 coherence (theta fr), T7-P7 coherence (beta fr), P3-O1 power (theta fr), F8-T8 power (alpha fr), T8-P8 coherence (alpha fr); E5_E60 cross correlation. Conclusions: In these two samples of youth with MDD, qEEG as a diagnostic biomarker continues to show promise. A classifier developed in a training set of youth with MDD also showed robust predictive value in a validation sample of youth with MDD. The expanded classifier, including a broader range of dyads, performed more robustly than the narrow, 4 item classifier. Both sets of MDD participants show differences in measures of connectivity in frontal regions, consistent with changes in the default mode network in depressed youth. Future longitudinal studies are warranted, especially in youth prior to the initiation of treatment. In addition, studies investigating how medications, severity of anxiety, and anhedonia impact qEEG results are indicated. Studies investigating the difference between MDD youth with and without suicidality are also indicated in the future based on this preliminary research. Keywords: Adolescent Depression, Biomarker, EEG Connectivity Disclosure: Nothing to disclose. P155. Baseline Alterations and Acute Effects of Escitalopram on Amygdala Functional Connectivity in Youth at High Risk for Bipolar Disorder Lu Lu, Maxwell Tallman, Thomas Blom, Kaitlyn Bruns, Jeffrey Welge, Luis Rodrigo Patino, Jeffrey Strawn, Qiyong Gong, John Sweeney, Kyle Hinman, Manpreet Singh, Melissa DelBello University of Cincinnati, Cincinnati, Ohio, United States Background: Youth with a family history of bipolar disorder, especially those who present with depression and/or anxiety symptoms, are at high risk for developing bipolar disorder and disrupted functional connectivity (FC) within the prefrontal-amygdala emotional network. Antidepressants are commonly used to treat depression and anxiety in youth. However, the neural effects of antidepressant exposure in these high-risk youth remain unknown. Our aims were 1) to compare resting state FC in depressed and/or anxious youth at high risk for developing bipolar disorder and healthy comparison youth and 2) to compare early changes in resting state FC in these high-risk youth following treatment with escitalopram and psychotherapy vs. placebo and psychotherapy. Methods: We conducted a two-site, double-blind, placebo-controlled trial of escitalopram in youth (12-17 years old) with depression and/or anxiety and a first-degree relative with bipolar I disorder (BD). At baseline, 121 high-risk youth and 55 healthy controls (HC) without any first- or second-degree relative with a mood or psychotic disorder were recruited. High-risk youth were then randomized to receive psychotherapy plus escitalopram (n = 77) or psychotherapy plus placebo (n = 44) for up to 16 weeks. A 3-Tesla scanner with an 8-channel phased-array head coil was used at each site to acquire resting-state functional and anatomical images at baseline in high-risk and HC youth, and at 4-weeks after treatment initiation in high-risk youth. Two 5-minute functional runs used TR = 2000 ms and slice thickness=3 mm. Image analysis was performed using SPM, CONN, and MATLAB. Preprocessing steps included discarding the initial ten volumes, slice-timing, realignment, structural segmentation and normalization, functional normalization, outlier detection, and smoothing. Denoising was then performed, which applied linear regression of confounders, band-pass filtering, and linear detrending to remove unwanted motion, physiological, and other artificial effects from the BOLD signal. Next, the FC map between the amygdala and voxels of the whole brain was generated for each youth, which was then taken to two second-level statistical analyses: 1) High-risk vs. HC t-tests of amygdala-based FC with age, sex and site as covariates using thresholds p < 0.005 at voxel level and p < 0.05 at cluster level with False discovery rate (FDR) correction and 2) treatment (escitalopram or placebo)-by-time (baseline or week 4) interactions using a full-factorial analysis with age, sex, and site as covariates, and using the same thresholds as the baseline analyses. Connectivity values were extracted from clusters with significant treatment-by-time interactions. Post-hoc analyses were performed to calculate the direction of FC changes from baseline to week 4 in each treatment group. Results: Baseline differences in amygdala-based FC between high-risk and HC youth: The baseline analyses included one hundred and six high-risk youth and 46 age- and sex-matched HC with usable data. Compared to HC, high-risk youth had decreased connectivity between left amygdala and left ventral prefrontal cortex extending into insula and superior temporal cortex (peak coordinate: x = -46, y = 16, z = -16; cluster size=122; t = 4.78; FDR-corrected p = 0.036), and decreased connectivity between the right amygdala and bilateral dorsal anterior cingulate cortex (peak coordinate: x = -4, y = 26, z = 14; cluster size=217; t = 5.48; FDR-corrected p = 0.002) and right dorsolateral prefrontal cortex (peak coordinate: x = 22, y = 52, z = 14; cluster size=129; t = 4.45; FDR-corrected p = 0.014) as compared to HC. Escitalopram effect on amygdala-based FC in high-risk youth: Eighty-four high-risk youth with usable baseline and week 4 data were included; 50 of them received escitalopram and 34 received placebo. Significant treatment-by-time interactions were found between right amygdala and bilateral supplementary motor cortex (peak coordinate: x = -2, y = -26, z = 62; cluster size=264; F = 20.18; FDR-corrected p = 0.011), left lateral occipital cortex including left fusiform gyrus (peak coordinate: x = -44, y = -78, z = 10; cluster size=424; F = 16.97; FDR-corrected p = 0.001) and right occipital fusiform gyrus (peak coordinate: x = 32, y = -64, z = -6; cluster size=197; F = 15.72; FDR-corrected p = 0.032). Post-hoc analyses showed these amygdala FCs significantly decreased in youth treated with escitalopram relative to youth who received placebo. Conclusions: We found that depressed and/or anxious youth at high-risk for bipolar disorder had decreased amygdala connectivity in regions that subserve emotion perception and regulation relative to healthy individuals. These findings suggest that brain functional connectivity within and between emotion processing and regulation networks are disrupted in youth at high risk for bipolar disorder. These alterations may serve as potential targets for early intervention in this population. Additionally, amygdala connectivity with sensory and motor cortices decreased significantly in high-risk youth after four weeks of treatment with escitalopram and psychotherapy relative to those receiving four-weeks of placebo and psychotherapy. Further analyses will examine whether the significant effects of escitalopram on the interface among emotional, visual and motor networks are associated with subsequent behavioral and mood changes resulting from antidepressant treatment in this population. Keywords: Bipolar Disorder, High Risk, Functional Connectivity Disclosures: Myriad, Medscape: Advisory Board (Self), Alkermes: Consultant, Contracted Research (Self), Lundbeck, Janssen: Contracted Research (Self), Janssen: Consultant (Self), Allergan, Shire: Contracted Research (Self) P156. Brain Development Trajectories in Adolescents With and Without Non-Suicidal Self-Injury Kathryn Cullen, Jessica Butts, Donovan Roediger, Zeynep Basgoze, Bonnie Klimes-Dougan, Bryon Mueller, Kelvin Lim University of Minnesota Medical School, Minneapolis, Minnesota, United States Background: Adolescence is a critical period for brain development, and is also notable for the onset of non-suicidal self-injury (NSSI), a risk factor for future suicide attempts. Examination of brain developmental processes associated with the onset and course of NSSI during adolescence promises to advance understanding of risk mechanisms and to guide intervention strategies. Methods: 164 adolescents ages 12-16 years (assigned female at birth) with and without a history of NSSI were enrolled in a longitudinal study that involved annual clinical and neuroimaging assessments for three years. NSSI frequency and severity was evaluated using the Self-Injurious Thoughts and Behaviors Interview. Brain imaging included a structural scan, a 12-minute resting-state fMRI scan and a 6-minute task fMRI scan to measure threat-related response in which participants matched emotion (fearful, angry) faces or neutral shapes. Functional data were parcellated using the Glasser atlas for cortical regions and the Harvard-Oxford atlas for subcortical regions. Given the relevance of the Sustained Threat domain to NSSI, following our prior work indicating a relationship between NSSI severity and threat-related circuitry at baseline (Başgöze et al., 2021), brain metrics for longitudinal analysis included amygdala activation in response to threat, and resting state functional connectivity (RSFC) between bilateral amygdala and ventromedial PFC (vmPFC). In addition to the standard methods of calculating RSFC, to further explore neural connectivity and allow examination of the direction of neural connections, we used causal discovery analysis to identify causal relationships (edges) among regions across the brain. Since very few participants were found to have causal edges involving the amygdala, these analyses focused on connections amongst key frontal regions implicated in emotion regulation – bilateral medial and dorsolateral frontal cortex (mPFC and dlPFC)– as well as the default mode network (DMN), which has been implicated in depression and self-related processing. For the task data, we focused on the activation in the emotion>shape contrast in bilateral amygdala. For longitudinal analyses, we first evaluated developmental associations on this set of brain metrics across adolescence in this NSSI-enriched sample by conducting separate regression analyses for each brain metric using generalized estimating equations (GEEs) to estimate the association of baseline age and change in age from baseline on connectivity strength. Second, to evaluate the impact of NSSI behavior on brain development, we added a term for baseline NSSI group and the interaction between baseline NSSI group (none, Mild [1-4 lifetime episodes], Moderate [more than 5 episodes and frequency of at least once per month], and Severe [more than 5 lifetime episodes and frequency of more than once per month]) and change in age from baseline. Results were considered statistically significant if the two-sided p-value was less than 0.05. P-values were not adjusted for multiple comparisons. Results: Usable clinical and imaging data was available for 140 participants at Time 1, 87 participants at Time 2, and 39 participants at Time 3. For the analyses examining overall developmental trajectories in this sample, baseline age and change in age from baseline were positively associated with the brain metrics (p < 0.05) in the models for right amygdala activation to threat and the causal edge from DMN to left dlPFC. When examining group by time effects, the trajectory of change over time for the Severe NSSI group differed significantly from the No NSSI group for causal edges DMN to left mPFC and right mPFC to DMN. The trajectory of change over time for the Moderate NSSI group differed significantly from the No NSSI group for causal edges DMN to right mPFC (which also had a main effect of age), and right mPFC to right dlPFC. When examining NSSI severity group without a significant interaction with change in age, the Mild NSSI group had significantly weaker connections than the No NSSI group in the edges from left dlPFC to DMN and from left mPFC to left dlPFC. Additionally, the Severe NSSI group had significantly lower RSFC from the amygdala to vmPFC (which also had a main effect for age) compared to the No NSSI Group. Conclusions: We report evidence for developmental change in key circuits relevant to emotion regulation and self-related processing across mid-adolescence. Severity of NSSI in adolescence is associated with neural signatures, some of which are present across time and some which manifest as change over time, characterizing distinct neurodevelopmental trajectories in key circuits. Understanding these patterns is critical for conceptualizing and testing novel treatments designed to restore healthy neurodevelopment. Keywords: Brain Development, Adolescence, Non-Suicidal Self-injury (NSSI) Disclosure: Nothing to disclose. P157. Developmental Change in Human Substantia Nigra: Preliminary Observations Early and Late in Life Assessed With Neuromelanin-Sensitive MRI Rami Al-Haddad, Synthia Guimond, Philippe Robaey, Pedro Rosa-Neto, Clifford Cassidy The University of Ottawa Institute of Mental Health Research, Ottawa, Canada Background: In recent years neuromelanin-sensitive MRI (NM-MRI) has been applied to assay the integrity and function of human catecholamine systems in vivo. To optimize use of the method it is important to account for the known accumulation of neuromelanin (NM) in the substantia nigra (SN) throughout the lifespan, which has been shown to have a large impact on the signal. This is relevant not only in late life, where the method has often been applied in the context of neurodegenerative illness, but also early in life, where the method allows the rare opportunity to directly assay dopamine system function in the context of pediatric disorders. While prior work has indicated the SN signal may follow an inverted-u pattern, peaking in middle age and declining slightly late in life, this work did not examine the SN in anatomical detail, a level of analysis that may be necessary to optimize the use of the method for mechanistic studies or as a biomarker. Methods: NM-MRI images were collected at two separate locations using the same turbo spin echo sequence. Sample 1 was collected at the uOttawa Institute of Mental Health Research and included individuals aged from age 7-30 years (n = 27) and from 52-82 years (n = 22). Sample 2 was collected at the Montreal Neurological Institute and individuals aged over 52 years old, all of whom had longitudinal data with 1-2 annual follow-up scans (n = 68). NM-MRI signal in the SN was examined using a pipeline that brings NM-MRI images to standardized space and performs intensity normalization relative to a reference region near the SN with low NM content, the crus cerebri. In Sample 1, voxelwise robust linear regression analyses were performed within the SN to predict NM-MRI signal based on age and sex. In Sample 2, linear regressions were performed for each participant’s longitudinal data to determine the slope of change in NM-MRI signal over time for all SN voxels, followed by 1-sample t-tests on these slopes. Permutation tests were applied for significance testing. Results: In order to consider developmental change in the SN both early and late in life, we divided Sample 1 into individuals younger than 30 and older than 52 years old. In the younger group, the whole SN showed a significant increase in signal with age (R = 0.63, p = 0.0005, partial correlation controlling for sex) and voxelwise analysis found a large set of voxels within the central SN that increased with age (1000 of 1879 voxels thresholded at p < 0.05, corrected p < 0.001, permutation test). The annual increase in signal here was equal to 0.24% of the reference region signal. Almost no SN voxels showed decreasing signal over this range of age (4 voxels). In the older individuals, a set of 172 voxels in central SN showed increasing signal with age (annual change=0.19%) and a set of 112 voxels in peripheral SN showed decreasing signal with age (annual change = -0.21%). When examining change in SN signal longitudinally over time in a separate sample collected at a different institute (Sample 2, with a similar age range to the older group from Sample 1), there was a large cluster of voxels where signal decreased over time (thresholded at p < 0.05, 515 voxels, corrected p < 0.0001, permutation test, median annual change across subjects = -0.40%) and a small cluster of voxels where signal increased over time at this threshold (9 voxels, median annual change across subjects=0.34%). Examining the spatial overlap between SN voxels where signal changed with age (Sample 1, older group) and SN voxels where signal changed over time (Sample 2) there was significantly more overlap than would be expected by chance both for increasing signal voxels (corrected p = 0.007, permutation test) and decreasing signal voxels (corrected p < 0.0001). Conclusions: We found evidence that the NM-MRI signal increases at a relatively rapid rate throughout the SN in early life. Even into later life, signal in a portion of the central core of the SN continues to increase while the size of the SN appears to shrink, perhaps due to loss of dopamine cells during normal aging. These findings provide rare insight into the development of the human dopamine system. They should support the use of NM-MRI as a biomarker by enabling anatomically detailed age-specific norms for signal within the SN. This is particularly relevant as the method may have key applications in both youth and aging populations (e.g. in pediatric disorders and neurodegenerative disorders). These findings are consistent with NM contributing significantly to the contrast given the known tendency of NM to accumulate linearly throughout the lifespan. In future, with an expanded and longitudinal sample, we will develop a model of developmental change in NM-MRI signal across the full lifespan and we will investigate the impact of other clinical and demographic factors that may need to be considered to optimize potential use of the method as a biomarker. Keywords: Dopamine, Brain Development, Neuromelanin-Sensitive MRI, Healthy Individuals, Substantia Nigra Disclosure: Terran Biosciences: Patent,Contracted Research (Self) P158. Sex Differences in the Microbiome-Gut-Brain Axis are Time-Of-Day Dependent Eldin Jasarevic* University of Pittsburgh, Pittsburgh, Maryland, United States Background: Circadian rhythms orchestrate a wide range of homeostatic processes, including metabolism, immune function, and the bidirectional communication between the gut and brain. Emerging evidence points to the gut microbiome and its metabolites as a novel class of entraintment signals, but the precise molecular mechanisms by which diurnal rhythms in microbial metabolites contribute to these processes remains unknown. Moreover, circadian rhythms are influenced by sex, suggesting that microbiome-gut-brain axis function is sex-specific and influenced by time-of-day. Methods: To examine the hypothesis that sex differences in the microbiome-gut-brain axis are time-of-day dependent, we leveraged a novel systems biology approach by combining longitudinal analyses with dietary manipulation, metabolomics, bulk and single-cell transcriptomics of gut and brain, single-cell immunophenotyping, and machine learning. We harvested tissues from C57Bl/6J and BALB/c male and female mice every 4 hours across the day-night cycle. Results: Bulk transcriptomic analyses of the intestinal tract revealed that transcriptional signatures were sex-specific and dependent on time-of-day (FDR < 0.05). Diurnal gene expression patterns in the gut were synchronized with the production and peripheral availability of microbial-derived short-chain fatty acids (SCFAs). Integrated single-cell immunophenotyping, metabolomics and transcriptomics revealed that time-of-day shifts in the peripheral availability of SCFAs were associated modifications to circulating immune composition and transcriptional signatures in key neural regions controlling whole-body metabolism, including the arcuate nucleus of the hypothalamus. These sex-specific time-of-day patterns were abolished in germ-free mice, suggesting that an intact gut microbiome is necessary for the synchronization of sex differences across the microbiome-gut-brain axis (p < 0.05 for all comparisons). Further, biosynthesis of SCFAs requires availability of soluble fibers in the diet. Thus, to determine whether dietary availability of soluble fibers and subsequent availability of SCFAs is a necessary time-of-day sex differences in the microbiome-gut-brain axis, mice were fed a refined high-fat low-fiber diet (HFD) or a grain-based chow (GBC) control for six weeks. Time-of-day effects on SCFA availability were disrupted in mice consuming HFD, suggesting that dietary availability of soluble fiber is essential is necessary for daily oscillations in cecal weights and SCFA abundance. Conclusions: Our results show that synchronization of diurnal feeding patterns with availability of key nutrients control sex differences in the microbiome-gut-brain axis and regulate host immunity and neuroendocrine function. Our findings motivate exploration of the host circadian clock and microbial circadian rhythmicity and highlight the need to consider sex and timing of sample collection in neuroscience experiments. Keywords: Microbiota-Gut-Brain Axis, Immunity and Neurodevelopment by Sex, Immunometabolism, Neurometabolism, Brain Regulation of Metabolism Disclosure: Nothing to disclose. P159. Social Deficits Induced by Combined Air Pollution and Maternal Stress are Prevented by Microbial or Dopaminergic Intervention Caroline Smith, Danielle Rendina, Marcy Kingsbury, Karen Malacon, Dang Nyugen, Jessica Tran, Benjamin Devlin, Madeline Clark, Ravikiran Raju, Lauren Burgett, Staci Bilbo Duke University, Durham, North Carolina, United States Background: Epidemiological data suggest that perinatal exposure to environmental toxicants, such as air pollution, is associated with risk for autism spectrum disorders (ASD). Psychosocial stressors may activate the maternal immune system, increasing sensitivity to these toxicants. Still, the mechanisms by which this synergism occurs have yet to be elucidated. ASD is a male-biased disorder and is defined by impairments in social interaction/communication and repetitive/restrictive behaviors. Importantly, the gut-brain axis has also been strongly implicated in ASD. Using a novel mouse model of combined prenatal diesel exhaust particles (DEP) and maternal stress (MS), we found that male offspring display reduced sociability, changes in the gut microbiome, and altered microglia and dopaminergic tone following DEP/MS. Methods: First, we aimed to test whether restoring control-typical gut microbiota could rescue social behavior following DEP/MS via a cross-fostering procedure. DEP/MS-exposed male and female pups were fostered on the day of birth to either a DEP/MS or a CON-exposed dam. Social behavior was assessed during adolescence. Second, we used a chemogenetic approach to test whether activation of the dopamine system could rescue social behavior during adolescence. DAT-Cre+ CON or DEP/MS males received stereotaxic microinjection of a control virus or an excitatory DREADD receptor virus into the ventral tegmental area. Clozapine-N-oxide was administered prior to behavioral testing. Finally, we conducted RNA sequencing of isolated microglia from CON and DEP/MS male offspring and compared gene expression patterns to those of germ-free mice, LPS-challenged mice, and across development (based on previously published datasets) using rank-rank hypergeometric overlap (RRHO) analysis. Results: We found that cross-fostering of DEP/MS-exposed male pups to CON dams on the day of birth prevented decreases in sociability (t(1,13)=3.334, p < 0.01). Second, we observed that DREADD activation of VTA dopamine neurons restored social preference in DEP/MS-exposed male offspring (F (2,26) =4.253; p < 0.05). Finally, we found that microglial gene expression patterns in DEP/MS-exposed males corresponded with gene expression signatures in germ-free mice. Interestingly, this was opposite of comparisons between DEP/MS-exposed males and males that were acutely treated with LPS. Conclusions: These results suggest that intervening at the level of either the gut microbiome (perinatally) or the dopamine system (acutely during adolescence) is sufficient to rescue social functioning following prenatal DEP/MS exposure in male offspring. Furthermore, our findings suggest DEP/MS induces a microglial phenotype that is similar to that of germ-free mice, potentially one that is immature and less immunocompetent. Keywords: Autism, Air Pollution, Microglia, Dopamine, microbiome Disclosure: Nothing to disclose. P160. Investigating Developmental Microglial-Parvalbumin Interneuron Interactions in Microglial-MyD88 Deficient Mice Julia Dziabis, Caroline Smith, Irene Jonathan, Benjamin Horvath, Marcy Kingsbury, Dang Nyugen, Neil Rogers, Justin Savage, Richa Hanamsagar, Staci Bilbo Duke University, Durham, North Carolina, United States Background: Parvalbumin+ interneurons (PVIs) are fast-spiking, GABAergic cells critical for coordinating firing activity to maintain normal brain function. Developing PVIs are susceptible to inflammation, making them a common cell type impacted in disorders such as schizophrenia, autism spectrum disorders, and major depressive disorder. Decades of studies also point to the impact of perinatal inflammation on the function of microglia, the resident immune cells of the brain, and their roles in disease-relevant synaptic alterations and behaviors in mice. Importantly, microglia interact closely with developing neurons, influencing their maturation and survival. Despite this, nearly all existing studies on microglia-neuron interactions in the early postnatal period focus only on excitatory neurons, with still very little known about microglia-interneuron interactions across development. We hypothesized that broad ablation of microglial inflammatory signaling would be protective for developing PVIs following an early life immune challenge. However, loss of microglial inflammatory signaling resulted in an increased density of PVIs across the male brain, suggesting a role for microglial-MyD88 dependent signaling in regulating PVI development in baseline conditions. Here, we aim to investigate the mechanisms linking microglial inflammatory signaling pathways and adult PVI density. Methods: Our lab developed a mouse in which the toll-like receptor adaptor protein MyD88 (myeloid differentiation primary response 88) is ablated specifically from microglia (Cx3cr1-CreBT-MyD88f/f), effectively blunting microglial proinflammatory signaling. In adulthood, we characterized social and cognitive behaviors and PVIs across the brain by immunohistochemistry. To investigate the role of microglial-MyD88 signaling in very early PVI development, we used RNAscope to identify future PVIs prior to the expression of the parvalbumin protein. Finally, PVI synaptic density changes across wild-type development were determined by co-labeling of PVI-specific presynaptic label synaptotagmin-2 (Syt2) and postsynaptic label gephyrin, which was quantified using software developed by the Eroglu Lab at Duke University. Results: We found that microglial-MyD88 loss had a significant effect on PVI density in the hippocampus (F(1,37) = 5.54, p = 0.024; n = 6-12/group) and frontal cortex of males (F(1,10) = 10.82, p = 0.0082; n = 3-5/group), but not females (F(1,12) = 0.01774, p = 0.8963; n = 3-5/group). Microglial-MyD88 genotype did not significantly impact the density of somatostatin (SST) interneurons, another abundant subtype of interneurons, in the frontal cortex of males (F(1,13) = 1.578, p = 0.231; n = 3-5/group) or females (F(1,13) = 0.746, p = 0.404; n = 3-5/group). Mice lacking microglial-MyD88 signaling have normal sociability at baseline (males: F(1,33) = 0.321, p = 0.575; females: F(1,31) = 1.659, p = 0.207; n = 8-10/group), but males show reduced spatial reference memory in a Y-maze task (t (1,22) = 2.591, p = 0.0167; n = 12/group). We next hypothesized that microglial-MyD88 loss impacts adult PVI density developmentally. Preliminary results suggest that microglial-MyD88 loss does not impact the early postnatal P0-P8 cell death period of PVI development in the male dorsal hippocampus (F(1,11) = 0.1723, p = 0.6861; n = 2-4/group). Wild-type characterization of male and female PVI cell density from P4-P42 revealed no sex differences (F(1,32) = 0.2732, p = 0.6048; n = 2-5/group), but males have an increased PVI synaptic density in the third postnatal week (F(1,42) = 46.43, p < 0.0001; n = 4-7/group), identifying a potential mechanism through which adult PVI density is regulated in a sex-specific manner. Ongoing work will characterize synaptic elimination by wild-type and MyD88-deficient microglia in males and females during the PVI synaptic pruning period to attempt to identify a sensitive temporal window of PVI density regulation in males. Conclusions: Microglia regulate developing PVIs in the male frontal cortex and dorsal hippocampus at baseline in a MyD88-dependent manner, with consequences for adult cognition. The early postnatal PVI cell death period is not altered by loss of microglial-MyD88, suggesting microglial regulation of the adult PVI population size happens after the first postnatal week. Future work will probe microglial phagocytosis of PVI synapses in the early adolescent period. Keywords: Microglia, Neurodevelopment, Parvalbumin Interneurons, Toll-Like Receptors (TLRs), Cognition Disclosure: Nothing to disclose. P161. Early-Life Inflammation and Depression in Adolescents Born Extremely Preterm Sohye Kim, Jean Frazier, David Cochran, Isha Jalnapurkar, Kyle Roell, Stephen Hooper, Peter Fonagy, Hudson Santos, Robert Joseph, Karl Kuban, Semsa Gogcu, Rebecca Fry, T. Michael O’Shea University of Massachusetts Medical School, Worcester, Massachusetts, United States Background: Adolescent depression is a major public health problem, with an estimated lifetime prevalence of 14% for major depressive disorder (MDD) between the ages of 15-18 years. Approximately 84% of depressed adolescents later experience depression in adulthood. Despite devastating clinical consequences of adolescent MDD, its etiology and pathophysiology remain poorly understood. Increasingly, data support a potential role for inflammation in the pathogenesis of depression, although it is not yet established whether inflammation is a cause, a correlate, and/or a consequence of MDD. A recent meta-analysis demonstrated that elevations of inflammatory biomarkers, specifically interleukin-6 (IL-6) and C-reactive protein (CRP), can predate the onset of depression. However, available prospective studies to date have focused primarily on adults and older adults, with the strongest associations between inflammation and depression observed in geriatric samples. Less is known about the longitudinal associations in pediatric samples and no study to date has examined the role of early-life inflammation (ELI) in subsequent depression. A systematic understanding of how ELI interacts with well-known risk and protective factors of depression is also lacking. In the present study, we examined a longitudinal association between ELI assessed during the neonatal period and depression at age 15 years in a cohort of adolescents who were born extremely preterm. Extremely preterm neonates are at high risk for experiencing neonatal inflammation and for developing later psychiatric disorders, including adolescent depression. Therefore, extremely preterm born individuals are an ideal group for an examination of these associations. We hypothesized that: (1) ELI will be associated with an increased risk of depression at age 15 years, (2) childhood adversity, a well-documented risk factor of depression, will amplify the risk of ELI on depression, and (3) social support, a well-documented protective factor of depression, will attenuate the risk of ELI on depression. Methods: The Extremely Low Gestational Age Newborns (ELGAN) Study is a multi-site longitudinal study of children born < 28 weeks of gestation. We measured six circulating inflammatory proteins (i.e., interleukin-6 [IL-6], tumor necrosis factor-alpha [TNF-α], intercellular adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8], serum amyloid A [SAA], and C-reactive protein [CRP]) in 1121 extremely preterm infants on postnatal days 1, 7, and 14. These proteins were chosen given their consistent associations with structural and/or functional neurologic outcomes in previous ELGAN studies. For each protein, we defined intermittent/sustained systemic inflammation (ISSI) as having top quartile levels within its gestational age stratum on ≥ 2 days during the first 14 postnatal days. At age 15 years, 670 surviving adolescents (344 male and 326 female) were screened for depression using the Child Behavior Checklist (CBCL) and the PROMIS Depression scale and assessed for the diagnosis of MDD using the Mini-International Neuropsychiatric Interview for Children and Adolescents (MINI-KID). We used multiple logistic and linear regression models to examine the association between the number of proteins with ISSI (0, 1-2, or ≥ 3 elevations) and depression at 15 years. Childhood adversity, as measured by the Yale-Vermont Adversity Scale, and adolescent social support, as measured by the PROMIS Family and Peer Relationships scales, were examined as moderators. Analyses were stratified by adolescent sex and adjusted for confounders as determined by directed acyclic graphs, including familial history of depression and non-infectious initiators of neonatal inflammation (i.e., small for gestational age status, maternal low socioeconomic status, maternal prenatal tobacco exposure, maternal obesity, and maternal hypertensive disorders). Associations were expressed as odds ratios (OR) and 95% confidence intervals (CI). For continuous outcomes, effect sizes were expressed as regression coefficients that quantify the difference in depression T-scores for participants with and without the exposure. Results: In our extremely preterm sample, elevated ELI protein levels were not significantly associated with the diagnosis of MDD (ISSI 1-2: OR = 1.30, 95% CI = 0.75-2.22; ISSI ≥ 3: OR = 0.89, 95% CI = 0.42-1.82) or CBCL/PROMIS depression scores at 15 years (ISSI 1-2: βs -0.88 to 0.01; ISSI ≥ 3: βs -1.64 to -1.17). As expected, childhood adversity was positively associated with the adolescent MDD diagnosis (OR = 1.13, 95% CI = 1.08-1.18) and CBCL/PROMIS depression scores (βs 0.21 to 0.49), while adolescent social support was negatively associated with the MDD diagnosis (OR = 0.91, 95% CI = 0.86-0.96) and depression scores (βs -0.38 to -0.69). However, the proposed moderators did not significantly interact with ELI to predict adolescent depression at age 15 years. Conclusions: We did not observe significant associations between neonatal systemic inflammation and depression measured at 15 years of age. Future research is needed to determine whether individual levels of inflammatory proteins or timing of inflammation would be better predictors for the onset of depression. Keywords: Inflammation, Neonatal, Adolescent Depression, Biomarker Disclosure: Nothing to disclose. P162. Carving Adversity at its Joints: Dimensions and Downstream Effects of Traumatic and Early Childhood Experiences (TRACES) Justin Russell, Sara Heyn, Ryan Herringa University of Wisconsin, Madison, Wisconsin, United States Background: Worldwide, over one billion children are at risk of enduring the chronic, pervasive sequelae of experiences so noxious to be characterized as ‘adversity’ – circumstances or events threatening physical or psychological well-being. The constellate biological and psychological effects of adverse experiences presage chronic increases for risk of mental illness, poverty, addiction, criminality, morbidity, mortality, suicidality, and critically, perpetuating adversity onto others. Traumatic and early life experiences (TRACES; Weems et al., 2021) are the single greatest preventable risk factor for psychopathology and impose an annual economic cost to the United States ($748bn) exceeding the annual economic output of nine in ten nations around the globe. Though decades of research evince the link between adversity and chronic dysfunction, the very nature of adversity as a concept remains a topic of heated debate – with competing models emphasizing the importance of discrete features (Smith and Pollak, 2020) or bi-dimensional characteristics (McLaughlin et al., 2020) in capturing neurobehavioral sequelae. The current work proposes a unifying reconceptualization of adversity as a multi-dimensional concept comprised of clusters of thematically-linked events likely to exert common downstream effects. A novel data-driven approach is applied to youth-reported exposure to over 100 different experiences reflecting various forms of adversity known to perpetuate maladjustment. These data were reduced to dimensional TRACES components and considered as predictors of status and maturational change in presence of psychiatric symptoms and cognitive development. Methods: Analyses drew data from the 11,876 youth (ages 9-10; 51% female) and their caregivers participating in baseline (Year 0) data collection as part of the Adolescent Brain Cognitive Development (ABCD) Study, 11,225 dyads repeating assessment in ABCD Year 1, 10,414 in ABCD Year 2, and 6,251 in Year 3. Fluid intelligence was operationalized as a latent factor derived from age-corrected standardized scores obtained from relevant tests completed in an abbreviated administration of the NIH Toolbox (Y0, Y2). Internalizing and externalizing problems were operationalized using summary scores from the parent-report ASEBA Child Behavior Checklist Categorical gathered at each wave of data collection (Y0-Y3). A comprehensive review of assessment instruments administered through Year 2 (Y2) identified 106 youth or parent-report questionnaire items capturing exposure to adverse experiences. For items presented at multiple waves, the most recent data were used, while scores from items repeated across caregiver and child reports were combined by taking the larger value. The resultant list included binary, ordinal, count, and continuous items. Categorical principal components analysis (CATPCA) can appropriately model each of these item types (in contrast to traditional PCA), is robust to non-normality, and reduces items to formative composites. Latent means (fluid intelligence) and path estimates (externalizing, internalizing; effects of adversity components) reflecting change were derived from models holding measurement invariance across time. Component scores were computed and entered as covarying predictors in separate latent difference score models of baseline status and developmental change in internalizing and externalizing problems, as well as fluid intelligence. Results: An eight-component solution exhibited the best fit to the data and the most intuitive interpretation and included dimensions reflecting caregiver problems (e.g., incarceration, addiction, mental illness), community violence, bullying/peer aggression, chronic pain, poverty, discrimination, family conflict, and interpersonal violence. On average, ABCD youth exhibited increases in fluid intelligence [α = 1.63 (SE = 0.18), Z = 9.01, p < .001), no change in externalizing problems, and an annual decline in internalizing problems [β = -0.11 (0.016), p < .001]. All adversity components, except for bullying/peer aggression (β = 0.01, p = .42), were associated with greater externalizing problems at baseline assessment (all p < .05), while community violence predicted increasing externalizing problems across time [β = 0.019 (0.008), p < .05]. Caregiver problems, community violence, bullying/peer aggression, discrimination, and family conflict each predicted greater levels of internalizing problems at baseline (all p < .001), while caregiver problems (p < .05), community violence, chronic pain, discrimination, and family conflict were associated with increased internalizing problems across time. Conclusions: The findings demonstrate the viability of an alternative conceptualization of traumatic and adverse childhood experiences, as well as the utility of considering the components of a dimensional model as predictors of altered maturational change in cognitive ability and psychiatric problems. Presented findings will additionally examine moderating effects of race, ethnicity, and sex at birth. In total, this work demonstrates the merits of a multidimensional model that may enhance our collective ability to understand adverse experiences by carving nature at its joints. Keywords: Childhood Adversity, Childhood Psychiatric Symptoms, Statistical Methods, Adolescent Brain and Cognitive Development Study, Longitudinal Analysis Disclosure: Nothing to disclose. P163. Sensitivity to Time and Treatment Effects of New Short Forms of the PANSS in an Outpatient Pediatric Randomized Controlled Trial Joan Busner, Eric Youngstrom, David Daniel, Joshua Langfus, Robert Findling Signant Health, Virginia Commonwealth University, Avondale, Pennsylvania, United States Background: Global regulatory initiatives have resulted in an increasing number of psychopharmacology trials in the pediatric age range. Among the challenges in ensuring valid and reliable data in such trials are developmental limitations in symptom description, the need to integrate and weight information from varied sources (including the patient, parents/caregivers and other informants, and the global shortage of child-trained clinical investigators (Busner, 2013; Farchione, 2013). Moreover, few efficacy measures have been developed and validated specifically for pediatric trials. As a result, measures designed for and validated in adults, such as the Positive and Negative Syndrome Scale (PANSS), are frequently used in adolescent schizophrenia trials. The PANSS is a complex 30-item measure that has been extensively studied and shown to pose ratings challenges even in the adult patients for whom it was designed (e.g., Daniel and Dries, 2013). Our group has developed a 10-item PANSS based on psychometric analyses of NIMH TEOSS study data. The 10-item PANSS was reliable and sensitive to treatment changes. In the present study we attempt to replicate the findings using an independent multi-site trial (Findling et al., in press). Will a 10 item PANSS optimized for pediatric trials perform similarly to the 30 item PANSS in an independent placebo-controlled multi-site trial? Methods: The 6-week, double-blind, parallel group, acute phase data from the Johnson and Johnson- sponsored completed, positive, paliperidone study (Singh et al., 2011) were accessed from the YODA secure data environment. The trial included 201 12-17 year olds randomly allocated to placebo or one of three fixed doses of paliperidone. Analyses were performed using the mirt, lavaan, sjstat, rstatix and psych packages in R, using the same syntax and methods as the prior analyses (Findling et al., in press), with mixed regressions using random intercepts and partial eta-squared as the effect size estimate for time, treatment, and time x treatment interaction effects. Results: The 10-item vs. 30-item versions had similar average inter-item correlations (.25 and .25), as well as similar partial eta-squared values for time – .37 (.32 to .41) versus .41 (.36 to .45), treatment (all .00) and time x treatment (.007 versus .003 for the full length). IRT models indicated similar reliability as in the development sample, with good precision across a similar range of severity. LOCF analyses found separation from placebo using both the 10 and 30-item versions on multiple, identical arms; ANCOVAs controlling for PANSS at phase entry produced similar eta-squareds at subsequent weeks (largest difference = .005, favoring 10-item version). Conclusions: The 10-item version of the PANSS replicated well in an independent, larger adolescent sample using double-blind RCT data. The similar sensitivity to treatment effects is particularly promising given the substantial reduction in scale length and corresponding decreases in required rater training, interview length, and respondent burden. Keywords: Child Psychopharmacology, Clinical Outcome Assessments, Pediatric Clinical Trials Disclosure: Signant Health: Stock / Equity (Self) P164. Ecological Momentary Assessment Identifies Frustration as a Central Node in Irritability in a Transdiagnostic Sample of Youth Wan-Ling Tseng, Reut Naim, Amanda Chue, Ashley Smith, Shannon Shaughnessy, Daniel Pine, Ellen Leibenluft, Katharina Kircanski, Melissa Brotman Yale Child Study Center, Yale University School of Medicine, New Haven, Connecticut, United States Background: Irritability, an increased proneness to anger relative to peers, is a transdiagnostic symptom commonly occurring with anxiety and other mood symptoms. However, little is known about the temporal and dynamic interplay between irritability-related clinical phenomena. Network analysis has the potential to improve our understanding of this complex organization of symptoms and related constructs. Unlike traditional conceptualizations of psychopathology, which posits that symptoms are manifestations of an underlying, latent cause or disorder, network approaches conceptualize disorders as systems of causally connected, interacting, and mutually-reinforcing symptoms (Borsboom and Cramer, 2013; Borsboom et al., 2021). Thus, network approaches are suitable to delineate the patterns of interrelations between irritability-related symptoms and behavioral, cognitive, and mood constructs. A better understanding of the temporal dynamics between irritability-related symptoms may inform targets and timing for interventions. Using network analysis with smartphone-based ecological momentary assessment (EMA) to increase ecological validity, this study examined how daily naturally-occurring irritability symptoms and other mood and anxiety symptoms are connected. This is the first study to use network analysis to investigate the symptomatology of irritability in naturalistic settings. Methods: Sample included 152 youth ages 8–18 years (Age: M ± SD = 12.28 ± 2.53; 69.70% male; 65.80% White/Caucasian, 9.90% African American, 4.60% Asian American, 2.60% American Indian, 13,20% Multi-race, 3.90% unknown) across several diagnostic/phenotypic groups enriched for irritability symptoms. The groups included disruptive mood dysregulation disorder (DMDD; n = 34), subthreshold DMDD (i.e., those with impairing irritability but failed to meet the full DMDD criteria; n = 22), attention-deficit/hyperactivity disorder (n = 35), anxiety (n = 28), and healthy comparisons (n = 33). Participants completed EMA three times a day for seven days (i.e., 21 data points per participant). EMA items included irritability and related symptoms (grouchiness/crankiness, annoyance/anger, frustration, feelings of unfairness) as well as other mood and anxiety symptoms (worry, happiness, sadness, mood lability), sampling across different affective chronometries (momentary vs. longer-lasting symptoms). Irritability was also assessed using parent-, child-, and clinician-reports of the Affective Reactivity Index (ARI). Network analysis using the multilevel vector autoregressive (mlVAR) model with EMA data estimated three networks: (1) temporal network, (2) contemporaneous within-subject network, and (3) between-subjects network of symptoms. Additionally, to evaluate whether and how EMA measures of irritability and related symptoms/constructs were associated with rating scale-based “trait-like” measures of irritability, we examined the mean level and variability (i.e., SD and root mean square of successive differences [RMSSD]) of EMA-rated irritability (i.e., four items: grouchiness/crankiness, annoyance/anger, frustration, feelings of unfairness) across 21 data points and their correlations with golden standard clinician-, child-, and parent-reports of irritability (i.e., ARI). Results: Frustration was identified as the most central node in the network of irritability-related symptoms/constructs (i.e., grouchiness, unfairness) and other mood and anxiety symptoms (i.e., mood changes, worry). Frustration at one timepoint also positively predicted mood changes in the next timepoint. Moreover, we found both differences and similarities in the within-subject and between-subject processes between irritability-related constructs/symptoms and other anxiety and mood symptoms. While anger/annoyance was positively related to unhappiness/sadness within individuals and within measurement occasions, anger/annoyance was positively related to unhappiness/sadness, mood lability, and worry between/across individuals. Finally, compared to variability (i.e., SD and RMSSD; r’s = .13–.41), the mean level of EMA-indexed irritability-related constructs were more strongly associated with retrospective “trait-like” measures of irritability (i.e., ARI; r’s = .23–.56). Conclusions: This study advances our understanding of symptom-level and temporal dynamics of irritability. Frustration emerged as the most central node in the network of irritability-related symptoms/constructs and other mood and anxiety symptoms. Frustration also predicted increases in mood changes at the subsequent timepoint. These findings have potential clinical implications, suggesting that frustration may be a primary treatment target for youth with irritability. Future experimental work or clinical trials are needed to systematically manipulate irritability-related features (e.g., frustration, unfairness) to further understand the causal relations among clinical variables. For example, clinical trials can be designed to test the efficacy of interventions/treatments targeting frustration given its high centrality. If the causal effect of frustration on other mood symptoms is validated, with the use of EMA, interventions could be developed and delivered right when youth become frustrated, and before frustration influences other mood symptoms downstream, to intervene in a timely and maximally effective way. Keywords: Pediatric Irritability, Frustration, Ecological Momentary Assessment, Network Analysis Disclosure: Nothing to disclose. P165. Interpersonal Functioning and Relationship to Self-Harm in Adolescents Engaging in Either Suicide Attempts or Non-Suicidal Self-Injury Julianne Tirpak, Emma Cho, Josephine Au, Jillian Russo, Courtney Kaplan, Maria Naclerio, Eileen Lee, Angela Salisbury, Camille Nommi, Daniel Dickstein McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Suicide is the second leading cause of death in children and young adults (Eaton et al., 2005). However, completed suicides represent just a portion of adolescents suffering from suicidal behavior. Nationwide, over 16% of high school students seriously consider suicide, and over 8% attempt suicide each year. Furthermore, non-suicidal self-injury (NSSI), defined as deliberate destruction of one’s body tissue without intent to die, is nevertheless strongly associated with suicide attempts (SA) in adolescents (Scott et al., 2015; Stewart et al, 2017). Interpersonal dysfunction is among the most robust predictors of the development and maintenance of both NSSI and suicidal attempts in this age group (King and Merchant, 2008), but it is a complex, multifaceted construct. The purpose of this study is to better understand what components of interpersonal functioning lead to NSSI vs. SA in adolescents. We hypothesized that higher levels of interpersonal sensitivity and deficits in social problem-solving strategies would be observed across both NSSI and SA groups, but not in healthy controls. Methods: This IRB-approved study compared self-reported interpersonal sensitivity (Interpersonal Sensitivity Measure [IPSM]), social problem-solving strategies (Social Problem Solving Inventory [SPSI]), and suicidal ideation (measured by the Beck Suicide Scale [BSS]), among three mutually exclusive, relatively homogenous groups of adolescents (ages 13-17) who: (1) engaged in NSSI without history of SA (n = 50, Mage = 15.35, SD = 1.34, 82% female), (2) made a SA without history of NSSI (n = 49, Mage = 15.80, SD = 1.23, 63% female), and (3) typically developing controls (TDCs) without history of psychopathology (n = 43, Mage = 15.46, SD = 1.30, 61% female). The analytic strategy included: (1) ANOVA to evaluate between-group differences in IPSM, SPSI, and BSS, (2) MANOVA to examine possible group and sex differences in IPSM and SPSI, and (3) logistic regression to ascertain the effects of IPSM and SPSI scores on group status. Results: ANOVA results suggest significant between-group differences in IPSM (F(2,139)= 34.89, p < 0.001) and SPSI (F(2,139) = 42.5, p < 0.001). Tukey post-hoc tests revealed that NSSI (100.25 ± 18.1, p < 0.001) and SA (84.50 ± 18.50, p = 0.001) groups had significantly higher interpersonal sensitivity than TDCs (72.0 ± 10.40). NSSI group scores were significantly higher than SA group scores (p < 0.001). For social problem-solving, the NSSI group (44.00 ± 14.90, p < 0.001) and SA group (50.60 ± 17.90, p < 0.001) had significantly lower scores than the TDC group (71.90 ± 11.00); however, there was no significant difference between the NSSI and SA groups (p = 0.07). Similarly, there was no significant difference in suicidal ideation (SI) between the NSSI and SA groups, t(97)=1.70, p = 0.093. SI was not reported in any participants in the TDC group. A 3X2 MANOVA indicated no significant interaction effect of group and biological sex differences on IPSM and SPSI total scores. The overall MANOVA model for group was significant [F(4,270) = 21.04, p < 0.001]. Univariate analyses showed significant group effects for both the IPSM [F(2,136) = 25.23, p < 0.001] and SPSI [F(2,136) = 34.06, p < 0.001] total scores. Specifically, the NSSI IPSM mean [MNSSI = 99.95] was significantly higher than the TDC mean [MTDC = 71.72, p < 0.001, Cohen’s d = 10.04] and the SA mean [MSA = 84.00, p < 0.001, Cohen’s d = 5.79]. The SA group IPSM mean total score was also significantly higher than the TDC group mean total score [p = 0.002, Cohen’s d = 4.90]. The TDC SPSI mean [MSPSI = 71.60] was significantly higher than both the NSSI group mean [MNSSI = 44.22, p < 0.001, Cohen’s d = 10.60] and the SA group mean [MSA = 50.22, p < 0.001, Cohen’s d = 9.28]. There was no significant difference between the SA group and NSSI group SPSI mean total scores. The logistic regression model was statistically significant, χ2(2) = 17.027, p < .001. The model explained 21.1% of the variance (Nagelkerke R2) in group membership, and correctly classified 66.7% of cases, demonstrating that higher levels of interpersonal sensitivity were associated with an increased likelihood of NSSI-only adolescents. Higher levels of social problem solving were not significantly associated with either NSSI or SA group membership. Conclusions: Our results suggest that higher interpersonal sensitivity is uniquely associated with adolescents’ likelihood of engaging in NSSI, but not SAs. Such findings are in line with existing research theoretical frameworks that emphasize interpersonal factors such as rejection sensitivity in the development and maintenance of NSSI (e.g., the Four-function model of NSSI). This study can inform future research that uses multimodal and longitudinal assessment (e.g., behavioral tasks, imaging, genetic mediators, etc.) to probe and evaluate different facets of interpersonal functioning to better understand unique interpersonal risk factors for suicide vs. self-harm in adolescents. Ultimately, greater understanding of interpersonal dysfunction can translate to evaluation of if/how current frontline psychosocial treatments for suicidal adolescents directly target these constructs. Keywords: Suicide Attempt, Non-Suicidal Self-injury (NSSI), Social Factors and Functioning, Suicide Behavior Severity, Self-Harm Disclosure: Nothing to disclose. P166. Multimodal Brain Imaging of Methylphenidate Treatment in Patients With ADHD Robyn Wiseman, Peter Barker, William Clarke, Kristin Bigos Johns Hopkins School of Medicine, Baltimore, Maryland, United States Background: Attention deficit hyperactivity disorder (ADHD) impacts a significant number of adult patients, with a small percentage receiving an accurate diagnosis and/or proper treatment. The goal of this double-blinded, placebo-controlled, crossover study was to identify ADHD-related signatures in the brains of adult patients and examine how brain activity, metabolites, and cognitive performance are altered with the commonly prescribed stimulant medication methylphenidate. Methods: The 7 participants were 20-36 years old, non-smoking, right-handed, not actively taking a stimulant medication, and had no other Axis 1 psychiatric disorders. We combined task-based 3T functional magnetic resonance imaging (fMRI) with high-resolution 7T magnetic resonance spectroscopy (MRS) to examine brain activity and brain chemistry, respectively. Two main fMRI tasks were utilized - the N-back working memory task and the flanker attention task to evaluate response inhibition. A standard cognitive battery including the NIH Toolbox Cognitive Battery was administered along with the Connors Adult ADHD Rating scale (CAARS), a self-report survey of ADHD symptoms. Results: As expected, methylphenidate level correlated with frontal cortical activity during working memory (p = 9.9e10-5, slope=0.08073 r2 = 0.932). The ADHD index, a CAARS measure of symptom severity, was shown to be sensitive to a single dose of methylphenidate (p = 0.021), and drug level positively correlated with frontal cortical activity during working memory (p = 0.0227, slope=6.165, r2 = 0.6067). Increased glutamate levels in the anterior cingulate cortex and the dorsolateral prefrontal cortex (dlPFC) were associated with positive changes in composite cognitive function score (p = 0.0047, slope=30.89, r2 = 0.95) and fluid cognitive composite score (p = 0.0066, slope=18.91, r2 = 0.94), respectively. We also observed negative correlations between age and both processing speed (p = 0.0171, slope = -2.266, r2 = 0.8852, uncorrected scores) and dlPFC glutamate levels (p = 0.0339, slope = -0.091, r2 = 0.82). Conclusions: The novelty of this study is in the combination of approaches used to probe ADHD neural pharmacology. By combining functional measurements with high-resolution spectra of key brain metabolites and cognitive data, we created a richer picture of how stimulant medications impact the brain of those with ADHD. The fact that we were able to capture significant and synchronous changes in cortical activity and glutamate levels suggests that multimodal brain imaging may be a viable, noninvasive tool for ADHD drug development efforts. Keywords: ADHD, fMRI, 7T MRS, Methylphenidate Disclosure: Nothing to disclose. P167. Viloxazine Increases Interstitial Levels of Norepinephrine and Serotonin in a Dose Dependent Fashion in Rat Medial Prefrontal Cortex Brittney Yegla, Jennie Garcia-Olivares, David Zweibaum, Frank Bymaster, Chungping Yu Supernus Pharmaceuticals Inc, Rockville, Maryland, United States Background: Viloxazine ER (viloxazine extended-release (ER) capsules; Qelbree®), a non-stimulant drug, FDA-approved for attention-deficit/hyperactivity disorder (ADHD) in adults and children (≥6 yrs), is pharmacologically distinct from other approved treatments. Most FDA-approved ADHD treatments share a primary mechanism of increasing norepinephrine (NE) and/or dopamine (DA) levels. Viloxazine increases NE but is only a weak norepinephrine (NE) reuptake inhibitor (IC50 = 0.3 μM at human NE transporters) and was found to have moderate activity at serotonin (5-HT) receptors. Prior in vivo micro-dialysis experiments utilizing viloxazine have demonstrated strong increases in NE, DA, and 5-HT in the rat prefrontal cortex (PFC). As the PFC is associated with ADHD pathophysiology and attentional control, monoamine (5HT, DA, and NE) increases in this area may be crucial to the therapeutic effects of viloxazine. However, the viloxazine dose used in these prior microdialysis experiments (50 mg/kg, ip, rat) produced higher unbound viloxazine brain concentrations than the unbound concentration estimated in plasma of pediatric ADHD subjects receiving viloxazine ER 400 mg/day in clinical trials. Consequently, the current microdialysis study was undertaken to further evaluate dose-dependent effects of viloxazine on prefrontal monoamine concentrations at therapeutically relevant concentrations of Qelbree for ADHD. Methods: Freely-moving Sprague-Dawley rats (male, 8 weeks old, 5-6/group) were implanted with I-shaped microdialysis probes into the PFC. Following recovery from surgery, the rats’ probes were connected to a microperfusion pump, which perfused artificial cerebrospinal fluid containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2. After a 2-hour baseline was established, viloxazine (1, 3, 10, or 30 mg/kg) was administered (ip). Dialysate samples were collected from the interstitial fluid (ISF) of the PFC and measured at 30-minute intervals for the 2-hour baseline and 4-hour post-viloxazine administration periods. The dialysate samples were evaluated by LC-MS/MS to determine the concentration of viloxazine and viloxazine-induced changes in NE, DA, 5-HT and their respective metabolites, 3,5-dihydroxyphenylglycine (DHPG) and 5-hydroxyindoleacetic acid (5-HIAA), and precursor, 3,4-dihydroxyphenylalanine (L-DOPA). Plasma samples were collected at 30- and 90-minutes post-dosing for determination of corresponding viloxazine plasma concentrations. Animal research was performed at Charles River Laboratories (South San Francisco, CA, USA) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 2011). The experiments were conducted under approved protocol US19002 by the site’s Animal Care and Use Committee. Results: Pharmacokinetic evaluation showed a dose-dependent increase in viloxazine plasma and ISF concentrations. Plasma concentrations at 30-minutes post-dose were 2.4 ± 1.0 µM and 5.7 ± 3.6 µM for 10 and 30 mg/kg doses, respectively, yielding calculated free viloxazine plasma concentrations of ~0.9 and ~2.4 µM, respectively (based on prior murine studies showing viloxazine is 40% unbound to plasma proteins). The determination of viloxazine concentration in the dialysate collected from ISF in PFC showed that the viloxazine Tmax occurred at 60 minutes, with Cmax of 1.2 ± 0.3 µM and 3.5 ± 1.6 µM for viloxazine doses of 10 and 30 mg/kg, respectively. Viloxazine administration resulted in significant dose-dependent effects on interstitial NE levels at all doses tested. The analysis of study results showed that viloxazine treated animals had significantly higher interstitial levels of NE compared to vehicle treated animals at t = 60 minutes, and the 30 mg/kg dose continued to show elevated NE over vehicle up to t = 180 min. The increase in NE interstitial level at 60 minutes reached 236%, 418%, and 558% over baseline for the 3, 10 and 30 mg/kg dose groups, respectively. Dose-dependent decreases in DHPG (NE metabolite) interstitial concentrations corresponded with NE interstitial increases, reflecting viloxazine’s mechanistic activity as a NET inhibitor. Viloxazine treatment also resulted in a dose-dependent elevation of interstitial 5-HT concentrations in the PFC. Following the 30 mg/kg dose of viloxazine, 5-HT elevations reached 213% over baseline at a Tmax of 60 minutes and also were higher than the vehicle group; however, the lack of coincident changes in 5-HIAA (5-HT metabolite) interstitial concentrations reaffirmed viloxazine’s lack of activity as a 5-HT reuptake inhibitor. Conclusions: The viloxazine-mediated interstitial increases in NE and 5-HT were directly related to viloxazine interstitial concentrations, demonstrating a clear pharmacokinetic/pharmacodynamic relationship in the PFC, a critical target region for the therapeutic effect in ADHD. These results add to our understanding of viloxazine’s mechanism of action and show that, at the unbound plasma concentrations produced by clinically effective doses of viloxazine ER, viloxazine is increasing interstitial monoamine levels in the PFC. Thus, induction of noradrenergic and serotonergic activity in the PFC may be implicated in viloxazine ER therapeutic effects in treating pediatric and adult patients with ADHD. Keywords: ADHD, Serotonin, Microdialysis, Viloxazine, PFC Disclosure: Supernus Pharmaceuticals Inc.: Employee (Self) P168. Adolescents Engage Orbitofrontal Cortex and Dorsomedial Striatal Neurons Differently During Response Inhibition Aqilah McCane, Bita Moghaddam Oregon Health and Sciences University, Portland, Oregon, United States Background: Adolescence is a vulnerable developmental period. Neuronal underpinning of this vulnerability is poorly understood but has been attributed to an immature frontal cortex and its connections to striatal and other subcortical areas that modulate action selection. We have observed that action-guided reward processing by lateral orbitofrontal cortex (OFC) and dorsomedial striatum (DMS) neurons differ between adolescents and adults. Here, we test the specific hypothesis that action-outcome associations and response inhibition are processed differently in DMS-OFC circuits in adolescents compared to adults. Methods: We used a Cued Response Inhibition Task (CRIT) to assess response inhibition in male and female adolescent or adult rats. To characterize the role of OFC to DMS projections in adults and adolescents in CRIT, we used DREADDs to inhibit DMS projecting OFC neurons. In addition, we simultaneously recorded single units and local field potentials (LFPs) in the OFC and DMS from adolescent and adult rats performing CRIT. The neural data was analyzed using multiple computational approaches to investigate the interaction and synchrony between two brain regions during specific behavioral events. We performed mixed design analysis of variance (ANOVA) for all dependent variables with group factors sex and age. Results: Relative to adults, adolescents make more premature responses and fewer correct responses in CRIT. We find that inhibiting the OFC- > DMS projections decreases correct responses and increases premature responses during CRIT, suggesting this pathway is necessary for successful execution of response inhibition. Adolescents exhibit a reduced response to inhibitory cue presentation in the OFC and an enhanced response to reward in the DMS. Adolescent spiking variability in the OFC was also increased during inhibitory cue presentation. Lastly, adolescents show reduced OFC and DMS theta power and reduced OFC-DMS theta synchrony during reward. Conclusions: These results indicate that while the OFC-DMS circuit is critical for response inhibition in both adults and adolescent, adolescents engage this circuit differently by tuning stronger to reward and weaker to inhibitory cues and action. These data expand our knowledge of how the adolescent brain differently guides behavior relative to adults and highlights the importance of OFC-DMS circuits for mediating response inhibition in adolescents. Keywords: In Vivo Electrophysiology, Corticostriatal Circuit, Neurodevelopmental Disorders, Local Field Potentials, Childhood-Onset Schizophrenia Disclosure: Nothing to disclose. P169. Maturation of Prefrontal Circuits Underlying Learned Threat Avoidance Cassandra Klune, Caitlin Goodpaster, Rita Chen, Nico Jones, Laura DeNardo* University of California Los Angeles, Los Angeles, California, United States Background: The medial prefrontal cortex (mPFC) plays a key role in evaluating and responding to threats. In the prelimbic (PL) subregion of mPFC, coordinated activity of output circuits targeting basolateral amygdala (BLA) and nucleus accumbens (NAc) determines whether animals approach or avoid threatening stimuli. In development, as animals establish independence from caregivers, PL circuits undergo an extended maturation. Circuit maturation may promote age-appropriate levels of exploration, allowing mPFC to ultimately encode a nuanced repertoire of threat responses. Yet the causal relationships between mPFC circuit development and maturation of learned threat responding remain poorly understood. Methods: We developed a modified version of platform-mediated avoidance (PMA) that is compatible with study of developing mice. In this assay, a conditioned tone prompts rodents to navigate to a safety platform. Sweet swelling odor pots that are just out of reach entice animals to explore in between tone presentations. We combined PMA with projection-specific optogenetic manipulations to test the hypothesis that maturation of PL-BLA and PL-NAc circuits determines age-specific avoidance behaviors. Results: We found that learned threat avoidance behaviors are developmentally regulated. While adult threat memories are strong and long-lasting, juveniles exhibit weak threat memories, and adolescents show a temporary suppression of threat memories (Time on platform during retrieval, age main effect: P = 0.02; trial # main effect: p = 0.23; interaction: P = 0.16; Tukey-corrected post-hoc test: P35 vs. P60: P = 0.017). When we combined PMA with projection-specific optogenetic manipulations to test the hypothesis that maturation of PL-BLA and PL-NAc circuits determines age-specific avoidance behaviors, we discovered that PL behavioral contributions to PMA are age-specific. Activating PL-BLA reduced avoidance in juveniles by increasing latency to enter the safety platform. At P35, the same manipulation had no net effect on time on platform, but promoted entries onto the safety platform while reducing the length of platform stays. Finally, activating PL-BLA mildly enhanced PMA in adults (Time on platform during retrieval, age main effect: P = 0.002, opsin main effect: P = 0.82, interaction: P = 0.002; Turkey corrected post-hoc test: P23: P = 0.01, P35: P = 0.99, P60: P = 0.04). In contrast, activating PL-NAc had no effect at P23 or P35, but reduced avoidance in adults by enhancing the latency to enter the safety platform (Time on platform during retrieval, age main effect: P = 0.07; trial # main effect: p = 0.13; interaction: P = 0.08; Tukey-corrected post-hoc test: P60: P = 0.01). Silencing PL-BLA at P23 had no effect on PMA (P = 0.2, Student’s t-test), while silencing PL-NAc at P35 enhanced avoidance (P = 0.02, Student’s t-test) by decreasing the latency to enter the safety platform and increasing the duration of platform stays. Conclusions: Together, these findings support in a model in which, instead of simply strengthening across development, pronounced rearrangements of frontolimbic circuits determine age-specific threat behaviors. In juvenile stages, PL circuits may not play a key role in avoidance; rather, BLA and NAc may control learned threat avoidance. In adolescence, changes in PL dynamics may favor activation of PL-NAc, which promotes exploration even during encounters with threats. In ongoing work, we are using fiber photometry to monitor neural dynamics across development. By performing circuit-specific activity manipulation and monitoring in development, our studies will be among the first to establish causal links between mPFC circuit dynamics and the maturation of threat-induced behaviors. By revealing the trajectory along which threat avoidance emerges, our research will establish important foundations for understanding how genetic and environmental insults alter developmental trajectories and shift the balance toward developmental disorders. Keywords: mPFC, Developmental Trajectory, Active Avoidance, Threat Conditioning, Frontolimbic Network Disclosure: Nothing to disclose. P170. Sex Differences in the Transcriptional Networks Underlying Playfulness Suggest a Distinct Function for Play in Males Compared to Females Ashley Marquardt, Jonathan VanRyzin, Mahashweta Basu, Seth Ament, Margaret McCarthy University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Social play is a dynamic, well-conserved behavior known to be sexually differentiated; in most species, males play more than females, a sex difference driven by the medial amygdala (MeA). To investigate whether the transcriptional signatures underlying play also differ by sex, we performed RNA-sequencing of MeA samples from high- and low-playing juvenile rats of both sexes. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified 22 co-expression modules, or networks of genes highly correlated in expression across samples. Of the 12 modules (for p < 0.05) significantly associated with play, almost all (11/12; ~92%) are sex-specific in expression, correlating with expression of play in one sex only. These data suggest there is a distinct transcriptomic profile associated with playfulness in the MeA of males compared to females, a noteworthy finding given the MeA regulates many sex-typical adult social behaviors. We propose that this is no coincidence: play-associated gene networks in the MeA are sex-specific because play modulates circuitry driving different adult behaviors depending on sex. To investigate this, we deprived male and female rats of play during the peak juvenile play window and assessed the effects on various behaviors in adulthood, predicting that this would result in impairments in later-life behavior that would differ by sex. Methods: For 2 weeks during the peak of the play period (postnatal day 26-42), juvenile rats experienced either control housing conditions or were deprived of play via two different methods: full social isolation, or by being placed in modified cages containing a perforated Plexiglass cage divider to prevent play while allowing for other social interaction (“play barriers”). All subjects (n = 11-13/group) were then returned to normal housing and later assayed in a battery of behavioral tests in adulthood. One- or two-way ANOVAs were conducted when appropriate followed by Tukey’s post-hoc tests. Results: Supporting our hypothesis, we found that preventing juvenile play impaired object memory (p = 0.004), decreased sexual behavior (p = 0.01), and enhanced social preference (p < 0.001) in adulthood in males only. There was no effect of play deprivation on anxiety-like behavior, sex preference, or social recognition in either sex, or on any assessed behavior, including maternal behavior, in adult females. Conclusions: Consistent with the proposed hypothesis, play deprivation indeed resulted in sex-specific effects on later-life behavior: males prevented from playing as juveniles had impairments in object memory and in two tests of sociosexual behavior in adulthood. Surprisingly, there were no effects on females across any assessed behavior, suggesting female resilience to juvenile isolation. We are currently repeating these experiments with a longer play deprivation window (postnatal days 21-45) to determine if this may reveal additional deficits in females. These findings, together with our previous RNA-sequencing results, provide novel insight into the ultimate function of play and how and why this may differ by sex. Keywords: Social Play Behavior, RNA Sequencing, Sex Differences, Medial Amygdala, Behavior Disclosure: Nothing to disclose. P171. Maternal Transmission of Preconception Stress and Impact on Offspring Hypothalamic Development Yasmine-Marie Cissé, Kristen Montgomery, Tracy Bale University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Cumulative stress –including adverse childhood experiences (ACEs), repeated trauma, or racial discrimination stress– experienced prior to pregnancy is a predictor of adverse perinatal and neurodevelopmental outcomes. Although adversity experience is not unique to women, pregnancy is a pervasive female-specific physiological challenge. However, little is known about the biological mechanisms involved in transmitting this lasting signal of stress history. We have developed a novel mouse model of maternal preconception stress (MPS) where female, but not male, offspring are hypersensitive to stress as adults. Our previous work has demonstrated a dramatic shift in circulating extracellular vesicle (EV) protein content towards proteins involved in metabolism in MPS dams, specifically in pregnancy. This shift is associated with impaired glucose tolerance in MPS dams at mid-gestation. We hypothesize that the increased metabolic demands of pregnancy unmasks programming, from preconception adverse experiences, that alter signaling at the maternal:fetal interface, leading to changes in offspring neurodevelopment in a sex-specific manner. Methods: Adult female mice were exposed to chronic stress from 4-10 weeks of age. Mice were provided two weeks post-stress recovery to minimize confounding effects of acute stress prior to mating. To assess lasting effects of MPS on the maternal and fetal compartments, placentas, apposing uterine tissue, and fetal brains were collected at mid-gestation, E12.5, coinciding with full differentiation of the placenta and development of the hypothalamus. Utilizing Sim1-Cre x Ribotag mice, RNA transcripts specific to hypothalamic neurons, enriched in the paraventricular nucleus (PVN), were isolated and sequenced to assess the effect of MPS on offspring hypothalamic development and adult function (PN2, PN21, and PN70). Body weight and food intake was tracked in a separate cohort of offspring that were challenged with a high-fat low-fiber diet (HFD) from 4-10 weeks of age. Results: At mid-gestation, reproductive tissues at the maternal:fetal interface in MPS dams showed broad changes in cellular and lipid metabolism in an offspring sex-specific. Female offspring showed a greater magnitude of differential hypothalamic gene expression in response to MPS and altered metabolic gene expression in PVN-enriched hypothalamic neurons across development, linking metabolic changes at the maternal:fetal interface to lasting offspring sex-specific effects on development of the hypothalamus. Unlike control female offspring, when challenged with a high-fat low-fiber diet, female MPS offspring were susceptible to elevated weight gain. Conclusions: These data highlight the lasting effects of cumulative stress experienced preconception that surface during pregnancy to alter metabolic signaling at the maternal:fetal interface, ultimately altering offspring sex-specific hypothalamic development and susceptibility to a metabolic challenge in adulthood, effects that may also indicate risk for other neurodevelopmental disorders. Keywords: Stress Models, Hypothalamic Development, RiboTag, RNA Sequencing Disclosure: Nothing to disclose. P172. A Prospectively Randomized Pharmacogenetic Trial of Pregabalin for the Treatment of Co-Occurring PTSD and AUD in Black/African American Adults Daniel Roche, Clayton H. Brown, David Gorelick, Chamindi Seneviratne, Bankole Johnson, Melanie Bennett Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) commonly co-occur. This comorbidity is associated with worse symptom severity and treatment outcomes for each disorder. Black/African American (B/AA) individuals are more likely than other racial groups to be exposed to traumatic events, experience more severe symptoms of PTSD, and face worse consequences from alcohol consumption. Because they have been historically excluded from treatment trials, treatments for PTSD and AUD that are effective for B/AA individuals are sorely needed. The anticonvulsant pregabalin, via its affinity for the alpha-2-delta auxiliary site of voltage gated calcium channels, may help restore balance to the dysregulated glutamate and GABA systems that appear to similarly underlie both PTSD and AUD. Pregabalin has shown preliminary efficacy in treating AUD, generalized anxiety disorder, and PTSD. The goal of this randomized, controlled trial was to test whether pregabalin, vs. placebo, reduces alcohol consumption and improves PTSD symptoms in B/AA individuals with co-occurring PTSD and AUD. Additionally, a personalized medicine approach was employed to examine whether variation of SLC6A1, i.e. the gene encoding the GABA transporter GAT-1, affected response to pregabalin. There is evidence that GAT-1 is downregulated in AUD and upregulated by pregabalin administration. In B/AA, variants at the SLC6A1 gene promoter region insertion (i.e., non-insertion/insertion or insertion/insertion (NI/I or I/I) compared with those of Non-insertion/Non-insertion (NI/NI) type have significantly higher levels of GAT-1 promoter activity. Methods: This study (NCT02884908) was a double-blind, placebo-controlled, and prospectively genotyped 12-week clinical trial to test the efficacy of pregabalin vs. placebo in reducing both alcohol consumption and PTSD symptoms. Participants were randomized to receive pregabalin 450 mg/day or placebo for 12 weeks in a 1:1 scheme with each medication group also stratified by SLC6A1 genotype (NI/I/II vs. NI/NI). Because of a low allelic frequency, individuals with the double copy insertion were combined into one group with those with the single copy (i.e., NI/I/II). At each of the 12 weekly visits, participants received standardized, guided interviews to enhance medication compliance and completed measures to assess alcohol consumption and PTSD symptoms. Primary outcomes were number of heavy drinking days (HDD) assessed by Timeline Followback Interview (TLFB) and severity of PTSD cluster B and E symptoms assessed by the PTSD Symptom Checklist for DSM-5 (PCL-5). Secondary outcomes were number of days abstinent and drinks per drinking day assessed by TLFB and PTSD total symptom severity as assessed by the PCL-5 and Clinician-Administered PTSD Scale for DSM-5 (CAPS-5). The study was actively recruiting from 07/2017 to 11/2021 with enrollment ceased for roughly one year (03/2020 – 02/2021) due to the COVID-19 pandemic. Power analyses indicated a need to enroll n = 307 and randomize n = 203 in order to detect a medium sized medication effect. In actuality, this study enrolled n = 149 and randomized n = 54 B/AA participants. A modified intent to treat analysis was performed which included n = 44 B/AA individuals who received at least one dose of medication (n = 26 pregabalin; n = 18 placebo). Repeated measures mixed effects models, adjusted for a pre-baseline assessment of the respective PTSD or drinking outcome, were used to compare change over the twelve weekly assessments between the two medication conditions. To test whether medication response differed by genotype, models were rerun with SLC6A1 genotype added as a main effect and interaction term. Results: Participants reported a significant reduction in alcohol use (HDD, days abstinent, and drinks per drinking day) and PTSD symptoms (B and E clusters and total PTSD symptom severity) over the 12-week trial. However, there was no difference between medication condition nor was there a pharmacogenetic effect on any of the primary or secondary outcomes. Conclusions: Pregabalin did not reduce alcohol use or improve PTSD symptoms in B/AA individuals with comorbid AUD and PTSD, and genetic variation in GAT-1 activity was not associated with treatment outcomes or medication efficacy. However, this study was severely underpowered, and these results, particularly the pharmacogenetic findings, should be viewed with caution given the small sample size. Further studies are needed to identify novel and targeted pharmacological treatments for B/AA with AUD and/or PTSD. Keywords: Alcohol Use Disorder - Treatment, Post Traumatic Stress Disorder, Randomized-Controlled Trial, Pharmacogenetics, African American Disclosure: Nothing to disclose. P173. Open Trial of Transcranial Direct Current Stimulation for Warriors Experiencing Chronic Pain (tDCS for Warriors) Sheila Rauch, Oluwatoyin Thompson, Anna Woodbury, Kathryn Black, Tahira Scott, Syreese Fuller, Jessica Maples-Keller, Barbara Rothbaum, Melba Hernandez-Tejada, Hyochol Ahn Atlanta VAHCS, Emory University, Atlanta, Georgia, United States Background: Chronic pain, or “pain that persists or recurs for more than 3 months”5 costs the United States hundreds of millions of dollars annually.2 Severe pain is about 1.5 times more prevalent in veterans than non-veterans.3 Among veterans, chronic pain is a robust risk factor predicting suicide across all service eras.6 Commonly prescribed medications to treat chronic pain such as opioids and NSAIDs pose increased risks to patients. This study explores the effectiveness of a safe, non-opioid, self-administered treatment - transcranial direct current stimulation (tDCS) - in the treatment of chronic pain in veterans completing an intensive outpatient treatment program for mental health concerns. tDCS is a non-invasive, painless brain stimulation treatment that uses direct electrical currents to stimulate specific parts of the brain and modulate neuronal activity. tDCS produces analgesic effects and improves pain system function through direct effects on the motor, somatosensory, and frontal cortices implicated in pain sensitivity.1,4 tDCS has yet to be examined for chronic pain in veterans. We investigate acceptability and effectiveness of tDCS for chronic pain in veterans completing the Emory Healthcare Veterans Program Intensive Outpatient Program (EHVP-IOP)7. Methods: To date, 8 veterans with chronic pain in the EHVP-IOP underwent staff-monitored tDCS sessions. Starting on Day 1, tDCS was administered with a constant current intensity of 2 mA for 20 minutes per daily session for up to 10 sessions over the 2 weeks using a Soterix 1x1 tDCS mini-CT Stimulator (Soterix Medical Inc., NY). Staff reviewed a Medical Pain Assessment. To measure the degree of chronic pain, participants completed the Defense and Veterans Pain Rating Scale (DVPRS) daily both before and after each session and overall. On days 1 and 12 they completed Patient-Reported Outcomes Measurement Information System (PROMIS) 3a and 8a questionaries. To assess for co-morbid conditions such as PTSD and depression, on days 1,3,5, 8, 10, and 12 participants completed the Posttraumatic Stress Disorder Checklist (PCL-5) and the Patient Health Questionnaire (PHQ-9). Post-treatment follow-up occurred at 1, 3, 6, 9, and 12 months. Results: We have currently enrolled 8 veterans in the EHVP-IOP plus tDCS program and will present all available veterans who enroll through mid-November (18 veterans estimated). Preliminary analyses show promise and address feasibility and acceptability. In brief, veterans find using the tDCS device with remote monitoring acceptable and can successfully self-administer under the tele video supervision of study staff with few reported issues. Worsening of headache occurred in one veteran who chose to discontinue. Significant and large reductions in PTSD are reported as expected with the EHVP-IOP program (t (5) = -2.79, p = .03, d = -1.3). Reductions in pain as measured with DVPRS show a small to medium effect size (t (5) = 1.00, p = .18, d = .41) that is not statistically significant possibly due to small sample size. Updated results will be presented in the poster including patient level pain data to illustrate veteran response over the two weeks of EHVP-IOP. Conclusions: Based on this open trial, tDCS is a feasible and acceptable pain intervention for concurrent use during mental health intensive outpatient treatment. Impact on pain is promising with small to moderate reductions in pain across the daily tDCS program. Additional controlled trials with large sample are needed to establish efficacy. Reported pain in the sample is heterogeneous in focus (fibromyalgia, knee, migraine, etc.). Additional analysis within subgroups is needed. Keywords: Pain therapeutics, veterans, Treatment, PTSD, Depression Disclosure: Nothing to disclose. P174. Chronic Stress Prior to Cocaine Exposure Produces Sex Differences in Cocaine Consumption and Memory Retrieval in Rats Roberto Morales-Silva, Ursula Gelpi-Domínguez, Joshua Perez-Torres, Yobet Perez-Perez, Genesis Rodriguez-Torres, Marian Sepulveda-Orengo* Ponce Health Sciences University, Ponce Research Institute, Ponce, Puerto Rico Background: A correlation has been recognized between post-traumatic stress disorder (PTSD) and substance use disorder (SUD), and stress has been associated with a higher likelihood of relapse to addiction. Moreover, stress is associated with a higher likelihood of relapse. Studies using different models of PTSD and a short-access cocaine self-administration paradigm in male rodents have produced varying results, depending on the specific experimental conditions. For example, a preclinical study revealed that rats exposed to modified single prolonged stress before cocaine short access self-administration (2 hours) showed a reduced cocaine self-administration, while rats exposed to single prolonged stress prior to cocaine showed reduced cue-induced primed reinstatement, without any effects on the acquisition or extinction of cocaine self-administration. On the other hand, early-life stress was shown to facilitate the acquisition of cocaine, but it did not alter the reinstatement of cocaine-seeking behavior. However, the effects of chronic stress and extended-access cocaine self-administration on cocaine-seeking in either male or female rats have not been reported. We hypothesize that chronic stress before cocaine self-administration will increase cocaine consumption in rats of both sexes. Additionally, we predict that forced abstinence will result in increased active lever presses in cue-reactivity after 30 days of abstinence, compared to 15 days, in both sexes, with male rats exposed to stress and cocaine showing higher incubation of cocaine craving, compared to female rats. Methods: We employed an unescapable footshocks paradigm for 5 days at an intensity of 0.50mA (presented randomly), followed by 6-hour sessions of extended-access cocaine self-administration for 10 days, and subsequently, by either a 15 or a 30-day period of forced abstinence in male and female rats. Subsequently, we examined cue-primed and cocaine-primed memory retrieval. Results: Our data show that chronic stress before cocaine acquisition decreases cocaine consumption in female rats, compared to males. In addition, cue-reactivity after 15-day withdrawal seems to decrease in both, male and female rats, without affecting cocaine-induced memory retrieval. Interestingly, after 30 days of withdrawal female rats show an increase in cue reactivity in the stressed group compared to non-stressed rats, while males show similar cue reactivity in both groups. Furthermore, stressed male rats show an increase in cocaine-primed memory retrieval compared with the non-stressed group after 30 days of withdrawal, while female rats did not show any difference. Conclusions: Our findings suggest that the effects of chronic stress before cocaine exposure on incubation of cocaine-seeking behavior depend on the withdrawal period’s length, indicating that there are different neurophysiological phases during forced abstinence. Moreover, chronic stress before extended-access cocaine self-administration produces sex differences in cocaine acquisition, consumption, cue-reactivity, and cocaine-primed memory retrieval. Our findings represent evidence that stress before cocaine exposure induces sex-dependent differences in cocaine-seeking behavior. Keywords: Cocaine Sex Differences, Chronic Stress, Incubation of Cocaine Craving Disclosure: Nothing to disclose. P175. Inhibition of Hippocampal and Thalamic Inputs to the Nucleus Accumbens Restores Dopamine System Function in a Rodent Model Used to Study PTSD and Comorbid Psychosis Hannah Elam, Alexandra McCoy, Angela Boley, Daniel Lodge UT Health Science Center at San Antonio, San Antonio, Texas, United States Background: Post-traumatic stress disorder (PTSD) is a psychiatric illness that afflicts approximately 8% of the United States population. In addition to core symptoms of the disorder, patients with PTSD commonly present with a comorbid diagnosis, including psychosis. Symptoms of psychosis are thought to be driven by increased mesolimbic dopamine transmission, however, no clear histopathology has been identified within these cells. Rather, hyperactivity in upstream brain regions that regulate ventral tegmental area (VTA) dopamine neuron activity contributes to psychosis-like behavior. Two such regions are the paraventricular nucleus of the thalamus (PVT) and ventral hippocampus (vHipp), which synergistically regulate VTA dopamine neuron activity through a multisynaptic circuit that begins with convergent inputs to the nucleus accumbens (NAc). We have previously demonstrated that activation of PVT-NAc or vHipp-NAc projections significantly increases VTA dopamine neuron population activity, defined as the number of neurons firing spontaneously. These data suggest that hyperactivity in either the PVT or vHipp, following stress, may contribute to psychosis-like behavior in a rodent model used to study PTSD. Interestingly, hippocampal and thalamic regulation of VTA population activity requires simultaneous activity from both regions, suggesting that targeting either region may be a novel site of intervention for comorbid psychosis related to PTSD. Methods: In this study, we induced stress-related pathophysiology in male Sprague Dawley rats using the two-day inescapable foot shock procedure. To selectively inhibit PVT-NAc or vHipp-NAc projections we used a chemogenetic approach. A Cre-dependent inhibitory DREADD was expressed in either the PVT or vHipp. Concurrently, a retrograde Cre virus was injected in the NAc. This allowed for selective inactivation of these projections following administration of Compound 21 (3 mg/kg; i.p.). To examine alterations in dopamine system function, we used in vivo electrophysiology. Results: Two-day inescapable foot shock resulted in increased dopamine neuron population activity, consistent with what is observed in other rodent models used to study psychosis. Chemogenetic inhibition of either PVT-NAc projections or vHipp-NAc projections, following stress, restored dopamine system function. Conclusions: These results demonstrate that aberrant dopamine neuron activity that is observed after stress can be reversed by inhibiting the activity of PVT-NAc or vHipp-NAc projections. Here, we provide evidence that the PVT or vHipp may be novel therapeutic targets for decreasing psychosis symptoms observed in PTSD. Keywords: Psychosis, Chemogenetics, Electrophysiology Disclosure: Nothing to disclose. P176. Effects of Cannabis Legalization in Canada on Cannabis Use Patterns in Mental Health Disorders Maryam Sorkhou, Elle Wadsworth, Samantha Goodman, David Hammond, Tony George University of Toronto, Toronto, Canada Background: Since the legalization of recreational cannabis use in Canada in 2018, one emerging public health concern is whether cannabis use will increase in people with mental health disorders. Relative to the general population, problematic cannabis use is elevated in individuals with mental health disorders, including those with psychotic-spectrum, mood, and anxiety disorders. Moreover, problematic cannabis use is associated with more severe psychopathology and poorer functional outcomes. Thus, there is a need to evaluate whether legalization has led to changes in cannabis use patterns among psychiatric populations. We explored whether there are significant changes in cannabis use patterns pre- and post-legalization in a nationally representative sample of Canadian adults experiencing a mental health condition within the previous 12-months of survey administration. Methods: Data came from Canadian respondents (N = 13,527) between Waves 1 (pre-legalization; Aug – Oct 2018) 2 (post-legalization; Sept – Oct 2019), and 3 (post-legalization; Sept – Oct 2020) from the International Cannabis Policy Study (ICPS). Binary logistic regressions and repeated measures ANOVAs examined changes in daily and 30-day cannabis use respectively, pre- and post-legalization among those who self-reported 12-month experiences of anxiety, depression, PTSD, schizophrenia/psychosis, or bipolar disorder. Results: Respondents reporting schizophrenia symptoms were significantly more likely to use cannabis daily post-legalization (OR = 5.9, CI = 1.9-15.9; p < .01;). Daily cannabis use did not increase in other diagnostic groups or in nonpsychiatric populations post-legalization. Similarly, past 30-day cannabis use increased post-legalization only among respondents reporting schizophrenia symptoms (p < .05). Conclusions: Our preliminary findings suggest that patients with schizophrenia have had selective increases in daily and 30-day cannabis use in the first two years of cannabis legalization in Canada. Keywords: Cannabis Use, Schizophrenia (SCZ), Depression and Anxiety, PTSD, Cannabis Use Disorder Disclosure: Nothing to disclose. P177. Risk for Antipsychotic Induced Weight Gain in a Large Population Health Sample is Attributable to Weight Dependent and Weight Independent Genetic Factors Ginger Nicol, Adam Locke, Tooraj Mirshahi, Daniel Mueller, Eric Lenze, John Newcomer, J. Philip Miller, Anne Justice Washington University, Saint Louis, Missouri, United States Background: Obesity and its metabolic consequences contribute substantially to accelerated aging and reduce lifespan in psychiatrically ill individuals. The risk for antipsychotic induced weight gain (AIWG) is thought to be highly genetic, but our understanding of these contributors remains limited. Characterizing the genetic variation that influences AIWG is critical for the development of precision treatments and decision support tools in Psychiatry. The present analysis represents the initial step in the NIH-funded multi-omic ORAcLE (Obesity Related to Antipsychotic Liability and Exposure) study, a multi-study consortium which aims to combine genetic and clinical data from large health systems, existing cohort studies, and randomized clinical trials both to validate previously reported genetic contributions to risk for AIWG and to identify new common variants that impact risk for AIWG. The Geisinger MyCode Community Health Initiative, a population health genomics project with genetic and clinical data on ~185,000 individuals, was used to test the initial analytic approach for the overall ORAcLE consortium. Using the current interim genome wide association study (GWAS) of BMI from the Genetic Investigation of ANthropometric Traits (GIANT) consortium, which includes >1.1 million European participants, we derived polygenic risk scores (PRS) for cross-sectional body mass index (BMI), then tested those PRS for effect on AIWG, as measured by longitudinal change in BMI during treatment with an atypical antipsychotic. Methods: Individuals in the MyCode data set with > 2 BMI measures > 30 days apart while prescribed an atypical antipsychotic medication were eligible for inclusion in the analyses. Non-physiologically plausible baseline and change values for weight, height or BMI were removed. Changes in height, weight or BMI between visits due to unit conversion error or injury, such as amputation were also removed. Weights collected during pregnancy and 6 months post-delivery, as well as post-bariatric surgery weights, were excluded from the analyses. We employed a linear mixed model, where BMI was regressed with intercept and time (years) as both fixed and random effects, to derive the slope of AIWG, stratified by sex and ancestry. We conducted analyses of overall change in BMI in the population of all eligible individuals, as well as for “gainers” only (i.e., only individuals with a positive AIWG slope). PRS weights were estimated in SBayesR. Association analyses between standardized PRS and AIWG were done using generalized linear models while accounting for families using general estimating equation (GEE). All models were adjusted for baseline age, age2, sex, baseline BMI, follow-up duration, and five genetically-informed principal components of ancestry [European (EUR), African Ancestry (AA), American Indian/Native American (AMR), East Asian (EAS), South Asian (SAS)]. Incremental R2 values were estimated in an unrelated subset of participants using linear regression. The association between PRS and baseline BMI was also assessed. Results: There were 8,493 participants (97% European ancestry, 68% female, 61% who gained weight while prescribed antipsychotic medication) with usable data who met criteria for inclusion in the analysis. There was an average of 33 observations (SD = 58) per individual participant, with a mean of 3.7 (SD = 3.7) years of follow-up, and mean AIWG of 0.18 kg/m2 per year (0.93). The final PRS included 1,080,060 variants and was significantly associated with baseline BMI (P = 1×10-305, β = 4.03, R2 = 14.4%). We observed significant relationships between the cross-sectional BMI PRS and AIWG among all individuals (R2 = 0.3%, P = 4×10-8, β = 0.069 BMI units/year) and among “gainers” (R2 = 0.3%, P = 6.5×10-6, β = 0.051 kg/m2/year), respectively. Finally, a small but significant difference (0.125 kg/m2/yr, P = 2×10-4) in adjusted mean AIWG between the lowest and highest quintiles of the PRS was also observed. Conclusions: These results demonstrate that genetic variants associated with cross-sectional measures of obesity, represented by a PRS derived from the GIANT Consortium’s ongoing BMI GWAS are associated with AIWG. This result extends observations from smaller studies that there is a clear heritable component to AIWG that is detectable using a PRS originally designed to detect genetic risk for obesity. The proportion of variance explained in AIWG by the cross-sectional BMI PRS was significant but small, suggesting that generalized risk for obesity, and its related pathways, might not be the primary mechanism for AIWG. This further motivates the primary goal of the consortium to bring together relevant genomic and clinical data to discover new genetic variants associated with AIWG. Our ongoing analyses in larger population samples and representing greater diversity will identify the modifiers specific to longitudinal AIWG and the affected molecular pathways. Keywords: Antipsychotic Induced Weight Gain, Population Genetics, Molecular Genetics, Human Genetics Disclosures: Alkermes, Novartis: Consultant (Self), LB Pharmaceuticals: Grant (Self) P178. Dissecting Hypothalamic Dysfunctions in the Olanzapine Induced Metabolic Syndrome Federica Veneziani, Aleksandra Marakhovskaia, Clementine Quintana, Lakshmi Rajakrishna, Jean-Martin Beaulieu University of Toronto Faculty of Medicine, Toronto, Canada Background: Olanzapine is a second- generation antipsychotic used for the treatment of schizophrenia and mood disorders. The mechanism of action of this drug includes the modulation of several G-protein coupled receptors (GPCR). Olanzapine chronic administration is burdened by an increased risk of metabolic syndrome, a clinical condition characterized by the co-occurrence of obesity, dyslipidemia, diabetes, and hypertension. Mechanisms by which second generation antipsychotics trigger metabolic syndrome remain unclear. However either central or peripheral signaling functions have been put forward. The hypothalamus is a major regulator of energy metabolism, therefore an involvement of this brain structure in metabolic syndrome induced by antipsychotic medications could be suspected. Here we examine the potential contribution of hypothalamic GPCRs in the onset of the metabolic dysfunction induced by antipsychotics. Methods: Animals: All animal procedures were performed in accordance with Canada Council of animal care guideline and were approved by the University of Toronto animal ethics Committee. The experiments were carried out on C57BL/6J wild-type female mice of approximately 7 weeks old. For the Olanzapine treatment pure Olanzapine was compounded into high fat diet. The Vehicle group received the high fat diet only. The mice were treated for 28 days. Stereotaxic Injection: The mice were divided into three batches: H1R- Knockout, CB1R-Knockout, and Control group (n = 24/each). The knockout (KO) was induced with a double viral CRISPR approach using AAV-MeCP2Cas9. The viruses were administered through hypothalamic stereotaxic injection. Phenotype characterization: For the phenotype characterization the following parameters were measured: food intake, weight gain, blood glucose, serum insulin, HDL, LDL, and Triglycerides. The Fatty liver disease was evaluated using the Oil-Red- O staining and Masson’s Trichrome staining. Statistical analysis The data were presented as means ± SEM. For comparison between multiple groups one-way ANOVA was used followed by Bonferroni correction. The differential gene expression (DEG) analysis, the pathways analysis, and the gene co-expression network analysis were performed using mattest matlab package, Ingenuity pathway analysis (Qiagen IPA) and WGCNA R-studio package respectively. Results: Analysis of hypothalamic gene expression in adult female mice allowed the identification of a transcriptomic signature of olanzapine induced metabolic syndrome. Then, using an unbiased approach we have identified two GPCRs: Histamine Receptor 1 (H1R) and Cannabinoid Receptor 1 (CB1R) that seem to play a crucial role. We thus implemented CRISPR/Cas9 mediated approaches to knockout each receptor in neurons of the hypothalamus. The hypothalamic- neuronal KO of H1R explains weight gain but not glucose impairment neither the changes in lipid metabolism. The mice with H1R-KO in hypothalamic neurons show a statistically significant increase in weight compared to controls (p < 0.001). The present weight gain appears fully comparable to the effect of the treatment with Olanzapine in control mice. Moreover, the treatment with Olanzapine in H1R-KO mice does not result in further increase in weight. Analysis of others metabolic parameters (glucose, insulin, triglycerides, LDL, HDL) indicate that H1R-KO in hypothalamic neurons seems not to lead by itself to glucose impairment or to a lipid dysfunction, with no statistical differences between H1R-KO mice and Control mice. The pathway analysis of the DEGs between H1R-KO and Control mice reveals that in H1R-KO mice the Neuropeptide Y (NPY) pathway is up-regulated. On the other hand, the comparison of the gene expression of H1R-KO mice treated with Olanzapine with H1R-KO mice treated with Vehicle highlights that Olanzapine not only increase the up-regulation of the NPY pathway but also induces a complete deregulation in the metabolic homeostasis inducing an up-regulation also of the Pro-opiomelanocortin (POMC) pathway. The KO of CB1R in hypothalamic neurons is able to restore the full phenotype of Olanzapine-induced Metabolic Syndrome. The CB1R KO mice do not show any difference in terms of weight gain compared with the controls. However the administration of Olanzapine to both groups reveals that CB1R-KO mice show less increase in weight compared with controls (p < 0,01). Moreover the Olanzapine treated CB1R-KO mice have a reduced glycemic dysfunction (p < 0,001), decreased levels of triglycerides (p < 0,001) and LDL (p < 0,001) and increased level of HDL (p < 0,0001) compared to the treated controls. The pathway analysis of the DEGs between CB1R-KO and Control mice both treated with Olanzapine reveals that in this case the NPY pathway is down- regulated in CB1R-KO mice. Moreover comparing the hypothalamic co-expression network of CB1-KO mice with the Control both treated with Olanzapine appears that the Knockout of CB1 receptor induces a dissociation of the expression of H1R and the main regulators of metabolic homeostasis (NPY, AGRP, POMC, NPY2R). Conclusions: These results suggest that metabolic syndrome is a complex clinical phenotype where central and peripheral deregulations may act together resulting in a lost of the metabolic homeostasis. The possibility of reverting Metabolic Syndrome targeting the hypothalamic CB1R on the other hand opens promising perspective for therapeutic development Keywords: Metabolic Side Effects, antipsychotic-Induced Metabolic Dysfunction, Metabolic Syndrome, Antipsychotic Induced Weight Gain, Cannabinoid Receptor Disclosure: Nothing to disclose. P179. Microglia are Implicated in Risk-Taking Behavior in a Mouse Cancer Model Lindsay Strehle, Corena Grant, Lauren Otto, Leah Pyter* Ohio State University, Columbus, Ohio, United States Background: Breast cancer is the most common cancer among females worldwide. Following diagnosis but before cancer treatment, up to 30% of these patients report mood disturbances (e.g., anxiety, depression), which reduce quality of life and inhibit curative treatment compliance. Remarkably, our mouse mammary tumor model recapitulates the negative affective-like behaviors observed in cancer populations, which coincide with increased proinflammatory mediators in the midbrain and hippocampus. Indeed, neuroinflammation is a proposed mechanism underlying the etiology of mood disorders and therefore may be contributing to cancer-associated mood disturbances by modulating neurotransmission (e.g., serotonin metabolism). However, the cellular mechanisms by which tumor-induced neurobehavioral changes occur remain unknown. We hypothesize that microglia are the primary cells driving tumor-induced neuroinflammation, which contributes to affective-like behavior and altered serotonin metabolism. Methods: Young adult female Balb/c mice were induced with an orthotopic, syngeneic, non-metastatic 67NR mammary tumor; tumor-free controls underwent a sham surgery. Experiment 1 assessed brain region-specific microglia morphology (Iba1 immunohistochemistry) and serotonin-related gene expression (Tph2, Th, Slc6a4), as well as gene expression of Percoll-enriched brain microglial cells (Il1b, Il6, Tnfa, Tlr4). Experiment 2 tested the necessity of microglia in tumor-induced behavioral and neuroinflammatory/serotonin outcomes via microglia depletion using a colony stimulating factor 1 receptor inhibitor (PLX-5622) in chow. Results: In Experiment 1, mammary tumors increased gene expression of midbrain serotonin transporter (Slc6a4) and a rate-limiting serotonin enzyme (Tph2) relative to tumor-free controls (t-test: p < 0.05). Specifically, within brain microglial cells, tumors increased the toll-like receptor 4 gene relative to controls (t-test: p < 0.05); microglial immunohistochemistry analyses are underway. In the open field test of Experiment 2, tumor-bearing mice unexpectedly increased locomotion in the risky center of the field compared to tumor-free mice; these tumor-induced risky behaviors were attenuated by PLX-5622 chow (2-way ANOVA interaction: p < 0.05). Similarly, tumor-bearing mice were less hesitant to approach a novel object, a risk-taking behavior that was reversed when microglia were depleted (2-way ANOVA interaction: p < 0.05). Conclusions: These results suggest that microglia may have region- and mediator-specific involvement in tumor-induced neuroinflammation and neurotransmission. This research will advance our understanding of the mechanisms underlying behavioral disorders associated with cancer in order to identify specific cellular and/or molecular targets to prevent or mitigate them. Keywords: Microglia, Risky Behaviors, Neuroinflammation, Serotonin, Cancer Disclosure: Nothing to disclose. P180. TAAR1 Agonist Ulotaront Improves Glycemic Control, Reduces Body Weight and Modulates Neurocircuits Governing Energy Balance and Feeding in Rodents Nina Dedic, Eva Hajos-Korcsok, Phil G. Jones, Colleen Synan, Serena Wu, Christoph Anacker, Steven P. Vickers, Jacob Hecksher-Sørensen, Snezana Milanovic, Linda J. Bristow, Kenneth S. Koblan Sunovion Pharmaceuticals, Inc., Marlborough, Massachusetts, United States Background: Ulotaront (SEP-363856) is a trace amine-associated receptor 1 (TAAR1) and 5-HT1A agonist currently in Phase 3 clinical trials for the treatment of schizophrenia. Metabolic Syndrome (e.g. central obesity, dyslipidemia, hypertension, hyperglycemia, etc.), which can be induced or exacerbated by the current class of antipsychotic drugs (APDs), is highly prevalent in schizophrenia patients. The need for novel treatments that lack APD class-specific metabolic side effects is therefore apparent. Ulotaront has no significant activity at receptors commonly associated with APD-induced metabolic changes (i.e. D2, 5-HT2C, H1 and M3). Recent preclinical evidence has identified TAAR1 as a novel regulator of metabolic control and a promising target for obesity and type 2 diabetes. Here we evaluated the risk-benefit profile of ulotaront for the treatment of schizophrenia by assessing its effects on metabolic parameters in rodent models of diabetes, obesity, and iatrogenic weight gain. Methods: Effects of 15-day and 35-day oral ulotaront treatment (0.3-10 mg/kg) on body weight, food intake and metabolic parameters were investigated in Sprague Dawley rats on high-fat diet (HFD) and in a mouse model of diet-induced obesity (DIO), respectively. In addition, body weight effects were determined in a rat (8-day treatment) and mouse (21-day treatment) model of olanzapine-, and corticosterone-induced body weight gain, respectively (1-10 mg/kg, po). Glucose tolerance was assessed in C57BL/6J and diabetic db/db mice following acute oral administration (0.3-10 mg/kg) as well as in DIO mice following 15 days of dosing (0.3-10 mg/kg, po). The acetaminophen absorption test and phenol red test were used to evaluate effects on gastric emptying in C57BL/6J mice (0.3 – 10 mg/kg, po). To obtain insights into the neurocircuits modulated by ulotaront, whole-brain 3D c-fos imaging was performed in C57BL/6J mice. Comparisons between groups were analyzed using One-Way ANOVA followed by appropriate post-hoc tests. Treatment effects over time were assessed with Repeated Measures ANOVA. Statistical comparisons of c-fos data were based on either ROIs of the Allen Brain atlas or evenly spaced voxels, using negative binomial regression. Results: Acute ulotaront administration dose-dependently reduced plasma glucose excursion in C57BL/6J (F(4, 47) = 15.07, p < 0.0001, n = 10/group) and diabetic db/db (F(4, 45) = 51.41, p < 0.0001, n = 10/group) mice during an oral glucose tolerance test (oGTT). Delayed gastric emptying was seen in response to ulotaront treatment in mice, which is likely the main mechanism driving reductions in glucose excursion during the oGTT (F(5, 56) = 29.90, p < 0.0001, n = 10/group). Administration of ulotaront to rats on HFD resulted in significant and dose-dependent reductions in body weight (F(4, 55) = 40.12, p < 0.0001, n = 12/group), food intake (F(4, 55) = 22.43, p < 0.0001, n = 12/group) and liver triglyceride content (F(4, 55) = 3.3, p < 0.02, n = 12/group) compared to vehicle controls. A more rapid reversal of olanzapine-induced weight gain (F(5, 67) = 17.04, p < 0.0001, n = 12-13/group) and food intake (F(5, 67) = 30.69, p < 0.0001, n = 12-13/group) was observed in rats switched to ulotaront treatment compared to vehicle alone. Consistent with the body weight-lowering effects in rats, chronic treatment with ulotaront normalized corticosterone-induced body weight gain in mice (F(4, 45) = 46.1, p < 0.0001, n = 8-12/group) and reduced body weights in the DIO model in a dose-dependent manner (F(4, 55) = 5.5, p = 0.0009, n = 12/group). In addition, chronic ulotaront treatment improved glycemic control in DIO mice as indicated by reduced fasting plasma glucose (F(4, 55) = 6.3, p = 0.003, n = 12/group) and insulin levels (F(4, 55) = 2.9, p = 0.03, n = 12/group) and increased glucose tolerance (F(4, 55) = 4.51, p = 0.003, n = 12/group). Whole-brain imaging revealed neuronal activation (increased c-fos expression) of appetite-regulating pathways related to meal termination (initiated by signaling in the hindbrain nucleus of the solitary tract and relayed through the lateral parabrachial nucleus). Neuronal activation was also seen in hypothalamic subregions associated with the regulation of food intake and integration of peripheral metabolic signals including the arcuate and paraventricular nucleus. The c-fos signature of ulotaront was distinct from that of the typical APD haloperidol. Conclusions: The current data indicate that ulotaront not only lacks APD-induced metabolic liabilities but can reduce body weight and improve glycemic control in rodent models. The underlying mechanisms likely include TAAR1-mediated peripheral effects on glucose homeostasis and gastric emptying, and/or direct modulation of homeostatic and hedonic neurocircuits regulating energy balance. The positive effects of ulotaront on metabolic parameters may suggest an improved risk-benefit profile compared to the established pharmacological class of APDs. In addition, the current preclinical results further support evaluation of TAAR1 agonists for the treatment of metabolic disorders. Keywords: TAAR1, Schizophrenia- Novel Treatment, Antipsychotic-Induced Metabolic Dysfunction, Metabolic Side-Effects Disclosure: Sunovion Pharmaceuticals: Employee (Self) P181. Serum Biomarkers of Liver Fibrosis Identify Changes to Striatal Metabolites Consistent With Lipid Accumulation Natalie Zahr, Edith Sullivan, Adolf Pfefferbaum Stanford University, Stanford, California, United States Background: The presence of hepatitis C virus (HCV) affects the levels of magnetic resonance spectroscopy (MRS)-detectable metabolites in the brain. In particular, the levels of striatal Cho (i.e., choline-containing compounds) were found to be higher in HCV seropositive relative to seronegative individuals. Methods: To determine whether liver fibrosis independent of HCV status would affect brain metabolite levels, MRS data collected in 3 regions of interest [ROIs, striatum (10.6 cc), cerebellum (9.8 cc), and pons (5.9 cc)] were evaluated for their relations with 2 serum-derived, liver fibrosis markers. Cutoff scores for liver fibrosis were aspartate aminotransferase to platelet ratio index (APRI) > 0.7 and a fibrosis score (FIB4) > 1.5 calculated in a cohort of 170 participants: 62 healthy controls (47.8 ± 19.9yrs), 37 individuals with alcohol use disorder (AUD) (48.5 ± 10.6yrs), 33 individuals seropositive for HIV (50.0 ± 9.1yrs), and 38 individuals with comorbidity for AUD + HIV (51.9 ± 8.6yrs). Results: Among all participants, 13 had APRI > 0.7 (1 control, 2 AUD, 4 HIV, 6 AUD + HIV) and 34 had FIB4 > 1.5 (4 controls, 5 AUD, 13 HIV, and 12 AUD + HIV). Sex did not differentiate the number of individuals with fibrosis (APRI p = .26; FIBR4 p = .30). Using nonparametric Wilcoxon-rank tests, an APRI > 0.7 was associated with higher striatal Cr (total creatine, Z = 3.3, p = .001), higher striatal Cho (Z = 4.2, p < .0001), and higher striatal mI (myoinositol, Z = 2.7, p = .008); metabolites in the other 2 ROIs did not differ by the APRI cutoff. FIB4 > 1.5 was associated higher striatal Cho (Z = 3.1, p = .002) and higher striatal (Z = 2.6, p = .009) and pontine (Z = 2.8, p = .005) mI. For striatal Cho, a multiple regression including HCV, APRI, and FIB4 explained 16.6% of the variance and was driven by the APRI score (p = .0009, alone contributing 14.7% to the variance). A similar model (i.e., including HCV, APRI, FIB4) resulted in the same pattern for striatal Cr (5.9% of the variance explained, driven by APRI p = .02); the model was not significant for striatal mI; pontine mI was more affected by HCV (11.1% of the variance explained, HCV p = .01). An APRI > 0.7 was associated with lower verbal fluency scores (F-A-S, Z = -2.7, p = .006; Freda’s, Z = -2.6, p = .01). A FIB4 > 1.5 was also associated with lower verbal (Freda’s, Z = -3.1, p = .002) and figural (RUFF, Z = -2.6, p = .009) fluency scores. Conclusions: Together, these data suggest that the presence of liver fibrosis can selectively affect striatal metabolite concentrations. High levels of striatal Cho and mI suggest the accumulation of lipids, which may contribute to T1 signal hyperintensities observed in basal ganglia structures in liver cirrhosis. Correlations between higher fibrosis scores and compromised fluency suggest functional ramifications of these neurometabolite abnormalities measured in vivo. Keywords: Liver Brain Axis, Alcoholic Liver Disease, HIV, Hepatitis C, Basal Ganglia Disclosure: Nothing to disclose. P182. Shape and Volume Alterations in Habit-Centered Basal Ganglia Structures Present in Atypical Anorexia Nervosa Lauren Breithaupt, Amanda Lyall, Felicia Petterway, Holly Carrington, Laura Holsen, Franziska Plessow, Yaen Chen, Jennifer Thomas, Madhusmita Misa, Elizabeth Lawson, Kamryn Eddy Massachusetts General Hospital, Boston, Massachusetts, United States Background: Anorexia nervosa (AN) is a serious eating disorder characterized by maladaptive restrictive eating. Restrictive eating patterns often develop as goal-directed dieting behaviors and transition to habitual restrictive eating, unmotivated by initial rewarding goals. The transition to habit-governed behaviors may perseverate as a result of structural alterations in the basal ganglia that occur as a consequence of extreme weight loss. It is unknown whether morphometry of the basal ganglia is altered in individuals with atypical anorexia nervosa (atypical AN) who do not have low-weight (BMI < 18.5 kg/m2) but are otherwise clinically similar to AN. Based on prior literature, we hypothesized that participants with AN would show lower volume and greater shape deformations in regions associated with habitual responding, including the bilateral putamen and caudate, compared to participants with atypical AN and healthy controls. Methods: The sample included 77 females with AN (n = 30, x̄ age = 19.7, x̄ duration of illness 5.0 years), atypical AN (n = 21, x̄ age = 18.3, x̄ duration of illness 3.6 years), and healthy controls (n = 26, x̄ age = 18.0). T1-weight images were processed using a standard internal pipeline, which included: visual quality control, manual masking, and outlier detection. The recon-all function of Freesurfer v7.1 was used for segmentation of the basal ganglia to extract regional volumes. Between-group clinical characteristics were assessed using chi-square tests, analysis of covariance (ANCOVA), and Tukey post-hoc tests. Group volumetric comparisons were corrected for age and head size, Bonferroni corrected, and p < .016 was considered statistically significant. Shape of the basal ganglia was processed using the ENIGMA Shape Pipeline to extract the logarithm of the Jacobian determinant. For group comparisons of basal ganglia shape, we conducted vertex-wise regression analyses for each vertex on each region of interest, with age and estimated total intracranial volume (eTIV) as covariates, in a pairwise manner. Benjamini-Hochberg correction for multiple comparisons was utilized with a false discovery rate of 5% (q = .05). Results: Contrary to our hypothesis, we found a significant main effect of group on the volume and shape of the bilateral putamen (Left: F2,72 = 6.88, p = .0018; Right: F2,72 = 7.23, p = .0013). Individuals with atypical AN had lower volume and greater shape deformations in these regions compared to both individuals with AN (R: p = .0013; L: p = .0014) and healthy controls (R: p = .0016; L: p = .002). We also found significant differences in the shape of the right caudate between atypical AN compared to AN, but not between atypical AN and HC. Conclusions: Our findings suggest structural brain differences between individuals with both AN and atypical AN who share similar clinical presentations. However, the group differences in basal ganglia structures are even more pronounced in those with atypical AN relative to AN. Gray matter differences in AN are often attributed to the severe low-weight status present among individuals with AN. Our study suggests that basal ganglia structural differences persist regardless of the low-weight clinical delineation. Our findings also complement clinical data demonstrating that habitual restriction occurs in atypical AN. These results underscore the importance of weight restoration based on individual history, in order to target the habitual features seen in both AN and atypical AN. Keywords: Eating Disorders, Basal Ganglia, Habit Disclosure: Nothing to disclose. P183. Using Causal Discovery to Identify Heterogeneity in the Causal Relationship Between Negative Affect and Binge-Eating and Compensatory Behavior in Bulimia Nervosa Kelvin Lim, Lisa Anderson, Erich Kummerfeld, Carol Peterson, Stephen Wonderlich, Ross Crosby, Scott Engel University of Minnesota, Minneapolis, Minnesota, United States Background: Negative affect has been implicated as a precipitant and maintenance factor of binge eating and purging behavior in bulimia nervosa (BN). This relationship has been supported by laboratory data and group analyses of ecological momentary assessment (EMA) data. However, treatment outcomes in BN intervention studies targeting negative emotions have been mixed, suggesting that there may be heterogeneity in these causal relationships at the individual level. Identifying individual patterns of negative emotion and BN behaviors can be used to more accurately target causal relationships that maintain eating disorder behaviors. We used causal discovery analysis of EMA data to identify the causal network structure that best fits the individual data for adults with BN. Methods: To examine individual causal relationships, we analyzed data from 128 adult women with a minimum of 60 EMA data points collected over 14 days. EMA data included measures of negative/positive affect, anger/hostility, binge eating, vomiting, and exercise. We used the Greedy Fast Causal Inference (GFCI) algorithm to learn the partial ancestral graph and to compute a hypothetical causal model for each participant from their data. Structural equation modeling was used to estimate effect sizes for each model. Counts for the number of unconfounded causal edges between variables of interest were computed across all participants. Results: We found a direct causal relationship with high negative affect preceding binge eating in 28/128 (22%) of the participants. Other causal relationships identified included low positive affect preceding binge eating: n = 5 (4%), anger/hostility preceding binge eating: n = 5 (4%), vomiting preceding binge eating: n = 45 (35%), and binge eating preceding vomiting: n = 26 (20%). Conclusions: Prior studies examining negative affect and binge eating in BN have been limited by group-level analyses. By using causal discovery, we found that only 22% of participants had a causal relationship between negative affect and binge eating. Causal discovery also revealed heterogeneity in the sequence of binge-eating behaviors, with binge eating following self-induced vomiting (35%) as well as preceding it (20%). Identifying causal models at the individual level may optimize precision medicine approaches in the design of future trials for eating disorder treatments. Keywords: Computational Psychiatry, Eating disorders, Ecological Momentary Assessment, Personalized Medicine Disclosure: Nothing to disclose. P184. Meal Challenge Revealed Differential Associations of Anorexia Nervosa Psychopathology With Soluble Epoxide Hydrolase-Derived Oxylipins Nhien Nguyen, Jun Yang, Christophe Morisseau, Dongyang Li, Eileen Lam, J. Bruce German, D. Blake Woodside, Bruce D. Hammock, Pei-an (Betty) Shih University of California, San Diego, La Jolla, California, United States Background: Oxylipins are bioactive lipid mediators derived from polyunsaturated fatty acids (PUFA) via cytochrome P450 (CYP), lipoxygenase (LOX), cyclooxygenase (COX), or non-enzymatic pathways. Soluble epoxide hydrolase (sEH) converts a class of CYP-derived oxylipins, epoxy fatty acids (epoxides), to corresponding diol fatty acids (diols). This process modulates important biological processes such as inflammation and metabolism. sEH-encoding gene was previously found to associate epidemiologically with anorexia nervosa (AN) risk. This study focused on clarifying the differential impact of sEH, PUFA, and oxylipins measured directly on AN risk and psychopathology. Methods: Blood samples were analyzed from 70 women with AN (age: 32 ± 12) and 96 age-matched control women (age: 29 ± 8) at both fasting and 2h after consumption of a high-fat study meal. Fasting and postprandial levels of 59 oxylipins from CYP, LOX, COX, and non-enzymatic pathways, sEH protein expression and enzyme activity, and 13 diol/epoxide ratios (as sEH in vivo activity markers) were examined. AN clinical phenotypes were studied using EDI-3 and EDE-Q (eating disorder severity scales), anxiety, depression, and food aversion. Statistical analyses were conducted using t-tests and covariate-adjusted models. Results: 17 fasting oxylipins (29% of 59 assayed), mostly derived from n-6 arachidonic acid (ARA, n = 9) and n-3 docosahexaenoic acid (DHA, n = 7), were significantly decreased in AN compared to controls (all p < 0.01). CYP-pathway oxylipins (n = 9 epoxides and 3 diols, 38% of total CYP oxylipins) were decreased in AN by 30% to 60%, while 5 LOX-pathway oxylipins (29% of total LOX oxylipins) were lowered by 42% to 82%. At the postprandial timepoint, levels of 6 CYP oxylipins (19% of total CYP oxylipins) and 3 LOX oxylipins (18% of total LOX oxylipins) were significantly different between groups. In AN, CYP epoxides were significantly elevated whereas CYP diols and 3 LOX oxylipins were lowered relative to controls (all p < 0.05). Significant between-group differences in the changes of oxylipin levels were found for 10 CYP epoxides (31% of total CYP oxylipins) (all p < 0.03), which all increased (range: 1 to 48 nmol/L) in AN but decreased (range: -2 to -306 nmol/L) in controls after eating. One LOX oxylipin increased in both groups but with a significantly greater magnitude in controls (p = 0.02). In AN, of the 17 fasting oxylipins associated with AN risk, none of the CYP epoxides were significantly associated with psychopathology using EDI-3 and EDE-Q scores. However, CYP diol derived from n-6 ARA (5,6-DiHETrE) was linked to higher psychopathology in 5 of the EDI-3 scale scores, whereas n-3 DHA-derived diols (10,11- and 4,5-DiHDPE) were associated with less disorder severity, depression, and food aversion (all p < 0.05). LOX-pathway’s 11- and 15-HETE were linked to less severe perfectionism score whereas 5-HETE was linked to higher interoceptive deficits score (all p < 0.05). None of the oxylipins were significantly associated with anxiety in either AN or controls. sEH protein expression and enzyme activity were 16% (p = 0.04) and 22% (p = 0.01) higher in AN compared to controls at the fasting timepoint only. After eating, sEH expression and activity increased by 56% and 63% in AN (p < 0.05) and by 111% and 155% in controls (p < 0.10). None of the fasting oxylipin ratios (sEH in vivo activity markers) differed significantly between groups. At the postprandial timepoint, 7 oxylipin ratios (54%) were 44% to 75% lowered in AN (all p < 0.05) compared to controls. sEH expression, activity, and oxylipin ratios were not significantly associated with EDI-3 or EDE-Q scores. Conclusions: sEH affects human health by converting PUFA-derived epoxides into their metabolic products, diol oxylipins. Association between sEH and AN was established previously at the genome wide level. Here, we present further evidence of sEH’s link to AN risk by demonstrating differential associations between sEH substrates (epoxides) and products (diols) with AN risk and phenotypes that are PUFA class-dependent. Direct measurement of sEH expression and activity confirmed higher sEH level in AN. Significant between-group differences in meal-induced oxylipin changes were marked by directionally opposite changes in sEH substrates (epoxides), which increased in AN but decreased in controls. Surprisingly, although both epoxide and diol oxylipins were associated with AN risk, only diols were associated with AN phenotypes. Moreover, n-6 diols were associated with worsening AN phenotypes while n-3 diols were associated with better AN phenotypes. Our results confirmed the role sEH plays in modulating not only AN risk but also eating disorder severity. sEH action on n-3 PUFA oxylipins versus n-6 PUFA oxylipins led to differential impact on AN phenotypes, highlighting the importance of accounting for PUFA source in sEH studies and the potential for targeted dietary guidance in the future. Keywords: Metabolites, Polyunsaturated Fatty Acids, Anorexia Nervosa Disclosure: Nothing to disclose. P185. Characterizing Midbrain Dopamine Function in Anorexia Nervosa Using Neuromelanin-Sensitive MRI Blair Uniacke, Karin Foerde, Monica Jablonski, Kenneth Wengler, Guillermo Horga, Jonathan Posner, Steinglass Joanna Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Anorexia nervosa (AN) is a devastating illness that most commonly emerges during adolescence and has a mortality rate among the highest of any psychiatric disorder. Several prior studies have identified altered dopamine function in AN, suggesting that abnormalities in the dopamine system may play a role in the pathophysiology of persistent maladaptive dietary restriction in AN. Although prior research utilizing positron emission tomography (PET) and lumbar puncture to assess metabolic byproducts of dopamine metabolism (e.g., homovanillic acid) broadly indicates dopamine dysfunction in AN, the nature and direction of this disturbance have not been clarified. This gap in knowledge may have arisen, at least in part, because AN often emerges and becomes entrenched during adolescence, when the use of in vivo assessments of dopamine function such as PET or lumbar puncture have limited appeal due to invasiveness, ethical considerations, cost, and methodology. Neuromelanin-sensitive magnetic resonance imaging (NM-MRI) is a high-resolution neuroimaging technique that provides a noninvasive proxy measure of dopamine function by measuring the concentration of neuromelanin, a byproduct of dopamine metabolism, in the substantia nigra (SN) and ventral tegmental area (VTA). This ongoing study examines dopamine in the SN and VTA in adolescents with AN as compared with healthy teens (HC). Methods: NM-MRI data were collected in 14 female, post-menarchal adolescents with AN and 10 HC (ages 14-18 years); data collection is ongoing. Extracted neuromelanin (NM) signal (contrast-to-noise ratio) was averaged across the SN pars compacta (SNpc; SN region with greatest concentration of DA neurons) and the VTA. Independent samples t-tests were used to compare between-group differences and Hedge’s g to measure effect size. Results: Relative to HC participants, adolescents with AN had lower NM signal within the SNpc (t1,22 = 2.07, p = 0.05; g = 0.86). Preliminary analyses also suggest lower NM signal in the VTA (t1,12.5 = 2.0, p = 0.067; g = 0.91), though this result does not reach significance in this small sample. Conclusions: Neuromelanin-sensitive MRI (NM-MRI) is a validated, non-invasive proxy measure of dopamine function. Our results indicate lower NM-MRI signal in the substantia nigra of adolescents with AN relative to healthy teens, suggesting reduced midbrain dopamine function in AN. NM-MRI may provide a noninvasive approach to measuring dopamine function which can be used safely and routinely in pediatric and non-pediatric populations with AN. Examining the relationship between NM-MRI signal in the midbrain and stage, severity, and trajectory of illness through longitudinal assessment in adolescents with AN will be a fruitful avenue for future investigation. Keywords: Anorexia Nervosa, Dopamine, Human Neuroimaging Disclosure: Nothing to disclose. P186. Translocator Protein Distribution Volume, A Positron Emission Tomography Marker of Gliosis in Anorexia Nervosa Mariel Lepra, Joeffre Braga, Blake Woodside, Stephen Kish, Pablo M. Rusjan, Thomas Chao, Michael Bagby, Stefan Kloiber, Ishrat Husain, Jeffrey Meyer Centre for Addiction and Mental Health, University of Toronto, Toronto, Canada Background: Anorexia Nervosa (AN) has a lifetime prevalence of ~0.8% and is arguably the most lethal psychiatric illness in the world, with a mortality rate 5.86% higher than in the general population. Unfortunately, there are no medications with indications for this disease, and a lack of therapeutic targets for AN in the brain. Presently it is unknown whether gliosis occurs in AN, but there are several plausible reasons why it may occur. Peripheral inflammation may cause gliosis and there is evidence that greater peripheral inflammation may occur episodically in AN. Anorexia nervosa occurs at prevalence rates as high as 20% in children with Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections and Pediatric Acute-onset Neuropsychiatric Syndrome. The prevalence of autoimmune diseases, which may lead to gliosis, is ~2 fold greater in AN. Inducing peripheral inflammation in humans, such as with vaccination or lipopolysaccharide administration, the latter which also raises gliosis marker translocator protein (TSPO) in the brain, induces the symptom of anorexia, and direct cerebral inflammatory stimulation in rodents also induces anorexia. In addition, inverse relationships between body mass index (BMI) and TSPO VT, an index of translocator protein density and gliosis, have been frequently reported in neuropsychiatric disease. TSPO VT can be measured using [18F]FEPPA, a radiopharmaceutical with excellent properties for use in positron emission tomography (PET) imaging. We hypothesized that TSPO VT will be elevated in circuitry implicated in AN, including the anterior cingulate cortex (ACC), insula, dorsal putamen, dorsal caudate, orbitofrontal cortex (OFC), ventral striatum, and thalamus. Methods: Eleven AN (9 F, 2 M, average age 25.09) untreated with antidepressant, antipsychotic or anti-inflammatory medication and eleven healthy controls (8 F, 3 M, average age 25.82) underwent [18F]FEPPA PET scanning with concurrent arterial sampling. Exclusionary criteria included cigarette smoking and substance use disorders. A two-tissue compartment model was applied to measure TSPO VT. Regions of interest included the ACC, insula, dorsal putamen, dorsal caudate, OFC, ventral striatum, and thalamus. Groups were compared using a repeated measures ANOVA (rmANOVA), with group (healthy or AN) as the independent variable and region of interest as the repeated measure. ANOVAs to compare individual regions between groups were also done. The genotype of a single nucleotide polymorphism of the TSPO gene (rs6971) was applied as a factor in all analyses. Results: There was an interaction between group and the a priori selected regions, indicating that some regions were more different between groups than others (rmANOVA, p = 0.04). ANOVAs comparing individual regions between groups found greater TSPO VT in ACC (32% difference, F1,19 = 8.1, p = 0.01) and thalamus (41% difference, F1,19 = 7.2, p = 0.01). Conclusions: To our knowledge, this is the first investigation of gliosis in AN. Differences in TSPO VT are more prominent in the anterior cingulate cortex and thalamus, which implies that gliosis is more elevated in these regions in AN. This suggests that anti-inflammatory approaches should be considered for development as treatment in AN and these two regions should be a focus for future investigations of inflammatory processes in AN. Keywords: Anorexia Nervosa, TSPO, Positron Emission Tomography Imaging, Gliosis, Eating Disorders Disclosure: Nothing to disclose. P187. Neural Correlates of Decreased Impulsivity During Delay Discounting Task in Obese Patients After Laparoscopic Sleeve Gastrectomy Wenchao Zhang, Guanya Li, Yang Hu, Jia Wang, Weibin Ji, Gang Ji, Peter Manza, Dardo Tomasi, Nora Volkow, Yi Zhang, Gene-Jack Wang National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: Individuals with obesity (OB) prefer immediate rewards of food-intake over the delayed reward of healthy well being achieved through diet management and physical activity more often than normal-weight individuals (NW). This may reflect deficits in intertemporal decision-making function and heightened impulsivity. Overeating resulting from greater impulsivity is an important factor contributing to the development and maintenance of obesity. Laparoscopic sleeve gastrectomy (LSG) is one of the most effective treatments for morbid obesity and produces sustained weight-loss. In addition, LSG improved problematic and disordered eating behaviors, such as enhanced control of food-intake and decreased frequency of snacking on high-calorie foods. However, it remains unclear whether these improved eating behaviors were associated with decreased impulsivity in OB after LSG and how LSG regulates associated brain functions. Therefore, the current study employed functional magnetic resonance imaging (fMRI) with a monetary delay discounting (DD) task to examine LSG-induced changes in impulsivity and its neural correlates. Methods: Twenty-nine OB underwent whole-brain fMRI scan with DD task before (PreLSG) and one-month after LSG (PostLSG). Thirty NW gender and age matched to the OB participants were recruited as controls and underwent the same fMRI scan. The discounting rate (K) based on the hyperbolic function and the area under curve (AUC), which is independent of theoretical assumptions regarding the discounting function were quantified as measures of impulsivity. Because the discounting rate did not follow a normal distribution, the log-transformed K (lg(K)) was calculated. Preprocessing of fMRI data was performed with Statistical Parametric Mapping 12. In the current study, two different statistical analyses were performed with the General Lineal Model (GLM). First, a GLM including hard- and easy-choice regressors was constructed for each participant with hard choices defined as those with similar subjective values for immediate and delayed reward options. Second, we constructed a GLM including SIR (choices for smaller immediate rewards) and LDR (choices for larger delayed rewards) condition regressors for each participant. Then, individual beta images responded to hard/easy-choices, SIR/LDR conditions and contrast images for “Hard - Easy” and “LDR-SIR” were calculated. To examine differences in activation between PreLSG and PostLSG, we performed second level analysis (paired t-tests) with those beta and contrast images. Clusters showing significant differences between PreLSG and PostLSG were selected as regions of interest (ROIs). In addition, whole-brain psychophysiological interaction (PPI) analysis was performed to investigate alterations in choice difficulty and subjective choice related functional connectivity with these ROIs as seeds. We also performed two-sample t-tests to compare the brain activations and functional connectivity between NW and LSG groups (PreLSG and PostLSG). Results: LSG significantly reduced discounting rate in OB. During the hard trials in DD task, LSG significantly decreased activations in left dorsolateral prefrontal cortex (DLPFC) (Brodmann area (BA) 9), dorsomedial prefrontal cortex (DMPFC), which on average was equivalent to that in NW, and additionally increased activations in bilateral posterior insula (INS). Changes in activation in the left DLPFC were negatively correlated with changes in cognitive control measured with the Three Factor Eating Questionnaire (TFEQ, r = -0.549, P = 0.004) and AUC (r = -0.484, P = 0.010). Changes in activation in the left DMPFC were positively correlated with change in hunger (TFEQ) (r = 0.513, P = 0.005). Changes in activation in bilateral INS were negatively correlated with changes in body mass index (BMI) (r = -0.551, P = 0.002) and disinhibition (TFEQ, r = -0.464, P = 0.011). In addition, LSG significantly decreased activations in the left DLPFC (BA46) and right caudate during SIR condition, which on average was equivalent to that in NW. Changes in the left DLPFC activation was positively correlated with changes in BMI (r = 0.555, P = 0.002), lg(K) (r = 0.467, P = 0.012), and negatively correlated with changes in cognitive control (TFEQ, r = -0.522, P = 0.004), AUC (r = -0.460, P = 0.012). In PPI analysis, there was increased functional connectivity between the right caudate and left DMPFC (BA32) in responses to LDR versus SIR choices in PostLSG compared with PreLSG and NW. Changes in functional connectivity between the right caudate and left DMPFC were positively correlated with changes in cognitive control (TFEQ, r = 0.588, P = 0.001) and negatively correlated with changes in disinhibition (TFEQ, r = -0.540, P = 0.004). Conclusions: These findings indicate that decreased impulsivity following LSG was associated with the normalization of activation in regions involved in executive control and reward evaluation, and compensatory effects through increasing interoception and prospection. This study may provide neurophysiological support for the development of nonoperative treatments such as brain stimulation for obese and overweight patients who do not qualify for LSG. Keywords: Obesity, Task fMRI, Bariatric Surgery Disclosure: Nothing to disclose. P188. Expectation of Reward and Prediction Error Response and its Longitudinal Association With Treatment Response Among Young Adult Females With Anorexia Nervosa Sasha Gorrell, Megan Shott, Guido Frank University of California, San Francisco, San Francisco, California, United States Background: Anorexia nervosa (AN) is a severe and often-chronic psychiatric illness with unknown etiology. Recent evidence from taste and monetary reward paradigms that investigated the reward prediction error suggests that dysregulation in reward processing may underpin symptoms and create a vicious cycle that contributes to chronicity and frequent relapse. However, whether conditioned reward expectation and receipt can predict long-term outcome has not been directly studied. Here we followed individuals who were treated for AN and tested whether brain imaging reward response could predict BMI. We anticipated that greater neural activity relative to expectation (higher arousal) and prediction error response (higher dopamine-related reward response) would predict lower BMI (poorer treatment response) at one-year follow-up. Methods: The sample comprised a sub-set of a larger study of young-adult female patients with AN who received eating disorder treatment in a specialized partial hospital or residential program (n = 35, Mage [SD] = 23[7]). Functional brain imaging was used to test brain response during a classical conditioning paradigm, where violations of learned associations between conditioned visual and unconditioned taste stimuli evoked the dopamine-related prediction error. Brain images were analyzed for expectation of the caloric stimulus as well as for prediction error response, which was comprised of the unexpected receipt or omission of the caloric taste stimulus sucrose. Regional brain response data were extracted for insula, orbitofrontal cortex and striatum. Study participants were contacted for long-term follow up assessment after discharge from the treatment program. Results: Follow-up assessment occurred 1648 days (SD ± 1198) after brain imaging. Mean follow-up BMI = 18.9 (SD ± 3.3); mean BMI change between assessment time points = 2.5 (SD ± 3.2). Patients self-reported Eating Disorder Examination-Questionnaire (EDE-Q) Restraint = 2.2 (SD ± 1.9), Eating Concern = 2.3 (SD ± 1.3), Shape Concern = 3.2 (SD ± 1.5) and Weight Concern = 2.9 (SD ± 1.7); participants also rated mood (Beck Depression Inventory [BDI] = 20.2 [SD ± 12.9]), and anxiety (State/Trait Anxiety Index [STAI] = 49.3 [SD ± 12.3]). Long-term follow up BMI was positively correlated with discharge BMI (r = .59, p < .001) and days from scan (r = .44, p = .009). Adjusting for multiple comparisons, no significant associations were found for prediction error or expectation and follow-up BDI, STAI or EDE-Q scores. A pattern emerged whereby significant associations (at p < .05) in prediction error-related activity and follow-up BMI were positive, whereas associations in expectation-related activity and long-term follow-up BMI were negative. Adjusting for multiple comparisons (p < .001), a significant correlation remained only for expectation-related activity in the right superior orbitofrontal cortex with BMI, r = -.58, p < .001, 95%CI = [0.77, 0.31]. Conclusions: Results indicate that expectation, but not prediction error is related to weight outcomes. The study suggests that expectation response in the superior orbitofrontal cortex may be particularly important for long-term outcome. The superior orbitofrontal cortex is part of the superior frontal gyrus and is involved in executive function and inhibitory control, but also mood regulation and personality traits. The superior orbitofrontal cortex could be mechanistically involved in the persistent and cognitive emotional aspects of AN and could be a biomarker for long-term outcome. Keywords: Anorexia Nervosa, Reward Expectancy, Superior Orbitofrontal Cortex Disclosure: Nothing to disclose. P189. Trait Anxiety is Associated With Amygdala Expectation and Caloric Taste Receipt Response Across Eating Disorders Guido Frank, Megan Shott, Skylar Swindle, Tyler Nguyen, Tamara Pryor, Joel Stoddard University of California San Diego, San Diego, California, United States Background: Anxious traits are elevated in eating disorders (EDs), are considered risk factors for ED development, and trait anxiety has been linked to ED psychopathology. How trait anxiety relates to ED neurobiology is not well understood. Here we investigated a transdiagnostic study sample across the ED spectrum to test response to caloric stimulus (sucrose) expectation and expected receipt, both contrasted against non-caloric taste stimulus, and the effects of anxious traits. We hypothesized that elevated trait anxiety in individuals with EDs would be associated with brain response to caloric taste stimulus expectation and receipt. The amygdala is a brain region central to expectation, vigilance, anxiety and threat. We expected that the ED sample would show elevated amygdala response to expectation of a high caloric sucrose solution stimulus compared to the non-caloric stimulus and that expectation response would negatively bias and reduce response in brain reward regions during expected taste receipt, which could interfere with food intake. Methods: In this study 179 individuals across the ED spectrum (anorexia nervosa n = 91; other specified EDs n = 34; bulimia nervosa n = 56; binge ED n = 16), and 120 healthy controls were assessed for anxious traits and learned to expect and receive caloric or neutral taste stimuli during fMRI of the brain. We developed first-level models to predict the response in each voxel as a function of the following conditions: (1) sucrose expectation: trials with CS predicting sucrose receipt contrasted against trials with CS predicting artificial saliva (non-caloric taste stimulus); (2) expected sucrose receipt: trials with expected US caloric sucrose receipt contrasted against trials with expected US non-caloric artificial saliva receipt. We extracted beta values from predefined regions of interest bilaterally. Group comparison studies were conducted with and without potential confounding covariates in the group-comparison (MANOVA, or MANCOVA for estimated marginal means). Regression analyses tested associations between behavior and brain activation. Moderator analysis (PROCESS, SPSS) was used to test the effects of anxiety on the relationship between sucrose amygdala expectation response (X) and reward circuitry taste receipt response (Y). Results were multiple comparisons controlled using false discovery rate (FDR). Results: Amygdala sucrose expectation response differed across groups (Wilk’s lambda=0.945, p = 0.023), and was higher on the left in anorexia nervosa compared to healthy controls (p = 0.002). Expected sucrose receipt response across taste reward regions was not different between groups. In the ED sample, trait anxiety negatively moderated the relationship between amygdala expectation and right dorsal (p = 0.0062) and ventral (p = 0.0046) anterior insula receipt response. A subgroup analysis showed similar results for anorexia nervosa, and partially in bulimia nervosa. Across EDs, appetitive motivation correlated positively with bilateral orbitofrontal cortex, caudate head, and ventral striatal sucrose receipt response (r = 0.215 to 0.179, p = 0.002 to 0.012). Across the study sample, trait anxiety showed an inverted-U-shaped relationship with right (r = 0.147, p = 0.034) and left (r = 0.162, p = 0.016) amygdala expectation response. Conclusions: Elevated amygdala response in AN and the combined ED sample suggests elevated arousal to food stimuli. The relationship between amygdala expectation and right insular stimulus receipt response is moderated by trait anxiety in individuals with EDs. The influence of trait anxiety on brain taste response supports the notion that anxious traits may interfere with normal reward and interoception processing and thus have an important role in perpetuating ED pathophysiology and psychopathology. The study raises the question whether modifying the effects of trait anxiety and associated arousal via psychopharmacologic or psychotherapeutic interventions could have an important role in facilitating ED-specific treatment. Keywords: Eating Disorders, Anorexia Nervosa, Reward Anticipation, Anxiety Disclosure: Nothing to disclose. P190. Attenuation of Anorexia-Related Phenotype in Recipients of Obese Adipose Tissue Transplant Stephanie Dulawa, Jie Zhang, Rizaldy Zapata, Avraham Libster, Olivia Osborn University of California - San Diego, La Jolla, California, United States Background: Anorexia nervosa (AN) is observed predominantly in girls and women, and is characterized by hypophagia, dangerously low body weight, and compulsive exercise. Recent large-scale human genome-wide studies have revealed that AN has a substantial metabolic component. Other recent studies point to adipose tissue as the location of ‘metabolic memory’ of earlier nutrition status. The activity-based anorexia (ABA) induces certain aspects of AN in rodents; mice are simultaneously exposed to running wheels and scheduled feeding, which induces hypophagia, weight loss, and hyperactivity. Here, we tested the hypothesis that transplanting adipose tissue from high fat diet (HFD)-fed obese mice into recipient mice would attenuate ABA-induced weight loss in recipients, possibly due to diffusible factors released by obese adipose tissue. Methods: Nine-week-old mice received an intra-abdominal perigonadal adipose transplant harvested from either normal chow-fed mice, or high fat diet-fed obese mice (n = 21/group). All donors and recipients were female C57Bl/6 mice. Four weeks post-surgery, recipient mice were tested in the ABA paradigm. All mice underwent 2 days of acclimatization to single housing and constant running wheel access. Then, mice entered a baseline phase (4 days) in which food and running wheels were available continuously. Finally, mice entered a restriction phase (14 days) in which running wheels were continuously available, but food was only available 3 hours daily starting at 0900h. Body weight, food intake, and running distance were measured daily. During restriction, mice were removed from the study (termed dropout) after losing 25% of their baseline body weight. The number of days in restriction until dropout provided a measure of survival. Finally, this same experiment was performed using a new cohort, but within metabolic chambers (n = 7/group). Additional dependent measures were VO2 (volume of oxygen consumed, ml/kg/hr), VCO2 (volume of carbon dioxide produced, ml/kg/hr), respiratory quotient (RQ), food and water intake, body weight, as well as activity (x beams, y beams, wheel running counts). Results: In the first study, no differences in body weight, food intake, or wheel running were observed between control adipose recipients and obese adipose recipients during baseline. However, during the restriction phase, obese adipose recipients remained in the ABA paradigm longer than control adipose recipients (p < .01). Despite the increase in survival time observed in obese adipose recipients, there were no differences in body weight, food intake, or wheel running between the groups. In the metabolic chamber study, no differences were found between groups for any measure. Conclusions: Our results indicate that transplanting adipose tissue from high fat diet (HFD)-fed obese mice into recipient mice extends survival time in the ABA paradigm, defined as retaining > 75% of baseline body weight. These findings are consistent with the hypothesis that diffusible factors released by obese adipose tissue prevents weight loss. Studies are underway to examine the mechanisms underlying this effect. Keywords: Food Intake, Wheel Running, Body Weight, Energy Metabolism, Transplantation Disclosure: Nothing to disclose. P191. A Hypothalamic to Infralimbic Melanocortin Circuit Regulates Food Intake and Food-Seeking Behavior Priyanka Das, Michaela Cooke, Angela Kim, Kate Callahan, Yan Li, Vadim Bolshakov, Kerry Ressler, Rachel Ross Albert Einstein College of Medicine, Bronx, New York, United States Background: The melanocortin-4-receptor (MC4R) is implicated in metabolism and energy expenditure, and mutations of the MC4R are strongly linked to obesity in humans and mice. Its activity is regulated by peptides released from arcuate feeding and satiety neurons, agouti related protein (AgRP) and pro-opiomelanocortin (POMC). The MC4R is highly expressed in the hypothalamus, but deletion of MC4R from this region does not recapitulate the obesity induced by global brain knockout, suggesting MC4Rs in other regions are involved. The MC4R is expressed in the infralimbic cortex (IL), an area of the brain involved in decision making and habitual activity. Human imaging data implicates this region in obesity-related food cue responses. We hypothesized that MC4R activity in the IL (IL-MC4R) is regulated by peptides released from hypothalamic neurons, and also influences food intake and food-seeking behavior Methods: We used a combination of genetically modified mice (male and female) with viral vectors to manipulate projections from molecularly defined melanocortinergic arcuate neurons (AgRP and POMC) at the terminal region in the infralimbic prefrontal cortex (IL), as well as the MC4R-expressing neurons in the IL directly. We used slice electrophysiology to test the pharmacologic function of the MC4R in the IL, and immunohistochemistry to further define the molecular identity of these ILMC4R neurons. Results: We found that MC4R agonists applied in slice depolarized the membrane and increased excitability of IL-MC4R neurons. These neurons are also glutamatergic and project to areas associated with food-motivated behavior. Using optogenetic manipulation, we found that terminal stimulation of AgRP axons in the IL increases food intake acutely. Lastly, we used viral-cre manipulation in male MC4Rlox/lox mice to selectively delete IL-MC4R and observed an increase in food intake and body weight, as well as a delay in food-seeking and consuming behavior in an open field environment. Conclusions: Our data highlights a novel population of MC4R-expressing neurons in the IL that receive functional input from hypothalamic feeding neurons, and influences food intake and other food-seeking behavior. Keywords: Neuroendocrine, Eating Disorders, Melanocortin, Infralimbic Cortex, Food Intake Disclosure: Nothing to disclose. P192. Agrp Neurons Coordinate the Mitigation of Activity-Based Anorexia Ames Sutton Hickey, Sean Duane, Laura Mickelsen, Ahmed Shamma, Anna Skillings, Chia Li, Michael Krashes National Institutes of Health, Bethesda, Maryland, United States Background: Anorexia nervosa (AN) is a debilitating and deadly disease characterized by low body mass index due to diminished food intake, and oftentimes concurrent hyperactivity. A high percentage of AN behavioral and metabolic phenotypes can be replicated in rodents given access to a voluntary running wheel and subject to food restriction, termed activity-based anorexia (ABA). Despite the well-documented bodyweight loss observed in AN human patients and ABA rodents, much less is understood regarding the neurobiological underpinnings of these maladaptive behaviors. Hunger-promoting hypothalamic agouti-related peptide (AgRP) neurons have been well characterized in their ability to regulate appetite, yet much less is known regarding their activity and function in the mediation of food intake during ABA. Methods: Female C57Bk6, AgRP-iCre, AgRP-iCre::hM3Dq, AgRP-iCre::hM4Di, or littermate controls, were used in these studies (n = 3-11). Mice were single housed and separated into one of three behavioral groups (Activity, FR, ABA) with access to ad libitum (Activity) or time restricted (FR, ABA) food and a voluntary running wheel (Activity, ABA only). Food access in FR and ABA cohorts was restricted to the first 3 hours of the dark cycle. Feeding information was collected using FED3 devices, and detection or manipulation of neural circuits was performed using in vivo fiber photometry or chemogenetics, respectively. Paired t-tests, unpaired t-tests, one-way ANOVAs followed by Bonferroni post-hoc tests (if applicable), two-way ANOVAs followed by Bonferroni post-hoc tests (if applicable), or two-way mixed ANOVAs followed by Bonferroni post-hoc tests (if applicable) were calculated as appropriate. Normality and homogeneity of variances were tested and, if necessary, accounted for using the Shapiro-Wilk and Levene’s tests, respectively. Significance was determined for p < 0.05. Results: We demonstrate that ABA mice decreased food intake due to increased interpellet interval retrieval and diminished meal number. Longitudinal activity recordings of AgRP neurons in ABA animals exhibited a maladaptive inhibitory response to food. We then demonstrated that ABA development or progression can be mitigated by chemogenetic AgRP activation through the reprioritization of food intake (increased meal number) over hyperactivity, but only during periods of food availability. Conclusions: Taken together, we have ascertained the unique capability, necessity and circadian timeframe required for AgRP neurons to ameliorate ABA despite disrupted neural activity responses to food. These results elucidate a potential neural target for the amelioration of behavioral maladaptations present in AN patients. Keywords: Feeding Behavior, Hypothalamus, Hyperactivity, Arcuate, Eating Disorders Disclosure: Nothing to disclose. P193. The Role of Insular Cortex NOS1 Neurons in Non-Homeostatic Feeding Maria Jose Olvera Caltzontzin, Darielle Lewis-Sanders, Yang-Sun Hwang, Sarah Stern Max Planck Institute, Jupiter, New York, United States Background: Feeding is a complex motivated behavior that is modulated by both homeostatic and non-homeostatic factors. Changes in feeding behavior can result in increased consumption leading to obesity, as well as to decreased feeding in the case of anorexia nervosa. Both cases have profound clinical implications and have been largely intractable to pharmaceutical interventions. We have recently identified the insular cortex as a brain region implicated in controlling learned feeding behaviors that may contribute to obesity and eating disorders. Specifically, Nos1 neurons in the insular cortex were required for the expression of a learned overconsumption task. However, little is known about how insular cortex Nos1 neurons code for feeding-related behaviors and the mechanism by which they control overconsumption. Here we used a combination of chemogenetics and calcium recordings to gain a comprehensive understanding of how Nos1 neurons contribute to non-homeostatic changes in feeding. Methods: Nos1-Cre mice (n = 6-10) were injected with a cre-dependent virus expressing the inhibitory DREADD, hM4Di (AAV-FLEX-hM4Di-mCherry) into the insular cortex. Mice were tested in a number of tasks including learned overconsumption, conditioned taste aversion, anxiety tasks (light-dark box, elevated zero maze, novelty suppressed feeding) and object recognition memory. Mice were also tested for homeostatic feeding of regular chow and palatable food (e.g. Ensure). Saline or CNO was injected 30 minutes prior to testing in both mCherry controls and hM4Di expressing mice. A separate cohort of Nos1-Cre mice (n = 4-6) were injected with a cre-dependent virus expressing the calcium indicator GCaMP in the insular cortex, and a fiber optic was implanted above the injection. Calcium recordings were done with the Neurophotometrics fiber photometry system during the previously mentioned tasks and data was analyzed with custom Matlab and Python scripts. Results: Inhibition of insular cortex Nos1 neurons resulted in changes in behavior only in associative learning feeding tasks. While inhibition of Nos1 neurons prevented learned overconsumption, it surprisingly enhanced the conditioned taste aversion. We therefore tested whether Nos1 neurons lead to increased anxiety or altered memory, but we did not observe any changes in anxiety-related behaviors, object recognition memory, palatability, or homeostatic feeding. We therefore used fiber photometry recordings to correlate Nos1 activity to behavior. We found strong correlations between Nos1 activity and food consumption bouts, but not correlation to food approach or investigation. We also found increased activity in Nos1 neurons when mice were placed in a novel context and when exposed to novel, noxious odors. Conclusions: We have found that Nos1 neurons are specifically involved in controlling the expression of learned feeding behaviors, but not anxiety, memory, or homeostatic feeding. Their calcium activity similarly correlates quite closely with food consumption bouts and olfactory and contextual cues that might be important for associating cues to food availability. Through Nos1 neurons, the insular cortex is therefore able to provide top-down control of complex feeding behaviors. Keywords: Insular Cortex, Nos1, Feeding Behavior Disclosure: Nothing to disclose. P194. The Neuropeptide PACAP Enables Glutamate Signaling Between Nucleus Accumbens Astrocytes and Neurons to Regulate Behavioral Control Gregory Simandl, Evan Hess, Linghai Kong, Sara Kassel, Nicholas Raddatz, Brian Maunze, Qing-song Liu, Sujean Choi, David Baker Biomedical Sciences, Marquette University, Milwaukee, Wisconsin, United States Background: Prior work implicates system xc- (Sxc-) mediated glutamate signaling between astrocytes and neurons in drug seeking and other forms of maladaptive behavior. Here, we investigated which forms of cognition are dependent on Sxc- and how neurons regulate glutamate release from astrocytes to enable sophisticated cognitive control of behavior. Our hypothesis was that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is released from cortico-striatal inputs to the nucleus accumbens where it regulates astrocyte to neuron signaling to enable cognitive control over behavior. Methods: Rat nucleus accumbens afferent and efferents were labeled by injecting the retrograde neural tracer Cholera Toxin b into the nucleus accumbens or substantia nigra/ventral pallidum, respectively. Fluorescent-activated cell sorting was used to isolate nucleus accumbens astrocytes, efferents, and afferents. mRNA was measured using PCR or in situ hybridization. System xc-dependent glutamate release from nucleus accumbens astrocytes was measured using a cystine-evoked glutamate release assay coupled with reverse-phase HPLC. Neuronal activity in nucleus accumbens medium spiny neurons were measured using slice electrophysiology. Cocaine self-administration and primed reinstatement was performed using a long-access protocol (12 days). Working memory, impulsivity, decision-making, and cognitive flexibility were measured using Bussey-Saksida Touchscreens. Results: Mutant rats lacking Sxc (MSxc rats) display significant deficits in decision making in the Rodent Gambling Task (N = 10/genotype; session x genotype x stimulus: F42,1008 = 1.387, p < 0.05), cognitive flexibility in set shifting paradigm (N = 6-7/genotype; Trials to criterion: Extradimensional Shift: t11 = 0.3.83, p < 0.05 relative to WT; Interdimensional Shift: t11 = 2.96, p < 0.05 * relative to WT; Perseverative Errors: t11 = 3.39, p < 0.01), increased impulsivity in 5-choice serial reaction time task (N = 6-7/genotype; t11 = 2.217, p < 0.05), increased levels of punished behaviors in Pavlovian conditioning (N = 9-10/genotype; session: F4,68 = 7.633, p < 0.05; genotype: F1,68 = 6.187, p < 0.05), and cocaine primed reinstatement (N = 13/genotype, t11 = 3.26, p < 0.05 * relative to WT). We also show that PACAP is expressed in nucleus accumbens inputs from the prefrontal cortex and that PACAP increases glutamate release from Sxc- in nucleus accumbens astrocytes (N = 6-12/genotype; genotype: F1,40 = 275.645, p < 0.05; treatment: F3,40 = 4.492, p < 0.05; genotype x treatment: F3,40 = 4.003, p < 0.05). To our surprise, we also found that PACAP acts at PAC1R expressed by MSNs to remove magnesium block from GluN2B-containing NMDA receptors, thereby enabling these receptors to be activated by Sxc- released glutamate. Lastly, intra-nucleus accumbens PACAP signaling blocks cocaine-primed reinstatement (treatment: F1,18 = 13.03, p < 0.05; test procedure: F1,18 = 45.89, p < 0.05; treatment x test procedure: F1,18 = 13.82, p < 0.05), and this effect requires intact Sxc- and GluN2B-containing NMDA receptor activity (treatment: F3,52 = 5.646, p < 0.05; genotype: F1,52 = 3.666, p > 0.05; treatment x genotype: F3,52 = 10.148, p < 0.05). Conclusions: PACAP is expressed in cortico-striatal inputs to the nucleus accumbens and it enables a form of astrocyte to neuron signaling by increasing glutamate release from Sxc- and enabling activation of GluN2B-containing NMDA receptors. The consequence of PACAP-enabled astrocyte to neuron signaling is improved behavioral control resulting in reduced cocaine-primed drug seeking, only when Sxc- is intact. Next steps will be to determine whether PACAP release from cortico-striatal inputs is regulating behaviors dependent on Sxc- mediated signaling between astrocytes and neurons. Keywords: Glutamate, Astrocyte, PACAP, Cognitive Control Disclosure: Nothing to disclose. P195. Dopamine D2 Receptors in Accumbens Cholinergic Interneurons Increase Delay-Based Impulsive Choice Julianna Cavallaro, Jenna Yeisley, Ruby Setara, Joseph Floeder, Basak Akdogan, Peter Balsam, Eduardo Gallo* Fordham University, Bronx, New York, United States Background: Impulsive choice, often characterized by excessive preference for small, short-term rewards over larger, long-term rewards, is a prominent feature of substance use and other neuropsychiatric disorders. Growing evidence implicates nucleus accumbens (NAc) dopamine and its actions on D2 receptors (D2Rs) in various forms of impulse control. Because several NAc cell types and afferents express D2Rs, it has been difficult to determine the specific neural mechanisms linking NAc D2Rs to impulsive choice. Among those cell types, the cholinergic interneurons (CINs), which express D2Rs, are key regulators of striatal plasticity and local dopamine release, but their role in impulsive behavior is unknown. Our recent work showed that increased D2R expression specifically in NAc CINs impairs learning to suppress actions in a Go/No-Go task. While this is consistent with increased action impulsivity, here we set out to determine the contribution of CIN D2Rs to impulsive choice. Methods: To determine whether increased D2R levels in NAc CINs contribute to impulsive choice, we first delivered Cre-dependent adeno-associated viruses (AAV) expressing D2R-EGFP or EGFP into the NAc of adult ChAT-Cre mice (8 mice/group, both sexes). Mice were trained on a delay discounting task that measures the choice between pressing a lever to obtain a small, immediate reward or pressing another lever to obtain a larger reward (3X) that was presented after increasing delays. After sessions in which both large and small rewards were delivered immediately following a lever press, delays to the large reward (2, 4, 6, 8, 10 s) were introduced across sessions in ascending order. We also used a probabilistic discounting task in a separate cohort of ChAT-Cre mice overexpressing either D2Rs or EGFP in NAc CINs (n = 8 mice/group). Mice had a choice between a small, certain reward and a large reward whose probability was progressively decreased (80, 60, 50, 40, 33, 20%). In both tasks, the mean percent of choices made on the “large” lever was analyzed using two-way repeated measures ANOVA. We further selectively inactivated the D2R gene in CINs using ChAT-IRES-Cre x Drd2flx/flx (CIN D2-KO) mice to measure the effect on both delay and probability discounting. Next, we determined whether CIN D2R upregulation altered the representation of time intervals using a peak interval task. After learning that lever presses were only reinforced after a 24-s fixed interval, mice received “peak” trials in which lever presses were not reinforced. Individual lever press rate data (peak location and width) were used to assess timing accuracy and precision. Furthermore, we used a temporal discrimination task to examine the ability to correctly categorize two auditory tones of different durations as short or long (2 vs. 8 s and 6 vs. 24 s). Results: In the absence of any delays to the large or small reward, D2R-overexpressing and control mice similarly increased their preference for the large reward. As delays to the large reward increased, both groups showed discounting of the large reward, as shown by reduced choice of the large reward. This discounting, however, was significantly steeper in D2R-overexpressing mice compared to controls (virus x delay interaction: F (5, 70) = 6.13, p < 0.05). We then tested whether this manipulation would also alter probabilistic discounting, another dimension of impulsive choice in which rewards are discounted as they become more uncertain. Indeed, we found that greater uncertainty increased discounting (increased small, certain reward choices) in both groups, but there was no significant effect of D2R upregulation. To determine whether D2Rs in CINs are required for impulsive choice, we measured delay and probability discounting in CIN-D2KO and control Drd2flx/flx mice. Both groups discounted the large reward after greater delays, but CIN-D2KO switched less readily to the small, immediate option, indicating decreased delay discounting. In contrast, we found no effect of CIN D2R deletion on probabilistic discounting. Furthermore, D2R-overexpressing and control mice showed comparable response rates and distribution near the target of 24 s in the peak interval task. The temporal discrimination task also revealed no group differences in correctly categorizing long vs short tones, even when durations were proportionally increased. Conclusions: Our findings show that increasing CIN D2R expression in the NAc leads to increased delay discounting without altering probabilistic discounting. This is supported by complementary data from CIN-D2KO mice showing that delay discounting, but not probabilistic discounting, is decreased compared to controls. These results suggest that CIN D2Rs promote impulsive choice involving delayed but not uncertain rewards. Because we observed no group differences in choices when both rewards were delivered immediately, the delay discounting effect is likely not due to different sensitivity to reward magnitude. Moreover, the lack of effect in peak interval and temporal discrimination tasks suggests that the ability to time intervals is not fundamentally altered by D2R upregulation. Together, these findings suggest that dopamine signaling via D2Rs expressed in CINs plays a key role in delay-based impulsive choice, providing new insight into the mechanisms by which NAc dopamine regulates CIN function and impulsive behavior. Keywords: Cholinergic Interneuron, D2 Dopamine Receptor, Impulsive Behavior, Nucleus Accumbens, Delay Discounting Disclosure: Nothing to disclose. P196. Optogenetic Stimulation of ESR1-Expressing Neurons in PVT Reveals a Putative Neural Substrate for Outgroup, but Not Ingroup, Aggression Brandy Briones, Marissa Borrego, Prabhat Aluri, Alondra Torres, Jason Siputro, Garret Stuber University of Washington, Seattle, Washington, United States Background: Navigating novel social environments is complex, multisensory, and cognitively demanding, where mammals have very limited initial information to guide their social behaviors. In some cases, this can lead to increased perceived threat and aggression towards an unfamiliar outgroup member, a phenomenon known as ingroup bias. Kinship and familiarity reduce aggression-related interactions in mice, however the neural circuits and mechanisms involved in ingroup versus outgroup social behavior biases remain to be determined. The Paraventricular Thalamus (PVT) is a critical node for integrating sensory features and emotional state information within novel and aversive contexts. However, the integration of social information in the PVT within these contexts and whether steroid hormones regulate PVT activity remains unresolved. Our study identifies a subset of steroid hormone receptor neurons in the posterior PVT involved in male outgroup aggression. Methods: Using a multiplexed hybridization chain reaction assay, we characterized sex steroid hormone-related gene expression to identify distinct cell clusters in the PVT of male and female mice (n = 3 male, n = 4 female). With a transgenic Esr1-Cre + /- mouse line, we virally-targeted these steroid hormone receptor-expressing neurons in the PVT to investigate the role of steroid hormone genes in social behaviors. After testing a variety of behavior assays with optogenetic stimulation, (e.g., real time place preference, open field, elevated plus maze, and freely moving social interaction), we uncovered a male-specific aggression phenotype and focused the remainder of our study on male-male aggression in the resident intruder paradigm (group sizes of 8-10). In a resident-intruder paradigm, we tested male outgroup aggression bias in wildtype mice (n = 8) and recapitulated the behavior in the absence of optogenetic manipulation. Results: Across the anterior-posterior axis of the PVT, we analyzed approximately 10,000 cells. Estrogen and androgen receptor-related genes, specifically Esr1, Esrra, and Rora, were highly expressed in the central and posterior areas of PVT, further corroborated by expression patterns of viral-labeled neurons in Esr1-Cre + /- mice. Additionally, Esr1, Esrra, and Rora were found to be highly co-expressed (>50%) within the same cell population, suggesting that sex steroid receptor-dependent activity in the PVT occurs in the same subset of cells, demonstrating the Esr1-cre + /- mouse a viable model for investigating sex steroid hormone modulation in the PVT during social behavior. During a resident intruder paradigm, optogenetic activation of Esr1+ PVT neurons in the resident mouse elicited an aggression phenotype specific to males, where male mice initiated aggressive attacks time-locked to light stimulation towards outgroup, but not ingroup, mice (p < 0.05). Additionally, we observed approximately 50% of wildtype C57BL/6 male mice with sexual experience exhibit outgroup, but not ingroup, aggression in the resident intruder paradigm in the absence of any optogenetic manipulation. Viral-labeling of Esr1+ PVT neurons in Esr1-Cre + /- mice also revealed dense projections to amygdala and extended amygdala regions, bed nucleus of the stria terminalis, ventromedial nucleus accumbens, insular cortex, and sensory cortices, providing us with downstream targets to dissect this behavior further. Conclusions: From the data we have collected thus far, our results suggest that activation of the central and posterior PVT, specifically in subsets of neurons expressing estrogen and androgen receptor-related genes, gate outgroup aggression. Future directions of our study will aim to investigate 1) in vivo PVT neural activity in Esr1+ neurons during freely moving social behavior, 2) whether estrogen and/or androgen receptors in the PVT are necessary for outgroup attack behavior, and 3) if circulating hormones alter the activity in this neuron population. Keywords: Paraventricular Nucleus of the Thalamus, Aggression, Ingroup Bias, Sex Steroid Hormone Receptors, Fluorescence in Situ Hybridization Disclosure: Nothing to disclose. P197. Distinct Behavioral and Neural Responses With Environmental Cues Associated With Addicted Behaviors Yui Asaoka, Moojun Won, Emi Ishikawa, Tomonari Morita, Yukiori Goto Kyoto University, Kyoto, Japan Background: Behavioral addiction, such as addiction to gambling, Internet use, and gaming, has been recognized, but its concept and definition are still undetermined. Some impulse control disorders, such as kleptomania and paraphilia, have been suggested to meet the criteria of addiction, but are not legitimately categorized as addictive disorder, due to insufficient studies for comprehensively understanding these disorders as a behavioral addiction. In this study, we investigated how environmental cues associated with the disorder were processed in patients with kleptomania to gain further insights about it as behavioral addiction. Methods: The images of the grocery store with (MKT + H) and without (MKT-H) a person, those of outside with (OUT + H) and without (OUT-H) a person, and those of foods (FOD) and stationary (STY) were presented to healthy adult subjects (n = 27; 11 males, 16 females) and patients (n = 11; 4 males, 7 females), who had been hospitalized for treatment of kleptomania with stealing foods in grocery stores. The areas of interest (AOIs) were set around the person within the MKT + H and OUT + H images. Their gazing patterns on the images were measured with eye-tracking, and oxy- and deoxy-hemoglobin changes while subjects gazed the images were simultaneously recorded at 10 regions within the prefrontal cortical (PFC) area with functional near infrared-spectroscopy. Results: Bayesian ANOVA supported no overall difference in any of the number, duration, and dispersion fixations, the number of blinks, and changes (expressed as coefficient of variance) of pupil diameters over the course of gazing between the patients and healthy subjects as well as between images. Bayesian t-test or Mann-Whitney U test supported, although weakly, the alternate hypothesis over the null hypothesis in duration (BF10 = 2.41, error % = 0.007) and dispersion (BF10 = 2.41, error % = 0.009) of fixations made on the AOI of the MKT + H image, and those (BF10 = 4.51, error % = 0.001 in duration; BF10 = 1.927, error %=0.018 in dispersion) on the OUT-H image. The number of recording sites within the PFC where oxy- or deoxy-hemoglobin responses significantly increased or decreased from the baseline (quantified as area under the curve, with significance defined as p < 0.05 with t-test) over the courses of gazing the images was overall less in patients than that of healthy subjects. In addition, the principal component analysis with PFC oxy- and deoxy-hemoglobin changes unveiled that the correlations were strong across all images (correlation coefficients in the range of 0.912 to 0.986) in the healthy subjects, whereas in the patients, the correlations were strong between images (correlation coefficients in the range of 0.865 to 0.987) except those between the MKT-H and any other images, where they were substantially lower in the range of 0.272 to 0.643. Conclusions: These results suggest that patients with kleptomania exhibit distinct behavioral and neuronal responses than healthy subjects to the environmental stimuli associated with their uncontrolled behaviors. Keywords: Addiction, Eye Tracking, fNIRS, Prefrontal Cortex Disclosure: Nothing to disclose. P198. Preliminary Evidence of Reduced Ventral Pallido-Striatal Synaptic Density in Opioid Use Disorder: A Pilot 11C-UCB-J PET Study Robin Bonomi, Mika Naganawa, Marcella Mignosa, Patrick Skosnik, Irina Esterlis, Nabeel Nabulsi, Marc Potenza, Richard Carson, Kelly Cosgrove, Robert Malison, Gustavo Angarita-Africano Yale University, New Haven, Connecticut, United States Background: Decreased dendritic spine density has been demonstrated within the brain reward pathway (nucleus accumbens and prefrontal cortex) in rodents following abstinence from opiates [1, 2]. Whether similar micro-architectural alterations are present in humans with opioid use disorder (OUD) is unknown. Positron Emission Tomography (PET) imaging using the radioligand 11C-UCB-J, which selectively binds to the synaptic vesicle protein 2A (SV2A), is a novel method for visualization and quantification of synaptic density in the living human brain [3, 4]. Recently, PET imaging studies with 11C-UCB-J demonstrated relatively decreased synaptic density in other substance use disorders [5, 6]. Methods: Individuals with OUD, as diagnosed by DSM-5 criteria, (N = 6, 36.7 + /-7.8 years, BMI: 28.3 + /- 7.1 kg/m2, all white male), underwent inpatient detoxification and monitored abstinence prior to PET imaging with 11C-UCB-J. Demographically matched healthy controls (N = 6, 38.5 + /- 8.7 years, BMI: 25.0 + /- 3 kg/m2, all white male) also participated in one 11C-UCB-J PET scan to assess SV2A density differences between the two populations. Female subjects also participated but unfortunately arterial line blood data was not able to be measured from these three individuals for a variety of reasons. The volume of distribution (VT) calculated using a one-tissue compartmental model with arterial input function with regions of interest analyzed and compared including the anterior cingulate (ACC), dorsomedial and ventromedial prefrontal cortex (PFC), lateral and medial orbitofrontal cortex (OFC), globus pallidus, and striatum. Groups were compared using independent two-sample, two-tailed t-tests. Results: Initial PET results showed a significantly lower VT in regions of the pallido-striatal system, including globus pallidus (VT: N = 6; -18%; p = 0.03), putamen (VT: N = 6; -12 %; p = 0.035), and ventral striatum (VT: N = 6; -13%; p = 0.07) in OUD participants. Additionally, a significant negative correlation was found between smoking tobacco (cigarettes/day) and globus pallidus VT values (N = 6, slope = -0.1596, P = 0.01), other regions were tested but not found to be significant. No significant correlation was found between opiate dose used and VT values for this sample cohort. Conclusions: These preliminary analyses 11C-UCB-J PET suggest decreased synaptic density within the striatum and globus pallidus among humans with OUD. To our knowledge, this represents the first in vivo clinical imaging demonstrating synaptic density differences in OUD as compared to HC participants. Though these preliminary findings align with literature preclinical work, there are several limitations such as small samples, no females, and potential confounding effects for nicotine. Limitations are being addressed with ongoing recruitment. Future analyses should employ partial volume corrections as well as in depth between groups’ comparison for demographics. If these preliminary differences within OUD participants are confirmed, they would support future studies of agents with synaptotropic effects [7-13]. References: 1. Robinson, T.E. and B. Kolb, Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats. Synapse, 1999. 33(2): p. 160-2. 2. Robinson, T.E., et al., Widespread but regionally specific effects of experimenter- versus self-administered morphine on dendritic spines in the nucleus accumbens, hippocampus, and neocortex of adult rats. Synapse, 2002. 46(4): p. 271-9. 3. Finnema, S.J., et al., Imaging synaptic density in the living human brain. Sci Transl Med, 2016. 8(348): p. 348ra96. 4. Nabulsi, N.B., et al., Synthesis and Preclinical Evaluation of 11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain. J Nucl Med, 2016. 57(5): p. 777-84. 5.Angarita, G.A., et al., Lower prefrontal cortical synaptic vesicle binding in cocaine use disorder: An exploratory (11) C-UCB-J positron emission tomography study in humans. Addict Biol, 2022. 27(2): p. e13123. 6. D’Souza, D.C., et al., Preliminary in vivo evidence of lower hippocampal synaptic density in cannabis use disorder. Mol Psychiatry, 2021. 26(7): p. 3192-3200. 7. Kelly, J.R., et al., The psychedelic renaissance: the next trip for psychiatry? Ir J Psychol Med, 2019: p. 1-5. 8. Ly, C., et al., Psychedelics Promote Structural and Functional Neural Plasticity. Cell Rep, 2018. 23(11): p. 3170-3182. 9. Olson, D.E., Psychoplastogens: A Promising Class of Plasticity-Promoting Neurotherapeutics. J Exp Neurosci, 2018. 12: p. 1179069518800508. 10. Duman, C.H. and R.S. Duman, Spine synapse remodeling in the pathophysiology and treatment of depression. Neurosci Lett, 2015. 601: p. 20-9. 11. de Vos, C.M.H., N.L. Mason, and K.P.C. Kuypers, Psychedelics and Neuroplasticity: A Systematic Review Unraveling the Biological Underpinnings of Psychedelics. Front Psychiatry, 2021. 12: p. 724606. 12. Shao, L.X., et al., Psilocybin induces rapid and persistent growth of dendritic spines in frontal cortex in vivo. Neuron, 2021. 109(16): p. 2535-2544.e4. 13. Raval, N.R., et al., A Single Dose of Psilocybin Increases Synaptic Density and Decreases 5-HT(2A) Receptor Density in the Pig Brain. Int J Mol Sci, 2021. 22(2). Keywords: PET Imaging, Opioid Use Disorder, Synaptic Density, PET Tracer, [11C]UCB-J Disclosure: Nothing to disclose. P199. Correlates and Patterns of Cannabis Use in an ADHD Sample Michael Van Ameringen, Bárbara T. M. Q. dos Santos, Beth Patterson, Maryam Rahat, Juliette Mojgani, Grace Lethbridge, Fernando Sumiya, Carolina Goldman Bergmann McMaster University, Hamilton, Canada Background: Attention-deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, and impulsivity. According to the National Comorbidity Survey Replication the 12-month ADHD prevalence rate of 4.4% for adults [1]. ADHD often leads to adverse outcomes across life domains, as well as an increase in comorbidity with other psychiatric conditions, such as anxiety and mood disorders and substance misuse [2,3]. Although effective the first line ADHD treatments are associated with side effects which often lead to poor treatment adherence [4]. Cannabis is one of the most commonly used psychoactive substances in the world; adult cannabis use in the last 12-months was globally estimated as 3.8%. It has been reported that cannabis has helped improve focus and hyperactivity in individuals with ADHD [5]. Among individuals with ADHD, the prevalence of cannabis use is higher than that in the general population, with estimates of 34-46% in treatment-seeking patients who use cannabis [6]. Although the relationship between ADHD and substance abuse is well-known, the literature examining the effects of cannabis on symptoms of ADHD is both scarce and equivocal. Few studies have examined the prevalence of cannabis use as a self-medication strategy in ADHD, and the effects of cannabis on specific symptoms. This cross-sectional, international survey study aimed to address this gap by asking individuals with ADHD about the frequency and benefits of cannabis use to treat ADHD symptoms. Methods: Adults (all sexes) aged 18 and over with a self-reported lifetime diagnosis of ADHD, completed an online survey, posted between August 2020 and February 2022 in English. After signing an electronic informed consent, a questionnaire battery was completed, containing the Barkley Adult ADHD Rating Scale-IV (BAARS-IV) Quick-Screen, the Generalized Anxiety Disorder-7 (GAD-7), and the Patient Health Questionnaire (PHQ-9) to assess depression severity. Demographics and information regarding current and past substance use, was also collected. Validated cut scores on the GAD-7 (≥ 15) and PHQ-9 (≥ 15) were used to estimate rates of moderate-severe GAD and Major Depressive Disorder (MDD). Statistical methods included descriptive statistics, Chi square for normally distributed variables and Mann-Whitney analyses for non-normal distributions (only the BAARS-IV was non-parametric). Results: The survey was completed by 838. Respondents were primarily female (71%), Caucasian (79%), and educated (41% with a graduate degree); with a mean age of 31.8 ± 9.9; primarily from Canada (56.2%), the U.S. (24.9%) and the U.K (4.4%). Most (76%) were receiving current pharmacotherapy for ADHD. The median BAARS-IV score was 16 (range 5.00 -20.00), indicating that most fell within the 98th to 99th percentiles. The mean scores on the GAD-7 (12.03 ± 5.3) and PHQ-9 (14.65 ± 5.8) were above the clinical threshold for diagnosis; 28.5% met threshold criteria for severe GAD, while 44.7% met criteria for moderately-severe MDD. Although lifetime rates of alcohol (89%) and tobacco (58.9%) use were similar to those in the general population, rates of cannabis use (77.3%), amphetamine/methamphetamine use (29%) and hallucinogens (33.6%) were nearly double the rates found in the general North American population[7,8]. Over half of the sample (55.6%) reported using cannabis in the past 6 months. Within this group of current users (n = 466), 51.1% reported daily or almost daily use of cannabis. Most (62.8%) of the total sample reported using substances to help manage their ADHD symptoms at some point in their life. Within this group (n = 526), 70% had used cannabis to manage their ADHD, reporting that they felt more calm (74.5%), got to sleep easier (70.1%) and felt less irritable or aggressive (59.3%). Although 50.6% reported that cannabis did not worsen their ADHD symptoms, respondents also said that cannabis worsened the ADHD symptoms of losing my train of thought (45.1%), having more difficulty staying focused (31.9%) and having more difficulty with organizing tasks and activities (27.8%). ADHD symptom severity was not associated with cannabis use or comorbid anxiety symptoms. However, those with moderate-severe MDD had significantly higher BAARS-IV scores (p < .0001). Individuals with moderate-severe GAD and MDD were significantly more likely to report current cannabis use (p < .01 – GAD; p < =.05 – MDD) as well as significantly higher frequency of current cannabis use (p < .00001 for both). Conclusions: In this online sample of respondents with ADHD, the rates of lifetime substance use (excluding alcohol and tobacco) were much higher than North American population norms. This is especially surprising given that a high proportion of the sample was female. Substance use is typically higher in male populations. Furthermore, although most of the sample reported using cannabis to treat their ADHD symptoms, the benefits are unclear as the symptoms noted to have improved aren’t core ADHD symptoms. It does appear that those with comorbid anxiety and depressive symptoms are using more cannabis than those without this comorbidity raising the possibility that cannabis maybe benefiting anxiety and depressive symptoms in this group. Keywords: ADHD, Cannabis Use, Anxiety, Major Depression Disclosures: Allergan, Almatica, Bausch Health, Brainsway, Elvium, Empowerpharm, Jazz, Lundbeck, Otsuka, Tilray, Vistagen: Advisory Board (Self), Biohaven, Otsuka: Contracted Research (Self), Canadian Institute for Health Research, Michael G. DeGroote Centre for Medicinal Cannabis Research, Elvium: Grant (Self), UptoDate: Honoraria (Self), Abbvie, Allergan, Elvium, Lundbeck, Otsuka, Pfizer, Sunovion, Takeda: Speakers Bureau (Self) P200. CADM2 is Implicated in Impulsive Personality Traits by Genome- and Phenome-Wide Association Studies in Humans, With Further Support From Studies of CADM2 Mutant Mice Sandra Sanchez-Roige, Mariela Jennings, Hayley Thorpe, Jazlene Mallari, Lieke van der Werf, Sevim Bianchi, Calvin Lee, Travis Mallard, Samuel Barnes, Jin Yi Wu, Amanda Barkley-Levenson, Ely Boussaty, Cedric Snethlage, Danielle Schafer, Zeljana Babic, Boyer W Winters, Katherine Watters, Thomas Biederer, James MacKillop, David Stephens, Sarah L Elson, Pierre Fontanillas, Jibran Khokhar, Jared Young, Abraham Palmer* University of California San Diego, La Jolla, California, United States Background: Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. Methods: We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509-133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed a phenome-wide study (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and tested them using a battery of analogous behavioral tasks. Results: In humans, impulsive personality traits showed modest chip-heritability (∼6-11%), and moderate genetic correlations (rg = .20-.50) with other personality traits, and various psychiatric and medical traits. We replicated associations from earlier GWAS of these traits and found novel associations including DRD2, CRHR1, FOXP2, TCF4, PTPRF. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Cadm2 mutant mice recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Conclusions: Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries, with further support from studies of Cadm2 mutant mice. Keywords: GWAS, Impulsivity, Mouse Genetics, Polysubstance Abuse Disclosures: Vivid Genomics: Advisory Board (Self), Own a patent: (Self) P201. Investigating Fatty Acid Amide Hydrolase in Comorbid Borderline Personality Disorder and Major Depression: A [11C]CURB Positron Emission Tomography Study Nathan Kolla, Michelle De Pol, Dorsa Rafiei University of Toronto, Toronto, Canada Background: Borderline personality disorder (BPD) is a debilitating psychiatric condition that is often comorbid with major depressive disorder (MDD). However, there is a lack of research investigating biomarkers that are common to both disorders. The endocannabinoid system (ECS) is a potent modulator of mood and stress circuits, and its dysregulation has been implicated in both animal and human models. Fatty acid amide hydrolase (FAAH), an enzyme of the ECS, plays a vital role in the metabolism of endogenous cannabinoids. Preclinical evidence suggests that FAAH levels are elevated within fronto-limbic brain regions in MDD, and a previous positron emission tomography (PET) study found elevated FAAH in the prefrontal cortex of humans with BPD. A FAAH gene variant (rs324420) also contributes to differential levels of FAAH expression. The ECS, however, has never been characterized in humans with comorbid BPD + MDD using in vivo imaging techniques. We hypothesized that brain FAAH would be elevated in the prefrontal cortex, hippocampus, and anterior cingulate cortex in humans with BPD + MDD compared to healthy controls using PET. Methods: Ten BPD + MDD patients, aged 18 to 65 years, and 10 age-matched healthy controls were included in this pilot study. There were nine females in the comorbid group and eight females in the healthy group. Eligible psychiatric participants were unmedicated and met criteria for a current major depressive episode in addition to BPD. Control participants were excluded if they had any history of psychiatric disorder. All participants had negative drug urinalysis testing on the scanning days. FAAH binding was measured using PET with the radiotracer [11C]CURB. Participants also underwent genotyping to determine the FAAH gene polymorphism. Mood and personality questionnaires were administered for exploratory correlational analyses. A linear mixed effects model was employed to determine the differences in FAAH binding between the two groups, with genotype as a fixed factor. Results: No significant differences in levels of brain FAAH were observed between BPD + MDD patients and controls in each examined brain region, including the prefrontal cortex, anterior cingulate cortex, and hippocampus. A whole brain analysis similarly did not find differences in the amygdala, temporal cortex, insula, thalamus, dorsal caudate, ventral striatum, and cerebellum between groups following Bonferroni correction. However, exploratory analyses revealed a significant positive correlation between FAAH binding in the ventrolateral prefrontal cortex (vlPFC) and impulsivity, as measured by the Barratt Impulsiveness Scale-11 (BIS-11), within the BPD + MDD group (r = 0.635, p = 0.048). Conclusions: Considering the lack of response of current therapeutics by patients with BPD and MDD, research for novel biomarkers of these conditions is necessary. Our pilot study is the first to investigate FAAH in comorbid BPD + MDD using [11C]CURB PET. No significant group differences related to FAAH binding were observed. However, our sample at this point is likely underpowered to observe an effect. Our exploratory analyses suggest that prefrontal FAAH may be related to impulsive behavior in BPD + MDD, which is relevant because impulsivity is a core feature of BPD. Faulty vlPFC functioning has been implicated in impulsive decision-making, and it is possible that elevated FAAH in the vlPFC impacts neurotransmission to lead to impulsivity. Further research, however, is required to elucidate this potential mechanism. Keywords: Borderline Personality Disorder, Major Depressive Disorder, Endocannabinoid System, Positron Emission Tomography (PET), Fatty Acid Amide Hydrolase, PET, Cannabis Disclosure: Nothing to disclose. P202. Sex-Dependent Effects in Dopaminergic Modulation of Risky Decision-Making in Rats Samantha Ayoub, Avraham Libster, Stephanie Dulawa, Jared Young University of California - San Diego, San Diego, California, United States Background: Dopamine (DA) modulates risky decision-making across species. Psychiatric disorders known to impact DA functioning (i.e., schizophrenia, bipolar disorder) exhibit worse performance in risky decision-making tasks, e.g., the Iowa Gambling Task, while DA agonist treatments, e.g., the DA-2 receptor (DA2R) agonist pramipexole (PPX), result in gambling problems. Using a risk-taking task, Zalocusky (2016) demonstrated the risk preference of male rats can be increased by systemic administration of PPX and decreased by optogenetic silencing of DA2R-expressing neurons in the nucleus accumbens (NAc). Sex-dependent differences were not examined however, and the role of sex or DA2R blockade in risky decision-making remains unclear. Here, we trained female and male rats in the same task to explore sex-differences in risk preference at baseline and in response to the pharmacological challenges of PPX, the DA2R antagonist sulpiride (SUL), and the dopamine transporter (DAT) inhibitor GBR-12909 (GBR). Methods: In operant boxes, rats could choose from one of two nose-poke holes, one that delivered a 50 µl strawberry milkshake reward (safe-option), and the other a 10 µl reward with a 75% probability or a 170 µl reward with a 25% probability (risky-option). Once trained to a stable baseline of risk preference, rats were treated with PPX (0.15 or 0.3 mg/kg; Experiment 1) or SUL (30 mg/kg; Experiment 2) for 3 days, each separated by a saline washout. In experiment 3, animals were tested after an injection of GBR (5 or 16 mg/kg) or Vehicle. The primary outcome variable was percent risk choice (%RC). Results: During baseline, females were less risk-adverse/more risk-prone than males. Experiment 1: collapsed across drug and saline tests, there was a main effect of drug on %RC change from baseline [F(1,18)=10.5, p < 0.01], with PPX increasing %RC. When analyzed across each testing day, a main effect of session [F(6,108)=3.6, p < 0.005] was observed, as was a sessionsexdrug interaction [F(6,108)=2.2, p < 0.05]. Post hoc analyses revealed females differed from males in the timing of their response to PPX based on the dose administered. Experiment 2: collapsed across drug and saline tests, there was a main effect of drug on %RC change from baseline [F(1,6)=20.0, p < 0.01], with SUL decreasing %RC. There was also a trend toward a drugsex interaction [F(1,6)=3.6, p = 0.11], with more pronounced attenuation of %RC by SUL in females. A similar pattern was observed when data was analyzed across all drug and saline tests. Experiment 3: there was a main effect of GBR on %RC change from baseline [F(2,26)=4.0, p < 0.05], with the high dose of GBR (16 mg/kg) increasing %RC compared to VEH and low dose GBR (5 mg/kg; p < 0.05). Conclusions: Together these data indicate a sex-specific modulation of baseline risk preferences, and that female rats may be more sensitive to DA manipulations on risky decisions, highlighting the necessity of tracking sex-based differences in such tasks. The data further support DAT inhibition (GBR) as recreating mania-like risk preference in rodents. Ongoing studies will determine whether DA2R NAc activity reveal a similar sex-specific change in DA during risk-preference. Understanding the neural basis of sex-based differences in risk, and DA modulation of risk, will enhance the management of psychiatric disorders that alter risky-decision making, particularly in those populations prescribed DA-based medications. Keywords: Risky Decision-Making, D2 Dopamine Receptor, Sex-Differences Disclosure: Nothing to disclose. P203. Chemogenetic and Optogenetic Manipulation of Neuronal Projections From the Lateral Hypothalamus to the Paraventricular Nucleus of the Thalamus Impact Cue-Motivated Behaviors Amanda Iglesias, Sara Westbrook, Stephen Chang, Justin Chung, Paolo Campus, Shelly Flagel University of Michigan, Ann Arbor, Michigan, United States Background: The survival of an organism is dependent on their ability to properly respond to cues in the environment. Associative learning processes underlie an individual’s response to such cues. For some, reward-paired cues are attributed with incentive motivational value (incentive salience), and can gain excessive control, leading to maladaptive behavior that is characteristic of psychopathology. To investigate the neural mechanisms that promote incentive learning, we utilize the goal-tracker (GT)/ sign-tracker (ST) animal model. With Pavlovian conditioned approach (PavCA) training, GTs attribute predictive value to reward-cues; whereas STs attribute predictive and incentive value to such cues. The attribution of incentive motivational value, or incentive salience, transforms the cue into an attractive and desirable stimulus. For STs, both food- and drug-associated cues gain excessive incentive value and elicit maladaptive behaviors. The GT/ST model, therefore, can be utilized to elucidate the neurobiological mechanisms that encode adaptive or maladaptive cue-driven behaviors. GTs and STs rely on distinct neurobiological mechanisms and the paraventricular nucleus of the thalamus (PVT) has emerged as a neural hub that mediates their characteristic differences in associative learning. Prior findings have led us to postulate that bottom-up projections to the PVT relay the incentive value of reward-associated cues, with the lateral hypothalamus (LH), which sends dense orexinergic (OXergic) projections to the PVT, acting as a critical neural node. Orexin (OX) is known to play a role in motivation, and administration of OX receptor antagonists into the PVT attenuates the incentive value of food-paired cues in STs. We hypothesize that OXergic transmission in the LH-PVT pathway encodes the incentive motivational value of reward cues and modulates the propensity to sign-track. Thus, we postulate that excitation of the pathway, via optogenetics, would elicit sign-tracking, and inhibition of the pathway following learning, via chemogenetics, would reduce sign-tracking behavior. Methods: Here we investigate the role of the LH-PVT pathway in encoding the value of reward cues via (1) optogenetic excitation and (2) chemogenetic inhibition during PavCA behavior. In PavCA sessions, an illuminated cue (lever) extends for 8 seconds then retracts and a food pellet is dispensed into a food cup. In the optogenetics study, we used transgenic OX-Cre Long Evans male and female rats (N = 11), which express Cre-recombinase in OX neurons, and infused a retrograde Cre-dependent excitatory optogenetic virus (pAAVrg-Ef1α-DIO-ChR2-EYFP; channelrhodopsin, ChR2) or empty vector (pAAVrg-Ef1α-DIO-EYFP; control) into the anterior PVT. Animals received laser-induced excitation of LH-PVT neurons during cue (lever) presentation on sessions 1-5. In the chemogenetic study, we utilized a dual-vector approach to selectively express an inhibitory (Gi) DREADD virus in the LH-PVT pathway in outbred Sprague-Dawley male rats (N = 46). A retrograde Cre virus (pAAVrg-hSyn-EGFP-Cre) was infused into the anterior and posterior PVT, while a Cre-dependent Gi virus (pAAV8-hSyn-DIO-hM4D-mCherry) was bilaterally infused into the LH. Following virus incubation, rats had 7 sessions of PavCA. After acquiring a conditioned response, rats received either vehicle or clozapine-N-oxide (CNO; 5 mg/kg) prior to sessions 5-7 of PavCA to activate the Gi-DREADDs. Results: In the optogenetics study, preliminary results indicate that OXergic LH-PVT pathway stimulation during cue presentation results in increased goal-tracking behavior in ChR2 (n = 6) rats compared to controls (n = 5). This effect appears to be more pronounced in male ChR2 (n = 3) rats relative to male EYFP (n = 2), female ChR2 (n = 3), and female EYFP (n = 3); however, studies are ongoing to increase the sample size. In the chemogenetics study, we assessed the impact of Gi DREADD activation on the expression of a learned conditioned response, session 5-7 of PavCA. A linear mixed-effects model analysis compared the effect of treatment (VEH vs. CNO) and phenotype (ST vs GT vs IR (intermediate responders)) for various behaviors in PavCA across sessions, and for all measures, there was a significant effect of phenotype (P < 0.005). When phenotypes were assessed independently using a repeated measures ANOVA, there was no effect of treatment on sessions 5-7 for either STs (VEH, n = 3; CNO, n = 5) or IRs (VEH, n = 7; CNO, n = 7). However, for GTs, there was a significant effect of treatment, such that CNO-treated GTs (n = 8) had less food-cup contacts (p = 0.034), a lower probability to approach the food-cup (p = 0.002), and a slower food-cup approach (p = 0.01) than VEH-treated GTs (n = 16). Conclusions: These findings suggest that the LH-PVT pathway plays an important role in goal-directed behaviors. Preliminary findings indicate that optogenetic stimulation of OXergic neurons in the LH-PVT pathway of transgenic male rats increases goal-tracking behavior. Further, chemogenetic inhibition of the LH-PVT pathway in outbred male rats decreases the expression of goal-directed behaviors for GTs, without affecting the behavior of STs or IRs. Thus, the LH-PVT pathway appears to modulate predictive value encoding and the expression of goal-directed behaviors both early in training and after a conditioned response has been acquired. Ongoing studies are increasing the sample size in both studies to further investigate a potential role for the LH-PVT pathway in incentive learning and any sex differences that might arise. Keywords: Individual Differences, Lateral Hypothalamus, Paraventricular Nucleus of the Thalamus, Incentive Salience, Pavlovian Conditioning Disclosure: Nothing to disclose. P204. Serotonin Modulation of Dorsal Striatal Medium Spiny Neurons During Reward Anticipation Ka Ng, Arati Sharma, Mitchell Spring, Kate Nautiyal* Dartmouth College, Hanover, New Hampshire, United States Background: Serotonin influences reward processing, threat detection, behavioral inhibition, and other components of motivation and behavioral control that are frequently disordered in a number of psychiatric disorders. In the dorsal striatum (DS), pharmacological manipulation of serotonin signaling disrupts behavioral control and the prospective encoding of rewards. Additionally, many serotonin receptors, including the serotonin 1B receptor (5-HT1BR) have been implicated in regulating reward-motivated and impulsive behaviors. However, the nature of the behavioral encoding/control of reward-related behavior by serotonin is not well understood, nor have the circuit-level mechanisms through which serotonin modulates reward-related behaviors in the dorsal striatum been identified. Methods: We used miniature microendoscopes for calcium imaging of dorsal striatal medium spiny neurons (MSNs) in adult mice during a simple appetitive operant task in the DIY-NAMIC system to analyze reward-related neural dynamics (n = 5; 3 male, 2 female). Additionally, mice lacking 5-HT1BR were also imaged to assess the effect of 5-HT1BR signaling on reward-related MSN activity. In parallel we used a G-protein coupled receptor activation based fluorescent sensor for serotonin (GRAB-5-HT) to measure serotonin release in the dorsal striatum of mice during reward trials delivered with the Davis lickometer (n = 10; 6 male, 4 female). These methods enable measurement of neural dynamics and serotonin signaling on a time scale compatible with single trial reward-related behaviors in Pavlovian and operant paradigms. Results: We analyzed changes in calcium event rates in D1 and D2 MSNs during the different phases of reward seeking behavior and in response to an increased palatable reward. We find that the majority of cells decrease their rate to reward-predicting cues and reward delivery. Using supervised learning algorithms, we were able to accurately classify behaviorally correct, incorrect, and omitted trials based on the neural data during the trials. Calculated accuracy was >99%, and dropped to <40% when training labels were shifted. We also used recursive feature elimination to determine the number and pattern of cells that contributed the most to the classifier performance. Overall, our modeling work indicates that MSN activity in the dorsal striatum uses a relatively small number of cells for ensemble encoding of earned reward. Serotonin levels as measured by the GRAB-5-HT sensor in the DS increased prior to reward receipt. Interestingly, the increase in serotonin signal preceded reward consumption and was closely aligned with the initiation of approach to the reward following the cue signaling reward availability. The majority of licks for the reward (96%) occurred within 2 seconds after the onset of identified significant serotonin rises. Additional studies varying reward concentration with 2 and 6-bottle paradigms are ongoing, but preliminary data suggests there is some increase in the size and duration of serotonin rises with increases in reward value. Additional studies with GRAB-5-HT imaging are focused on parsing out the role of serotonin in encoding expected reward value, anticipation, and motivation. Conclusions: Our results are important for understanding reward value coding in the DS and its modulation by serotonin. Current analysis of calcium imaging data is aimed at identifying the effect of 5-HT1B on MSN-reward related neural activity and determine how classifiers may change with the absence of this serotonin signaling. Characterizing real-time serotonin release and its influence on MSN neural activity and reward-related behaviors is important to understand how serotonin modulates reward processing and behavioral control. Keywords: Serotonin, Reward, GRAB-5HT, Dorsal Striatum Disclosure: Nothing to disclose. P205. Transcriptional Effects of Neuropsychiatric Copy Number Variants in Postmortem Brain Anton Schulmann, Nirmala Akula, Stefano Marenco, Pavan Auluck, Armin Raznahan, Siyuan Liu, Alexandra DeCasien, Qing Xu, Ningping Feng, Bhaskar Kolachana, Barbara Lipska, Xueying (Sherry) Jiang, Asya Khlebodorova, Justin Lack, Francis McMahon NIMH, Bethesda, Maryland, United States Background: Copy number variants (CNVs) are structural variants, where a large stretch of genomic DNA – typically spanning tens of kilobases – is either duplicated or deleted. Several CNVs have been associated with increased risk of neuropsychiatric disorders such as major depression, bipolar disorder, schizophrenia, and autism. These CNVs, while relatively rare, are highly penetrant and pleiotropic, but their impact on gene expression in brain has been little studied. Here we assessed gene expression changes in postmortem brain of carriers of neuropsychiatric CNVs compared to matched controls. Methods: We identified 13 verified carriers of neuropsychiatric CNVs aged 10 or older from SNP genotyping array data across three postmortem brain repositories. Specimens included mirror CNVs on 22q11.2, 16p11.2, 1q21.1, 15q11.2 (BP1-BP2), and the 7q11.23 Williams-Beuren syndrome deletion. Whenever available, two brain regions – dorsolateral prefrontal cortex and anterior cingulate cortex – were profiled from a carrier and two matched controls, resulting in a total sample size of 62. RNA-sequencing data were quantified at the gene level and analyzed using linear mixed models to account for nested experimental design. Multivariate adaptive shrinkage was used to assess convergent effects across multiple CNVs. Results: While few genes passed false-discovery rate of 5%, most genes within the respective CNV regions were underexpressed in deletion carriers and overexpressed in duplication carriers, as expected. Multivariate adaptive shrinkage showed moderate to low magnitude of differential gene expression effect sharing between CNVs, for the most part below 20%. The most prominent gene expression changes were observed in the 22q11.2 deletion and the least prominent ones in the 16p11.2 duplication, consistent with phenotypic severity. Conclusions: We present the largest study of neuropsychiatric CNVs in postmortem human brain to date. Despite limited sample size of individual CNVs, initial assessment of differential gene expression showed prominent expression changes for genes within each CNV region that were consistent with copy number. Only moderate to low levels of convergence were found between different CNVs. Future analyses are directed toward functional enrichment and gene co-expression analyses of the shared and CNV-specific effects. Keywords: Copy Number Variants, Human Postmortem Brain Tissue, RNA-Sequencing, Dorsolateral Prefrontal Cortex (DLPFC), Anterior Cingulate Cortex (ACC) Disclosure: Nothing to disclose. P206. Cell-Type-Specific Regulation of FosB Gene Expression in Nucleus Accumbens Moderates Effects of Chronic Stress on Sleep and Diurnal Rhythms Dominika Burek, Mykel Robble, Andrew Hall, Elisa Taylor-Yeremeeva, Eric Nestler, William Carlezon Harvard Medical School McLean Hospital, Belmont, Massachusetts, United States Background: There is considerable evidence that Fosb in nucleus accumbens (NAc) medium spiny neurons (MSNs) regulates resilience to chronic social defeat stress (CSDS). Promoting Fosb function in dopamine type 1 receptor-expressing MSNs (D1-MSNs) confers resilience to CSDS as measured with social interaction and sucrose preference tests, whereas repressing Fosb in D1-MSNs confers susceptibility. D1- and D2-MSNs also differentially regulate sleep architecture, such that DREADD-mediated inhibition of D1-MSNs increases duration of REM sleep. However, the ways in which changes in Fosb function in MSNs within the NAc affect CSDS-induced alterations in sleep architecture remains unknown. We hypothesized that enhancing Fosb function in D1-MSNs, through use of a construct that promotes histone acetylation at the Fosb promoter with a targeted zinc finger protein (ZFP) fused to p65, would prevent CSDS-induced alterations in sleep architecture, reflecting a pro-resilient phenotype. Methods: Male Drd1-Cre mice were injected with a viral vector (AAV(DJ)-Fosb-ZFP-p65) that induces the endogenous Fosb gene in the NAc to physiologically relevant levels, or the corresponding control vector in which the ZFP lacks a functional domain (AAV(DJ)-Fosb-ZFP-ASSB). Two weeks later, mice were implanted with wireless transmitters to measure EEG, EMG, body temperature, and locomotor activity, followed by a week for recovery. Continuous telemetry was collected for 2 days of baseline, 10 days of CSDS, and 2 days of post-stress recovery. Active wake, REM sleep, and slow wave sleep (SWS) vigilance states, as well as activity and body temperature, were quantified as percent of baseline across all 14 days of the experiment. Data were analyzed using two-way repeated measures ANOVA and Sidak’s post-hoc multiple comparisons tests. Results: Elevated Fosb function (induced by Fosb-ZFP-p65) in NAc D1-MSNs prevented stress-induced alterations in REM duration observed in control mice (group: F1,2 = 26.95, p = 0.0352). In addition, Fosb-ZFP-p65 increased measures of slow-wave sleep during the light phase in the latter days of the CSDS regimen, an effect that persisted during recovery (group x time: F13,26 = 2.594, p = 0.0187). Body temperatures fell below baseline throughout the CSDS regimen, until recovery (time: F1.905,3.80 9 = 2.594, p = 0.0243). Conclusions: Enhancement of Fosb function in D1-MSNs within the NAc mitigated the effects of CSDS on REM duration and SWS bouts, reflecting a potential pro-resilient effect. Across all groups, CSDS-induced changes in REM sleep and body temperature differed in direction and magnitude from those seen in naïve mice exposed to a similar regimen, raising the possibility that microinjections of these viral vectors into the NAc can produce non-specific changes in these endpoints. Future studies will characterize the ways in which repression of Fosb function in D1-MSNs, or corresponding changes within D2-MSNs, affect these same endpoints. Keywords: Sleep, Social Defeat Stress, Telemetry, deltaFosB Disclosure: Nothing to disclose. P207. Contribution of the Integrated Stress Response to Behavioral Phenotypes Associated With Loss of TRNA Methylation in the Rodent Cortex Jennifer Blaze, Behnam Javidfar, Schahram Akbarian Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: NSUN2, a mammalian tRNA methyltransferase, is expressed at high levels in brain and has been linked to neurodevelopmental defects in humans and mice due to its regulatory role in protein synthesis. We recently showed that there is a potent effect of Nsun2 depletion, via loss of tRNA methylation, on codon-specific tRNA expression and amino acid levels, producing proteomic shifts that impair synaptic transmission, cognition, and depressive-like behavior. Because dysregulation of amino acids and tRNA expression are known to produce the integrated stress response (ISR), we sought to explore whether this may be a mechanism by which loss of tRNA methylation alters behavioral outcomes. Methods: We used Cre-driven conditional knockout of Nsun2 in the postnatal mouse cortex to deplete tRNA methylation levels and used western blot to measure hallmarks of the ISR, including Atf4 and phosphorylated Eif2a protein expression (n = 5 KO/5 WT males). Further, we injected Nsun2 KO mice with ISRIB (n = 4 KO males), an inhibitor of the ISR, or vehicle (n = 3 KO males) to alter the molecular phenotype. We also conducted behavioral testing (forced swim test) for depressive-like behavior on wild-type mice treated with tunicamycin (n = 10 males), a potent activator of the integrated stress response, or vehicle (n = 10 males). Data was analyzed using two-tailed t-tests. Results: Nsun2 KO mice showed a marked increase in ATF4 expression (p < 0.01) and phosphorylated Eif2a expression (p < 0.01) compared to WT control mice. These molecular hallmarks were reversed after systemic treatment with ISRIB. Surprisingly, injecting wildtype mice with tunicamycin did not produce the same antidepressant phenotype as the Nsun2 KO but instead produced a pro-depressant phenotype in the forced swim test (time immobile: p < 0.05), suggesting another mechanism may be mediating changes in depressive-like behavior in the Nsun2 KO brain. Conclusions: Data suggest that the ISR is activated following tRNA dysregulation but likely is not related to depression-related behavior in the Nsun2 KO mouse. Future studies will investigate alternate mechanisms including neuronal glycine content as potential molecular mechanisms for altered depressive-like behavior following loss of Nsun2-mediated tRNA methylation. Keywords: Epigenetic, Noncoding RNA, Depressive-Like Behavior Disclosure: Nothing to disclose. P208. Enhancement of Cortical Plasticity on GABAergic and Glutamatergic Synapses by Targeting M1-Type Muscarinic Receptors in Somatostatin Interneurons Mediates Stress-Relevant Behavioral Outcomes Manoela Viar Fogaca, Min Wu, Chan Li, Xiao-Yuan Li, Ronald Duman, Marina Picciotto Yale University Medical School, New Haven, Connecticut, United States Background: Major depressive disorder (MDD) and chronic stress impair both glutamate and GABA function in the mPFC, leading to altered connectivity and network dysfunction in cortical and limbic brain regions. Rapid-acting antidepressants, including ketamine and scopolamine, restore these deficits by targeting somatostatin (SST) interneurons in the mPFC, resulting in interdependent homeostatic enhancement of glutamate- and GABA-related plasticity, which restores the integrity of signal transfer to target regions. In particular, scopolamine, a non-selective muscarinic receptor antagonist, produces rapid antidepressant-like effects by targeting M1-type acetylcholine receptors (M1R) on SST interneurons, but these effects have largely been studied in male mice and have involved relatively short-term manipulations. Methods: We generated mice with conditional M1R deletion in SST interneurons (M1f/fSstCre+) as an animal model to study long-term consequences of M1R deletion on GABAergic and glutamatergic synapses in the mPFC of male and female mice that lead to stress-relevant behavioral outcomes. We combined pharmacological, molecular, electrophysiological, chemogenetic and behavioral approaches to investigate whether the antidepressant-like effects of scopolamine can be mimicked (baseline behaviors) or occluded (scopolamine challenge) in male and female M1f/fSstCre+ mice. Results were analyzed by Student’s 2 tailed t-test and one- or two-way ANOVA, as appropriate, followed by post-hoc analysis, p ≤ 0.05. Results: Deletion of M1R in SST interneurons results in baseline decreases in stress-relevant behaviors in female mice accompanied by increased synaptic strength onto SST cells and enhancement of pre- and postsynaptic glutamate- and GABA-related signaling in the mPFC, but these baseline changes were not seen in male M1f/fSstCre+ mice. In contrast, both female and male M1f/fSstCre+ mice were resilient to stress following CUS exposure, with a more prominent effect in tests that evaluate coping strategies and motivation, which were positively correlated with levels of proteins relevant to glutamate and GABA signaling in the mPFC. In addition, SST M1R deletion in both male and female mice prevented the behavioral and molecular effects induced by scopolamine. Selective inhibition of SST interneurons in the mPFC of female M1f/fSstCre+ mice reversed the baseline behavioral changes, confirming that the mPFC somatostatin neurons are critical for stress-relevant effects of muscarinic receptor signaling. Conclusions: Taken together, these results suggest that enhancement of both GABA and glutamate plasticity is necessary to restore signal integrity in the mPFC and other limbic regions, culminating in antidepressant-like states and stress resilience. These findings support the idea that restoring optimal excitation/inhibition function via M1R blockade in SST interneurons could represent a promising strategy for antidepressant development. Keywords: Depression, Scopolamine, Antidepressant, Anxiety, Somatostatin Disclosure: Nothing to disclose. P209. Microglial BDNF Regulates Synaptic Plasticity and Behavior in Response to Stress and Ketamine Administration Samuel Woodburn, Helina Asrat, David Dadosky, James Flurer, Hana Schwierling, Justin Bollinger, Lauren Vollmer, Eric Wohleb* University of Cincinnati College of Medicine, Cincinnati, Ohio, United States Background: Seminal studies demonstrate that brain-derived neurotrophic factor (BDNF) is a potent regulator of synaptic plasticity in the prefrontal cortex (PFC) and influences behavioral responses to stress and antidepressants. Prior studies indicate that neurons are the primary source of BDNF, but more recent work suggests that microglia produce BDNF as well. Still, it is unclear whether microglial BDNF is important for the effects of stress and antidepressant treatments. Methods: In the first set of studies, mice with microglia-specific depletion of BDNF (Cx3cr1Cre/+:Bdnffl/fl) and genotype controls (Cx3cr1Cre/+:Bdnf + /+) were exposed to a shortened chronic unpredictable stress (CUS) paradigm (7 days). In separate studies, wild-type or transgenic mice ((Cx3cr1Cre/+:Bdnffl/fl; Cx3cr1Cre/+:Bdnf + /+) were administered a subanesthetic dose of ketamine (10 mg/kg, i.p.). In both experimental designs, molecular and histological analyses assessed markers of synaptic plasticity and microglia phenotype in the PFC as well as behavioral and cognitive outcomes. Results: Flow cytometry and RT-PCR confirmed Bdnf depletion in sorted PFC microglia and revealed shifts in phenotypic markers, including reduced CSF1R and P2Y12 levels, due to CUS and BDNF depletion. Protein analyses of PFC synaptoneurosomes showed that Cx3cr1Cre/+:Bdnffl/fl mice had lower baseline expression of GluN2B in the PFC compared to genotype controls. Further, 7 days of CUS additionally reduced GluN2A expression in Cx3cr1Cre/+:Bdnffl/fl mice. Behavioral and cognitive testing showed that this coincided with exacerbated stress effects on temporal object recognition. Further studies showed that behavioral and synaptic changes caused by ketamine (10 mg/kg, i.p.) coincide with a 2-fold increase in Bdnf transcript in sorted PFC microglia. To test whether microglial BDNF signaling is important for these effects, we administered ketamine to Cx3cr1CreER/+:Bdnffl/fl mice and genotype controls. Protein analyses of PFC synaptoneurosomes again indicated that microglial BDNF depletion caused a reduction in GluN2B expression. Assessment in the forced swim test showed that ketamine reduced immobility in Cx3cr1CreER/+:Bdnf + /+ mice, but these effects were completely blocked in Cx3cr1CreER/+:Bdnffl/fl mice. Conclusions: Collectively, our results show that microglial BDNF regulates expression of select glutamate receptor subunits in the PFC, which directs behavioral responses to stress and ketamine administration. These data indicate that microglia have an underappreciated role in promoting tolerance to stress and potential therapeutic utility in antidepressant treatment. Keywords: Microglia, Brain-Derived Neurotrophic Factor, Stress, Ketamine, Synapses Disclosure: Nothing to disclose. P210. Increased Plasma Lactate and Lactate-to-Pyruvate Ratio in Patients With Treatment Resistant Depression Giselli Scaini, Krista Wartchow, Tina Li, Bashar Asir, Giovana Zunta-Soares, Albert Fenoy, Jair Soares, Joao de Quevedo University of Texas Health Science Center at Houston, Houston, Texas, United States Background: Major Depressive Disorder (MDD) affects roughly 264 million people worldwide, which translates to approximately 4.5% of the global population, being recognized by the World Health Organization (WHO) as one of the major causes of disability worldwide. Antidepressant medication or psychotherapy are common treatments for MDD; however, not all patients respond to treatment. Treatment-resistant depression (TRD) is a form of depression where standard medications tend to provide little to no relief. Mitochondria are organelles responsible, both directly and indirectly, for multiple cellular functions and signaling cascades, being considered a central platform in the execution of diverse cellular events. In this framework, it is particularly intriguing to think of the mitochondria as an active regulator of many of the biological phenomena involved in depression and the efficacy of or resistance to the most widely used pharmacological treatments. This study aims to evaluate biochemical markers associated with mitochondrial function in patients with MDD and TRD. Methods: In this interim analysis of an ongoing pilot study, we included 17 healthy controls, 11 patients with MDD, and 14 patients with TRD. All subjects underwent a comprehensive clinical interview and diagnosis of MDD and TRD according to the DSM-IV-TR. Depressive symptoms and functional status were assessed with the Montgomery Asberg Depression Scale (MADRS) and Global Assessment of Functioning (GAF). Quantitative analysis of lactate and pyruvate plasma levels was performed using commercial kits. Results: One-Way ANCOVA, after controlling for age and gender, showed that TRD patients had higher plasma levels of lactate and pyruvate when compared to healthy controls and MDD patients. Moreover, the lactate-to-pyruvate ratio was significantly different between TRD patients and healthy controls but not between TRD and MDD patients. Additionally, we evaluated the lactate-to-pyruvate ratio in a mitochondrial disease cohort for comparison/validation purposes. Our results showed that the lactate-to-pyruvate ratio is higher in patients with mitochondrial diseases than in other groups. Another notable finding was that subjects with higher lactate-to-pyruvate ratio had higher MADRS scores and worse functional status. Conclusions: In summary, our findings corroborate previous studies and support the notion that mitochondrial dysfunction is integral to the pathogenesis of MDD and may play a role in clinical and functional outcomes. Although preliminary, our results suggest that the lactate-to-pyruvate ratio might serve as one of the diagnostic bases of TRD. However, in this preliminary study, we cannot rule out the possibility of a type I error since it is a cross-sectional study with a small sample size. Therefore, our results should be seen as exploratory and require replication and validation. Keywords: Major Depressive Disorder, Treatment Resistant Depression, Mitochondrial Dysfunction, Plasma Lactate and Pyruvate, Lactate-to-Pyruvate Ratio Disclosure: Nothing to disclose. P211. Cell-Type and Sex-Specific Molecular Signatures of Stress Resilience and Susceptibility in VTA Catherine Pena, Adelaide Minerva Princeton Neuroscience Institute, Princeton, New Jersey, United States Background: Mounting behavioral, circuit, and molecular-level evidence in humans and rodents shows that resilience to stress and depression is not an absence of susceptibility-associated changes, but an active process itself. The ventral tegmental area (VTA) has been strongly linked with depression and other psychiatric disorders. While a majority of studies have focused on changes within dopaminergic regions, there is mounting evidence for involvement of other cell types in stress response and resilience. We therefore sought to characterize the cell-type-specific molecular signatures associated with resilience and susceptibility to chronic stress within VTA. Methods: Male and female mice were subject to chronic social defeat stress and tested on a battery of behavioral tests. VTA tissue was harvested 15-minutes after re-exposure to an aggressive encounter and processed for single-nucleus RNA-seq on a 10X chromium platform, to examine transcriptional signatures of cellular activity. Tissue from two mice of the same sex and group (control/susceptible/resilient) were randomly pooled for each sample, such that average social behavior was similar across samples within each group. Bioinformatic processing and analysis was performed with Cell Ranger (10X) and the Seurat package in R. Results: We sequenced 185,908 nuclei from 16 samples, with an average of ~13,000 nuclei/sample, ~22,000 reads/nucleus, ~900 genes/nucleus, and ~72% of reads mapped to the genome. Clusters for expected VTA cell types were identified using UMAP. We show that resilience is associated with greater activation (immediate early gene expression) of dopamine, oligodendrocyte, endothelial, and GABAergic cells, while mixed glutamate/GABA cells were more active in susceptible mice. We present sex-specific characterization of excitatory/inhibitory balance, plasticity-related gene expression changes, neuroimmune, and neurovascular alterations associated with individual response to stress. Conclusions: Distinct cell populations contribute to resilience or susceptibility to social stress. An imbalance in how each cell population adapts to stress may contribute to sex-specific vulnerability to psychiatric-disease-associated behavioral and physiological phenotypes. Keywords: Ventral Tegmental Area (VTA), Social Defeat Stress, Resilience, Single-Nucleus RNA Sequencing Disclosure: Nothing to disclose. P212. Chronic Variable Mild Stress Alters Avoidance Behavior in Mice but has no Influence on the Transcriptome of the Anterodorsal Bed Nucleus of the Stria Terminalis and the M-Current of NPY Neurons Thomas Degroat, Kimberly Wiersielis, Katherine Denny, Jessica Tollkuhn, Benjamin A. Samuels, Troy Roepke* Rutgers University, New Brunswick, New Jersey, United States Background: In humans, chronic stress leads to the development of mood disorders such as major depressive disorder and post-traumatic stress disorder. It has also been shown that women are more susceptible to the development of these disorders, suggesting a sex-related difference in how we process stress. The anterodorsal bed nucleus of the stria terminalis (abBNST) is a brain region that is essential for the central stress response. It is considered a part of the extended amygdala and serves as a relay between the amygdala and the prefrontal cortex. This region also has sexually dimorphic in expression of aromatase and estrogen receptors. Therefore, the adBNST may play a major role in the sex difference observed. We have previously demonstrated that chronic stress alters corticotropin-releasing hormone (CRH) expression and the M-current, a potassium current, in CRH neurons in the BNST from male mice. Other BNST neurons express Neuropeptide Y (NPY) which may plays a role in the stress response. We hypothesized that the CVMS paradigm would results in alterations in behavior of both male and female mice, sex-dependent differences in the transcriptome, and that adBNST NPY neurons would be affected by chronic stress, leading to a decreased M-current. Methods: To study the effects of chronic stress on the adBNST, we used both wild-type and NPY-GFP male and female mice. Mice experienced six weeks of a chronic variable mild stress (CVMS) paradigm or no stress prior to behavior testing, adBNST tissue collection for RNA sequencing, and whole-cell patch clamp electrophysiology. The behavior tests conducted were the open field test (OFT), elevated plus maze (EPM), light dark box (LDB), and novelty suppressed feeding (NSF). Results: Our results show that stress did cause sex-dependent differences in behavior depending on the parameters of the test. In the OFT, CVMS mice spent less time in the center (main effect of stress P = .0019) and more time in the corners (main effect of stress P = .0209). In the EPM, CVMS exposed mice spent more time in the closed arms than controls (main effect of stress P = .0011) and females had more open arm crossings than males. In the LDB, no effects were observed other than male mice had more stretch attend postures than females (main effect of stress P = .0377; interaction P = .0002). In the NSF, no effects were observed except that stressed mice weighed less than non-stressed mice (main effect of stress P = .0136). Serum corticosterone levels are currently being analyzed from blood collected before and after stress. RNA sequencing of the adBNST from stressed and unstressed mice found no effects of stress on any gene, potentially due to high variability between samples, but did find differences in expression between the sexes for ~30 genes. CVMS exposure did not affect neuronal excitability or the M-current in the adBNST NPY neurons, nor was there a sex-related difference. There was no effect on resting membrane potential yet there was an effect of stress on input resistance (P = .0286). The max peak of the M-current in control males was 30.4 pA, in stress males max peak was 34.0 pA (t-test: P = 0.5175), in females, control max peak was 23.4 pA, and stress max peak was 26.8 pA (t-test: P = 0.6979). Conclusions: Our data suggests that CVMS is an effector of behavior in mice, but that adBNST NPY neurons may not be an important regulator of chronic stress or a mediator of the sex differences in the response to chronic stress. Keywords: Chronic Unpredictable Mild Stress, BNST, Approach/Avoidance, Sex Differences, Neuropeptide Y Disclosure: Nothing to disclose. P213. The Involvement of SKA2, a Novel Glucocorticoid Receptor Interaction Partner, in Stress-Related Psychiatric Disorders Jakob Hartmann, Thomas Bajaj, Claudia Klengel, Lindsay Rexrode, Kenneth McCullough, Nina Dedic, William Carlezon, Torsten Klengel, Sabina Berretta, Harry Pantazopoulos, Nils Gassen, Kerry Ressler McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Mood and anxiety disorders represent a major disease and social burden worldwide, but the underlying molecular mechanisms are still poorly understood. In recent years, evidence has emerged for the crucial role of genes involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, especially in the context of stress-related psychopathologies such as anxiety and depression. The glucocorticoid receptor (GR) is the main mediator of the HPA axis negative feedback loop in response to stress. The SKA2 gene, encoding the spindle and kinetochore associated complex subunit 2, has previously been identified as a GR interaction partner in peripheral cells in vitro. Interestingly, single nucleotide polymorphisms and DNA methylation within the SKA2 gene, as well as gene expression alterations, have been associated with posttraumatic stress disorder and suicide risk in human patients. Yet, little is known about the underlying molecular mechanisms and the role of SKA2 in the brain. Here, we set out to further investigate the function of SKA2 in the CNS of humans and mice in the context of stress-associated psychiatric disorders and behaviors. Methods: We conducted Immunohistochemistry (IHC) to study the expression pattern of SKA2 in human postmortem hippocampus and amygdala samples. Additional co-immunoprecipitation (co-IP) assays addressed whether SKA2 physically interacts with the GR in the mouse brain and in human postmortem brain samples. Moreover we performed GR reporter gene activity (MMTV-Luc) assays and qPCR following Ska2 perturbation to investigate the receptors activity and downstream effects in neuronal cell culture. Western blot (WB) analysis were performed to examine SKA2 protein expression in basolateral amygdala (BLA) and hippocampus samples of individuals with bipolar disorder and matched controls. Stress-induced changes in SKA2 expression in mice were investigated via WB following fear conditioning. Results: IHC analyses in human postmortem hippocampus and amygdala samples revealed a prominent expression of SKA2 in Camk2a-positive neurons. Moreover, co-IPs revealed a physical interaction between SKA2 and GR in the mouse brain and human postmortem samples. In addition, Ska2 knockdown in hippocampal neuroblastoma (HT-22) cells led to significantly reduced GR reporter gene assay activity, while overexpression of Ska2 resulted in the opposite effect (2-Way ANOVA, interaction: F(14,42) = 16.38, p < 0.0001). Along these lines, Ska2 knockdown led to significantly altered mRNA expression of GR target genes Fkbp5 (t-test, T(21) = 2.683, p < 0.05) and Id3 (t-test, T(22) = 5.762, p < 0.001). Interestingly, we detected significantly increased SKA2 protein levels in the BLA of individuals with bipolar disorder compared to matched controls (ANCOVA, F = 5.83, p < 0.025, n = 14 per group). Moreover, hippocampal SKA2 expression is increased in bipolar disorder, when ‘presence of psychoactive drugs in the blood at death’ is included as co-variate (ANCOVA, p < 0.01, n = 20 per group). Detailed mapping and co-labeling studies in mice also revealed a distinct pattern of SKA2 expression in neurons of the BLA, as well as in the hippocampus, medio-dorsal thalamus, the paraventricular nucleus of the hypothalamus and throughout the cortex. Most of the SKA2-positive neurons also expressed the GR. It has previously been reported that the synthetic glucocorticoid dexamethasone decreases SKA2 protein levels in human lung epithelial cells. Consequently, we assessed whether acute stress is able to modulate SKA2 expression in the mouse brain. Using WB, we found dynamic changes of SKA2 expression 8 and 12, but not 24 hours after stress (fear conditioning, 5 tone/foot shock pairings). Stressed mice showed significantly decreased SKA2 protein levels in the hippocampus (8 hrs: T(8) = 2.626, p < 0.05, T(8) = 3.358, p < 0.01, n = 4 (ctrl), n = 4 (stress)), while no expression changes were observed in the prefrontal cortex. Conclusions: Our findings reveal SKA2 as a novel GR interaction partner in brain regions associated with emotion processing and cognition. Further experiments showed that Ska2 gene expression is dynamically regulated by stress exposure and that SKA2 is able to positively modulate GR signaling and its downstream targets, providing a mechanistic link to its association with stress-related psychopathologies. Collectively, our data point to an important, and thus far unappreciated, role of SKA2 in stress-related psychiatric disorders, which is relevant to our understanding of the molecular mechanisms underlying such diseases. Keywords: PTSD, Bipolar Disorder, Suicide Risk Factors, HPA Axis, Glucocorticoid Receptor Disclosure: Nothing to disclose. P214. Sex and Region Differences in Microglia and Dendritic Spines in the Human Brain Marianne Seney, Tyler Myers, David Volk, Kenneth Fish University of Pittsburgh Medical Center, Western Psychiatric Institute and Clinic, Pittsburgh, Pennsylvania, United States Background: Microglia are resident macrophages of the brain, performing roles related to brain homeostasis, including trophic support, phagocytosis of apoptotic cells and debris, as well as brain protection and repair. In healthy adults, microglia exhibit a ramified morphology, sampling the local environment by extending and retracting processes. Various stimuli, including infection, trauma, or altered neuronal activity, induce changes in microglia shape, gene expression, and function. Evidence from rodent studies reveal that microglia density, morphology, gene expression, and proliferation vary based on brain region and sex. In addition to being the resident macrophages of the brain, microglia participate in elimination and formation of synapses on pyramidal cells. The primary source of excitatory synaptic input onto pyramidal cells are dendritic spines, which are morphological protrusions on PC dendrites. Similar to microglia, evidence suggests brain region and sex differences in dendritic spine density. Notably, most studies describing brain region and sex differences in microglia and dendritic spines were performed in rodents. Whether similar differences exist in the human brain has not been fully studied, especially with consideration of whether there are correlations between microglia reactivity and dendritic spine density within the same subjects. Here, we simultaneously assess dendritic spine density and microglia reactivity state in the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of human subjects unaffected by psychiatric or neurological disorders. Methods: For DLPFC and ACC separately (N = 5/sex; same subjects used for DLPFC and ACC), 2 equally spaced coronal sections from the immersion-fixed left hemisphere were processed for immunofluorescence. Dendritic spines are identified by labeling for Shank3, a spine scaffolding protein, and f-actin, which is concentrated in dendritic spines, using the phallotoxin phalloidin. Using the colocalization of two dendritic spine markers provides a robust approach for identifying spines. In the same sections, microglia are labeled using antibodies against the microglia specific marker TMEM119 to assess microglia morphology and CD68 to assess microglia phagocytic activity. NeuroLucida 360 was used to mask fluorescent signal in 3 dimensions. Microglia complexity was assessed using Sholl analysis; an average of 55 microglia were analyzed per subject per brain region. Two-way repeated measures ANOVA was used to assess the number of microglia process intersections at 10 micron radii surrounding each microglia soma; Tukey’s multiple comparisons test was used for posthoc analyses. Unpaired t-tests were used to assess sex differences in colocalized shank + /phalloidin+ puncta (putative dendritic spines) and one-way ANOVA was used to assess layer differences in colocalized shank + /phalloidin+ puncta, with Tukey’s multiple comparisons test used for posthoc analyses. Results: We observed striking differences in microglia between the DLPFC and ACC. Microglia in the ACC exhibited a more activated morphology (more amoeboid; fewer, less complex processes) compared to microglia in the DLPFC. We note that assay methods, imaging, and processing were identical between the ACC and DLPFC. Thus, technical differences are not driving the robust morphological differences between microglia in the ACC and DLPFC. Within the ACC, we observed sex differences in microglia reactivity and in dendritic spine density. Specifically, Sholl analysis indicated that female microglia exhibit less complexity (main effect of sex: p < 0.01; sex-by-distance from soma: p < 0.0001), and female microglia had shorter process length (p < 0.05); both findings suggest that female microglia are more reactive. Interestingly, females also had lower density of shank + /phalloidin+ puncta (putative dendritic spines) within 12µm proximity of microglia (p < 0.01). When males and females were combined, we also observed cortical layer differences in microglia complexity (layer-by-distance from soma: p < 0.01) as well as in microglia process length, with layer 2 microglia exhibiting a more reactive profile compared to other layers. Additionally, layer 3 had more dendritic spines than other layers (p < 0.05 for comparisons to other layers). Conclusions: Our findings suggest a high level of microglia heterogeneity in the human cortex in subjects without psychiatric or neurodegenerative disorders. In terms of microglia reactivity, we found noted differences based on brain region (ACC > DLPFC), sex (female > male), and cortical layer (layer 2 > other cortical layers). Assessment of dendritic spine density in the proximity of microglia within the ACC revealed striking sex differences (male > female), which is consistent with females having more reactive microglia. Ongoing studies are expanding this analysis to psychiatric disease cohorts. Keywords: Sex Difference, Human Microglia, Dendritic Spines, Human Postmortem Brain Disclosure: Nothing to disclose. P215. GM-1020: A Novel, Orally Bioavailable NMDA Receptor Antagonist With Rapid and Robust Antidepressant Effects and Reduced Ataxia in Rodents Laszlo Kiss, Adam Klein, Eric Austin, Dino Dvorak, Silvia Gatti, Mariusz Papp, Gerard Marek, Jonathan Sporn, Zoe Hughes, Andrew Kruegel Gilgamesh Pharmaceuticals, New York, New York, United States Background: The discovery of the rapid antidepressant effects of the NMDA receptor (NMDAR) antagonist (R,S)-ketamine has ushered in a new wave of interest in identify molecules that can achieve the rapid and robust efficacy of (R,S)-ketamine but improve on some of its shortcomings. At the commonly administered antidepressant dose of (R,S)-ketamine (0.5 mg/kg, iv infusion), patients experience dissociative side effects and ataxia, which restrict safe administration to a supervised, in-clinic setting and limits the clinical uptake of this treatment. In addition, (R,S)-ketamine has poor oral bioavailability that necessitates parenteral dosing. Spravato®, an intranasal formulation of the more potent S-isomer of ketamine, has been approved for patients with treatment-resistant depression, but still requires supervised administration due to its dissociative effects. Early clinical data with intravenous R-ketamine indicate that this less potent enantiomer retains antidepressant efficacy at non-dissociative doses, suggesting an improved therapeutic index is possible for this mechanistic class. However, there remains an unmet need for a non-dissociative, orally bioavailable NMDAR antagonist with rapid and robust antidepressant efficacy. GM-1020 is an orally bioavailable, NMDAR antagonist with robust, durable antidepressant effects and attenuated side effects in rodents. Methods: The binding affinity of GM-1020 at NMDAR was determined using displacement of [3H]MK-801 from rat cortical tissue homogenates. Functional antagonism was demonstrated using automated patch clamp in oocytes expressing human GRIN1/GRIN2A- or GRIN1/GRIN2B-containing NMDAR. The metabolism of GM-1020 was evaluated in rat, mouse, minipig, dog, cynomolgus monkey, and human liver microsomes and hepatocytes. Oral bioavailability of GM-1020 was determined by comparing PK after IV and PO administration in rat, mouse, mini-pig, and cynomolgus monkey. GM-1020 was assessed in a paradigm of Chronic Mild Stress (CMS), wherein male Wistar Han rats (n = 8/group) were subjected to stressors (e.g., wet bedding, tilted cage) for a period of 10 weeks. After two weeks of CMS, animals were dosed once per week with GM-1020 (0.75 - 9 mg/kg, ip), (R,S)-ketamine (10 mg/kg, ip), or vehicle for 5 weeks. Rats continued to be exposed to stress for an additional 3 weeks after the final dose to determine the durability of the antidepressant effect. Each week, anhedonia was assessed by measuring sucrose intake (24 h after dosing). Anxiety levels in the elevated plus maze (EPM) and memory impairment in the novel object recognition (NOR) test were assessed 48 and 72 h after the first dose, respectively. Acute effects of GM-1020 (1- 32 mg/kg, sc) on locomotor activity were assessed in male C57BL/6 mice in an open field test. The effects of GM-1020 (1- 32 mg/kg, sc) and (R,S)-ketamine (3.2- 32 mg/kg, sc) on motor coordination were assessed using an accelerating rotarod in mice (n = 12/group). Results: GM-1020 bound to NMDAR in rat cortical tissue with low-micromolar affinity (Ki 3.25 µM). GM-1020 exhibited channel blocking properties of glutamate/glycine-induced NMDAR currents in the absence of Mg2+ at GRIN2A- (IC50 6.45 µM) or GRIN2B-containing (IC50 4.16 µM) NMDAR. Oral bioavailability (%F) was determined to be 27-43% in non-clinical species. Microsomal clearance in vitro was found to be a good predictor of %F (32-52%); human microsomal clearance predicted human %F of >60%. Rats exposed to CMS showed a robust anhedonic phenotype evidenced by a reduction in sucrose intake throughout the study in vehicle treated rats (P = 0.0003). GM-1020 reversed the effects of CMS on sucrose intake at doses ≥1.5 mg/kg (P < 0.001 vs stress vehicle group). CMS also resulted in increased anxiety in the EPM and memory impairment in the NOR test. Both effects were reversed by administration of GM-1020 (≥1.5 mg/kg; P < 0.001 vs stress vehicle group). Similarly, (R,S)-ketamine (10 mg/kg) reversed the anhedonia, anxiety and memory impairment caused by CMS. Efficacy of GM-1020 was maintained for 14 days, and ketamine for 7 days, after the final dose. Evaluation of higher doses of GM-1020 (1-32 mg/kg) for effects on motor activity showed no change in distance moved in the open field (0-60 min post dose), while GM-1020 (≥10 mg/kg) produced a decrease in latency to fall off the rotarod 5 minutes after dosing (P < 0.05 vs vehicle). The dose of GM-1020 resulting in a 50% reduction in latency to fall was ~20 mg/kg, whereas for (R,S)-ketamine, it was ~6 mg/kg. Conclusions: GM-1020 has a good oral bioavailability in non-clinical species and predicted bioavailability of >60% in humans. In the CMS model of depression, the minimum antidepressant-like dose of GM-1020 was 1.5 mg/kg, with robust efficacy observed after the first dose and maintained for 14 days after the final dose. The ED50 for ataxia was ~13-fold higher, while no effect on locomotion was observed even at >20-fold higher doses than the minimum efficacious dose in CMS. These data indicate that pre-clinically, GM-1020 shows large separation between antidepressant doses and those causing ataxia. Literature reports with (R,S)-ketamine indicate that in rodents, ataxia is observed at doses only 3-fold higher than antidepressant doses, whereas in humans, the antidepressant dose of (R,S)-ketamine is associated with ataxia and dissociation. This supports further development of GM-1020 as a putative orally bioavailable, rapid-acting, robust antidepressant with an improved therapeutic index with respect to dissociation and ataxia. Keywords: NMDA Antagonists, Depression and Anxiety, Rapid-Acting Antidepressant Disclosure: Gilgamesh Pharmaceuticals: Employee (Self) P216. Baseline Differences and Treatment-Responsiveness to Reelin and (2R,6R)-HNK of IPSC-Derived Neurons From Participants With Treatment-Resistant Depression Jenessa Johnston, Brandi Quintanilla, Peixiong Yuan, Mani Yavi, Hector Caruncho, Bashkim Kadriu, Carlos Zarate Jr University of Victoria, Victoria, Canada Background: Treatment-resistant depression (TRD) is of great clinical concern, with patients often not responding to first-line antidepressant treatments. Subanesthetic doses of ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, has been found to have rapid and robust antidepressant effects in patients with TRD. However, certain psychoactive effects necessitate administration under medical supervision. Recent research suggests that the fast-acting antidepressant effects of ketamine are mediated through transient activation of the mammalian target of rapamycin (mTOR), which promotes an upregulation of proteins which strengthen synaptic signaling, such as surface insertion of GluA1. (2R,6R)-hydroxynorketamine (HNK) is a major metabolite of ketamine which, at therapeutic levels, does not antagonize NMDARs. Preliminary findings demonstrate that (2R,6R)-HNK may have similar effects to ketamine without the associated side effects. Reelin, a glycoprotein essential for proper cortical development, is an important mediator of synaptic plasticity and also parallels some of the fast-acting antidepressant-like behavioural and biological effects of ketamine, warranting further investigation. Methods: Inducible pluripotent stem cells (iPSCs) were programmed from peripheral mononuclear blood cells collected from subjects with treatment-resistant depression (n = 5) and healthy controls (HCs) (n = 2) from both males and females. The STEMdiffTM embryoid body protocol was followed for 19 days to develop single-cell neural progenitor cells. Cells were supplemented and cultured for 10 weeks, then divided into 5 different conditions (vehicle + DMSO; 5nM, 10nM, and 50nM reelin; 1μM (2R,6R)-HNK) at 2 timepoints (1 hour; 24 hours) to assess short and long-term effects of reelin and (2R,6R)-HNK. Western blotting analyses were used to quantify mTOR, phosphorylated-mTOR (p-mTOR), post-synaptic density 95 (PSD95), GluA1, Synapsin 1 (Syn-I), Disabled-1 (Dab1), tyrosine kinase receptor B (TrkB), extracellular signal-regulated kinase (ERK), and p-ERK. Results: Baseline differences of mTOR (p < 0.05), GluA1 (p < 0.05), TrkB (p < 0.05), p-ERK (p < 0.01), and ERK (p < 0.001) were observed between MDD cell lines and healthy controls. At 1 hr in cell lines from subjects with TRD, (2R,6R)-HNK and reelin significantly increased levels of PSD-95 (R50nM, p < 0.05; HNK1μM, p < 0.01), Syn-I (R10nM, p < 0.05; R50nM, p < 0.01; HNK1μM, p < 0.01), Dab1 (R10nM, p < 0.05; R50nM, p < 0.01; HNK1μM, p < 0.01), and p-ERK (R50nM, p < 0.05; HNK1μM, p < 0.05). With 24 hr application, these effects were reversed, demonstrating a significant down-regulation of expression of PSD-95 (R10nM, p < 0.05; R50nM, p < 0.01) and Syn-I (R10nM, p < 0.05; R50nM, p < 0.01; HNK1μM, p < 0.01). However, in HC, no significant differences were found in any treatment or time group. Conclusions: High rates of TRD have necessitated the search for novel antidepressants that have lower long-term side effects, higher efficacy rates, and rapid therapeutic effects. This exploratory study shows both reelin and (2R,6R)-HNK hold promise in activating cellular pathways essential for fast-acting antidepressant responses. The differences between TRD and HC cell lines and their responsiveness towards treatment warrants further investigation. In addition, a limitation on the translatability of novel antidepressants has been due to the lack of in vitro or in vivo models that can encompass the heterogeneity of depression. While still preliminary, (iPSCs) could provide individualized options for evaluating treatment responsiveness. This study is a step forward in developing innovative antidepressant therapeutics and improving translation in pharmacological research using novel methodologies. Keywords: Treatment Resistant Depression, (2R,6R)-hydroxynorketamine, Reelin, Induced Pluripotent Stem Cells (iPSCs) Disclosure: Nothing to disclose. P217. Cell-Type-Specific Dynamics of Histone 3.3 Lysine 27 Methylation Confers Susceptibility to Stress Across the Lifespan Angélica Torres-Berrío, Molly Estill, Aarthi Ramakrishnan, Hope Kronman, Angélica Minier-Toribio, Orna Issler, Caleb J. Browne, Freddyson J. Martínez-Rivera, Yentl Y. van der Zee, Eric Parise, Sherlyn Ramirez, Simone Sidoli, Li Shen, Eric Nestler Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: A life history of stress is the strongest risk factor for several psychiatric disorders. Indeed, stress is known to “scar” the brain, leading to persistent transcriptional alterations in regions involved in reward and mood regulation, such as the nucleus accumbens (NAc). Here, we characterized the enduring changes in histone modifications in the NAc of mice exposed to stress, either in early life or in adulthood, and assessed their role in mediating life-long behavioral and transcriptional dysregulation. Methods: We exposed female and male mice to early life stress (ELS), a paradigm known to heighten vulnerability to stress in adulthood, by combining maternal separation and reduced bedding during postnatal days (PND) 10 to PND 17. In parallel, an independent cohort of adult mice was subjected to chronic social defeat stress (CSDS), a validated model for the study of depression-like behaviors that separate mouse populations into susceptible (SUS) and resilient (RES) based on a social interaction test (SIT) which is predictive of numerous other behavioral abnormalities. NAc tissue at PND21 and PND60 from mice exposed to ELS or from SUS and RES mice exposed to CSDS was processed for histone profiling via mass spectrometry. Finally, we mapped the genome-wide enrichment of the most changed histone modifications in both stress models using Cleavage Under Targets and Release Using Nuclease (CUT and RUN) followed by sequencing. Results: Our results show that stress alters the methylation (me) dynamics of lysine (K) 27 of the histone variant H3.3—the predominant form of H3 present in adult brain neurons. We found a consistent increase in the abundance of H3.3K27me1, and decrease in the abundance of H3.3K27me2, in the NAc immediately after ELS exposure (PND21), effects that lasted into adulthood (PND60). Strikingly, we also observed that SUS mice to CSDS exhibit the same opposite pattern of H3.3K27me1/me2 abundance in the NAc. Genomic distribution shows that H3.3K27me1 is primarily enriched in gene bodies and proximal promoters, and correlates positively with gene expression, whereas H3.3K27me2 negatively correlates with gene expression and is weakly deposited across intergenic regions. Finally, we observed that changes in H3.3K27me1 and H3.3K27me2 are associated with elevated expression of SUZ-12, an important regulator of H3K27 methyltransferase activity. Indeed, cell-specific overexpression of SUZ-12 increases H3.3K27me1 expression in D1 medium spiny neurons and leads to stress-induced persistent social, behavioral, and cognitive alterations. Conclusions: Together, these results suggest that H3.3K27me1 and H3.3K27me2 are important epigenetic “scars” that mediate stress susceptibility across the lifespan, and point at SUZ-12 as a novel epigenetic target for therapeutic interventions. Keywords: Histone Variants, Chronic Stress, Psychiatric Disorders, Cognitive Function Disclosure: Nothing to disclose. P218. Psychedelics Show an Antidepressant Biosignature but a Sustained Response Aksu Gunay, Jeffrey Schappi, Anastasia Kotsouris, Mark Rasenick University of Illinois College of Medicine, Chicago, Illinois, United States Background: Previous data suggest that the heterotrimeric G protein, Gsalpha (Gsα) is ensconced predominantly in lipid rafts in subjects with major depressive disorder (MDD), resulting in impaired stimulation of adenylyl cyclase. Both diminished Gsα-adenylyl cyclase coupling and an increase in the proportion of Gsα in lipid rafts have been observed in MDD. Antidepressants accumulate slowly in lipid rafts and evoke translocation of Gsα out of lipid rafts toward a more productive association with adenylyl cyclase, resulting in sustained cAMP elevation and sequelae such as increased brain-derived neurotrophic factor. This is also observed in cellular model systems and rodents. We hypothesize that heightened accumulation of Gsα in lipid rafts is a biomarker for depression, and that the translocation of Gsα from those rafts is a biomarker for clinical response to antidepressants. "Rapid-acting" antidepressant compounds, such as ketamine, have the same effect, but on an accelerated timescale. Some clinical data suggest that psychedelics, at hallucinogenic doses, evoke a long-lasting antidepressant response after a single dose Methods: This study sought to compare the effects of monoamine-centric antidepressants, ketamine and psychedelics on cellular features of antidepressant response using cultured cells (C6 glioma, SK N SH neuroblastoma or astrocytes induced from fibroblasts derived from MDD or control subjects) exposed to fluoxetine, desipramine or ketamine (all 10 µM) or LSD (10 nM) or psilocin (psilocybin metabolite-50nM). cAMP was determined by Alpha Screen or fluorescent reporters. Results: While the antidepressant biomarker activity (Gs translocation and augmented Gs activation of adenylyl cyclase) was seen with all compounds to which antidepressant activity has been attributed, there were distinct differences in the response profile for the compounds. Fluoxetine and desipramine required three-day exposure to translocate Gsα from rafts and increase Gsα-activated adenylyl cyclase. The effects of ketamine, while the most rapid in onset (15 minute exposure), declined over a 24-hour period, returning to pre-drug values after that time. However, both LSD and psilocin (1 hr exposure followed by washout) gave a prolonged antidepressant biosignature. All experiments were repeated 4-6 times in triplicate. Conclusions: Antidepressants, psychedelics and ketamine all show an antidepressant biosignature, revealing a mobilization of Gsα from lipid rafts and an increased signaling of GPCRs working through Gsα. Previous studies in postmortem brain and in platelets showed that this biomarker was enriched in lipid rafts from depressed subjects and reverted toward control values after effective treatment (as indicated by HAM-D). The similarities and differences in the actions of these drugs at the cellular level may lead to a better molecular understanding of the distinct kinetic features of their actions. Keywords: GPCR, cAMP Signalling, Ketamine, Depression, Psychedelics Disclosure: Pax Neuroscience: Founder (Self) P219. Sex Hormone-Driven X Chromosome Dynamics in the Adult Female Brain Devin Rocks, Mamta Shukla, Laila Ouldibbat, Silvia Finnemann, Achyuth Kalluchi, Jordan Rowley, Marija Kundakovic* Fordham University, Bronx, New York, United States Background: Sex differences in brain physiology and disease result from the interplay of sex hormones and sex-chromosome-linked genes. From puberty to menopause, cyclical sex-hormone fluctuations represent a female-unique experience, which is associated with substantial brain plasticity and increased female risk for brain disorders such as anxiety and depression. One of the X chromosomes in females is typically inactivated by the process involving chromatin condensation, although there are genes that escape this process known as “X escapee genes”. However, whether ovarian hormones can dynamically change 3D chromatin organization and gene expression of the X chromosome in the adult female brain is unknown and this knowledge is needed to understand female-specific brain dynamics and disease risk. Methods: We examined 3D compartmental organization of the X chromosome in adult ventral hippocampal (vHIP) neurons across the estrous cycle and by sex using an unbiased chromatin conformation capture (Hi-C) method combined with candidate-loci DNA fluorescence in situ hybridization (FISH). Hi-C was performed in triplicates (N = 6 animals/group) on vHIP neurons isolated from 11 week-old male and female mice by fluorescence-activated nuclei sorting. We included females in two estrous cycle stages: proestrus (high estrogen-low progesterone) and diestrus (low estrogen-high progesterone), mimicking the human follicular and luteal phase, respectively. To address the effect of estrogen on the X chromosome 3D organization, we treated ovariectomized (OVX) mice with estradiol (5 µg, 4 hours) or vehicle and performed Hi-C assay on vHIP neurons (5 biological replicates or N = 10 animals/group). Compartmental analysis was performed by calculating the eigenvector on the Pearson correlation matrix in 25 kb bins. Motif analysis of differential compartmental regions was performed using meme-chip. The role of 3D genome organization in gene regulation was assessed by integrating Hi-C data with chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) data (N = 6 animals/group). Results: We show that ~16% of the X chromosome changes compartmental organization in vHIP neurons across the estrous cycle. With naturally rising estrogen levels in female mice, the X chromosome acquires an interaction profile more similar to males, and is likely to undergo a decondensation event and increase in volume during proestrus. We confirmed X chromosome compartmental dynamics across the cycle using FISH, as a complementary approach. We also found associated changes in the expression of neuronal function-relevant X-linked genes, describing for the first time estrous cycle-dependent X escapee genes. The estrous cycle-dependent compartments are enriched for estrogen response elements, implying the role of estrogen and its receptors in X chromosome dynamics across the cycle. This is further confirmed by estrogen replacement in OVX mice, which reproduces around 25% of the cycle-dependent compartmental changes in the X chromosome. Conclusions: Overall, our data show unexpectedly high dynamics of the X chromosome in response to cycling sex hormones, with an indication that inactive X chromosome may undergo a decondensation event during the high-estrogenic phase of the ovarian cycle. Rhythmic “breathing” of the inactive X-chromosome could induce global changes in cellular environment affecting neuronal gene regulation and function, with critical implications for female physiology and disease risk. Our findings also open an intriguing possibility of hormonally-mediated X chromosome re-activation that may be of interest for the treatments of X-linked developmental disorders such as Rett syndrome. Keywords: Sex Hormones, X chromosome, 3D Genome, Brain Plasticity, Epigenetic Disclosure: Nothing to disclose. P220. Interleukin-6 Decreases Dopamine Availability and Gene Pathways Related to Vesicular Transport and Release in Human Pluripotent Stem Cell Derived Dopaminergic Neurons Andrew Miller, Michael Lucido, Chong Chong Xu, Yiqi Huang, Weibo Niu, Zhexing Wen Emory University School of Medicine, Atlanta, Georgia, United States Background: Chronic inflammation may impair dopamine (DA) neurotransmission in ventral striatum leading to motivational deficits including anhedonia, a core symptom of depression. For example, inflammatory cytokines such as interleukin (IL)-6 have been shown to decrease DA release in ventral striatum in laboratory animals and humans, and in the context of stress and inflammation, IL-6 has direct access to striatal DA’ergic neurons through disruption of the blood-brain-barrier. Methods: To study the direct effects of IL-6 on DA neurons, in vitro studies were conducted on DA neurons derived from human induced pluripotent stem cells (hiPSCs) of healthy female volunteers. Using standard protocols, hiPSCs were differentiated into neurons, ~80% of which expressed tyrosine hydroxylase or PITX3, indicative of DA neuron differentiation. Experiments (n = 3 or more per condition) were conducted in midbrain organoids or 2D cultures, and results between and among conditions were compared using Student’s t-tests or one-way analysis of variance, respectively. Routine procedures were used for protein identification using immunohistochemistry as well as for RNA extraction and sequencing. Results: iPSC-derived DA neurons exhibited positive staining for IL-6 receptors on the cell body and axonal projections. IL-6 treatment (5ng/ml) for 24 hours reduced DA availability in culture supernatants as measured by high performance liquid chromatography in the absence of neurotoxicity (as determined by caspase staining). Transcriptomic analyses of hiPSC-derived DA neurons treated with IL-6 (100 pg/ml) for 24 hours revealed significantly down-regulated gene pathways involving “vesicle-mediated transport”, “synaptic vesicle exocytosis”, “signal release from synapse”, and “neurotransmitter secretion” (all -log(10)p > 5). These IL-6-induced molecular signatures were reversed by 24 hour co-treatment of cells with IL-6 (100 pg/ml) and baricitinib (200nM), an inhibitor of Janus Kinase (JAK) 1 and 2, which are pivotal signaling molecules in the response to IL-6. Finally, IL-6 treatment (100pg/ml) for 24 hours increased the relative expression of the short (versus long) form of the DA receptor 2 (p < 0.001), which is preferentially expressed on presynaptic DA neurons and inhibits DA vesicular release. Conclusions: These data indicate that IL-6 has direct effects on DA’ergic neurons that can reduce DA vesicular transport and release as well as increase DAergic feedback inhibition. These effects were reversible by the JAK inhibitor baricitinib. Taken together, these findings support the use of iPSCs to reveal mechanistic insights into effects of inflammation on DA neurotransmission, while providing a platform for drug discovery. Keywords: Dopamine, Neurons, Inflammation, IL-6, Induced Pluripotent Stem Cells (iPSCs) Disclosure: Cerevel Therapeutics: Consultant (Self) P221. Characterizing Disease-Associated Missense Mutations in the GABA Transporter, GAT1 Benjamin Throesch, Michelle Wennerholm, Pascal Bonaventure, Grace Woodruff Janssen Research and Development, San Diego, California, United States Background: Dysregulation of neural circuit excitatory/inhibitory balance may underlie multiple neuropsychiatric disorders. Genes involved in GABAergic neurotransmission, the main regulator of inhibition in the central nervous system, have been increasingly implicated in disease. SLC6A1 encodes the GABA transporter GAT1, one of two GABA transporters expressed in the brain. GAT1 primarily localizes to presynaptic terminals of GABAergic neurons, where it regulates signaling through the re-uptake of GABA from the synaptic cleft. Knockout mouse studies have demonstrated impairment of GAT1 leads to abnormal GABA receptor signaling, alterations to hippocampal oscillations, abnormal social behavior, impairments in learning and memory, as well as deficits in prepulse inhibition. To date, over 85 rare mutations in the coding sequence of GAT1 have been identified in patients and associated with a wide variety of disorders including epilepsy, neurodevelopmental delay (NDD), autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), schizophrenia (SCZ), and Tourette Syndrome (TS). Previous studies have investigated a select cohort of variants primarily associated with epilepsy, ASD, and/or NDD and found them to all be loss-of-functions mutations. Here, we set out to characterize an extensive list of missense mutations across the complete spectrum of disorders associated with GAT1. Methods: The effects of 42 missense mutations in GAT1 on GABA transport were evaluated using a radiometric GABA uptake assay in transiently transfected HEK293T cells. From this characterization, three mutations (T46M, R211C, and G297R) were chosen for further evaluation. Subcellular localization and protein expression was measured using confocal imaging in transfected MG-63 cells and western blot in transfected HEK293T cells. Transporter kinetics and affinity to two selective GAT1 antagonists (Tiagabine and NNC-711) were assessed by radiometric GABA uptake. CRISPR/Cas9 gene-editing was then used to introduce these mutations into human induced pluripotent stem cells (hiPSCs). Gene-edited hiPSCs were differentiated into cortical, GABAergic neurons and GABA transport was measured by the radiometric GABA uptake assay. Data were analyzed using a one-way ANOVA with a Dunnett correction for multiple comparisons and graphed as the mean + /- SEM. The significance level was set at p < 0.05. Results: Radiouptake analysis in transiently transfected HEK293Ts demonstrated that 41/42 (97.6%) mutations significantly impacted GABA uptake (n = 9, 3 independent transfections, 3 replicates per transfection). Most variants (39/42, 92.9%) were characterized as loss-of-function, but two mutations (4.8%) were gain-of-function. To gain insight on how GAT1 function correlates with clinical phenotypes, we compared the level of GABA uptake for all mutations associated with a variety of disorders. Mutations associated with epilepsy, NDD, ASD, and ADHD showed on average severe loss-of-function (<25% of WT levels), whereas those associated with SCZ were only mildly impacted (71.5%). The unique mutation associated with TS showed a robust increase in function (293.7%). Protein expression, membrane localization, and ER retention were not significantly different in the selected mutations (T46M, R211C, and G297R) as measured by confocal imaging (n = 12, 4 independent transfections, 3 replicates per transfection) and western blot (n = 6). Mutation T46M showed increased GABA transport velocity, decreased GABA affinity, but no difference in affinity for GAT1 antagonists. R211C demonstrated decreased transport velocity, increased GABA affinity, and increased affinity for GAT1 antagonists. G297R had no active transport (n = 9, 3 independent experiments). Preliminary experiments in hiPSC-derived GABAergic neurons show the neurons broadly recapitulate alterations to GABA transport found in transfected HEK293T cells (n = 12, 2 independent differentiations, 6 replicates per differentiation). Conclusions: Using a radiometric GABA uptake assay, we functionally assessed 42 missense mutations in the GABA transporter, GAT1. Consistent with previous studies, most of the mutations were loss-of-function, yet two variants demonstrated enhanced GABA uptake. Our findings indicate bi-directional modulation of GABA uptake is relevant for neuropsychiatric disease. These changes to GABA transport could not be explained by protein expression or localization, but by transporter kinetics and affinity for GABA. Furthermore, we established hiPSC-derived GABAergic neurons can be used to model alterations to GABA transport in human neurons. Future experiments will focus on the role these mutations play on neuronal signaling using gene-edited, hiPSC-derived GABAergic neurons. Keywords: SLC6A1/GAT1, GABA, hIPSCs, Disease Modeling Disclosure: Johnson and Johnson: Employee (Self) P222. A KRAB-Zinc Finger Transcription Factor Promotes Social Behaviors by Coordinating the Anti-Pathogen Immune Response in Prefrontal Cortex Natalie Truby, Richard Kim, Gabriella Silva, Joseph Picone, Xiaohong Cui, Peter Hamilton* Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia, United States Background: Social behaviors are central to the health of society and the individual and are disrupted in a number of psychiatric illnesses. However, the neurobiological origins of complex social behaviors are incompletely understood. Earlier work employing co-expression analyses of RNA-sequencing (RNAseq) datasets of limbic brain areas from mice subjected to chronic social stress identified one member of the Kruppel-associated box (KRAB) zinc finger protein (KZFP) transcription factor (TF) gene family, Zfp189, as a key protective factor against stress-induced social deficits. Here, to more fully interrogate how ZFP189 may function in the PFC to govern social behaviors, we designed and synthesized novel synthetic ZFP189 TF variants each capable of exerting distinct forms of transcriptional control at in vivo ZFP189 target genes. These ZFP189 variants were virally delivered to mouse PFC and subsequent effect on pyramidal neuron dendritic spine morphology, social behaviors, and transcriptional response were characterized. Methods: Three ZFP189 variants were synthesized: ZFP189-WT which is identical to the endogenously expressed ZFP189 and contains a transcriptionally repressive N-terminal KRAB domain and a C-terminal Cys2-His2 DNA-binding domain; ZFP189-VPR in which the endogenous KRAB domain is replaced with the robust transcriptional activator VP64-p65-Rta (VPR) yet retains the endogenous ZFP189 DNA-binding domain; and ZFP189-DN in which any transcriptional regulatory domain is entirely removed. Each variant was sub-cloned into identical (p1005) expression vectors and gene-targeted regulation was characterized in combination with a ZFP189 response element (RE) luciferase assay in Neuro2a cells (n = 3 in technical triplicate). Expression vectors were then packaged in herpes simplex virus (HSV) to enable brain-region targeted expression. Both male and female C57Bl/6J mice, aged 8-10 weeks, were utilized in these studies. All mice were used in protocols approved by the IACUC. HSV-ZFP189-WT, -ZFP189-VPR, or -ZFP189-DN were delivered by stereotaxic surgery bi-laterally to PFC (15°; +1.8 AP; ± 0.75 ML; -2.7 DV, 1uL). Three days after surgery dendritic spine morphology, social interaction, or RNAseq was performed. For dendritic spine morphology, mice were transcardially perfused with 4% paraformaldehyde, brains were sectioned coronally at 40um, and sections were imaged on a Zeiss 880 Airyscan confocal microscope. For social behaviors, a three-chamber social interaction test was performed involving a choice between an empty cage and novel mouse followed by a choice between a novel vs familiar mouse. Sample sizes are 10-15 mice per group and statistical analyses were performed with one-way ANOVAs, including a post-test comparing the test groups (HSV-ZFP189WT, -ZFP189VPR) versus the control group (HSV-ZFP189DN). Finally, we extracted RNA from virally infected PFC tissues, performed ribosomal RNA depletion, prepared libraries, performed RNAseq in the following configuration: 2x150 paired-end reads on an Illumina sequencing platform (HiSeq 2500) at a depth of >30M reads per sample (n = 8 mice for each condition). Differentially expressed genes (DEGs) were generated using DESeq2 in comparing either test group (HSV-ZFP189-WT, -ZFP189-VPR) to the control group (HSV-ZFP189-DN) with 5% FDR adjusted p-value < 0.05 and absolute log2 fold change > 1 DEGs called as significant. Results: By co-transfecting the ZFP189 RE luciferase plasmid and iterations of our ZFP189 variants, we are able to observe, relative to GFP control: ZFP189-VPR induces robust targeted gene activation (P < 0.0001), ZFP189-WT induces gene-targeted repression (P < 0.05), and ZFP189-DN exerts no regulatory control in this assay (P > 0.5). In virally delivering each of these ZFP189 variants to mouse PFC, we observe that both HSV-ZFP189-VPR and HSV-ZFP189-WT significantly potentiate mature mushroom spine morphology on transduced pyramidal neurons (P < 0.0001). In three chamber social interaction testing, in contrast to all other groups, mice expressing ZFP189-VPR intra-PFC show no preference for social interaction with a novel mouse over investigating an empty cage, nor do they show a preference for social interaction with the novel mouse over a familiar mouse (P < 0.05). By excising virally manipulated PFC and performing RNAseq, we identify significant DEGs regulated by either ZFP189-WT or ZFP189-VPR. Interestingly, in performing gene ontology analyses on these DEGs, we observe an opposing effect on the genes that comprise the anti-pathogen immune response, with particular opposite impact to the gene products involved in interferon gamma signaling. Conclusions: By designing synthetic ZFP189 TFs capable of exerting opposing forms of transcriptional control in brain, we are able to identify that ZFP189-mediated transcriptional regulation in PFC governs social behaviors in rodents. By inverting the natural, repressive gene-regulation of ZFP189-WT to gene-activation with ZFP189-VPR, we observe that mice demonstrate pronounced social deficits with no other detectable behavioral abnormalities. In combining our transcriptional manipulations with RNAseq transcriptome profiling, we detect that ZFP189-VPR consequently down-regulates, and ZFP189-WT up-regulates, immune genes that comprise the anti-pathogen response, suggesting that the PFC function of ZFP189 has evolved to regulate cortical neuroplasticity as a means of connecting brain immune function to social behaviors. Keywords: Adaptive Immunity, Social Behavior, Brain Transcription, Artificial Transcription Factors Disclosure: Nothing to disclose. P223. Patient-Derived Medium Spiny Neurons as a Model of Circadian Rhythms in Bipolar Disorder Kayla Rohr, Michael McCarthy* University of California, San Diego, San Diego, California, United States Background: Bipolar disorder (BD) is a serious mental illness characterized by recurrent mood episodes of depression and mania and disrupted circadian rhythms. BD patients show alterations in reward processing in the striatum, a brain structure in which the largest neuronal population is composed of GABA-producing medium spiny neurons (MSNs) that express D1 and D2 dopamine receptors. In animal models and humans, MSNs are critically important for directing motivated behavior and are the targets of antipsychotic drugs commonly used to treat BD. Moreover, genetic mutations to clock genes in animals alter dopamine signaling to MSNs. Existing human induced pluripotent stem cell (iPSC) models of BD have focused primarily on glutamatergic neurons. Methods: We generated GABAergic MSNs from BD patient and healthy control-derived stem cells using a dual SMAD inhibition protocol. PCR was used to confirm the presence of MSN distinct markers. Expression of the clock genes BMAL1, CRY and PER was evaluated over a 24 h time course as a measure of cellular circadian rhythms. The cell population was characterized further using immunohistochemistry to quantify the percentage of neurons that express GABA versus glutamate. Results: Our protocol yields cell populations highly enriched for GABAergic neurons that express a variety of MSN-specific markers, dopamine receptors, and clock genes. Conclusions: BD patient-derived medium spiny neurons will provide a novel model to study BD and advance previous work examining circadian rhythm abnormalities in other cellular models. Keywords: Bipolar Disorder, iPSC, GABA, Dopamine Receptor Disclosure: Alkermes: Consultant (Self) P224. Cytomegalovirus Antibodies are Associated With Increased Risk of Neuroinflammation, White Matter Damage, and Mental Illness: Evidence From Postmortem Samples Haixia Zheng, Maree Webster, Cynthia Weickert, Martin Paulus, Robert Yolken, Jonathan Savitz Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Herpesviruses such as cytomegalovirus (CMV) have attracted the psychiatric field’s attention not only because they are neurotrophic and may cause neurological disease but because they can be reactivated by stress and inflammation. Importantly, lytic viral replication can in turn worsen inflammation, raising the possibility that poorly controlled CMV infections may contribute to the inflammation underlying the development of some cases of mental illness. We recently published some of the first work linking CMV infections to neuroimaging abnormalities in the context of major depressive disorder (MDD). Whether these findings result from a direct effect of viral damage to the brain or are from indirect inflammatory processes is unclear. Here, we investigated whether CMV infection increases the propensity of having mental illness, neuroinflammation, and brain cellular alterations using postmortem samples. Methods: Postmortem brain data (n = 364) was obtained from the Stanley Medical Research Institute’s brain collection (n = 114 with schizophrenia; n = 78 with bipolar; n = 87 with depression; n = 85 controls). IgG antibodies to CMV were measured using ELISAs. Neuroinflammation was determined on a recursive two-step cluster analysis using expression data for 8 inflammation-related genes in the dorsolateral prefrontal gyrus (DLPFC). Eighty-two cases (n = 30 with schizophrenia; n = 23 with bipolar disorder; n = 29 controls) were divided into “high” (n = 30) or “low” inflammation groups (n = 52). The density of astrocytes, oligodendrocytes, microglia, and neurons were quantified in white matter underlying DLPFC for 51 samples (n = 19 with schizophrenia; n = 18 with bipolar disorder; n = 14 controls). Results: Relative to CMV negative (CMV-) samples, CMV positive (CMV+) samples were 2.12 times more likely to have mental illness (Odds ratio [OR] = 2.12, 95%CI = 1.20~3.80, p = 0.01) with controlling for age, sex, race, postmortem interval, and brain PH. Post-hoc analysis revealed that CMV infection did not significantly increase the odds of having schizophrenia. However, CMV seropositivity significantly increased the odds of having mood disorders (bipolar: OR = 2.49, 95%CI = 1.32~ 4.76, p = 0.004; depression: OR = 2.62, 95%CI: 1.41~ 4.95, p = 0.002). Furthermore, every 1 standard deviation increase of CMV antibody level was associated with 79% increase in odds of having neuroinflammation (OR = 1.79, 95%CI = 1.01~3.32, p = 0.05). Although not statistically significant, CMV + cases also had lower neuron density (Cohen’s d = -0.51) and lower astrocyte density (Cohen’s d = -0.42) in white matter. Conclusions: Anti-viral IgG antibody levels are a surrogate marker of viral reactivation, with higher antibody levels generally indicative of an active infection. Together these results are therefore consistent with the possibility that the reactivation of CMV is a contributing factor to increased neuroinflammation, cellular deficits, and major affective disorders. Nevertheless, it is also possible that the arrow of causality is reversed and that reactivation of CMV could be due to cellular damage or increased cytokines. Experimental designs, potentially with anti-herpesvirus medications, are required to evaluate the potential pathophysiological effects of CMV. Keywords: Postmortem Human Brain, Mental Illness, CMV, Neurons, Astrocytes, Neuroinflammation Disclosure: Nothing to disclose. P225. Using Computational Approaches and Exosomes to Integrate Biological Networks, Environmental Factors and Clinical Outcomes: Multidimensional Predictors of Antidepressant Response Benedetta Bigio, Olivia Barnhill, Paolo de Angelis, Josh Dobbin, Katie Watson Lin, Marin Kautz, Natalie Rasgon, James Murrough, Bruce McEwen, Carla Nasca NYU School of Medicine, New York, New York, United States Background: There is a dearth of new mechanistic models for the development of objective tools to diagnose depression and monitor antidepressant responses. In rodent models, boosting mitochondrial metabolism of acetylcarnitine (LAC) leads to a rapid and persistent antidepressant response by epigenetic regulation of glutamatergic function in key brain circuits; we also reported decreased LAC levels in phenotypes of treatment resistant depression associated with childhood trauma. Methods: Here, we used computational approaches and efficiently leveraged biological samples from previous randomized, placebo-controlled trials to determine the emergence of the pivotal mitochondrial metabolite LAC in relation to previously described individual predictors of antidepressant responses. Results: Our newest data showed the lowest central levels of LAC as assessed by using exosomes enriched for L1CAM, a protein highly expressed in the brain, in subjects characterized by the highest severity of depressive symptoms. We also found the lowest peripheral LAC levels in those subjects with depression characterized by elevated BMI, high reported rates of emotional abuse and decreased leucocytes telomere length (n = 47). Furthermore, we used prediction profilers to show that decreased baseline LAC levels, elevated BMI and high reported rates of emotional abuse predict lack of antidepressant response to pioglitazone (p = 0.02, r = 0.86) with a stronger ability than each individual measure alone. Conclusions: The current findings suggest that integrated factors spanning central and peripheral mitochondrial metabolism, cellular aging, metabolic function and childhood trauma contribute to define potential new mitochondrial phenotypes of depression. The further novelty of combining the exosome technology with computational approaches to study in-vivo molecular mechanisms of mitochondrial metabolism otherwise inaccessible in the human brain will aid in building individualized biological models of depression. Keywords: Mitochondria, Epigenetics, Machine Learning Disclosure: Nothing to disclose. P226. Astrocyte- And Neuron-Specific CD44 Exon Variant Expression and Hyaluronan Levels in Postmortem Brains of Mood Disorders Man Choi (Ada) Ho, Stacey Winham, Sebastian Armasu, Craig Stockmeier, Grazyna Rajkowska, Mark Frye, Marin Veldic* Mayo Clinic, Rochester, Minnesota, United States Background: Cell adhesion molecule CD44 and its ligand hyaluronan play critical roles in the central nervous system, including the regulation of glutamate neurotransmission and neuroplasticity. We previously reported that CD44 gene expression was associated in silico with SNP rs3829280, which was associated with anterior cingulate glutamate levels of major depressive disorder (MDD) and bipolar disorder (BD) patients. Since the relationship between CD44 and mood disorders has not been explored in human brain tissues, we conducted this study to validate the association between rs3829280 and CD44 gene expression and examine the associations of CD44 and hyaluronan in brain tissues of mood disorder patients. Methods: All procedures were conducted according to the Declaration of Helsinki and approved by the Institutional Review Board of the University of Mississippi Medical Center, Jackson, MS, and the University Hospital Cleveland Medical Center, Cleveland, OH. Subjects’ psychopathology was determined by a board-certified clinical psychologist and a board-certified psychiatrist. We selected gray matter tissues of the dorsolateral prefrontal cortex (DLPFC; BA9) and anterior temporal pole (TP; BA38) of subjects meeting DSM-IV criteria for MDD (n = 10) or BD (n = 20) and controls without a psychiatric diagnosis (n = 10) matched by sex and age. rs3829280 genotypes were determined by TaqMan genotyping assay, CD44 exon variant expression levels (astrocyte-specific “short” variant; neuron-specific v4, v5, and v10 variants) were ascertained by RT-qPCR, and hyaluronan was measured by immunoassay. We have previously performed genome-wide DNA methylomics analysis on the same brain tissue samples using the Illumina 850K Human MethylEPIC array. All statistical analyses were performed using SPSS v.25 and in a brain-region-specific manner. Exon variant expression levels and hyaluronan levels were Log10 transformed for analysis. To examine the association between SLC1A2 rs3829280 and CD44 exon variant expression, we compared CD44 exon variant expression levels between SLC1A2 rs3829280 genotypes by ANCOVA with age, sex, and subject group as covariates. To test the associations of CD44 exon variant expression and hyaluronan levels with mood disorders, we compared CD44 exon variant gene expression levels and hyaluronan levels between subject groups by ANCOVA with age and sex as covariates. The correlations between CD44 protein and hyaluronan levels were tested by partial correlation, controlling for age and sex, for each subject group. Comparisons that attained nominal statistical significance at unadjusted p < 0.05 were highlighted, but statistical significance was set at Bonferroni-corrected p < 0.05 to account for multiple comparisons. The Log10 values of the methylomic levels (β value) of 60 CpG site probes mapped to CD44 were compared between groups by ANCOVA with age and sex as covariates. Comparisons that attained nominal statistical significance at unadjusted p < 0.05 were highlighted, but statistical significance was set at Bonferroni-corrected p < 0.05 to account for multiple comparisons. CD44 CpG sites that showed between-group differences were entered into a linear regression model to test for their relationship with the gene expression levels of CD44 exon variants, controlled for age and sex. Results: CD44 exon variant expressions in both DLPFC and TF were not different between rs3829280 A-allele carriers and non-carriers. In DLPFC, nominal statistical significance was observed in between-group comparisons of CD44s (F2,40 = 4.185; p = 0.023), CD44v4 (F2,40 = 3.861; p = 0.031), CD44v5 (F2,40 = 3.937, p = 0.029), and CD44v10 (F2,40 = 4.923; p = 0.013) expression levels, which was mainly due to lower levels in the BD patients compared to the controls (posthoc p < 0.05). However, comparisons did not survive multiple comparison correction (Bonferroni-corrected p > 0.05). In DLPFC, hyaluronan levels were marginally different between groups but without reaching the statistically significant difference (F2,40 = 3.157; p = 0.055) due to relatively higher levels in the BD group. In the DLPFC of the BD group, nominal negative correlations were found between hyaluronan levels and CD44 exon variant expressions: CD44s (r = -.575; p = 0.013), CD44v4 (r = -.504; p = 0.033), and CD44v5 (r = -.535; p = 0.022). Out of 60 CpG sites mapped to CD44, three CpG sites showed between-group differential methylation levels in the DLPFC at nominal level cg01766065 (F2,40 = 3.378; p = 0.046), cg23186333 (F2,40 = 3.605; p = 0.038), cg11478974 (F2,40 = 3.493; p = 0.041), and cg22768101 (F2,40 = 5.100; p = 0.011). Nevertheless, none of these findings remained significant after multiple testing correction. Conclusions: Although we found no association between rs3829280 and CD44 exon variant expressions in human DLPFC and TP, our other findings suggest that astrocytic- and neuronal-related CD44 exon variants and hyaluronan in DLPFC may play a role in the neuropathophysiology of BD, but a larger sample size would be needed. Future studies may investigate how neuronal- and astrocytic-specific CD44 deficiencies and the balance between hyaluronan synthesis and degradation are related to BD symptoms and the mechanisms underlying treatment efficacy. Keywords: CD44, Bipolar Disorder, Major Depressive Disorder, Postmortem Human Brain Disclosure: Nothing to disclose. P227. Lipidomic Signatures Informs Intravenous Racemic Ketamine Associated Remission in Treatment-Resistant Depression: A Pilot Study Balwinder Singh, Siamak MahmoudianDehkordi, Jennifer Vande Voort, Xianlin Han, John Port, Mark Frye, Rima Kaddurah Daouk Mayo Clinic College of Medicine, Rochester, Minnesota, United States Background: Ketamine, a novel rapid-acting antidepressant, is increasingly being used for treatment-resistant depression (TRD), while its mechanism of action (MOA) is actively investigated. In a preliminary study leveraging an established pharmacometabolomic approach targeting acylcarnitines (ACs), we demonstrated rapid utilization of short-chain ACs within 40 minutes of ketamine treatment followed by restoration within 24 hours; this change in short-chain ACs with ketamine is consistent with our previous work identifying similar change but at 8-weeks with slower-acting SSRI treatment. In this abstract, we report novel findings of change in triglycerides using a comprehensive metabolomic platform. We investigated an association between the change in baseline peripheral/blood metabolites after a 40-minute intravenous (IV) racemic ketamine infusion followed by recovery (change in metabolites in 24 hrs) with ketamine treatment response, in nine adults with treatment-refractory major depression. Methods: This is an ongoing open-label, non-randomized, feasibility trial where 14 adults (18-65 years of age) with treatment-resistant major depression and moderately severe/severe symptoms of depression (PHQ-9 score ≥ 15), who provided written informed consent are enrolled. This study is approved by our Institutional Review Board. TRD is defined by the failure of at least two previous antidepressant treatments of at least 8 weeks duration within the index depressive episode. All patients received a single IV ketamine infusion (at the rate of 0.5 mg/kg body weight over 40 minutes) while in the MRI scanner, and the functional MRS (fMRS) sequence was completed. Patients were monitored for an additional 60 minutes after completing the fMRS scan and ketamine infusion. We are reporting data for nine subjects (2 subjects – data not yet analyzed, 3 subjects – data could not be used due to errors in the consent form). The fMRS data was available for five subjects. Peripheral metabolites were measured at baseline, at the end of a 40-minute infusion, 60 min after the completion of infusion, and 24 hours post-infusion. A comprehensive non-targeted metabolomics platform, the Biocrates MxP® Quant 500 kit provided measurements of up to 630 metabolites from 26 biochemical classes including lipids (e.g., glycerophospholipids, sphingolipids, triglycerides [TGs]), and small molecules such as amino acids, biogenic amines, carboxylic acids, indoles, and bile acids. Montgomery-Asberg Depression Rating Scale (MADRS) was used to measure depression symptoms. Early changes in the metabolites (baseline to end of infusion) and late changes (100 minutes to 24 hours post-infusion) were measured. Spearman correlation was conducted to test for a relationship between percent change in peripheral metabolites, quantified as a log2 change, with change in MADRS (baseline to 24 hours). Results: Nine middle-aged (mean age 43 ± 16 years) subjects (3 males/6 females), had moderate depression (MADRS score 23 ± 5). Several metabolites including ceramides, TGs, sphingolipids, and cortisol showed significant changes within 40 min. The comprehensive metabolomic platform showed a significant decrease in 93 Triacylglycerols (TAGs), CE(16:0), CE(17:0), lysoPC a C28:0, PC aa C30:0, PC aa C32:3, PC aa C36:1, PC ae C34:3, PC ae C38:1, PC ae C40:1 within 40 min of ketamine infusion. A significant increase in sphingolipids/glycerophospholipids, TAGs, ceramides/ glycosylceramides, and cholesterol esters was observed within 24 hours (recovery phase). Several TGs, TG(14:0_36:4), TG(18:2_32:1), TG(20:2_34:3), TG(16:0_36:5), and ceramides correlated with change in MADRS. Early changes in blood TGs and other lipids were noted and TG change correlated with central GABA changes, thus suggesting a role of TGs and lipid metabolism in ketamine’s rapid MOA. Conclusions: This new data suggests rapid utilization of triglycerides with ketamine treatment followed by recovery within 24 hours, highlighting a possible role of lipid metabolism in ketamine’s MOA. Lipid peroxidation plays a vital role in CNS homeostasis and a lipid-based pathway is possibly involved in depression pathology, activated by stress and inflammation. The rapid reduction of TAG levels in plasma likely is a consequence of energy deficiency after ketamine shock since fatty acid synthesis and lipoprotein assembly is an energy-consuming process, leading to the reduction of fatty acid biosynthesis and lipoprotein assembly/release. This can be tested by measurement of apolipoprotein and cholesterol/cholesterol-ester levels. TAG accumulation in the recovery phase represents the compensatory effects of energy mobilization and storage. Changes in metabolites that correlate with response to ketamine at 24 hours provide novel insights into the mechanism of ketamine’s action. These changes when linked to brain imaging data inform how effects peripherally, such as in liver and many other organs, impact brain metabolic state and affect mood and other functions. These preliminary findings need to be investigated further to elicit the role of triglycerides and ketamine response. Keywords: IV- Ketamine, Metabolomics Signature, Treatment Resistant Depression, Open Label Trials Disclosure: Nothing to disclose. P228. Efficacy of Lumateperone in Pooled Short-Term Late-Phase Clinical Trials for the Treatment of Major Depressive Episodes Associated With Bipolar II Disorder Suresh Durgam, Hassan Lakkis, Susan G. Kozauer, Richard Chen, Ian D’Souza, Roger S. McIntyre Intra-Cellular Therapies, Inc., New York, New York, United States Background: Bipolar II disorder is associated with significant functional impairment, mood instability, and long periods of potentially disabling symptoms of depression. In December 2021, lumateperone was the second antipsychotic FDA-approved as monotherapy and first approved as adjunctive therapy to treat depressive episodes associated with bipolar II disorder. Lumateperone is also FDA-approved to treat schizophrenia and depressive episodes associated with bipolar I disorder as monotherapy and as adjunctive therapy with lithium or valproate. Lumateperone is mechanistically novel, simultaneously modulating serotonin, dopamine, and glutamate neurotransmission. A robust late phase clinical trial program established the safety and efficacy of lumateperone 42 mg in patients with a major depressive episode (MDE) associated with bipolar I or bipolar II disorder. This analysis evaluated the efficacy of lumateperone 42 mg across pooled short-term studies in patients with depressive episodes associated with bipolar II disorder. Methods: All trials enrolled men and women (18-75 years) with a clinical diagnosis of bipolar I or II disorder who were experiencing an MDE (Montgomery-Åsberg Depression Rating Scale [MADRS] Total score ≥20 and Clinical Global Impression Scale-Bipolar Version-Severity [CGI-BP-S] score ≥4 at screening and baseline). Patients in each trial were stratified by their diagnosis of bipolar I or bipolar II disorder. Lumateperone 42 mg was administered once daily in the evening. This post hoc analysis pooled efficacy data for the intent-to-treat (ITT) population and subgroup of patients with bipolar II disorder from 3 short-term, 6-week, placebo-controlled studies. Study 401 (NCT02600494) and Study 404 (NCT03249376) investigated lumateperone 42-mg monotherapy and Study 402 (NCT02600507) assessed lumateperone 42 mg adjunctive to lithium or valproate. Assessments included change from baseline in MADRS Total score, CGI-BP-S Total score, CGI-BP-S subscores (Depression, Overall Bipolar Illness, and Mania), and Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q-SF) percent score. Results: The pooled ITT population comprised 1,067 patients with bipolar I or bipolar II disorder (placebo, 539; lumateperone 42 mg, 528). Lumateperone 42 mg significantly improved symptoms of depression in the ITT population compared with placebo, as measured by the change from baseline to Day 43 in MADRS Total score (least squares mean difference vs placebo [LSMD], − 2.5; 95% confidence interval [CI] − 3.8, −1.2; P < .001). In the 174 patients (16.3%) with bipolar II disorder (placebo, 87; lumateperone 42 mg, 87) lumateperone also significantly improved MADRS Total score from baseline to Day 43 compared with placebo (LSMD, − 4.0; 95% CI − 7.2, −0.7; P < .05), with significant improvements beginning at Day 22 (P < .05). In patients with bipolar II disorder lumateperone significantly improved illness severity at Day 43 compared with placebo as measured by CGI-BP-S Total score (LSMD, − 1.0; 95% CI − 1.8, −0.2; P < .05), CGI-BP-S Depression subscore (LSMD, − 0.5; 95% CI − 0.9, −0.1; P < .05), and CGI-BP-S Overall Bipolar Illness subscore (LSMD, − 0.5; 95% CI − 0.9, −0.1; P < .05); change from baseline to Day 43 in CGI-BP-S Mania subscore was minimal with lumateperone treatment compared with placebo (LSMD, − 0.0; 95% CI − 0.2, 0.1; P = .61). Lumateperone 42 mg also significantly improved quality of life in the bipolar II disorder subgroup, as measured by Q-LES-Q-SF percent score compared with placebo at Day 43 (LSMD, 6.3; 95% CI 1.1, 11.4; P < .05). Conclusions: In patients with bipolar II disorder experiencing an MDE, short-term treatment with lumateperone 42 mg as monotherapy or as adjunctive therapy significantly improved symptoms of depression, disease severity, and quality of life. Keywords: Lumateperone, Bipolar II Disorder, Bipolar Depression Disclosures: Intra-Cellular Therapies, Inc.: Employee, Stock/Equity (Self) P229. ANC-501, A Novel V1B Receptor Antagonist: Association of MDD Response With MADRS Subscales Stephen Kanes, Philip Perera, Ilan Zipkin, Lara Dennie Ancora Bio, Villanova, Pennsylvania, United States Background: Depression is ranked as a leading cause of disability worldwide by the World Health Organization. Major depressive disorder (MDD) is a psychiatric disorder that is characterized by the occurrence of one or more major depressive episodes, along with an absence of any history of manic, mixed, or hypomanic episodes. In addition to the high mortality rate due to suicide, depressed patients are more likely to develop coronary artery disease and type 2 diabetes among many other related health conditions. Stress mediated by the hypothalamus-pituitary-adrenal (HPA) axis has been hypothesized to be a pivotal factor in the pathophysiology of depression. Specifically, both corticotropin-releasing factor (CRF) and arginine vasopressin (AVP), both of which are produced in the paraventricular nucleus of the hypothalamus are considered primary factors in the regulation of HPA axis activity. Receptor subtypes for these neuropeptides, which may be involved in the regulation of HPA axis activity, have attracted much attention as potential targets for the treatment of depression and anxiety. ANC-501 (formerly TS-121) is an investigational new drug with antagonistic activity of the vasopressin receptor 1b (V1b receptor), which plays a role in the modulation of stress and mood. Based on nonclinical and early clinical studies, ANC-501 appears to be a promising candidate for clinical development with a novel mode of action that may benefit MDD patients. Methods: The initial phase 2 study of ANC-501 was a randomized, double-blind, placebo-controlled phase 2 study, enrolled 51 MDD patients (43 of whom completed the study) randomly assigned to either ANC-501 10 mg, 50 mg or placebo for 6 weeks treatment period with a 2-week follow-up period. The primary endpoint was change from baseline on the Montgomery-Åsberg Depression Rating Scale (MADRS) score at week 6. A key secondary was the change in baseline at the end of the follow-up period. Repeated Measures analysis (MMRM)/non-parametric methods were used to compare the difference between the three treatment groups on the primary endpoint (change from baseline in MADRS score). To further characterize these patients, we performed an exploratory post-hoc analysis of individual baseline MADRS item scores in the high cortisol patients treated with ANC-501 to determine which symptom(s) may be associated with subsequent response at the end of the treatment or follow-up period. Association reported as Pearson’s r uncorrected for multiplicity. Results: The changes from baseline in MADRS score at week 6 (Least Square Mean [95% Confidence interval] were: ANC 501 10 mg (-9.0 [-13.9, -4.1]), ANC-501 50 mg (-9.0 [-13.4, -4.5]), and placebo (-6.4 [-10.7, -2.2]). ANC-501 groups showed greater numerical reductions in MADRS score change from baseline compared to placebo, though these reductions did not achieve statistical significance. However, higher baseline urinary and hair cortisol levels were associated with a greater separation between TS-121 groups and the placebo group in the primary endpoint. In MDD patients with elevated urinary cortisol, baseline MADRS items 4-reduced sleep (r (9) = .61, p = .045) and 9-pessimistic thoughts (r (9) =.60, p = .049) were each correlated with the change from baseline in total MADRS score at the end of 6 weeks of treatment. Interestingly, items 1-apparent sadness (r(9)=.71, p = .013), 3-inner tension (r(9)=.73, p = .011), 5-reduced appetite (r(9)=.72, p = .014), 7-lassitude (r(9)=.71, p = .014), 8-inability to feel (r(9)=0.66, p = .03), and item 9-pessimistic thoughts (r(9)=.76, p = .005) were each strongly correlated with the subsequently change from baseline in total MADRS score at the end of 8 weeks (6 weeks of treatment and 2 weeks off-treatment follow-up). Conclusions: In this trial, post-hoc analysis of the MDD patients with elevated cortisol and who responded to V1b antagonism were characterized by a specific sub-group of baseline symptoms broadly consistent with Melancholic depression, a subtype of depression previously shown to have HPA axis disruption. While this exploratory analysis is limited by a small sample size, these data, if replicated in subsequent studies, may characterize a group of MDD patients whose symptoms are uniquely responsive to V1b antagonism, supporting a personalized, biomarker-driven approach to the treatment of MDD. A phase 2 study of ANC-501 in MDD patients with elevated cortisol is ongoing. Keywords: Depression, Vasopressin 1b Antagonist, Arginine Vasopressin, Adjunctive Treatment, Precision Psychiatry Disclosure: Ancora Bio: Employee (Self) P230. Tractography Analysis of a Clinical Trial of Subcallosal Cingulate Deep Brain Stimulation for Depression Ki Sueng Choi, Juna Khang, Patricio Riva Posse, Lyndahl Himes, Binith Cheeran, Helen Mayberg Emory University, Atlanta, Georgia, United States Background: Open-label studies of subcallosal cingulate deep brain stimulation (SCC DBS) for treatment-resistant depression have demonstrated sustained response and remission rates that are clinically significant. Validation of a patient-specific tractography-based targeting approach has further streamlined lead implantation surgery with improved clinical outcomes. This is in contrast to the halted multi-site, double-blind, randomized sham-controlled clinical trial (BROADEN), where long-term response rates matched open-label results (50% responders at 2 years) but where the predefined 6-month endpoint fell short of projections. Heterogeneity in patient selection, as well as variability in targeting, accuracy of lead placement, and programming are potential contributors. As subjects in BROADEN were implanted using anatomical rather than tractographic methods, we hypothesize that response variability may be explained by differences in white matter activation pathways (WMAP) impacted by trial-and-error adjustments during the open-label follow-up period. We test this hypothesis here using a retrospective normative tractography analysis. Methods: Fifty-five patients (of 90 enrolled) completed 2 years of open-label SCC DBS. WMAP was calculated for 47 patients (with adequate imaging data) using Lead DBS (https://www.lead-dbs.org). All 47 patients were assigned to one of four outcome groups: remitter (HDRS-17 ≤ 7, n = 16), responder (HDRS-17 improvement ≥40%, n = 8), low-responders (HDRS-17 improvement ≤40%, n = 13), and non-responders (no HDRS-17 improvement at any time over 2 years, n = 10). Standard imaging processing pipelines were used for generating common WM activation pathways. The anatomical location of active contacts was first compared among four groups. Then, common activation pathways were calculated using the HCP dMRI dataset, producing whole-brain common group activation pathways by Lead Group Analysis. Results: Group tracts explained differences in outcomes. Remitters show complete WMAP of three bundles: forceps minor (FM), uncinate fasciculus (UF), and cingulum bundle (CB). Responders show similar CB and UF to remitters, but incomplete FM activation. Critically, non-responders showed no activation and low-responders showed incomplete activation of the left CB. There were no anatomical location differences in active stimulation contacts among the groups. Conclusions: These findings provide evidence that poor outcomes in BROADEN were due to suboptimal lead implantation that could not be appreciated by anatomical location alone. As with independent experimental studies, patient-specific tractography-based targeting may be necessary to standardize and optimize SCC DBS. Keywords: DBS, Diffusion Tensor Imaging (DTI), Treatment-Resistant Depression Disclosure: Janssen Pharmaceuticals: Advisory Board (Self) P231. Effects of Acute Estradiol or Progesterone Administration on Perimenstrual Exacerbation of Suicidal Ideation, Depression, and Perceived Stress: Preliminary Analysis of a Three-Period Crossover Randomized Controlled Trial Jordan Barone, Jessica Peters, Tory Eisenlohr-Moul* University of Illinois at Chicago, Chicago, Illinois, United States Background: Suicide appears routinely among the top five causes of death worldwide among females of reproductive age. Perimenstrual (around menses) withdrawal from the ovarian steroids estradiol (E2) and progesterone (P4) correlates with suicidal ideation (SI) and behavior and therefore may represent a time-varying trigger for acute suicide risk. Our prior crossover placebo controlled RCT provided the first experimental evidence that administration of transdermal E2 (.1mg/day) and oral micronized P4 (200mg/day) in the perimenstrual weeks (days 7-21 following LH surge) reduces exacerbation of depressed mood, perceived stress, and suicidal ideation in female outpatients with SI. These beneficial effects were strongest around the onset of menses, suggesting a pathophysiologic role of perimenstrual E2 or P4 withdrawal in cyclical symptom exacerbation. The next step is to clarify whether E2 or P4 alone may be responsible for these effects. Methods: The present three-period, double-blind, crossover randomized controlled trial (R00MH109667; NCT03498313) extended this work by testing the unique effects of perimenstrual E2 or P4 administration using the same doses/routes in the same timeframe. Inclusion criteria included endorsement of past-month suicidal ideation; seeing a mental health professional at least every 3 months; aged 18-45; no history of serious or chronic nonpsychiatric illness; no family or personal history of recurrent blood clots or known thrombophilia; regular menstrual cycles (25-35 days); not pregnant or breastfeeding and at least one year postpartum; not using hormonal medications or devices; BMI 18-34.99 kg/m2; not regularly using nicotine; no history of hospitalization for manic episode or psychosis; and a current substance use disorder that would interfere with the study. Medication use was allowed. After a baseline month, participants completed three subsequent experimental intervals, separated by two washout cycles, with each interval spanning the two weeks surrounding expected menses onset (with medication taken from days +7 to +21 following a positive 40mIU/ml urine luteinizing hormone test). Daily surveys evaluated depressed mood, perceived stress, and suicidal ideation (1=Minimal or Absent, 6=Extreme), and calls evaluated imminent suicide risk; no suicide attempts occurred. We hypothesized that experimental E2 and P4 would both reduce perimenstrual exacerbation of symptoms, such that the symptom trajectory in the PBO condition would be characterized by increases in symptoms from the early luteal phase (when EP levels are rising/moderate) to the perimenstrual phase, when dual EP withdrawal is most pronounced, and that these changes would be prevented in both the E2 and P4 conditions. Results: Forty-five participants were randomized; 25 completed all three conditions, 34 completed two conditions, and 42 completed one condition. 34 participants with two conditions were included in the per-protocol sample for the present analyses. Power analyses indicated that the smallest detectible effect size (Given: person N = 34, daily N = 2,234; effective/corrected N = 67; ICC = .50, α = .05, 1- β = .80) was f2 = .09. Even though a clear perimenstrual exacerbation pattern was observed for all outcomes (including SI) in the pre-experimental baseline cycles, there was a mean perimenstrual decrease in SI across all experimental conditions, suggesting a marked placebo effect on SI that was not present in the prior trial. Relative to placebo, E2 administration reduced perimenstrual increases in depression (Est = -.41, SE = .17, p = .01; Effect size note: response scale range=5) and perceived stress (Est = -.33, SE = .15 p = .03), although a corresponding greater reduction in suicidal ideation severity did not reach statistical significance (Est = -.15, SE = .10, p = .17). In contrast, relative to placebo, P4 administration did not significantly alter perimenstrual changes in depression or perceived stress (p’s > .30), and showed less robust perimenstrual reductions (i.e., less robust placebo effect) in suicidal ideation severity (Est = .26, SE = .12, p = .03). Conclusions: These preliminary analyses suggest that the beneficial effects of dual E2 and P4 administration observed in the first trial may have been due to a benefit of E2 specifically. These may represent critical mediators of acute perimenstrual suicide risk. Though preliminary, results combine to suggest a potential pathophysiologic role of E2 withdrawal in perimenstrual exacerbation of depression, perceived stress, and suicidal ideation. Forthcoming intent-to-treat analyses will provide information about the potential usefulness of perimenstrual E2 as a clinical intervention. Further, additional per-protocol analyses of these trial data are warranted to determine the robustness of our findings to the inclusion of covariates such as daily medication changes, the COVID-19 pandemic, and physical pain. Larger clinical trials with pre-selection of patients for perimenstrual exacerbation, as well as appropriate attention to placebo mitigation methods, are warranted to determine the underlying causes of steroid withdrawal sensitivity—and particularly E2 withdrawal sensitivity—across the menstrual cycle. Keywords: Estradiol, Progesterone, Reproductive Affective Disorder, Suicide, Depression Disclosure: Nothing to disclose. P232. Metabolic Profile of Lumateperone in Late-Phase Clinical Trials for the Treatment of Major Depressive Episodes Associated With Bipolar I or Bipolar II Disorder Sharon Mates, Suresh Durgam, Susan G. Kozauer, Jason Huo, Robert E. Davis, Christoph U. Correll Intra-Cellular Therapies, Inc., New York, New York, United States Background: Atypical antipsychotics are correlated with a range of metabolic adverse effects including weight gain and increased risk of diabetes, hyperglycemia, and hyperlipidemia. Identifying treatment options with a favorable metabolic profile is important to improve patient outcomes and to reduce patient morbidity and mortality. Lumateperone is an FDA-approved antipsychotic to treat schizophrenia and depressive episodes associated with bipolar I or bipolar II disorder as monotherapy and as adjunctive therapy with lithium or valproate. Lumateperone is mechanistically novel, simultaneously modulating serotonin, dopamine, and glutamate neurotransmission. A robust late phase clinical trial program established the safety and efficacy of lumateperone 42 mg in patients with a major depressive episode (MDE) associated with bipolar I or bipolar II disorder. This analysis evaluated the metabolic impact of lumateperone 42 mg across short- and long-term studies in patients with bipolar depression. Methods: All trials enrolled men and women (18-75 years) with a clinical diagnosis of bipolar I or II disorder who were experiencing an MDE (Montgomery-Åsberg Depression Rating Scale [MADRS] Total score ≥20 and Clinical Global Impression Scale-Bipolar Version-Severity [CGI-BP-S] score ≥4 at screening and baseline). Lumateperone 42 mg was administered once daily in the evening. Safety data were reported for 3 trial groups: (1) data pooled from 2 short-term, 6-week, placebo-controlled studies of lumateperone 42-mg monotherapy (Study 401 [NCT02600494]; Study 404 [NCT03249376]); (2) a 6-month open-label extension period (OLE) of Study 401 that evaluated long-term effects of lumateperone 42-mg monotherapy; (3) a Phase 3 placebo-controlled study (Study 402, NCT02600507) that investigated lumateperone 42-mg therapy adjunctive with lithium or valproate in patients with bipolar depression. Metabolic assessments included changes in weight, body mass index (BMI), waist circumference, and cardiometabolic laboratory parameters. Potentially clinically significant (PCS) weight change defined as ≥7% change from baseline was assessed. Subgroup analyses were conducted in patients who were classified by baseline BMI as underweight (<18.5 kg/m2), normal weight (≥18.5 to <25), overweight (≥25 to <30), or obese (≥30). Results: The short-term safety population comprised 746 patients in pooled monotherapy trials (placebo, 374; lumateperone 42 mg, 372) and 352 patients in the adjunctive study (adjunctive placebo, 175; adjunctive lumateperone 42 mg, 177). In the short-term studies, mean changes from baseline to end of treatment with lumateperone were similar to placebo for weight (kg; placebo, 0.19; lumateperone, 0.06; adjunctive placebo, 0.23; adjunctive lumateperone, 0.00), BMI (kg/m2; placebo, 0.07; lumateperone, 0.02; adjunctive placebo, 0.09; adjunctive lumateperone, 0.00), and waist circumference (cm; placebo, 0.03; lumateperone, 0.18; adjunctive placebo, 0.11; adjunctive lumateperone, 0.06). No patients treated with lumateperone had PCS weight gain during treatment. PCS weight loss was rare in all groups (<1%); PCS weight loss with lumateperone monotherapy or adjunctive therapy was only observed in those who were overweight (2 patients) or obese (2 patients) at baseline. Mean change from baseline to the end of treatment with lumateperone was similar to placebo for total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, glucose, or insulin in the monotherapy and adjunctive therapy trials. The long-term OLE safety population comprised 127 patients. There was no mean increase from baseline to the end of treatment in weight (−0.01 kg), BMI ( − 0.01 kg/m2), or waist circumference (−1.46 cm) with lumateperone 42 mg. At any time during the 175-day treatment period, 3.4% of patients (2 normal BMI, 2 obese patients at baseline) experienced PCS weight increase and 6.0% had PCS weight loss (1 normal weight, 3 overweight, 3 obese patients at baseline). There were no clinically significant changes from baseline to end of treatment in total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, glucose, or insulin. Conclusions: In patients with bipolar I or bipolar II disorder experiencing an MDE, lumateperone 42-mg monotherapy or adjunctive therapy had a favorable metabolic profile for both acute and long-term treatment. Keywords: Lumateperone, Bipolar Disorder, Metabolic Profile Disclosure: Intra-Cellular Therapies, Inc.: Employee, Stock/Equity (Self) P233. Personalized Connectivity-Based Accelerated TMS Therapy for Bipolar Depression: Preliminary Outcomes Yvette Sheline, Walid Makhoul, Greg Bond, Connor Law, Eugene Cha, Kevin Lynch, Nicholas Balderston, Zafiris Daskalakis, Robin Cash Center for Neuromodulation in Depression and Stress, University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Bipolar disorder (BD) is prevalent, severe and under studied, and is often excluded from clinical trials. The depressive phase of Bipolar disorder I or II accounts for the majority of time a patient is ill –72% in BD-I and 81% in BD-II. Many patients fail to respond adequately or cannot tolerate pharmacotherapy due to adverse side effects. Thus, accessibility to alternative treatment options is important to help achieve and sustain remission. One treatment option, transcranial magnetic stimulation, (TMS) has demonstrated efficacy in treatment resistant major depression, however there have been relatively few studies in Bipolar Disorder, and none to our knowledge utilizing intensive high dose spaced theta-burst rTMS (HDS-TBS). Thus, the objective of this clinical trial is to determine the clinical and functional connectivity effects of intensive TMS in depressed patients with Bipolar Disorder I/II. Methods: To date, a total of 10 patients have been screened using the Structured interview for DSM-5, the Montgomery-Asberg Depression Rating Scale (MADRS), the Young Mania Rating Scale (YMRS), and screened for dementia (MMSE > 24). Following informed consent, participants were randomized to active or sham treatment using HDS-TBS. Inclusion criteria: Bipolar depression, DSM 5 criteria; Age 18-70; Right or left-handed; All genders; Treatment resistant depression: 2+ failed antidepressant trials (< 50% reduction in symptoms) using ATHF criteria; Not in a mixed bipolar episode (YMRS ≤12) ; Not rapid cycling (> 4 episodes in the past year); Stable medication regimen, including mood stabilizer; MADRS ≥ 20. Treatment sessions: iTBS was delivered to individually targeted left dorsolateral prefrontal cortex (DLPFC) site at 90% resting motor threshold (RMT). The stimulation pattern and total number of pulses: triplet 50 Hz bursts, repeated at 200 ms (5 Hz) intervals, 2 s on, 8 s off; 1800 pulses per session; total duration 10 min. Ten iTBS sessions/day x 5 sequential days. Neuroimaging sessions were conducted prior to iTBS treatment (‘pre’), and 24 hours following day 5 (‘post’). Sham iTBS: Both active and sham iTBS used a Magventure Cool B65 A/P coil. The sham treatment blocks the magnetic field with an internal spacer, allowing the appropriate coil surface (active vs. sham) to be placed against the scalp. During sham iTBS, brief electric pulses were delivered to the scalp simultaneous to the TMS pulse to mimic the scalp sensation. Neuroimaging procedures: MRI data was acquired on a 3 Tesla Siemens Prisma scanner using a 64-channel head coil (Erlangen, Germany). Our acquisitions were optimized for Siemens Prisma scanners, rigorously tested as part of the human connectome project (HCP) and consisted of T1, T2 and rs-fMRI (2x 8 min) scans. During the visits, patients were co-registered to the T1 scan using fiducial points at the Nasion, Tragi and nose tip. TMS pulses were delivered to the target at the optimal orientation, and the accuracy of this targeted stimulation was monitored and tracked by the Brainsight software. Neuronavigation and Targeting: DLPFC TMS targets were individually optimized based on anticorrelated functional connectivity with the subgenual anterior cingulate cortex (SGACC), methods detailed in Cash et al (2021). Coil orientation was defined individually such that the e-field at the cortical target was maximized (Balderston et al). Prior to the iTBS visits, target coordinates and orientation vectors were generated from the fMRI data and the e-field models and loaded into the Brainsight neuronavigation system along with the subject’s reconstructed T1 image. Scalp and cortical surfaces were generated from the T1. During the visits, the subject was co-registered to the T1 using fiducial points and the accuracy of targeted stimulation was monitored and tracked by the Brainsight software. Results: 7 participants meeting inclusion and exclusion criteria were included. Participants had a history of a mean of 5 failed treatment trials with antidepressants, (range 3-8) and a mean of 1 augmentation trial, as well as treatment with mood stabilizers. MADRS scores decreased from baseline (mean 30.8 + /-5.5, range 23-50) to post-treatment (mean 15 + /- 10.4, range 0-26). All participants tolerated the therapy without adverse events. Functional connectivity between SGACC and individually targeted DLPFC stimulation sites decreased (more anti-correlated) from baseline mean z = -0.02 + /- 0.24 to post-treatment mean z = -0.07 + /- 0.19. The decrease in MADRS scores was correlated with decrease in correlation (increased anticorrelation) of SGACC-individually targeted DLPFC pre- to post-treatment calculated as r = 0.79. Conclusions: We remain blinded to treatment status in this small sample of treatment refractory depressed bipolar patients but the study indicates the potential for being able to discriminate treatment effects. Some patients experienced a large decrease in depressive symptoms while others did not. Overall, the connectivity of SGACC to individually targeted DLPFC decreased (became more anti-correlated) and the decreases in MADRS pre- to post-treatment positively correlated with the changes in functional connectivity, suggesting a possible mechanism for treatment responses. The treatment was well tolerated with no adverse events. We will continue to recruit participants to this study and expect to have a larger sample to report. Keywords: Bipolar Depression, TMS, Connectivity, Subgenual Anterior Cingulate Cortex, DLPFC Disclosure: Nothing to disclose. P234. An Open Label Pilot of The Safety and Efficacy of Comp360 Psilocybin in Patients With Severe Treatment-Resistant Depression Scott Aaronson, Tammy Miller, Samuel Rudow, MacKenzie Forbes, Audrey Shoultz, Trisha Suppes* Stanford University School of Medicine and VA Palo Alto Health Care System, Palo Alto, California, United States Background: Psychedelics are rapidly gaining ground as an intervention of interest for severe mood and anxiety disorders. The focus for most of this research is for patients who have failed four or fewer adequate interventions. Few trials of any intervention have looked at patients with depression who have failed more than four treatments in the current episode. This is a pilot study looking at the open label administration of 25 mg of COMP360 (synthetic psilocybin) in twelve patients with documented failures of at least five treatments in the current major depression episode. Methods: Over 100 patients were screened to identify 12 patients ((n = 12 (6 men and 6 women)) with age a mean of 40.5 + /- SD 9.2 years) with documented major depressive disorder with failures of at least five treatments in the current episode. Failures could include FDA approved medications, neurostimulation and up to one failed trial of psychotherapy. Potential participants with a history of psychosis or of concurrent substance use disorders were excluded. Subjects with active suicidal ideation were excluded. Participants with a history of post-traumatic stress disorder (PTSD) were included provided the primary diagnosis was major depressive disorder and not PTSD. Participants were dosed with a single 25 mg dose of a proprietary synthetic psilocybin (COMP360) after withdrawal from antidepressants and most psychotropic medication (benzodiazepine related hypnotics were allowed) (subjects were drug free for at least two weeks prior to dosing). All subjects had three sessions of psychological preparation followed by a nine-hour dosing session, and then at least three psychological integration sessions. Subjects were followed for 12 weeks post dose. Subjects were monitored throughout the study by regular visits with the treating psychiatrist as well as a therapist. The primary outcome measure was the MADRS at three weeks post dose. Response was defined as a MADRS score reduction of ≥50% since baseline and remission as a score of ≤10. Subjects were also followed by CSSRS scores to monitor for safety. This trial is registered at clinicaltrials.gov under NCT04433858. Results: There were no unexpected adverse events. For participants with co-morbid PTSD histories, the dosing sessions were often quite emotional though no interventions with medication or hospitalization were necessary. Nor overall was there evidence of psychological difficulties with the dosing sessions. At one week post dose 9 of 12 (75%) participants met both response and remission criteria. By the primary outcome measure at week 3 eight of 12 (67%) met response and 5 of 12 (41.7%) met remission criteria. At week 12, 7of 12 (58%) were responders and 3 of 12 (25%) were still remitters. There was no evidence of an increase in suicidal ideation or adverse events post dosing. Curiously, the five participants determined to be co-morbid for PTSD at baseline were the five participants who did not meet response criteria at week 12. Those participants were also more likely to report minimal psychedelic or mystical impact or effect during the dosing sessions. Conclusions: Psilocybin may have efficacy across the spectrum of depression treatment resistance. Presence of PTSD may identify participants less likely to achieve durable response from this single dose strategy. These results are supportive of continued investigation into the use of psychedelic therapy in even the most treatment resistant patients who have not benefitted from even the most aggressive treatment interventions. Keywords: Depression, Treatment Resistant Depression, Psychedelic Medicine, Psilocybin Disclosures: Compass Pathways: Grant (Self), Zylorion: Advisory Board (Self), Merck: Contracted Research, Consultant (Self), Sunovion, Servier: Honoraria (Self), Impel, Intracellular Therapies: Consultant (Self) P235. Improvements in Cognitive and Physical Functioning Outcomes in Depressed Patients Treated With AXS-05 (Dextromethorphan-Bupropion): Results From the EVOLVE Open-Label, Long-Term Study Amanda Jones, Maurizio Fava, Zachariah Thomas, Caroline Streicher, Shawn Alter, Herriot Tabuteau Axsome Therapeutics, New York, New York, United States Background: Cognitive dysfunction and fatigue are common in major depressive disorder (MDD). Data from STARD suggest that sad mood, concentration difficulty, and fatigue are the symptoms of MDD that contribute the most to overall impairment. Treatments that rapidly improve mood and related symptoms may result in improved clinical and functional outcomes. AXS-05 (dextromethorphan-bupropion) is a novel, oral, investigational, NMDA receptor antagonist with multimodal activity being developed for MDD. The dextromethorphan component of AXS-05 is an antagonist of the NMDA receptor, an ionotropic glutamate receptor, and a sigma-1 receptor agonist. The bupropion component of AXS-05 serves primarily to increase the bioavailability of dextromethorphan, and is a norepinephrine and dopamine reuptake inhibitor. Here we report the impact of AXS-05 on cognitive and physical functioning outcomes in MDD, as assessed by the Massachusetts General Hospital Cognitive and Physical Functioning Questionnaire (CPFQ). The CPFQ is a 7-item patient-rated scale used to measure cognitive and executive dysfunction in mood and anxiety disorders and includes assessments pertaining to cognition, energy, wakefulness, and motivation. Methods: EVOLVE (Evaluation of NMDA Modulation for Depressive Episodes) was an open-label, US trial, in which patients were treated with AXS-05 (dextromethorphan HBr 45 mg- bupropion HCl 105 mg) twice daily for up to 15 months. Eligible patients had either rolled in following completion of a prior AXS-05 study or were directly enrolled and had a DSM-5 diagnosis of MDD, a MADRS score of ≥25, and had been treated with at least 1 prior antidepressant in the current major depressive episode. A total of 186 patients were enrolled, consisting of 35 roll-over and 146 directly enrolled. Efficacy endpoints included MADRS, CPFQ, and the Sheehan Disability Scale (SDS). The primary efficacy analysis was the change from baseline to Week 6 (primary timepoint) and Weeks 1 and 2 (key secondary timepoints). Here we present the results for the directly enrolled patients. Results: Treatment with AXS-05 resulted in meaningful improvement in the CPFQ. Mean baseline CPFQ scores were 28.4. Mean reductions from baseline to Weeks 1, 2, and 6 were 2.0 ± 4.28 (p < 0.001), 4.4 ± 5.61 (p < 0.001), and 7.5 ± 6.58 (p < 0.001), respectively. Improvements on the CPFQ were durable through Month 6 (-9.5 ± 6.48; p < 0.001) and Month 12 (-8.5 ± 6.90; p < 0.001). A ≥ 1 category improvement in CPFQ was achieved by 40.0%, 58.8%, and 75.8% of patients at Weeks 1, 2, and 6, respectively. At Months 6 and 12, 82.1% and 77.1% of patients had achieved ≥ 1 category improvement in CPFQ. Rapid, durable, and statistically significant improvements were also seen in the MADRS total score and the SDS, starting at Week 1 and every subsequent timepoint measured. Long-term treatment with AXS-05 was well tolerated. The most commonly reported adverse events were COVID-19 infection (8.9%), nausea (8.9%), headache (7.5%), dry mouth (6.2%), insomnia (5.5%) and dizziness (5.5%). Conclusions: Treatment with AXS-05 resulted in rapid and sustained improvement in the CPFQ, depression symptoms, and functioning in patients who failed one prior antidepressant in the current major depressive episode. These data provide additional evidence of the long-term efficacy and safety of AXS-05 in MDD. This research was supported by Axsome Therapeutics. Keywords: Major Depressive Disorder (MDD), NMDA Receptor Antagonist, Cognition, Rapid-acting Antidepressant Disclosure: Axsome Therapeutics: Stock / Equity (Self) P236. An Open Label Study of COMP360 (Synthetic Psilocybin) in Bipolar Type II Depression Scott Aaronson, Trisha Suppes, Tammy Miller, Samuel Rudow, MacKenzie Forbes, Audrey Shoultz Sheppard Pratt Health System, Baltimore, Maryland, United States Background: Treatment options are limited for patients with bipolar depression. With accelerating interest in the use of psychedelics in difficult to treat mood and anxiety disorders and early evidence of efficacy along with safety, the question is raised as to whether some patients with cyclical mood disorders may also benefit from psychedelic therapy. The single biggest concern is whether use of psilocybin could precipitate a manic or hypomanic episode in a vulnerable population and second is whether there is evidence for efficacy. For this pilot study, we chose patients with a history of bipolar type II depression and followed a similar protocol of treatment used in earlier studies in unipolar depression. Methods: Thirteen adults (5 Males/8 Females) age: (avg 36.1 SD 11.5) with bipolar type II depression who had failed at least two medications in the current episode, without rapid cycling by history, and no history of mania or psychoses were recruited to be dosed with a single 25 mg dose of a proprietary synthetic psilocybin (COMP360) after withdrawal from psychotropic medication (subjects were drug free for at least two weeks prior to dosing). Subjects with active suicidal ideation were excluded. All subjects had three sessions of psychological preparation followed by a nine-hour dosing session, and then at least three psychological integration sessions. Subjects were followed for 12 weeks post dose. Subjects were monitored throughout the study by regular visits with the treating psychiatrist as well as therapist. The primary outcome measure was the MADRS at three weeks post dose. Response was defined as a MADRS score reduction of ≥50%since baseline and remission as a score of ≤10. Subjects were also followed by CSSRS and YMRS scores to monitor for safety. This trial is registered at clinical trials.gov under NCT04433845. Results: There were no unexpected adverse events or evidence of psychological difficulties with the dosing sessions. No subjects developed hypomanic or mood instability following the dosing session. At one week after dosing 10 of 13 (76.9%) met remission criteria and 11 of 13 (84.6%) met response criteria. At three weeks, our primary outcome measure, 10 of 13 (76.9%) met both remission and response criteria. At 12 weeks 10 of 12 (83.3%)met both remission and response criteria. One subject was never a responder, though 8 subjects were in remission for all 12 weeks and 2 subjects were in remission from week 2 to week 12. One subject initially remitted but relapsed into depression at week 6, and one responded, relapsed into depression at week 3, resumed antidepressant medication at week 6 which discontinued them from the study as a non-responder at week 6. No subject showed evidence of mania or hypomania by YMRS score at any visit after dosing, and no significant disruption to sleep was reported. No subject reported an increase in suicidal ideation after dosing. One subject was dropped from the study prior to dosing due to worsening bipolar symptoms during withdrawal from mood stabilizing drugs and was not included in this analysis. Most subjects stayed off medication for 12 weeks post dose with three resuming mood stabilizing medication after the three week primary outcome measure for prophylaxis and not due to any symptomatic depression worsening. Conclusions: In this small, open-label pilot study, most subjects reported significant improvement in chronic depressive symptoms with durability lasting for the three months they were followed post dosing. The relative improvement was notably greater in treatment-resistant depression patients with bipolar II disorder versus patients with major depression. Further exploration into the efficacy of psychedelic therapy within this poorly served psychiatrically ill population is warranted. Keywords: Psychedelics, Psilocybin Therapy, Bipolar Type II Depression, Clinical Trial Disclosure: Compass Pathways: Grant (Self), LivaNova, Neuronetics, Genomind, Janssen, Compass Pathways, Sage Therapeutics: Consultant (Self) P237. Clinical Response to High-Dose Eicosapentaenoic Acid (EPA) Supplementation is Greater in MDD Patients Who Achieve Higher Plasma Concentrations of EPA-Derived Pro-Resolving Lipid Mediators Stefania Lamon-Fava, Minying Liu, Boadie Dunlop, Becky Kinkead, Schettler Pamela, Jennifer Felger, Thomas Ziegler, Andrew Nierenberg, Maurizio Fava, David Mischoulon, Mark Rapaport Tufts University, Boston, Massachusetts, United States Background: Chronic inflammation has been implicated in the pathophysiology of major depressive disorder (MDD). Chronic inflammation is characterized by an impaired resolution of inflammation. We hypothesize that activation of the resolution of inflammation through ω-3 fatty acid supplementation may be a successful therapeutic strategy for the treatment of MDD. Methods: MDD patients with body mass index >25 kg/m2 and plasma high-sensitivity C-reactive protein ≥3 μg/mL (n = 61) were enrolled in a 12-week randomized trial consisting of 4 parallel arms: eicosapentaenoic acid (EPA) 1 g/d, 2 g/d, and 4 g/d, and placebo. The supplement contained EPA and docosahexaenoic acid (DHA) in a 3.9:1 ratio. Depression symptoms were assessed using the clinician-rated IDS-C30 scale. Plasma fatty acids and pro-resolving lipid mediators (SPMs) were measured in 42 study completers at baseline and at the end of treatment by liquid chromatography/mass spectrometry. Results: The response rate (defined as ≥50% reduction in IDS-C30 score) was higher in the 4 g/d EPA arm than in the placebo and EPA 1 g/d and 2 g/d arms (64%, 40%, 38% and 36%, respectively). In the 4 g/d EPA arm, responders had significantly greater increases in 18-hydroxyeicosapentaenoic acid (18-HEPE), a direct precursor of the SPM resolvin E1-3, than non-responders. In addition, at week 12, resolvin E2 and E3 were detectable in plasma in a greater number of responders than non-responders. Within the 4 g/d EPA arm, the increase in 18-HEPE was significantly associated with reductions in plasma hs-CRP concentrations and IDS-C30 scores. Conclusions: Response rates were higher among MDD patients randomized to EPA 4 g/d supplementation and in those who showed a greater ability to activate the synthesis of 18-HEPE and its SPMs. The inverse association of 18-HEPE with both systemic inflammation and symptoms of depression highlights the activation of the resolution of inflammation as a likely mechanism in the treatment of MDD with ω-3 fatty acid supplementation. Keywords: EPA, Major Depressive Disorder (MDD), Inflammation Disclosure: Nothing to disclose. P238. Evaluation of Mania and Hypomania in Late-Phase Clinical Trials of Lumateperone in the Treatment of Major Depressive Episodes Associated With Bipolar I or Bipolar II Disorder Mauricio Tohen, Susan G Kozauer, Yifan Mo, Willie R Earley, Suresh Durgam University of New Mexico Health Science Center, Albuquerque, New Mexico, United States Background: Although atypical antipsychotics are efficacious in treating bipolar disorder, some have been associated with case reports of switching to mania/hypomania. Thus, identifying antipsychotic treatment options with a low risk for inducing mania/hypomania is important to optimize patient outcomes. Lumateperone is an FDA-approved antipsychotic to treat schizophrenia and depressive episodes associated with bipolar I or bipolar II disorder as monotherapy and as adjunctive therapy with lithium or valproate. Lumateperone is mechanistically novel, simultaneously modulating serotonin, dopamine, and glutamate neurotransmission. A robust late phase clinical trial program established the safety and efficacy of lumateperone 42 mg in patients with a major depressive episode (MDE) associated with bipolar I or bipolar II disorder. This analysis evaluated measures of mania and hypomania across short- and long-term studies of lumateperone in patients with bipolar depression. Methods: All trials enrolled men and women (18-75 years) with a clinical diagnosis of bipolar I or II disorder who were experiencing an MDE (Montgomery-Åsberg Depression Rating Scale [MADRS] Total score ≥20 and Clinical Global Impression Scale-Bipolar Version-Severity [CGI-BP-S] score ≥4 at screening and baseline). Lumateperone 42 mg was administered once daily in the evening. Mania and hypomania data were reported for 3 trial groups: (1) data pooled from 2 short-term, 6-week, placebo-controlled studies of lumateperone 42-mg monotherapy (Study 401 [NCT02600494]; Study 404 [NCT03249376]); (2) a 6-month open-label extension period (OLE) of Study 401 that evaluated long-term effects of lumateperone 42-mg monotherapy; (3) a Phase 3 placebo-controlled study (Study 402, NCT02600507) that investigated lumateperone 42-mg therapy adjunctive with lithium or valproate in patients with bipolar depression. Assessments included the incidence and severity of treatment-emergent adverse events (TEAEs) of mania/hypomania in the safety population (all patients who received ≥1 dose of study drug). Young Mania Rating Scale (YMRS) Total score and CGI-BP-S Mania subscore were measured in the intent-to-treat (ITT) population (all randomized patients who received ≥1 dose of study drug and had a valid baseline and ≥1 valid post-baseline MADRS assessment) in placebo-controlled studies. Change from baseline was analyzed using a mixed-effects model for repeated measures in the placebo-controlled studies and using a last observation carried forward approach for the OLE. Results: The short-term safety population comprised 746 patients in pooled monotherapy trials (placebo, 374; lumateperone 42 mg, 372) and 352 patients in the adjunctive study (adjunctive placebo, 175; adjunctive lumateperone 42 mg, 177). TEAEs of mania/hypomania were reported in 11 patients (placebo, 5 [1.3%]; lumateperone, 6 [1.6%]) in the pooled monotherapy groups and in 2 patients (adjunctive placebo, 1 [0.6%]; adjunctive lumateperone, 1 [0.6%]) who received adjunctive treatment. Mania/hypomania TEAEs were mild or moderate in severity. There was 1 serious TEAE of mania reported in the lumateperone 42 mg monotherapy group and no serious TEAEs of hypomania. The ITT population comprised 719 patients in pooled monotherapy trials (placebo, 365; lumateperone 42 mg, 354) and 348 patients in the adjunctive study (adjunctive placebo, 174; adjunctive lumateperone 42 mg, 174). Mean change in YMRS Total score from baseline to Day 43 was similar with lumateperone and placebo in monotherapy trials (least squares mean difference vs placebo [LSMD], − 0.5; 95% confidence interval [CI] − 1.0, 0.1; P = .10)) and the adjunctive study (LSMD, − 0.2; 95% CI − 0.8, 0.4; P = .56)). Change from baseline to Day 43 in CGI-BP-S Mania subscore with lumateperone was also similar to placebo for monotherapy trials (LSMD, − 0.0; 95% CI − 0.1, 0.1; P = 0.77) and the adjunctive study (LSMD, 0.0; 95% CI − 0.1, 0.1; P = .83). The long-term OLE safety population comprised 127 patients treated with lumateperone 42 mg. One patient (0.8%) had a TEAE of mild mania which was not serious. No patients had TEAEs of hypomania. There was not a significant mean change from baseline to end of treatment in YMRS score with lumateperone treatment (−0.5; 95% CI − 1.7, 0.6; P = .37). Lumateperone 42 mg significantly improved CGI-BP-S Mania subscore from baseline to end of treatment (mean change from baseline: 0.2; 95% CI 0.0, 0.4; P < 0.05). Conclusions: In patients with bipolar I or bipolar II disorder experiencing an MDE, lumateperone 42-mg monotherapy or adjunctive therapy was associated with low rates of treatment-emergent mania and hypomania that were similar to those of placebo-treated patients for both acute and long-term treatment. Keywords: Lumateperone, Bipolar Disorder, Mania, Hypomania Disclosures: Abbott, Abbvie, AstraZeneca, Alkermes, Allergan, Bristol Myers Squibb, GlaxoSmithKline, Lilly, Johnson and Johnson, Otsuka, Merck, Gedeon Richtr Plc, Sunovion, Intra-Cellular Therapies Inc., Forest, Roche, Elan, Lundbeck, Teva, Pamlab, Minerva, Neurocrine, Pfizer, Wiley Publishing: Honoraria (Self) P239. Sham Response in Transcranial Magnetic Stimulation Depression Trials is Increasing Over Time Zhi-De Deng, Megan Hynd, Zeynab Rezaee, André Brunoni, Sarah Lisanby National Institute of Mental Health, Bethesda, Maryland, United States Background: Psychophysiological and clinical studies with transcranial magnetic stimulation (TMS) require a valid control stimulation condition to achieve blinding of experimental conditions, hence allowing for the differentiation between active and placebo effects. A common approach is to use a sham intervention or active stimulation of a control site. The ideal sham condition should reproduce the ancillary aspects of TMS without producing significant direct brain stimulation. Thus, the ideal sham TMS system should be identical in appearance to the active system. In addition, the auditory (coil clicking) and somatic (coil vibration and scalp nerve and muscle activation) sensations should match between active and sham modes. Various coil manipulations and dedicated sham systems have been proposed, including coil tilt techniques, reverse current coils, spacers, field-blocking shields, synchronous electrical stimulation, and multi-coil configurations. These sham strategies differ in their ability to mask TMS operators and subjects. Our objective for this study was twofold, 1) to characterize and compare the physical aspects of different sham strategies, including electrical stimulation, and 2) to evaluate clinical research usage of the various strategies and factors influencing sham response in clinical TMS depression trials. Methods: To assess the sham TMS effects across clinical trials, studies were aggregated from Mutz, Edgcumbe, Brunoni, and Fu (2018) and updated with an independent search of PubMed up to the year 2021. Inclusion criteria for the review included the following: randomized, sham-controlled trial, primary diagnosis of major depressive disorder, and patients 12¬–70 years old. Two independent raters extracted the data. We calculated the sham and active response as percent change in the primary clinical outcome from baseline. We performed a univariate regression analysis in R. The regression model for the sham response included the following covariates: year of publication, mean sample age, percentage females, baseline depression severity, active percent change in depression score, sham strategy, blinding assessment, number of sessions, targeting method, stimulation site, stimulation protocol, and device manufacturer. We also ran an exploratory linear model for the active response, with the following covariates: year of publication, mean sample age, percentage females, baseline depression severity, active percent change in depression score, coil type, blinding assessment, number of sessions, targeting method, stimulation site, stimulation protocol, and device manufacturer. Results: We compiled 79 randomized, controlled TMS trials in patients with major depressive disorder, with total n = 1647 patients in the sham control arms and total n = 2128 patients in the active arms. Mean sample size was 21 patients in the sham groups and 19 patients in the active groups. The average age of the sham samples was 46.2 years and the mean percentage of the sample that was female was 56%. For the active samples, the average age was 46.4 years and 58% percent were female. Of the sham strategies used, the 90-degree coil tilt and dedicated sham coil were the most common (35% and 31%, respectively) followed by the 45-degree tilt method (27%). Synchronous electrical stimulation in addition to a dedicated sham coil was only used in 3 out of the 79 trials. For predictors of sham TMS response, year of publication (F(1, 76) = 6.18, p = 0.015), sham strategy (F(6, 71) = 1.806, p = 0.11), number of sessions (F(1, 76) = 5.45, p = 0.022), and percent change in depression score in the active arm (F(1, 71) = 17.22, p < 0.001) significantly influenced the sham percent change in depressions score. Post hoc comparison showed that the 45-degree coil tilt method produced the highest sham response, possibly due to the undesired residual electric field induced in the brain from this technique. For predictors of active TMS response, device manufacturer (F(5, 96) = 3.895, p = 0.003), targeting method (F(4, 97) = 3.817, p = 0.006), and sham percent change (F(1, 72) = 15.84, p < 0.001) are associated with change in depression score in the active arms. Conclusions: As seen with medications, there is a trend of rising sham response in TMS depression trials over the past 25 years. This time effect is not significant with active TMS. Therefore, the therapeutic effect size of TMS, that is, the difference between active TMS and placebo response, may be shrinking over time, requiring larger sample sizes in clinical trials to detect clinical benefits. It also argues for developing novel forms of TMS with fewer ancillary effects, e.g. controllable pulse TMS and quiet-TMS, which may be easier to blind with sham interventions that may have a lower placebo response. Keywords: Repetitive Transcranial Magnetic Stimulation (rTMS), Depression, Placebo Response, Meta-Analysis Disclosure: Nothing to disclose. P240. Predicting Depression Outcomes Through the Influence of Therapeutic Alliance and the Psychedelic Experience Using Path Modelling in a Phase IIb Randomized Controlled Trial of COMP360 Psilocybin Therapy Guy Goodwin, Hollie Simmons, Lindsey Marwood, Sunil Mistry, Danielle Schlosser, Joyce Tsai, Ekaterina Malievskaia COMPASS Pathways, London, United Kingdom Background: A recent phase IIb clinical trial of COMP360 (COMPASS Pathways’ proprietary synthetic psilocybin formulation) psilocybin therapy demonstrated efficacy in treatment-resistant depression (TRD), with significant improvements in depressive symptom severity following a single 25mg dose. Evidence suggests that therapeutic alliance and subjective psychedelic experience during COMP360 psilocybin administration are potentially important elements of the treatment (1, 2). Understanding how these components relate to COMP360 psilocybin therapy response is important in order to optimize the treatment paradigm, clarify underlying mechanisms, and help identify patients most likely to benefit. A previous study in participants with major depressive disorder treated with either COMP360 or escitalopram with psychological support, found that depressive symptom severity reduction was significantly predicted by acute psychedelic effects, measured by the Emotional Breakthrough Inventory (EBI) and Mystical Experience Questionnaire (MEQ) scores (1). Here we aim to compare these findings with data from our larger trial of COMP360 psilocybin therapy. Methods: This was a phase IIb, international, multicenter, randomized, fixed-dose, parallel group, double-blind trial investigating the safety and efficacy of a single dose of COMP360 psilocybin 25mg or 10mg compared to a 1mg dose in participants with TRD (COMP 001). COMP360 was administered alongside psychological support from trained therapists, delivered pre-, during and post-treatment. The primary efficacy endpoint was change from baseline in Montgomery–Åsberg Depression Rating Scale (MADRS) total score at Week 3. Subjective psychedelic experience was measured using the Five-Dimensional Altered States of Consciousness rating scale (5D-ASC), assessed at the end of the administration day, and EBI total scores, assessed the day following COMP360 psilocybin administration. Therapeutic alliance, measured by the Scale to Assess the Therapeutic Relationship Patient Version (STAR-P), was hypothesized to predict depressive symptom severity at Week 3 (absolute MADRS total score), through its interaction with the psychedelic experience (5D-ASC and EBI). Path analysis, a type of multiple regression evaluating relationships between variables to assess causality, was conducted on data from the 25mg dose group – the optimal therapeutic dose. Saturated paths were modelled, with each path including three variables: STAR-P total score, either a dimension score from the 5D-ASC or EBI total score, and MADRS total score at Week 3 as the outcome. Due to expected multicollinearity between the psychedelic experience variables, separate paths were fitted for EBI total score and for each of the five dimensions of the 5D-ASC. The path analyses used maximum likelihood estimation as the estimation method. Results: N = 233 participants (mean age 39.8 years; n = 121 female; 94% psilocybin-naïve) were randomized to COMP360 25 mg (n = 79), 10 mg (n = 75), or 1 mg (n = 79). The week 3 MADRS total score was significantly predicted by: EBI total score (β [standardized effect]=-0.57, R2[variance explained]=0.35, p < 0.0001), and the following 5D-ASC dimensions: visual restructuralization (β = -0.54, R2 = 0.30, p < 0.0001), oceanic boundlessness (β = -0.53, R2 = 0.29, p < 0.0001), and auditory alterations (β = -0.24, R2 = 0.09, p = 0.027). These variables all had a positive effect on the MADRS total score at Week 3 corresponding to a reduction of the total score. The absolute standardized effects and variance explained were largest for EBI total score, indicating it as the most reliable predictor of MADRS total scores at Week 3. The indirect effect of STAR-P on depression outcome was not significant for any path. Conclusions: Path analyses conducted on data from our phase IIb trial found that EBI total scores, and scores from three dimensions of the 5D-ASC were significant predictors of depressive symptom severity at Week 3 following COMP360 psilocybin therapy. The indirect effect of therapeutic alliance was not significant for any of the psychedelic experience paths. The outcome of this path analysis, using data from a larger, more robust TRD sample than in Murphy et al. (1), confirms previous reports: that subjective psychedelic effects were significant predictors of depressive symptom severity; if, as is very likely, intensity of psychedelic experience reflects drug level, it also echoes the dose related response seen in primary analysis of the COMP 001 trial. In contrast, indirect effects of therapeutic alliance here were non-significant. We cannot rule out the possibility that this may be due to differences in the therapy model, but effects in smaller studies are often not confirmed in larger samples. Further research is warranted to understand how we can use these findings to enhance the COMP360 psilocybin therapy treatment paradigm, and identify not only those who may be likely to benefit from this treatment, but also those who may not, and thus what can be improved to facilitate greater benefit. References: 1. Murphy R, Kettner H, Zeifman R, Giribaldi B, Kartner L, Martell J, et al. Therapeutic Alliance and Rapport Modulate Responses to Psilocybin Assisted Therapy for Depression. Frontiers in Pharmacology. 2022;12. 2. Griffiths, Johnson MW, Richards WA, Richards BD, McCann U, Jesse R. Psilocybin occasioned mystical-type experiences: immediate and persisting dose-related effects. Psychopharmacology (Berlin). 2011;218(4):649-65. Keywords: Psilocybin Therapy, Major Depressive Disorder, Randomised Controlled Trial, Subjective Effects, Therapeutic Alliance Disclosure: COMPASS Pathways Ltd: Employee (Self) P241. Dose-Dependent Acute Subjective Psychedelic Effects Following COMP360 Psilocybin Across Three Clinical Studies and its Relationship to Therapeutic Response Guy Goodwin, David Feifel, David J. Hellerstein, John R. Kelly, Lindsey Marwood, Sunil Mistry, Ania Nowakowska, Veronica O’Keane, James Rucker, Claudia Sisa, Joyce Tsai, Samuel Williams, Allan H. Young, Ekaterina Malievskaia University of California, San Diego, La Jolla, California, United States Background: Dose-dependent subjective psychedelic effects have been reported following psilocybin administration (1) and a positron emission tomography study found correlations between 5HT2a receptor occupancy, subjective psychedelic experience, and plasma levels of psilocin (the active metabolite of psilocybin) (2). In developing COMP360 (COMPASS Pathways’ proprietary synthetic psilocybin formulation) therapy for treatment-resistant depression (TRD), a phase IIb study (COMP 001) demonstrated significant efficacy for a 25mg dose as compared to 10mg dose. This dose-response relationship is an important finding in support of a central pharmacological effect of psilocybin, independent of expectation, in reducing depressive symptoms. Here we describe the Five-Dimensional Altered States of Consciousness (5D-ASC) questionnaire, assessing subjective psychedelic experience, and the Emotional Breakthrough Inventory (EBI), assessing emotional experience, and their relationships to changes in depressive symptom severity for the three doses employed in COMP001 and for comparison, the results in studies COMP 002 and 003. COMP 002 investigated the subjective effects of 25mg, 10mg, and placebo in healthy volunteers and COMP 003 investigated the effects of 25mg in TRD participants who continued to take a selective serotonin reuptake inhibitor (SSRI). Since serotonergic drugs downregulate 5HT2A receptor number and function in rodents (3), it was hypothesized that the action of psilocybin would also be attenuated in these participants. Methods: In all studies a single dose of COMP360 was given with psychological support from trained therapists pre-, during, and post-study drug administration. Participants completed the 5D-ASC (94 questions organized into five dimensions: oceanic boundlessness, anxious ego dissolution, visual restructuralization, auditory alterations, and reduction of vigilance) at the end of the administration day. In the TRD studies, the EBI was completed one day post-COMP360 administration and the Montgomery-Åsberg Depression Rating Scale (MADRS) was completed at multiple timepoints with the primary efficacy endpoint as the change from baseline in MADRS total score at Week 3. Results: In COMP 001, 233 participants (n = 121 female) were randomized to COMP360 25mg (n = 79), 10mg (n = 75), or 1mg (n = 79). In COMP 002, 89 participants (n = 41 female) were randomized to COMP360 25mg (n = 30), 10mg (n = 30), or placebo (n = 29). In COMP 003, 19 participants (n = 13 female) received open-label COMP360 25mg adjunct to a SSRI. Clear dose-dependent subjective psychedelic effects were observed in COMP 001 and COMP 002. At the same doses, 5D-ASC dimension scores for COMP360 25mg were generally similar across all three studies. In COMP 003, dimension scores indicated a psychedelic experience did occur, with an intensity between that observed for COMP360 25mg and 10mg in COMP 001. In COMP 001, moderate correlations were observed between the change from Baseline in MADRS total score at Week 3 and scores on three dimensions of the 5D-ASC; for COMP360 25mg, 10mg and 1mg, respectively; oceanic boundlessness (Pearson correlation coefficient; -0.508, -0.485, -0.477), visual restructuralization (-0.516, -0.431, -0.410), and auditory alterations (-0.293, -0.224, -0.358). Moderate correlations were also seen between the change from Baseline in MADRS total score at Week 3 and EBI total score; for COMP360 25mg, 10mg and 1mg, respectively (-0.614, -0.363, -0.424). Conclusions: Improvement in depression symptoms correlated with three dimensions of the of the 5D-ASC and the EBI. This would be expected if a given dose produces a range of psilocin levels, receptor effects and subjective experience. It supports the core pharmacological action that underlies the clinical effect of psilocybin. COMP360 adjunct to an SSRI did not appear to block either the psychedelic effect of psilocybin or its clinical benefit. In COMP 001, the 10mg arm produced a psychedelic experience but it was broadly ineffective in reducing depressive symptom severity, as compared to the 25mg dose. This suggests that simply knowing one has received an active dose (unblinding) and having a psychedelic experience is insufficient in improving symptoms, and thus points towards the validity of a pharmacological mechanism of action. These dose-dependent results from the 5D-ASC, and the correlations observed between changes in depressive symptom severity and 5D-ASC dimension and EBI total scores, warrant additional research to further understand the role that subjective psychedelic effects play in achieving therapeutic response. References: 1. Hirschfeld T, Schmidt TT. Dose–response relationships of psilocybin-induced subjective experiences in humans. Journal of Psychopharmacology. 2021;35(4):384-97. 2. Madsen MK, Burmester D, Stenbæk DS, Kristiansen S, Dyssegaard A, Lehel S, et al. Psilocybin occupancy of brain serotonin 2A receptors correlates with psilocin levels and subjective experience: a [11C]Cimbi-36 PET study in humans. European Neuropsychopharmacology. 2019;29:S304-S5. 3. Bonson K. Chronic Administration of Serotonergic Antidepressants Attenuates the Subjective Effects of LSD in Humans. Neuropsychopharmacology. 1996;14(6):425-36. Keywords: Psilocybin Therapy, Major Depressive Disorder, Subjective Effects, Randomised Controlled Trial Disclosure: COMPASS Pathways Ltd., MindMed, Relmada, Perception Neuroscience, Atai, Tripp, Neurolief, Brainsway: Contracted Research (Self) P242. Improvement in Depressive Symptoms With Zuranolone Treatment in Patients With Major Depressive Disorder With or Without Elevated Anxiety: Pooled Analysis From the MDD-201B, MOUNTAIN, and WATERFALL Studies Andrew Czysz, Charles Nemeroff, Alan Schatzberg, Robert Lasser, Lawrence Park, Youssef Toubouti, Fiona Forrestal, Ramon Iovin, Jeffery Jonas Sage Therapeutics, Dallas, Texas, United States Background: Patients having major depressive disorder (MDD) with elevated anxiety often have more severe depression and are less responsive to standard-of-care antidepressants than those without elevated anxiety. Zuranolone, a positive allosteric modulator of both synaptic and extrasynaptic GABA(A) receptors and a neuroactive steroid, is thought to upregulate GABA(A) receptor expression and enhance inhibitory GABAergic signaling. Zuranolone is hypothesized to rapidly restore network balance in brain areas dysregulated in depression. Zuranolone is investigational and in clinical development as an oral, once-daily, 14-day treatment course for adults with MDD. Clinical trials have demonstrated that treatment with zuranolone (30 or 50 mg) vs placebo leads to improvements in depressive symptoms as measured by change from baseline (CFB) in the 17-item Hamilton Rating Scale for Depression total score (HAMD-17). This pooled analysis of 3 completed studies of zuranolone evaluated the effect of zuranolone on depressive symptoms in patients having MDD with or without elevated anxiety at baseline. Methods: MDD-201B (NCT03000530; zuranolone 30 mg), MOUNTAIN (NCT03672175; zuranolone 20 or 30 mg [20 mg Cohort not included here]), and WATERFALL (NCT04442490; zuranolone 50 mg) were randomized, double-blind, placebo-controlled trials. MDD with elevated anxiety was defined as having a Hamilton Anxiety Rating Scale (HAM-A) total score ≥20 at baseline. The primary outcome (CFB in HAMD-17 at Day 15) and secondary outcome (CFB in HAM-A at Day 15) in individual studies were analyzed in a pooled analysis using the Mixed Model for Repeated Measures. Only patients with non-missing HAM-A scores at baseline were included. Analyses were not adjusted for multiplicity and p values reported here are nominal. Safety and tolerability were evaluated by incidence of treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs). Results: Of the 1007 patients included in this analysis, 508 (50.4%) met the criterion for MDD with elevated anxiety (zuranolone: n = 260; placebo: n = 248) and 499 did not (zuranolone: n = 245; placebo: n = 254). In the MDD with elevated anxiety subgroup, mean (SD) HAMD-17 (zuranolone vs placebo) was 27.3 (2.8) vs 27.6 (2.9) at baseline and 12.4 (8.0) vs 15.1 (8.2) at Day 15. This represented a least squares mean (LSM) (SE) CFB of −14.8 (0.6) vs −12.5 (0.6) (p = 0.002) at Day 15. In the MDD without elevated anxiety subgroup, mean (SD) HAMD-17 (zuranolone vs placebo) was 25.2 (2.2) vs 25.2 (2.1) at baseline and 12.1 (7.9) vs 13.6 (7.6) at Day 15. This represented a LSM (SE) CFB of −13.0 (0.6) vs −11.4 (0.6) (p = 0.038) at Day 15. In both subgroups, zuranolone separated from placebo at the first measured time point (Day 3; p < 0.0001). In the MDD with elevated anxiety subgroup, LSM (SE) CFB in HAM-A (zuranolone vs placebo) was −13.5 (0.6) vs −11.5 (0.6) at Day 15 (p < 0.0027). The proportions of patients who reported TEAEs were similar in both subgroups (MDD with elevated anxiety: zuranolone, 62.3% vs placebo, 47.6%; MDD without elevated anxiety: zuranolone, 55.5% vs placebo, 48.0%). Most patients reported TEAEs that were mild or moderate in severity, regardless of elevated anxiety status at baseline (MDD with elevated anxiety: zuranolone, 96.3% vs placebo, 97.5%; MDD without elevated anxiety: zuranolone, 96.3% vs placebo, 98.4%). Rates of SAEs were low and consistent across treatment groups, regardless of elevated anxiety status at baseline (MDD with elevated anxiety: zuranolone, 1.5% vs placebo, 0.4%; MDD without elevated anxiety: zuranolone, 0.8% vs placebo, 1.2%). Conclusions: In this pooled analysis of 3 randomized, double-blind studies—MDD-201B, MOUNTAIN, and WATERFALL—improvement in depressive symptoms with zuranolone was observed in patients with MDD, regardless of elevated anxiety status at baseline. Zuranolone was generally well tolerated and had a consistent safety and tolerability profile in patients having MDD with or without elevated anxiety at baseline. This analysis supports zuranolone as a potential treatment for the difficult-to-treat subpopulation of patients who have MDD with elevated anxiety. Keywords: Zuranolone, Major Depression Disorder, Neuroactive Steroid, Anxiety, Positive Allosteric Modulators Disclosures: Sage Therapeutics, Inc.: Employee, Stock/Equity (Self) P243. Lumateperone in the Treatment of Major Depressive Episodes Associated With Bipolar I or Bipolar II Disorder: Evaluation of Extrapyramidal and Motor Symptoms in Late-Phase Clinical Trials Tobie Escher, Suresh Durgam, Susan G. Kozauer, Richard Chen, John M. Kane Intra-Cellular Therapies, Inc., Indianapolis, Indiana, United States Background: Atypical antipsychotics exhibiting high dopamine D2 receptor occupancy (65%-80%) are associated with an increased risk of extrapyramidal symptoms (EPS). Lumateperone is an FDA-approved antipsychotic to treat schizophrenia and depressive episodes associated with bipolar I or bipolar II disorder as monotherapy and as adjunctive therapy with lithium or valproate. Lumateperone is mechanistically novel, simultaneously modulating serotonin, dopamine, and glutamate neurotransmission, with a low D2 receptor occupancy (~39%) at the recommended 42-mg dose. A robust late-phase clinical trial program established the safety and efficacy of lumateperone 42 mg as monotherapy and as adjunctive therapy with lithium or valproate in patients with a major depressive episode (MDE) associated with bipolar I or bipolar II disorder. This analysis evaluated the incidence of EPS across short- and long-term studies of lumateperone in patients with bipolar depression. Methods: All trials enrolled men and women (18-75 years) with a clinical diagnosis of bipolar I or II disorder who were experiencing an MDE (Montgomery-Åsberg Depression Rating Scale [MADRS] Total score ≥20 and Clinical Global Impression Scale-Bipolar Version-Severity [CGI-BP-S] score ≥4 at screening and baseline). Lumateperone 42 mg was administered once daily in the evening. EPS data were reported for 3 trial groups: (1) data pooled from 2 short-term, 6-week, placebo-controlled studies of lumateperone 42-mg monotherapy (Study 401 [NCT02600494]; Study 404 [NCT03249376]); (2) a 6-month open-label extension period (OLE) of Study 401 that evaluated long-term effects of lumateperone 42-mg monotherapy; (3) a Phase 3 placebo-controlled study (Study 402, NCT02600507) that investigated lumateperone 42-mg therapy adjunctive with lithium or valproate in patients with bipolar depression. Incidence and severity of EPS-related treatment-emergent adverse events (TEAEs) were reported according to narrow standard MedDRA query. EPS assessment scales included the clinician-rated Abnormal Involuntary Movement Scale (AIMS), the Barnes Akathisia Rating Scale (BARS), and the Simpson Angus Scale (SAS). Patients without akathisia (BARS ≤ 2) or parkinsonism (SAS ≤ 3) at baseline were evaluated for the emergence of BARS-confirmed akathisia (BARS > 2) and SAS-confirmed parkinsonism (SAS > 3) during treatment. Results: The short-term safety population comprised 746 patients in pooled monotherapy trials (placebo, 374; lumateperone, 372) and 352 patients in the adjunctive study (adjunctive placebo, 175; adjunctive lumateperone, 177). In the short-term studies, the only EPS-related TEAEs reported were 1 patient (0.3%) with mild dyskinesia on lumateperone monotherapy, 1 patient (0.6%) with mild akathisia on adjunctive lumateperone, and 1 patient (0.3%) with severe akathisia on placebo in the monotherapy trials. Benztropine and propranolol use was rare, with less than 1% of patients in any group receiving these treatments. Mean change from baseline in AIMS, BARS, and SAS scores were similar across groups for both monotherapy and adjunctive therapy. In patients without SAS-assessed parkinsonism at baseline (SAS ≤ 3), SAS-confirmed incidences of parkinsonism (SAS > 3) were rare in monotherapy (placebo, 1/347 [0.3%]; lumateperone, 2/327 [0.6%]) and adjunctive therapy (placebo, 1/168 [0.6%]; lumateperone, 2/168 [1.2%]). In patients without BARS-assessed akathisia at baseline (BARS ≤ 2), BARS-confirmed akathisia (BARS > 2) was also rare for monotherapy (placebo, 2/353 [0.6%]; lumateperone, 0/334 [0%]) and adjunctive therapy (placebo, 2/170 [1.2%]; lumateperone, 2/168 [1.2%]). The long-term OLE safety population comprised 127 patients. As observed in short-term trials, long-term treatment with lumateperone was associated with a low incidence of EPS-related TEAEs, with 2 patients (1.6%) experiencing akathisia of mild and moderate severity. Rates of concomitant benztropine (1.6%) and propranolol (0%) use were low. There were no notable changes in AIMS, BARS, or SAS scores during 6 months of treatment and no SAS-confirmed incidences of parkinsonism (SAS > 3, 0/122 [0%]). BARS-confirmed akathisia (BARS > 2) was reported in 3 of 122 patients (2.5%) without BARS-assessed akathisia at baseline (BARS ≤ 2). Conclusions: In patients with bipolar I or bipolar II disorder experiencing an MDE, lumateperone 42-mg monotherapy or adjunctive therapy had a favorable EPS profile in both acute and long-term treatment. Keywords: Lumateperone, Bipolar Disorder, Extrapyramidal Symptoms Disclosure: Intra-Cellular Therapies, Inc.: Employee, Stock/Equity (Self) P244. Psilocybin Assisted Therapy for Treatment-Resistant Depression: A Phase II, Randomized, Feasibility Study Joshua Rosenblat, Roger McIntyre University of Toronto, Toronto, Canada Background: Emerging preliminary evidence suggests that psilocybin-assisted therapy may be associated with rapid, robust and potentially enduring antidepressant effects in individuals with major depressive disorder (MDD). Given several challenging regulatory and logistical barriers, there have been no investigator-initiated psilocybin trials in Canada for several decades. Our study aimed to assess the feasibility, safety, and efficacy of single and repeat doses of psilocybin (25 mg) in persons with treatment-resistant depression (including both MDD and bipolar II disorder). The primary objective was to evaluate the feasibility of repeated high-doses of psilocybin in adults with treatment-resistant depression. The secondary objectives were to assess the antidepressant efficacy and tolerability of psilocybin-assisted therapy in a real-world, complex patient population with minimal exclusion criteria and no upper limit for degree of treatment resistance. Exploratory objectives of this pilot trial were to determine the effects of psilocybin on quality of life, wellbeing, functioning, disability, cognitive function, anxiety, anhedonia and mystical experiences. Methods: In this phase 2 randomized, wait-list controlled clinical trial, participants were randomized to immediate (n = 15) or delayed treatment (n = 15). Participants in the delayed treatment group (waitlist control condition) waited two weeks after enrolment before commencing the study interventions. The study intervention combines up to three high dose of oral synthetic psilocybin (25 mg; supplied by Usona) with psychotherapy over a 6-month period. With considerations for cost-effectiveness and scalability in mind, an ultra-brief four-session therapy model was evaluated (i.e., four sessions total for each psilocybin dose, including preparation, dosing and integration sessions). Within the four-session model, psychotherapy consists of one 2 hour preparatory session (one week before dosing session), one 6-8 hour supportive psilocybin dosing session and two integration therapy sessions (2 hours each session; one day after and one week after the dosing session). Participants received a single 25 mg dose of psilocybin with accompanying psychotherapy and were assessed weekly for six weeks and then biweekly for 18 weeks. Participants who responded and then relapsed could receive up to two repeated doses (e.g., maximum of three doses total, including first dose) of psilocybin over the course of the 6-month trial. Eligibility Criteria: Male and female outpatients, between the ages of 18 and 70, who met DSM-5-defined criteria for a current major depressive episode as part of major depressive or bipolar II disorder. Participants were required to have previously failed two or more first-line treatments with no upper limit to failed treatment trials. Personal and family history of psychosis, active suicidality requiring admission and moderate/severe substance use disorders were key exclusion criteria. Personality disorders, passive suicidality and history of hypomania were NOT exclusion criteria. Primary Outcome: The primary outcome of the study was feasibility. Feasibility was judged based on meeting regulatory requirements, recruitment, retention, safety, tolerability and preliminary efficacy. This trial received Health Canada and research ethics board approval and was registered with ClinicalTrials.gov: NCT05029466. Results: At the time of abstract submission, 22/30 participants (100% MDD to date) were enrolled and randomized with the trial to be completed by December 2022 (final trial results to be presented at ACNP). To date, feasibility results have been positive with all federal and local regulatory requirements met, rapid recruitment (159 patients referred for screening in first 6 weeks) and adequate retention (21/22 retained to primary endpoint). The safety and tolerability profile was positive, with zero serious adverse events (SAEs), zero suicide attempts, one dropout due to acute psychedelic effects, no psychedelic effects persisting past 12 hours and no emergency medications required during dosing sessions. 19/22 requested to receive a second dose believing benefits outweighed side effects. Preliminary efficacy testing was positive with statistically and clinically significant antidepressant effects observed with psilocybin treatment, as measured by the Montgomery–Åsberg Depression Rating Scale (MADRS) (p < 0.01 at all time points compared to baseline and wait list control). Significant reductions of suicidal thoughts and anxiety were also observed with subjective improvements in overall function, quality of life and wellbeing. Mean MADRS scores reduced by 8.9 points (SD = 7) 2-weeks after the first psilocybin dose (primary endpoint), as compared to 3.7 (5.1) in the waitlist control group, with a significant between group difference (p < 0.01). Greater reductions were observed 2-weeks after the second dosing session with an overall mean MADRS reduction of 17.5 (7.9), significantly greater than the MADRS reduction with the first dose (p < 0.01). Analysis of third dose effects are still pending. Conclusions: Interim results supported feasibility with adequate recruitment, retention and safety. Preliminary efficacy results suggest that the ultra-brief psilocybin-assisted therapy intervention was beneficial in an ultra-refractory patient population. Two-doses were associated with greater reduction in depressive symptoms than a single dosing session. Keywords: Psilocybin, Psychedelics, Psychotherapy, Treatment Resistant Depression, Major Depressive Disorder (MDD) Disclosures: Baxia Scientific Copr: Employee, Consultant (Self) P245. Adjunctive Simvastatin for Treatment-Resistant Depression: A 12-Week, Multicentre, Randomized Controlled Trial Ishrat Husain, Imran Chaudhry, Ameer Khoso, Tayyeba Kiran, Nawaz Khan, John Hodsoll, Mohammed Omair Husain, Haider Naqvi, Fareed Minhas, Jeffrey H. Meyer, Moin Ansari, Benoit H. Mulsant, Nusrat Husain, Allan H. Young Centre for Addiction and Mental Health, University of Toronto, Toronto, Canada Background: Small clinical trials of adjunctive statin use in major depressive disorder (MDD) have reported improvement in depressive symptoms. However, there are no appropriately powered clinical trials assessing the antidepressant efficacy of these repurposed agents in treatment-resistant depression (TRD). Methods: This 12-week, randomised, double blind, placebo-controlled trial, was conducted across five centres in Pakistan. It aimed to assess the efficacy of adjunctive simvastatin 20 mg compared to placebo for reduction of depressive symptoms in TRD. The study involved adults (age: 18-65) with a DSM-5 major depressive episode that had failed to respond to at least two adequate trials of antidepressants. Participants were enrolled between March 1, 2019 and Feb 28, 2021; the modified intent-to-treat analysis was performed from February 1 to June 15, 2022 using mixed models. Participants were randomised to receive: (i) simvastatin 20 mg plus standard care or (ii) placebo plus standard care. The primary outcome measure was mean change from baseline to week 12 on the Montgomery-Äsberg Depression Rating Scale (MADRS) scores. Secondary outcomes included changes in scores on the 24-item Hamilton Rating Scale for Depression, the Clinical Global Impression scale, the 7-item Generalized Anxiety Disorder scale and change in body mass index from baseline to week 12. C-reactive protein and plasma lipids measured were measured at baseline and week 12. The trial was registered on Clinicatrials.gov (NCT03435744) on Feb 16, 2018. Results: A total of 150 participants were randomised to simvastatin (n = 77) or placebo (n = 73) across study sites. Overall baseline-to–end point reduction in MADRS total score was observed in both groups however, the reduction in depressive symptoms did not differ significantly between groups (mean adjusted difference [95% confidence interval] for simvastatin vs. placebo: -0·86 (-3·96, 2·25), t(df) 0·5 (350·3), p = 0·589). There were no significant group differences in changes in any of the secondary outcomes and no evidence for differences in adverse effects between groups. A planned secondary analysis indicated that changes in plasma C-reactive protein and lipids from baseline to end-point did not mediate response to simvastatin. Conclusions: There was no evidence that adjunctive simvastatin was superior to placebo for reduction of depressive symptoms in TRD. This large RCT casts doubt on the potential therapeutic benefits of adjunctive statins as repurposed augmentation strategies in TRD. Keywords: Major Depression Disorder, Treatment Resistant Depression, Simvastatin, Statins, Clinical Trial Disclosure: Mindset Pharma, Wake Network: Advisory Board (Self), Psyched Therapeutics: Consultant (Self), COMPASS Pathways: Contracted Research (Self) P246. Effects of Acute Perimenstrual Estradiol and Progesterone Administration on Gabaergic Neuroactive Steroids and Cytokines: Preliminary Evidence From a Double-Blind Crossover Randomized Trial in Patients With Suicidal Ideation Jordan Barone, A. Leslie Morrow, David Rubinow, Susan Girdler, Tory Eisenlohr-Moul University of Illinois At Chicago College of Medicine, Chicago, Illinois, United States Background: More females than males attempt suicide, and these attempts and resulting deaths are most likely to occur around the onset of menses (perimenstrually), when estradiol (E2) and progesterone (P4) drop rapidly. Our recent crossover double-blind placebo-controlled trial of perimenstrual hormone administration (K99MH109667; CT.gov: NCT03720847) provides the first experimental evidence that administration of E2 and P4 in the weeks surrounding menses can reduce perimenstrual exacerbation of depression and suicidality in females with suicidal ideation (SI). Based on these findings and previous human and animal research, we hypothesized that this therapeutic effect of perimenstrual steroid administration (vs. placebo) may have been due to prevention of perimenstrual withdrawal from GABAergic neuroactive steroids (NAS). We also hypothesized that withdrawal from GABAergic NAS may have prevented depression-inducing perimenstrual increases in cytokine levels. We lastly hypothesized that within-person changes in GABAergic NAS and cytokines would covary with suicidal ideation. We evaluated banked serum samples from the same clinical trial to examine these two candidate biological pathways (reduced GABAergic NAS and increased cytokine levels) that might provide clues as to the mechanisms by which E2 and P4 administration (vs. placebo) reduced perimenstrual suicidal ideation. Methods: 25 naturally cycling participants with past-month SI completed a counterbalanced crossover double-blind RCT, comparing active E2 + P4 stabilization to placebo in the two weeks before and during menses. Participants wore a transdermal 17β-E2 patch (.1mg/day; or placebo) and took oral micronized progesterone (200mg/day, dosed 100mg BID; or placebo) from days +7 to +21 following a positive LH-surge ovulation test (day 0). Participants completed daily symptom ratings for the duration of study participation, and underwent a blood draw on days +7, +14, and +22 (timed relative to the ovulation test). NAS were quantified with gas chromatography/mass spectrometry, and cytokines were analyzed with high sensitivity multiplex assay kits. We ran three-level multilevel models in R (version 4.0.2), where log-transformed biomarkers and a composite SI score were nested within study interval and participant. Monte Carlo simulations estimating L1-to-L1 effects, assuming moderate outcome clustering (ICC = .3), indicate that our frequentist models achieve 80% power to detect roughly medium-sized effects of the intervention (f~.30, accounting for roughly 9% of the variance in the outcome) and roughly medium-to-large effects when looking at associations of variables in the placebo condition (f~.37, accounting for roughly 13% of the variance in the outcome). Results: We found a significant interaction between experimental condition and cycle phase predicting NAS, such that in the placebo condition (natural E2 + P4 withdrawal), P4-derived GABAergic NAS dropped from the midluteal to the perimenstrual visit, while the E2 + P4 administration prevented this decline (and in fact caused an increase) for 3α5α-tetrahydroprogesterone (3α5αTHP; β=2.97, SE = 0.22, p < 0.001), 3α5β-tetrahydroprogesterone (3α5βTHP; β=2.76, SE = 0.27, p < 0.001), and 3α5α-tetrahydrodeoxycorticosterone (THDOC; β=2.48, SE = 0.19, p < 0.001). We also found a trending interaction for experimental condition and cycle phase predicting IL-6, such that there was a midluteal-to-perimenstrual increase in IL-6 under placebo that was prevented in the E2 + P4 stabilization (β=-0.28, SE = 0.15, p = 0.07). Within the placebo condition only, between-person increases in IL-1β predicted higher concurrent SI (β = 2.07, SE = 0.75, p < 0.05), and within-person increases in IL-6 predicted higher concurrent SI at trend-level (B = 0.28, SE = 0.14, p = 0.06). However, within-person variance in 3α5αTHP, 3α5βTHP, THDOC, IL-1β, and IL-6 did not significantly predict concurrent SI (all p’s > 0.2) after accounting for experimental effects. Conclusions: In the first experimental RCT showing that perimenstrual administration of E2 + P4 reduces perimenstrual exacerbation of suicidal ideation in female outpatients, we used banked serum samples for preliminary analyses exploring the effects of the intervention on peripheral GABAergic NAS and cytokines as potential biological mechanisms. First, we demonstrated proof-of-concept that most hypothesized outcomes of interest (3α5αTHP, 3α5βTHP, THDOC, and IL-6) do change predictably across the natural menstrual cycle, and these changes can be prevented or reversed with experimental E2 + P4 administration. Second, we found that increased overall levels of IL-1β and natural increases in IL-6 (in the placebo condition) predicted increased concurrent SI. Third, we found that, after controlling for experimental effects of E2 + P4, neither overall levels nor person-centered increases in NAS or cytokines predicted SI. Given the primary trial’s demonstration of a causal relationship between E2 + P4 withdrawal and perimenstrual SI, we recommend future translational studies with larger samples combined with imaging or transcriptomic approaches to further probe inflammatory signaling, sensitivity of the GABAergic system (e.g., through differential gene expression of GABA receptor subunits), or other biological mechanisms that could underlie the effects of steroid withdrawal on depression and SI. Keywords: Ovarian Hormones, Neuroactive Steroid, Cytokines, Suicidal Ideation Disclosure: Nothing to disclose. P247. Chronic Stress Affects Negative Cognitive Bias and Related Inflammation in a Sex-Specific Manner in Rats Travis Hodges, Liisa Galea Mount Holyoke College, South Hadley, Massachusetts, United States Background: Major depressive disorder (MDD) is more common in women than in men, and women have more severe symptoms of MDD. Cognitive symptoms of MDD include negative cognitive bias. Negative cognitive bias is an increased perception of neutral situations or objects as negative. Elevated inflammation has been linked to MDD and cognitive impairment. MDD can be modelled in rodents using a chronic unpredictable stress (CUS) paradigm. We examined whether there were sex differences in cognitive bias after CUS and in neuroinflammation by examining nine cytokine concentrations in the ventral hippocampus and basolateral amygdala. Methods: Adult male and female Sprague-Dawley rats (N = 40) underwent either 2 weeks of CUS or no stress followed by an 18-day fear-based cognitive bias task. On the last day of cognitive bias testing, brains were collected and electrochemiluminescence was used to measure cytokine (IFN-γ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-α) and chemokine (C-X-C motif ligand 1; CXCL1) levels in the ventral hippocampus and basolateral amygdala. Results: On day 1 of cognitive bias training, male and female rats that underwent CUS displayed a more freezing (greater fearful response) than controls. By the end of cognitive bias training (day 16), rats exposed to CUS displayed a potentiated fear response in all contexts compared to non-stressed rats, and males displayed more freezing than did females. CUS increased freezing in the ambiguous context. In addition, CUS increased positive relationships between ambiguous freezing and inflammation in the basolateral amygdala in females, and CUS increased negative relationships between ambiguous freezing and inflammation in the ventral hippocampus of males. Conclusions: These findings indicate that CUS potentiates the freezing response to a neutral context, increasing negative cognitive bias in both sexes and suggests a sex-specific role of neuroinflammation in negative cognitive bias. Keywords: Cognitive Impariment, Stress, Sex Differences Disclosure: Nothing to disclose. P248. Stress-Resilient Mice Optimize Both Subjective Value and Food Security on an Economic Foraging Task Eric Nestler, Romain Durand-de Cuttoli, Freddyson J. Martínez-Rivera, Long Li, Angelica Minier-Toribio, Scott Russo, Brian Sweis Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Economic stress can often serve as a “second-hit” for those who have already accumulated a history of stressful experiences. This can precipitate significant changes in behavior that may be adaptive or maladaptive depending on one’s unique stress-response predispositions. How an individual recovers from a setback is a core feature of resilience but is seldom captured in animal studies. Methods: Here, we challenged mice in a novel two-hit stress model by first exposing animals to chronic social defeat stress (first hit) – a protocol known to separate individual differences in stress-resilient versus stress-susceptible phenotypes. Mice were then tested longitudinally across two months on the neuroeconomic task termed “Restaurant Row.” On this task, mice had a limited time period each day to forage for their sole source of food by navigating a maze with four uniquely flavored and contextualized feeding sites, or “restaurants.” Flavors were used to modulate subjective value determined by revealed preferences without assuming reward value (as opposed to varying pellet number in each restaurant, as this would come with added complexity of opportunity cost due to increased handling time per pellet). Each restaurant had a separate offer zone (OZ) and wait zone (WZ). Upon entry into the OZ, a tone sounded whose pitch indicated how long of a delay mice would have to wait in a cued countdown should they choose to enter the WZ. Tones were randomly selected from a range offer costs. OZ and WZ choices as a function of both cued offer cost and restaurant flavor demonstrate that animals understand the structure of the task while interacting with individual differences in subjective value. Mice were trained on this task for 60 consecutive days, across which the task grew increasingly difficult as the range of offer delays encountered grew more expensive. Results: An abrupt transition into a reward-scarce environment on this task 3 weeks into testing elicits an economic crisis (second hit) precipitating a massive drop in food intake that mice must respond to in order to survive. This recovery process occurs across a timescale on the order of weeks as mice learn to develop new decision strategies. We found that stress-resilient mice mounted the most robust behavioral response to this economic challenge and readily renormalized food intake back to baseline levels faster compared to stress-susceptible and non-defeated control mice. This was achieved through an efficient increase in effort expenditure and a redistribution in how time was allocated among competing opportunities. Interestingly, stress-resilient mice learned to accomplish this while simultaneously maximizing subjective value by re-establishing flavor preferences that approximated yield previously obtained in a reward-rich environment. Conclusions: These findings suggest that a resilient individual’s capacity to “bounce back” following economic stress while foraging entails the development of a multi-pronged strategy that not only ensures food security necessary for survival but also prioritizes other aspects of well-being including subjective value, highlighting a motivational balance preserved in stress-resilient individuals that may be impaired in depression. Keywords: Behavioral Economics, Social Defeat Stress, Stress Resilience and Susceptibility Disclosure: Nothing to disclose. P249. Linking Brain-Wide Activity Patterns During Neuroeconomic Decision Making to Aggression Brian Sweis, Antonio Aubry, Long Li, Julian Sackey, Farzana Yasmin, Salma Elhassa, Sanjana Ahmed, Eric Nestler, Scott Russo, Romain Durand-de Cuttoli Icahn School of Medicine at Mount Sinai, Minneapolis, Minnesota, United States Background: Aggression is an evolutionarily conserved response to a perceived threat that spans a large range of diverse behaviors and computational processes, including those that may be adaptive and protective as well as those that may be pathological and dangerous to others. It is becoming increasingly clear that the neural circuits recruited during the expression of aggressive behaviors, in addition to circuits known to subserve social interactions themselves, are critical nodes of the brain’s reward system. An active area of investigation is exploring to what degree pathological aggression may “hijack” key reward circuits in the brain, contributing to maladaptive reinforcement of aggression. Such studies involve the exploration of circuit engagement during social interactions directly as well as circuit engagement during general reward-related processing even for non-social targets (e.g., natural rewards like food). To date, the majority of aggression studies in rodents generally rely on (i) simple, naturalistic social behavioral assays (e.g., home-cage social behavior or social defeat-based interaction screening), (ii) are slowly extending into different ways to quantify social reward (e.g., social-aggression conditioned place preference or aggression self-administration), and (iii) are beginning to relate individual aggression profiles to more generalized behavioral and physiological signatures involved in the processing of non-social rewards. Because the brain has evolved to use multiple decision-making systems, simple tests of reward value may be unable to access computational subtleties that may be altered in the brains of aggressive individuals. Methods: Here, we aimed to characterize complex decision making profiles of animals screened for aggression in the home cage and then subsequently tested on a naturalistic, neuroeconomic decision-making task, termed “Restaurant Row.” This spatial task has been translated for use across species and is capable of operationalizing reward value along many dimensions and across numerous discrete behavioral computations that can reflect dissociable circuit functions. First, individual differences in aggressive behavior were quantified in 40 male outbred Swiss Webster mice. Next, mice were trained on the Restaurant Row task for 60 consecutive days. On this task, mice had a limited time period each day to forage for their sole source of food by navigating a maze with four uniquely flavored and contextualized feeding sites, or “restaurants.” Each restaurant had a separate offer zone (OZ) and wait zone (WZ). Upon entry into the OZ, a tone sounded whose pitch indicated how long of a delay mice would have to wait in a cued countdown should they choose to enter the WZ. This task produces a rich behavioral dataset accessing numerous features of reward cost, subjective value, and choice process. On the final day of testing, mice were allowed to engage the task for a short period of time before being removed and prepared for a time-sensitive tissue extraction and perfusion protocol in preparation for whole brain tissue clearing and staining. This approach, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, or iDISCO + , allows for brain-wide tissue staining for c-Fos expression, an activity-dependent immediate early gene. The timing of this protocol was intended to capture a snapshot of brain-wide activation patterns reflecting task engagement. Whole-brain imaging of cleared tissue was performed using light-sheet microscopy. Results: We found that aggression levels were generally stable within an individual measured across a 2-month period. We measured c-Fos expression levels in 275 distinct brain regions. Overall, we found that the majority of brain regions revealed decreased levels of c-Fos expression in highly aggressive animals versus non-aggressive animals. This initial finding is surprising given previous data from our lab and others tend to find overall increased activation in aggressive mice, although, measured following engagement of social interaction tests rather than a reward-based task for non-social targets as measured here. Using an unbiased, open-ended analysis approach, top region hits revealed strong negative correlations between aggression score and c-Fos expression, regions that lie in the medial wall of the prefrontal cortex (mPFC), including the anterior cingulate, prelimbic, and infralimbic cortex and are known to be engaged by the Restaurant Row task. Using this open-ended approach, we also found that several regions across the limbic system covaried with numerous key metrics from the Restaurant Row task, suggesting specific regions may be interacting in order to give rise to complex decision-making profiles. Using a focused analysis, we found that c-Fos expression in the mPFC scaled with individual differences in subjective value. Conclusions: These data provide fodder for future interrogation of sets of circuits that integrate only certain aspects of reward-related information processing. These data reveal how brain-wide activity signatures of aggression may drastically change, and even reverse direction from hyperactivation to hypoactivation, depending on the nature of the task engaged (e.g., social vs food reward) and the type of decision being processed. Lastly, these findings set the stage for exploring a deeper understanding of circuit-computation-specific functions of multiple valuation algorithms and aggression in a brain-wide manner. Keywords: Neuroeconomics, Whole-Brain Rodent Imaging, Decision Making, Reward, Aggression Disclosure: Nothing to disclose. P250. A Synaptic Substrate for Ketamine-Mediated Amelioration of Stress-Induced Anhedonia Federica Lucantonio, Shuwen Li, Jaden Lu, Michael R Tadross, Carlos Zarate, Marco Pignatelli Washington University in Saint Louis, St Louis, Missouri, United States Background: There is a major unmet need for the treatment of depression because current pharmacotherapies for depression require prolonged administration for clinical improvement and they are often associated with high non-response rate. In contrast, recent clinical evidence has shown that a single sub-anesthetic dose of ketamine (K), a noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, induces a robust antidepressant effect within hours of treatment in 70% of treatment-resistant patients. Particularly, such behavioral amelioration occurs within the context of anhedonia. Anhedonia –defined as diminished pleasure from, or interest in, previously rewarding activities– is a core symptom of depression which is commonly precipitated by exposure to chronic stress and is well-suited to study in laboratory animals. Specifically, hedonic deficits can be induced in rodents by chronic stress, which is able to affect the two main components of anhedonia: consummatory (subjective pleasure) and motivational (anticipation of and drive towards rewarding stimuli). The current study investigates the effect of K on both subtypes with a special focus on the Nucleus Accumbens (NAc). Indeed, the NAc plays a major role in the generation of motivated behaviors and stress determines opposing physiological changes within the two main populations of GABAergic medium spiny neurons (MSNs) residing within the NAc, D1 and D2 dopamine receptor expressing-MSNs. Accordingly, in this work we identify specific cellular elements of, and inputs to, the NAc recruited by in vivo K administration and responsible for its anti-anhedonic effects. Methods: Male and female mice were implanted with corticosterone (CORT) pellets (40 mg/kg). Behavioral and electrophysiological experiments were performed 21 days following implantation of the pellets. We assessed hedonic deficits with three-chamber sociability test, sucrose preference test and sucrose self-administration. Specifically, to investigate K’s anti-anhedonic effects, half of the mice received a single injection of K (10 mg/kg, IP) 24 hours before each test, while the other half received saline (n = 10-12 mice/test/treatment). To characterize α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission from molecular specified-MSNs in CORT-treated mice exposed to K, we performed in vitro whole cell recordings in transgenic mice expressing fluorescent proteins under the control of the D1 or D2 promoters (D1-TdTomato and A2A-TdTomato, respectively) (n = 10-15 cells from 8 mice/treatment). To test necessity of AMPAR-mediated synaptic plasticity on molecular specified-MSNs for the K’s anti-anhedonic effects, we employed DART technology in D1cre or A2Acre mice (n = 10-12 mice/test/condition). To identify specific inputs to the NAc recruited by K, we performed in vitro whole cell recordings in D1-TdTomato and A2A-TdTomato mice while optogenetically interrogating each monosynaptic input to the NAc (n = 10-15 cells from 6-8 mice/treatment/input). To test necessity of monosynaptic inputs to the NAc for K-mediated amelioration of stress-induced anhedonia, we used inhibitory DREADDs to inactivate each specific monosynaptic input to the NAc (n = 10-12 mice/test/condition/input). All experimental protocols were approved by the Washington University Institutional Animal Care and Use Committee with compliance to the National Institutes of Health guidelines. Results: Chronic CORT treated-mice displayed a reduction in sucrose preference ratio, time spent with a social target and breaking point compared to placebo-treated mice measured in the sucrose preference, social interaction and sucrose self-administration tests, respectively. K was able to rescue such hedonic deficits. Moreover, recordings from NAc slices obtained from CORT-exposed mice 24 h after a systemic saline injection showed decreased amplitude and frequency of spontaneous excitatory post-synaptic currents and reduced AMPAR to NMDAR ratio in accumbal D1-MSNs compared to placebo-treated mice. A single injection of K was able to rescue such reduced excitatory transmission within the NAc. Importantly, specific AMPAR blockade within D1-MSNs prevented K-operated amelioration of stress-induced anhedonia. Finally, we found that information flow from prefrontal cortex and ventral hippocampus to the NAc is enhanced by K exposure, however each input plays a unique role in K-induced anti-anhedonic effects. Conclusions: These results show circuit, cellular and synaptic mechanisms by which K triggers long-lasting amelioration in stress-induced anhedonia. These results have major implications for the rational design of more targeted, effective and safer medications for the treatment of anhedonia. Keywords: Ketamine, Anhedonia, Synaptic Plasticity, Nucleus Accumbens Disclosure: Nothing to disclose. P251. Sidekick-1, A Novel Regulator of Stress Resilience in the Prefrontal Cortex Vedrana Cvetkovska, Pedro do Couto Bordignon, Joelle Lopez, Emily Cha, Sophia Cumplido-Wilson, Rosemary Bagot McGill University, Montreal, Canada Background: Stress is a major risk factor for depression, yet some individuals remain resilient. Both environmental and genetic factors contribute to the dysregulation of neural circuits in depression and genes that regulate the formation, maintenance, and plasticity of synapses are of particular interest as molecular hubs for regulation of circuit function. We previously identified Sidekick-1 (Sdk1) as an affective circuit hub gene in a network regulating stress resilience and overexpression of Sdk1 in the prefrontal cortex (PFC) increased resilience to stress in male mice. Separately, several genome-wide association studies in humans found SDK1 variants associated with depression, suggesting an evolutionarily conserved role in regulation of emotional behavior. Sdk1 is a cell surface molecule implicated in circuit formation in the developing retina. However, little is known about cell-specific expression patterns of Sdk1 in the adult brain and its function within affective circuits. Here we probe Sdk1 cell-type and layer-specific expression in the PFC, conservation between mice and humans, its modulation by stress, and effects on reward learning in both male and female mice. Methods: We used chronic social defeat stress to generate susceptible and resilient male mice, and chronic witness defeat stress or subchronic variable stress in female mice. Quantitative real-time PCR determined bulk Sdk1 expression levels in the PFC, nucleus accumbens and ventral hippocampus of control and stressed mice (male control n = 13, stress n = 17; female control n = 8, stress n = 10). We assessed expression pattern of Sdk1 mRNA in the PFC control and stressed mice with multiplex RNAScope fluorescent in situ hybridization using probes for Slc17a7 (vGlut1) and Slc32a1 (vGAT) to identify excitatory and inhibitory neurons, respectively (male control n = 9, stress n = 15; female control n = 7, stress n = 7). We used publicly available scRNAseq datasets (Allen Brain Map) from mouse and human whole cortex to identify cell-type specific expression of Sdk1. Reward learning was assessed in a probabilistic reversal learning task. Results: We found that Sdk1 is expressed in the PFC, nucleus accumbens and ventral hippocampus of adult male and female mice, with expression highest in the PFC (p < 0.05 male; p < 0.0001 female). RNAScope in situ hybridization in the PFC showed that Sdk1 was expressed in subpopulations of vGlut1-positive neurons and vGAT-positive neurons. The highest level of Sdk1 expression was observed in deep layer 6 (p < 0.0001) with moderate expression in layers 2/3 and 5. Patterns of cell-type and layer-specific Sdk1 expression were conserved in scRNAseq analysis of human PFC. De novo clustering of scRNAseq data from mouse PFC showed that Sdk1-expressing cells were found across clusters, with several clusters of excitatory or inhibitory neurons marked by high numbers of Sdk1-expressing cells. Stress modulated Sdk1 expression in the PFC in a sex-specific manner. Cell-type specific overexpression of Sdk1 in the PFC modulated reward learning in the probabilistic reversal learning task with sex-specific effects. Conclusions: In this study we characterized the prefrontal expression of Sdk1, a novel regulator of stress resilience. We found that Sdk1 is modulated by stress and is expressed in subsets of both excitatory and inhibitory neurons, with a significant enrichment in deep layers of the PFC. Cell-type and cortical layer specificity was conserved between mice and humans suggesting an evolutionarily conserved function. The probabilistic reversal learning task allows us to probe reward learning in animals where prefrontal Sdk1 expression is altered. Future work will address the circuit-specific effects of Sdk1 in behavioral adaptation to stress. As a cell surface receptor, Sdk1 is a potential target for pharmacological intervention. Understanding how Sdk1 confers resilience may lead to development of new mechanistically-informed treatments for depression. Keywords: PFC, Depression, Stress Disclosure: Nothing to disclose. P252. Behind the Veil of Magic Mushrooms: Exploring the Correlation Between Subjective Experiences With 10 Psychedelic Mushrooms and Tryptamine Content Robert Morlock, Sean Johnston, Anthony Milewski, Leonard Lerer, Karin Blakolmer* BYAS-PEB, Chicago, Illinois, United States Background: Psilocybin containing “magic” or psychedelic mushrooms (PM) affect perception, mood, behavior, and consciousness. The neuroplastic, immune modulation, and anti-inflammatory effects of psilocybin are currently being explored for a range of conditions including depression, PTSD, anxiety, and substance abuse. There is anecdotal evidence of distinctive, subjective experiences with different PM and this may be due to variations in psilocybin content and effects of non-psychoactive tryptamines. This phenomenon, the entourage effect, may be the result of the pharmacological interaction between the tryptamines. This study aimed to establish the concentration of these tryptamines in a variety of popular PM that are associated with distinct subjective experiences in the non-clinical setting, and to explore whether there is a correlation between the type of subjective experience and the tryptamine composition of the mushrooms. Methods: A cross-sectional on-line survey was fielded July 22-July 25, 2022. Participants were recruited from online psychedelic user groups and a general national sample of adults in the US reporting PM experience. The national survey included a fictional PM for quality control. Participants were asked to rate their general overall experience, intensity of visual and physical effects by PM on a 0-to-10-point scale. Endpoints are assessed descriptively for participants in psychedelic user groups, the general national sample and both groups combined. For analysis, Psilocybe subtropicalis, Panaeolus bisporus, Psilocybe tampanensis, Psilocybe cubensis - “Enigma”, Psilocybe natalensis, Panaeolus cyanescens, Psilocybe cubensis - American Mystic (PE) were cultivated from spores. Psilocybe stunzii, Psilocybe ovoideocystidiata, and Psilocybe azurescens were obtained from field collections. Samples of dried ground mushrooms were extracted via methanol. Psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin and aeruginascin were quantified using a Waters Acquity H-Class UPLC and Xevo TQ-S Micro MS (Waters Corporation, Milford, MA, USA). Results: The national survey screened out 840 (88.4%) potential participants for selecting a fictional PM (86.5%) and other quality control screens (1.8%). A total of 149 individuals completed the survey with 37 (22%) individuals from psychedelic user groups and 112 (78%) individuals from the general national sample. Participants in the PM user groups, relative to those in the national sample were more likely to be male (77.4% vs. 45.5%) and older with a greater percentage between 43-52 years old (32.3% vs 16.0%). They tended to give lower ratings to their mushroom experiences. Overall, Psilocybe cubensis (75; 50.3%), Psilocybe subtropicalis (60; 40.3%) and Panaeolus cyanescens (60; 40.3%) were the 3 most common PM. Participants rated Psilocybe azurescens and Psilocybe ovoideocystidiata with the highest overall effect with an average score of 7.4; Psilocybe natalensis and Psilocybe ovoideocystidiata had the highest impact on visual effects with an average score of 6.8; and Psilocybe azurescens had the highest average physical effect of 6.7. For the majority of PM there was a wide range of ratings from 0 to 10. Psilocybin, the most abundant component, ranged from 0.03% (Psilocybe ovoideocystidiata) to 1.79% (Psilocybe subtropicalis) dry wt. %. The total content of other tryptamines varied proportionally to the combined psilocybin and psilocin content. Of note is that strong body and weak visual effects seem to be recorded for mushrooms with a relatively high baeocystin content while mushrooms with an equal ratio of psilocybin versus the other tryptamines seem to produce more visual effects. Conclusions: Despite interobserver variation in the assessment of experiences with 10 different PM, a pattern emerged pointing towards distinct visual and physical experiences with different mushrooms. This may be explained by the ratio of non-psychedelic tryptamines to psilocybin and the amount of baeocystin. The large number of participants screened out by control measures highlights the challenges in conducting such surveys. Keywords: Psychedelics, Psilocybin, Baeocystin Disclosure: BYAS-PEB: Consultant (Self) P253. Instrumental Control Over Stress Recruits Distinct Circuits in Female Rats Connor McNulty, Isabella Fallon, Jose Amat, Gianni Bonnici, David Root, Steven Maier, Michael Baratta* The University of Colorado Boulder, Boulder, Colorado, United States Background: Exposure to adverse life events is strongly linked to negative mental health outcomes, in which prevalence is often higher in women than men. Thus, investigating sex differences in experiential factors that promote stress susceptibility/resilience, and their circuit implementation, is of clinical interest. In male rats, instrumental control over stressor recruits a corticostriatal system involving the prelimbic cortex (PL) and dorsomedial striatum (DMS) that potently blunts a number of stress outcomes. In contrast, the stress-buffering effects of control are completely absent in females, and the mechanisms underlying this absence are not understood. Methods: For manipulation of controllability, Sprague-Dawley rats were run in a triadic design. One subject of each triad received escapable shock (ES), a second received yoked-inescapable shock (IS), and a third received no shock (HC). Following stress treatment, the number of Fos-immunoreactive neurons was determined for the DMS and dorsolateral striatum (DLS), and subsequently converted into the number of counts per unit area (mm2) for each region of interest (EXP1: n = 7-8/group/female only). In EXP2, stress-induced dopamine (DA) levels in the medial prefrontal cortex (mPFC) were assessed with in vivo microdialysis (n = 5-7/group/male and female). Multiplex-fluorescence in situ hybridization was used to quantify expression of the following mRNA transcripts in the ventral tegmental area (VTA) to PL pathway: tyrosine hydroxylase (TH), c-fos, and viral-encoded GFP (EXP3: n = 3-5/group/male and female). Intra-PL microinfusion of SCH 23390 or saline was delivered 30 min prior to ES/IS/HC (EXP4: n = 9-11/group/female only). Results: EXP1: At the level of the DMS, both female ES and IS led to a similar level of Fos expression (p = 0.291). In contrast, female ES robustly increased Fos immunoreactivity in the DLS (p < 0.001) compared to IS. EXP2: To examine whether the impact of PFC DA efflux differs in male and female ES subjects, a microdialysis probe was targeted to the mPFC. Male ES led to only a transient increase in DA, with DA returning to baseline levels by the second sample collection. ES females showed a different pattern - DA efflux was potentiated throughout the entire stress session (stress x sex x time interaction: F4,80 = 3.070, p = 0.021; Repeated Measures ANOVA). EXP3: Fluorescent in situ hybridization revealed that female ES led to a greater activation of PL-projecting VTA DA neurons compared to male ES (sex x stress interaction: F1,12 = 6.138, p = 0.0291; 2-way ANOVA). EXP4: Blockade of PL D1 receptors with SCH 23390 shifted Fos expression from the DLS to the DMS (t11 = 2.28, p = 0.043; independent t-test), thereby enabling behavioral protection in females with behavioral control. Conclusions: Behavioral control over adverse events, a key aspect of coping, recruits an instrumental learning system in females that differs from males. Our data provide novel insight into the influence of mPFC DA over striatal activity during instrumental learning involving aversive stimuli, and its subsequent impact on behavioral outcome. The implication is that the exercise of behavioral control at a procedural level is not the critical factor in determining its impact, rather the impact depends on the circuitry that is recruited during the acquisition of the controlling response. Keywords: Medial Prefrontal Cortex, Dopamine, Stress Resilience, Dorsal Striatum, Stress Coping Disclosure: Nothing to disclose. P254. Gm-2505 is a Novel 5-Ht2a Receptor Agonist and 5-Ht Releaser That Induces Rapid, Robust, and Durable Antidepressant Effects at Doses Associated With Decreased Power in Low Frequency EEG Bands in Rats Zoe Hughes, Adam Klein, Eric Austin, Dino Dvorak, Silvia Gatti, Laszlo Kiss, Gerard Marek, Jonathan Sporn, Andrew Kruegel Gilgamesh Pharmaceuticals, Newton Centre, Massachusetts, United States Background: Prototypical psychedelic agents such as psilocybin, LSD, and DMT continue to show promise as rapid acting antidepressants in clinical trials. These agents, which are 5-HT2A receptor agonists, produce profound alterations in human consciousness, with variable durations of action ranging from very long (8-12 h for LSD) to very short (~10 min for DMT). The antidepressant efficacy of this class of agents is durable and continues long past their clearance, making weekly, or even monthly dosing the preferred treatment paradigm. Here we describe GM-2505, a novel 5-HT2A receptor agonist with 5-HT releasing activity. It was designed to have an intermediate half-life in humans, allowing for sufficient target engagement to provide antidepressant effects without requiring an extended-duration in-clinic experience. Methods: The in vitro profile of GM-2505 was determined using competition binding and calcium flux (FLIPR) assays in cells expressing heterologous human 5-HT2A, 5-HT1A, 5-HT2C and 5-HT2B receptors. The effect of GM-2505 on 5-HT release from rat brain synaptosomes was also assessed. The brain and plasma PK of GM-2505 (1 mg/kg, sc and iv) were determined in Sprague Dawley (SD) rats. 5-HT2A receptor agonist activity in vivo was measured by counting wet dog shakes (WDS) and head twitches (HT) in the first 20 min after dosing rats (n = 6/group) with GM-2505 (0.03-10 mg/kg, sc). A study in mice (n = 6/group) determined the effect of pretreatment with the 5-HT2A antagonist, MDL100,907 (0.1 mg/kg, sc) on HT caused by GM-2505 (0.1–10 mg/kg, sc). GM-2505 was assessed in a paradigm of Chronic Mild Stress (CMS), whereby Wistar Kyoto rats (n = 8/group) were subjected to stressors (e.g., wet bedding, tilted cage) for a period of 10 weeks. After 2 weeks of CMS, animals were dosed once per week with GM-2505 (0.3 or 1 mg/kg, sc) or vehicle for 5 weeks. Rats continued to be exposed to stress for an additional 3 weeks after the final dose to test the durability of the antidepressant effect. Each week, anhedonia was assessed by measuring sucrose intake (24 h after dosing, weeks 3-7). Anxiety levels in the elevated plus maze (EPM) and memory impairment in the novel object recognition (NOR) test were assessed in the 3rd week of treatment, 48 and 72 h after dosing, respectively. The effects of GM-2505 (0.3-3 mg/kg, sc) on quantitative EEG (qEEG) were measured in a cohort of 8 freely moving SD rats implanted with 2 cortical electrodes connected to a DSI transmitter. Rats were tested once per week using a within-subjects Latin square design. Results: At human 5-HT2A receptors, GM-2505 bound with high affinity (Ki 4.9 nM and 140 nM, for DOI and ketanserin, respectively) and potent and efficacious agonist activity (EC50 15.0 nM, EMAX 80.6%). GM-2505 was also a potent agonist of 5-HT2C (EC50 9.5 nM), much less potent at 5-HT1A (EC50 16918 nM) while an antagonist at 5-HT2B (IC50 5.8 nM). GM-2505 activated the release of 5-HT from rat synaptosomes with efficacy of 66.8% and potency of 15.7 nM. GM-2505 (1 mg/kg, sc) achieved a Cmax of 41.3 ng/mL in rat plasma and 188 ng/g in rat brain at Tmax of 30 min and 15 min, respectively. After iv administration (1 mg/kg), the C0 was 159.9 ng/mL in plasma and 1507 ng/g in brain with a T1/2 of ~25 min. This profile was longer than DMT-like and shorter than psilocin-like comparator compounds. GM-2505 produced HT and WDS in rats with an ED50 of 0.07 mg/kg and peak effect at 1 mg/kg. In mice, the GM-2505 ED50 was 0.32 mg/kg for HT, with the peak response at 3.2 mg/kg. HT was absent in mice pretreated with MDL100,907. GM-2505 showed the expected inverted U-shaped dose response for HT in both rats and mice. Exposure of rats to the chronic mild stress paradigm caused a phenotype of anhedonia, anxiety and cognitive impairment, which was reversed by GM-2505 (0.3 and 1 mg/kg). 24 h after the first dose, GM-2505 fully reversed the stress-induced deficit in sucrose intake (P < 0.05 GM-2505 Stress vs Vehicle Stress group); this effect was maintained through 5 weeks of once weekly dosing and did not fully dissipate until 22 days after the final dose. CMS-induced anxiety and cognitive impairment were apparent in vehicle treated rats (EPM and NOR: P < 0.05 Vehicle Ctrl vs Vehicle Stress). Both deficits were reversed by GM-2505 (0.3 and 1 mg/kg): increase in % time in the open (P < 0.05 GM-2505 Ctrl vs GM-2505 Stress) and an increase in NOR index (P < 0.05 GM-2505 Ctrl vs GM-2505 Stress). In qEEG, GM-2505 (0.3-3 mg/kg) produced dose-dependent reductions in power in the lower frequency bands (delta, theta, alpha and beta). There was a strong relationship between the time course of this effect and the PK of GM-2505. Conclusions: Consistent with its in vitro activity as a potent 5-HT2A receptor agonist and 5-HT releaser, GM-2505 produced a robust 5-HT2A-mediated behavioral response in vivo. The PK of GM-2505 in rats was intermediate between DMT and psilocin analogs. In the CMS paradigm, doses of GM-2505 (at and below peak HT/WDS response) had robust antidepressant-like effects 24 h after the initial dose, which were sustained with once weekly dosing for 5 weeks. The durability of this effect was evident in the levels of sucrose intake in GM-2505-treated CMS rats not reverting to vehicle levels until 3 weeks after the final dose. Antidepressant doses of GM-2505 were associated with acute reductions in qEEG power in low frequency bands, an effect reported for psilocybin and DMT at clinically efficacious doses. Together, these data indicate that GM-2505 is a novel 5-HT2A receptor agonist and 5-HT releaser that holds promise as a rapid-acting, robust antidepressant. Keywords: Fast-acting Antidepressant, 5-HT2A Receptors, Psychedelic Medicine Disclosure: Gilgamesh Pharmaceuticals: Employee (Self) P255. Functional Connectivity Differences Related to Age of Mood Disorder Onset Melinda Westlund Schreiner, Scott Langenecker University of Utah, Salt Lake City, Utah, United States Background: Mood disorders often begin in adolescence, with this earlier onset being associated with increased likelihood of negative psychosocial outcomes. While studies have demonstrated the presence of neurobiological anomalies in adolescents with mood disorders, limited research has evaluated how these effects present in young adulthood. The cognitive control, default mode, and salience and emotion networks (CCN, DMN, and SEN respectively) have all been implicated in both adults and adolescents with mood disorders. These networks may be particularly impacted by early onset mood disorders as the presence of the disease may hinder development. Such disruptions may then contribute to the effective management of symptoms that may otherwise lead to functional impairment. Methods: Adults ages 18-30 with remitted mood disorder completed a resting-state fMRI scan. We examined functional connectivity of the cognitive control network associated with age of mood disorder onset. For the CCN, we used the dorsolateral prefrontal cortex, inferior frontal gyrus, and dorsal anterior cingulate cortex as seeds. For the DMN, we used the posterior cingulate cortex, dorsal medial prefrontal cortex, and temporoparietal junction. For the SEN, we used the amygdala, subgenual anterior cingulate, and anterior insula. We included age at time of scanning, sex, and movement parameters as covariates in all analyses. We reported results that exceeded an uncorrected p < .001 and voxel size of 75. Results: A total of 138 participants (97 female) were included in analyses. Average age of participants at the time of the scan was 22.54 (SD = 3.10), with an average age of mood disorder onset of 15.99 (SD = 3.93) (range = 5-26 years). Younger age at mood disorder onset was negatively associated with connectivity of CCN regions with right superior and medial temporal gyrus, right primary motor and visual association cortices, left secondary visual cortex, and bilateral fusiform. Younger mood disorder onset was also negatively associated with DMN connectivity with the right temporal pole and primary sensory cortex. There were no significant relations between age of onset and SEN connectivity. Conclusions: Individuals experiencing remission from mood disorders who have an earlier age of onset demonstrate decreased CCN and DMN involvement with regions implicated in social and emotional cognition and perception. As adolescence is a key period of rapid neurodevelopment, conditions such as depression can have significant effects that may disrupt typical neurobiological trajectories. By evaluating the effect of when mood disorders develop, longitudinal data that incorporates degree of functional impairment may aid in our understanding of what neurobiological changes have deleterious effects. Furthermore, we can also better identify which neurobiological processes may contribute to effective adaptation to the illness. These processes may aid in reducing illness recurrence and/or serve as a buffer against negative outcomes. Keywords: Mood Disorders, Connectivity, Onset Disclosure: Nothing to disclose. P256. Dynamic Resting State Default Mode and Frontoparietal Network Alterations in Youth at High Familial Risk for Major Depressive Disorder Emily Belleau, Rebecca Kremens, Erin Bondy, Angela Pisoni, Randy Auerbach, Diego Pizzagalli McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Major Depressive Disorder (MDD) is characterized by altered functioning of the default mode network (DMN) and the frontoparietal network (FPN). Although most of this work has focused on examining “static” functional network properties, the assessment of “dynamic” network properties may be particularly valuable in furthering our understanding of DMN-FPN imbalances in MDD. Co-activation pattern (CAP) analyses allows for the examination of brain dynamics by clustering the data into distinct co-activation patterns of brain activity at each time point. Prior studies have found that adults with MDD spend more time in a CAP consisting of DMN and FPN regions as well as transition more frequently between a DMN-FPN CAP with other DMN CAPs relative to healthy controls. However, it remains unclear whether dynamic functional DMN-FPN alterations are a scar of prior episodes or rather, reflect a premorbid risk factor present in healthy youth at familial risk for MDD. Further, it remains unclear whether DMN-FPN alterations predict future depressive symptoms and/or increased stress sensitivity. Methods: Participants included youth with (High-Risk Group, n = 27) and without (Low-Risk Group, n = 62) a maternal history of MDD. Youth and their mothers completed structured clinical interviews. All youth and Low-Risk mothers were free of lifetime psychiatric disorders. Mothers of High-Risk youth had a history of MDD. Youth participants also completed self-report questionnaires on depressive symptoms (Mood and Feelings Questionnaire, MAFQ), anxiety symptoms (Multidimensional Anxiety Scale for Children, MASC), and perceived stress (Perceived Stress Scale, PSS) at baseline and follow-up assessments every 3 months for 2 years. Additionally, resting state fMRI data were acquired at the baseline assessment for all youth. A CAP analysis was conducted on the resting state fMRI data. The optimal number of CAPs was determined using consensus clustering, which provides an index of clustering quality. Consensus clustering results indicated an optimal k of 9 and the 9 CAPs included a: 1) CAP with visual system activation, DMN deactivation, 2) CAP with dorsal medial prefrontal cortex activation, visual system deactivation, 3) CAP with DMN activation, FPN and dorsal attention network deactivation, 4)CAP with DMN activation, somatosensory network deactivation, 5) CAP with visual system activation, somatosensory network deactivation, 6)CAP with salience and dorsal attention networks, DMN and visual system deactivation 7) CAP with salience network activation, FPN deactivation 8) CAP with posterior DMN and FPN activation and 9) CAP with salience network activation, DMN deactivation. Given prior CAP studies on MDD, our analyses centered on CAP 8, the DMN-FPN CAP as well as transitions between CAP 8 with other DMN CAPs (CAPs 3 and 4). Both “Time in CAP 8” (i.e., the total number of volumes the participant spent in CAP 8) and “CAP 8 Persistence” (i.e., number of times each participant remained in CAP 8 from t to t + 1) was computed. Additionally, the number of transitions between CAP 8 and CAP 3 as well as CAP 8 and CAP 4 were calculated. Four ANCOVAs controlling for MAFQ and MASC scores were conducted to examine group differences in Time in CAP 8, CAP 8 Persistence, CAP 8 óCAP3 transition frequencies, and CAP 8ó CAP 4 transition frequencies. CAP metrics showing differences between the High-Risk and Low-Risk group served as predictors in multilevel models (MLM) predicting depression symptoms or perceived stress across the follow-up assessments. Restricted maximum likelihood estimation was used, and random intercepts and slopes were specified for each of the models. Results: The High-Risk group spent more time in CAP 8 (DMN-FPN) compared to the Low-Risk group, F(1,85) = 4.53, p = 0.036, ηp2 = 0.05. Additionally, the High-Risk group showed higher persistence in CAP 8 compared to the Low-Risk group, F(1,85) = 5.22, p = 0.025, ηp2 = 0.06. However, the groups did not significantly differ with respect to CAP 8 óCAP3 and CAP 8 óCAP4 transition frequencies, ps >0.3. Given group differences in Time in CAP 8 and CAP 8 Persistence, we conducted two MLMs predicting depressive symptoms, with each including Group, Time in CAP 8/CAP 8 Persistence, and study visit as predictors. We conducted two additional MLMs using the same set of variables predicting PSS scores. To correct for multiple comparisons, the p threshold was set to p < 0.025 for each set of omnibus MLMs. A significant Group x Visit x CAP 8 Persistence interaction predicting PSS scores emerged, p = 0.013. Follow-up MLMs within each group separately showed a Visit x CAP 8 Persistence interaction amongst the High-Risk Group, p = 0.042, but not the Low-Risk Group, p = 0.570. Those in the High-Risk group exhibiting high CAP 8 Persistence, reported increasing levels of perceived stress over time, whereas those exhibiting low CAP 8 Persistence had persistently lower PSS scores across time. No other effects were significant. Conclusions: Dynamic DMN and FPN alterations are present in healthy youth at high familial risk for MDD, potentially reflecting a premorbid vulnerability factor versus a scar of MDD. Additionally, persisting in a DMN-FPN CAP predicted stress sensitivity over time amongst the high-risk youth. High-Risk youth showing high levels of DMN-FPN CAP persistence show increasing levels of stress sensitivity over time, which may enhance future vulnerability to MDD. Dynamic dysfunction in the DMN and FPN may serve as a biomarker of risk for MDD. Keywords: Adolescent Depression, Resting-State fMRI, Co-Activation Pattern Analysis, Familial Risk for MDD, Stress Disclosure: Nothing to disclose. P257. The Effect of Selective Slow Wave Sleep Deprivation on Mood, Rumination, and Default Mode Network Connectivity in Adolescent Depression Adriane Soehner, Mary Phillips University of Pittsburgh School of Medicine, Pittsburgh, PA, Pennsylvania, United States Background: Safe, rapid-acting treatment strategies are urgently needed for depressed youth. In depressed adults, selective slow wave sleep deprivation (SW-SD) rapidly improved depression severity without disrupting sleep duration, but the effects of SW-SD on mood and the underlying neuro-affective mechanisms remain untested in youth. Sleep deprivation reduces default mode network (DMN) connectivity and heightened anterior-posterior DMN connectivity is associated with depression and rumination. In a pilot study, we tested the hypothesis that SW-SD would improve depression, reduce rumination, and stabilize DMN connectivity in adolescent depression. Methods: Eighteen adolescent participants in a depressive episode (Ages 13-19y, Mean Age = 17.5 ± 1.5yr; 14 Females) completed 3 consecutive nights of overnight polysomnographic sleep monitoring: Baseline (Night 1), SW-SD (Night 2), and Recovery (Night 3). On the SW-SD night, acoustic stimulation suppressed slow wave sleep (SWS) without impacting habitual sleep duration. After each night, clinician-rated depression severity (modified Child Depression Rating Scale) and self-rated rumination severity (modified Ruminative Response Scale) were assessed. Ten participants completed resting-state fMRI scans the mornings after Baseline and SW-SD; connectivity between anterior (medial prefrontal cortex) and posterior (posterior cingulate cortex) DMN nodes was estimated. Linear mixed-effects models adjusted for age, sex, SSRI medication, and baseline depression severity. Results: There was a significant effect of night on percent slow wave sleep (SWS%; F[1,13]=71.4, p < .0001) and rumination (F[1,13]=3.73, p = 0.0048) but not clinician-rated depression severity (F[1,13]=0.9, p = 0.91). SWS% was lower on the SW-SD night relative to Baseline (p < .0001) and Recovery (p < .0001) but did not differ between Baseline and Recovery (p = 0.778). SW-SD reduced rumination relative to Baseline (p = 0.027) and the effect was sustained through Recovery sleep (p = 0.017). Anterior-posterior DMN connectivity also declined after SW-SD relative to Baseline (F[1,5]=6.13, p = 0.0351). In exploratory analyses, reduction in %SWS (SW-SD minus Baseline) was associated with trend-level reductions in rumination (B = 0.47, p = 0.104) and Anterior-Posterior DMN connectivity (B = 0.72, p = 0.072), and there was a significant relationship between changes in rumination and DMN connectivity (B = 0.74, p = 0.049). Conclusions: SW-SD acutely reduced next-day rumination in depressed adolescents, but not overall depressed mood. Rumination improvement following SW-SD may be supported by stabilization of DMN connectivity. These preliminary data implicate SWS manipulation as a therapeutic approach for modifying DMN-mediated psychological processes, such as rumination, in adolescents. Keywords: Depression, Slow Wave Sleep, Adolescent, Default Mode Network, Rumination Disclosure: Nothing to disclose. P258. Ventral Visual Stream Functional Coupling During Implicit Emotional Face Perception is Associated With Internalizing Symptoms: A Double Dissociation by Face Valence at Baseline and Six Months Post-Scan E. Kale Edmiston, Jay Fournier, Maya C. Schumer, Michele Bertocci, Haris Aslam, Alexander Skeba, Tyler J. Brady, Simona Graur, Richelle Stiffler, Henry Chase, Mary Phillips University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Internalizing symptoms are characterized by alterations in emotional processing, including perceptual biases for negative face stimuli. Both depressive and anxiety disorders are associated with biases for negative stimuli. However, depressive symptoms are also associated with impaired emotional processing that is not specific to negative stimuli, including reduced cortical response to positive stimuli. Given shared perceptual biases for negative emotional faces, and distinct perceptual biases for positive affective faces, there are likely shared and distinct neural circuits associated with depression and anxiety symptoms. The anterior lateral fusiform gyrus is associated with face perception and its response to emotional faces is modulated by bottom-up signals from more posterior portions of the ventral visual stream, as well as more anterior temporal, limbic, and prefrontal regions. The fusiform gyrus offers a potential novel target for intervention in internalizing disorders given its key role in emotional face perception. Furthermore, it remains unclear how fusiform functional coupling is related to symptom improvement over time. In this emotional face functional MRI study, we hypothesized that fusiform coupling during happy face perception would be associated with depression symptoms, accounting for anxiety symptoms, while fusiform coupling during sad face perception would be associated with internalizing symptoms generally. Methods: Sixty-one adults (mean age 23.13 ± 2.86, 36 female, 3 non-binary) seeking treatment for psychological distress underwent 3T MRI scanning and completed an implicit emotional face processing task in the scanner. Participants were administered the Hamilton Depression and Anxiety Rating Scales. Clinical scores were transformed and summed for a general internalizing symptom score. The transformed anxiety scores were also subtracted from the depression scores to create a metric of the relative contributions of depressive and anxiety symptoms (i.e., HAMD-HAMA). We modeled functional coupling during implicit perception of sad and happy emotional faces. Using an anterior lateral fusiform gyrus seed consistent with the fusiform face area, generalized psychophysiological interaction analyses were performed at the whole brain level (p < 0.001, uncorrected, k = 20) with summed internalizing symptom scores, HAMD-HAMA scores, gender, and age included in all models. A subsample of 36 participants (mean age 23.56 ± 3.11, 23 female, 1 non-binary) returned 6 months post-scan and were administered the HAMD and HAMA once more. We modeled anterior lateral fusiform gyrus coupling during sad and happy face perception with summed HAMD and HAMA scores at follow-up, controlling for baseline internalizing symptoms and demographic characteristics (p < 0.001, uncorrected, k = 20). Results: During perception of sad faces, there was a positive association between internalizing symptom severity and coupling of the fusiform gyrus with the temporal pole (T = 6.79, k = 32), right inferior temporal gyrus (T = 6.47, k = 27), and orbitofrontal cortex (T = 5.59, k = 77). In contrast, for the happy face condition, there was a negative association between internalizing symptoms and fusiform coupling with the temporal pole (T = 5.68, k = 48) and left superior parietal cortex (T = 4.20, k = 117). There were no significant associations for the HAMA-HAMD variable in either task condition. Internalizing symptom severity six months post scan was negatively associated with coupling of the fusiform gyrus and medial prefrontal cortex (mPFC, T = 4.74, k = 109) during happy face perception. The relationship was such that greater coupling was associated with more internalizing symptom improvement at follow up. There were no significant associations during sad face perception and follow up scores. Conclusions: These findings indicate a potential double dissociation between internalizing psychopathology and fusiform-temporal cortex coupling during sad versus happy emotional face perception. The positive association during sad face perception and the negative correlation during happy face perception could be related to the relative salience of negative and positive emotional stimuli in internalizing disorders. These findings lend support to the importance of perceptual processes and the ventral visual stream in internalizing disorders. Furthermore, greater fusiform-mPFC coupling during happy face perception predicted more improvement six months post scan, highlighting the importance of neural response to positive affective stimuli to symptom change over time. Keywords: Internalizing Disorders, Ventral Visual Stream, Longitudinal Imaging Disclosure: Nothing to disclose. P259. Approach-Avoidance Behavior and Conflict in Remitted Depression: Neural Correlates and Latent Decision-Making Processes Manuel Kuhn, Jacob Blank, David Steinberger, Yinru Long, Genevieve Nowicki, Zeyang Yu, Swarag Thaikkandi, Mads L. Pedersen, Todd M. Herrington, Michael J. Frank, Diego A. Pizzagalli McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Abnormalities in approach-avoidance (Ap-Av) behaviors are implicated in a host of psychiatric disorders. Conflict between approach and avoidance typically arises when a decision has both rewarding and aversive consequences and is particularly central for major depressive disorder (MDD) and anxiety disorders. Rodent and human studies have implicated the amygdala, hippocampus, striatum, orbitofrontal cortex (OFC), and medial prefrontal cortex (mPFC) in Ap-Av behavior and conflict. Moreover, recent work has provided converging evidence for mPFC recruitment during Ap-Av conflict and aversive responsiveness across non-human primates and humans (Ironside et al., 2020). Importantly, in our recent neuroimaging study, individuals with current MDD showed reduced approach-related dorsolateral prefrontal cortex (dlPFC) activation as well as reduced avoidance-related pregenual anterior cingulate cortex (pgACC) activation. Whether aberrant Ap-Av-related neural responses persist after recovery from MDD remains unclear; understanding such mechanisms may provide important insights into relapse risk for individuals with a history of depression. Methods: Preliminary analyses from this ongoing study were based on 51 symptom-free and unmedicated individuals, including 17 remitted MDD (rMDD, age mean=25.2, m/f: 0/17, BDI mean= 0.55) and 34 healthy controls (HC, age mean=25.9, m/f: 10/24, BDI mean=0.59). Participants performed a novel adaptive Ap-Av conflict (aAAC) task during functional magnetic resonance imaging (fMRI). In this task, participants were offered a combination of monetary reward and aversive electrical stimulation parametrically spanning a continuum of reward and aversion across ten levels. Participants decided via joystick movements to either receive the reward and aversive stimulation (approach) or to forgo the offer (avoid), modeling Ap-Av behavior. For each trial, prior Ap-Av decisions were used to determine the next trial’s offer in order to adaptively sample closely around individuals’ decision boundaries; this strategy allowed to induce variable amounts of decision conflict while sampling the individual’s reward-aversion space. Measures of Ap-Av behavior and conflict were assessed in a multimodal and integrative approach utilizing skin conductance response (SCR) measurements, fMRI, and computational modeling (i.e., drift-diffusion modeling, DDM) to probe neural and latent decision-making processes. Results: General successful task performance and conflict induction in the aAAC task emerged in the form of a sigmoidal take probability and higher reaction times around the individual decision boundaries. Similarly, SCRs were higher in approach compared to avoidance trials [t(44) = 4,14, p < 0.001] and parametrically followed reaction times, with higher SCRs in slow response trials which is indicative of high conflict [t(44) = 3.57, p < 0.001]. fMRI analyses revealed expected Ap-Av-related neural patterns such as higher dorsal anterior cingulate (dACC) and striatal activation during approach trials and higher mPFC and OFC activation during avoid trials (all FWESVC < 0.001). Further analyses showed that dACC and dlPFC recruitment was strongly related to the degree of conflict as parametrically defined by distance to the decision boundary and increased reaction times, suggesting successful conflict induction (all FWESVC < 0.001). Group comparisons revealed that rMDD participants reported higher general trait avoidance scores [t(48) = 2.43, p = 0.02] as assessed by the Cognitive and Behavioral Avoidance Scale. On a neural level, decreased avoidance-related pgACC activation [t(49) = 2.29, p = 0.027] emerged exclusively in rMDD participants paralleling observations in individuals with current MDD (Ironside et al., 2020). In addition, rMDD participants more strongly recruited the mPFC with increasing conflict than HCs [t(49) = 3.01, p = 0.002]. These observations were complemented by posterior distributions on DDM model parameters showing that rMDD participants were more sensitive to the impact of aversiveness on evidence accumulation toward avoidance (rMDD<HC, 98% posterior samples). Additionally, whereas HCs showed an overall starting point bias toward approach (>99% posterior samples; independent of reward and aversiveness), rMDD participants showed no such bias. Finally, replicating previous findings in MDD patients (Pedersen et al., 2021), rMDD and HC showed opposite influences of accumbens activity on this starting point bias (HC > rMDD, 92% posterior samples). This was reflected in HC greater accumbens activity on a trial-to-trial basis related to an increased bias to approach, whereas increased accumbens activity in rMDD was related to an increased bias to avoid. Conclusions: Persistent aberrant approach-avoidance behavior and conflict processing may be important vulnerability factors for asymptomatic individuals with a history of depression and may index a heightened risk of relapse. Using a novel adaptive Ap-Av conflict task, we provide preliminary support for residual abnormalities in Ap-Av-related neural and latent decision-making processes in rMDD, which parallel alterations previously observed in patients with MDD. Elucidating neural and latent mechanisms linked to persistent abnormal Ap-Av decision-making promises to advance the development of new prevention and treatment strategies in remitted and acute depression. Keywords: Approach-Avoidance Conflict, Major Depression, Decision-Making, Functional MRI (fMRI), Remitted Depression Disclosure: Nothing to disclose. P260. Contributions of Prenatal Exposure to Air Pollution and Childhood Housing Insecurity to Adolescent Hippocampal Volumes and Depression Bruce Ramphal, Julie Herbstman, Amy Margolis Harvard Medical School, Cambridge, Massachusetts, United States Background: Housing insecurity and exposure to air pollution are common, modifiable threats to child development that often disproportionately affect low-income communities of color. Some studies have demonstrated that material hardship and prenatal air pollution exposure have compounding impacts on child neurodevelopment. Separately, housing stress and air pollution have been linked with risk for depression. Here, we sought to parse the shared and unique contributions of lifetime housing insecurity and prenatal air pollution exposure on adolescent depression and hippocampal volume as a potential mechanism. Methods: The Mothers and Newborns longitudinal birth cohort recruited largely low-income Black and/or Latinx women in northern Manhattan between 1998 and 2006. Exclusion criteria included maternal smoking during pregnancy, gestational diabetes, hypertension, or HIV. Beginning in the third trimester of pregnancy, mothers completed questionnaires assessing their environmental exposures, housing status, and other characteristics every two years. At birth, maternal blood was assessed for the presence of benzo[a]pyrene (a polycyclic aromatic hydrocarbon (PAH)) DNA adducts, which are DNA lesions due to air pollution exposure. Children underwent structural MRI at one timepoint between age 14 and 21 years, and the Kiddie Schedule for Affective Disorders and Schizophrenia (KSADS) was conducted. Multiple regression (n = 197) examined the associations between hippocampal volumes or depressed mood during adolescence and either the presence of PAH DNA adducts at birth, cumulative housing insecurity, or their interaction. Results: Housing insecurity over the first 14 years of life (β = 0.68, p = .004) and the presence of PAH DNA adducts at birth were associated with increased risk for depressed mood (β = 1.54, p = .009) independently, but did not interact. This interaction was significant on left hippocampal body volume (β = .30, p = .008). Conclusions: The current findings provide evidence that childhood housing insecurity and prenatal air pollution exposure, two ubiquitous environmental insults, have unique and convergent effects on adolescent depression and hippocampal structure. These findings are consistent with previous studies establishing associations between depression and both housing insecurity and air pollution exposure. Keywords: Housing, Hippocampus, Depression Disclosure: Nothing to disclose. P261. Cognitive Effort Preference in Veterans With Suicide Attempt Histories James Bjork, Chelsea Sawyers Rooney, Lisa Straub, David Garavito, Andrew Westbrook Virginia Commonwealth University, Richmond, Virginia, United States Background: Suicide attempts (SA) are increasing in the United States, especially in veterans. Critical to suicide prevention is understanding individual differences that demarcate the subset of suicide ideators who attempt suicide. Cognitive theories attribute SA to schema (gist)-based negative interpretations of environmental events. In light of over-general autobiographical memory and facile solutions in problem-solving tasks in SA survivors, aversion to expending cognitive effort itself may be a neurobehavioral marker of SA risk. Methods: In veterans receiving care for mood disorder, we compared choices in a cognitive effort discounting task and evidence-gathering in a beads task between veterans with (SAHx + ; n = 26) versus without (SAHx-; n = 22) a history of SA. Groups did not differ in current depressed mood or in a proxy metric of premorbid intelligence. Results: Compared to SAHx- participants, SAHx+ participants self-reported significantly more severe cognitive problems in most domains, and also eschewed choice to earn higher monetary reward if earning it required a slightly (but not greatly) increased working memory (WM) demand relative to an easy WM task. There was no group difference, however, in extent of evidence-gathering before declaring a conclusion in a beads task. Conclusions: These preliminary data suggest that aversion to expenditure of cognitive effort, potentially as a component of cognitive difficulties, may be a marker for SA risk. Keywords: Suicide Risk Factors, Cognitive Effort, Veterans, Mood Disorder Disclosure: Nothing to disclose. P262. Oscillatory Dynamics of Frontal-Striatal Circuits Underlie Symptom Dimensions Within Major Depressive Disorder Justin Riddle, Moria Smoski, Crystal Schiller, David Rubinow, Flavio Frohlich University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: Patients with major depressive disorder (MDD) present with a heterogenous collection of symptoms. Factor analysis of depressive symptoms has consistently found two prominent dimensions within depression: anhedonia and anxiety. However, the neural oscillatory basis for how these symptom dimensions of depression arise from specific circuits in the brain is unknown. Previous research suggests that reward-based decision-making may elicit systematic differences in neural activity as a function of depressive symptoms. Methods: In our first experiment (sub-study of NCT03449979), 42 participants in a major depressive episode and 41 age and sex-matched euthymic control participants (N = 83; 66 women) performed a streamlined version of the expenditure of effort for reward task (S-EEfRT) with EEG. Behavior was quantified along two dimensions: goal-directed behavior was defined as willingness to exert effort for an uncertain reward and reward-evaluation was the degree to which effort was strategically exerted for higher incentives. We analyzed interregional phase-amplitude coupling between prefrontal cortex (PFC) and posterior regions, and related coupling strength to behavior and symptom dimensions. In our follow-up experiment (NCT05084924), 48 participants with MDD performed the S-EEfRT during EEG and functional MRI. This dataset was used to replicate the findings from the first experiment and to further investigate the contributions of frontal-striatal circuitry to behavior in the S-EEfRT. Task-evoked functional connectivity in fMRI was quantified between the striatum, dorsal caudate versus nucleus accumbens, and the PFC, dorsolateral versus ventromedial. Results: In experiment 1 (N = 79 analyzed), anhedonia displayed a significant negative relationship with goal-directed behavior (t = -2.19, p = 0.03) and with delta-beta coupling from PFC to left motor cortex (t = -2.24, p = 0.03). Similarly, anxiety was positively related to theta-gamma coupling between PFC and posterior visual areas (p < 0.05, k = 11) and with anxiety symptoms (t = 2.15, p = 0.04). We did not find a significant effect of biological sex in our analyses. In experiment 2, we replicated the presence of delta-beta coupling from PFC to motor cortex (t = 2.242, p = 0.047) and its relation to goal-directed behavior (r = 0.265) and symptoms of anhedonia (r = -0.373). Our task-evoked functional connectivity analysis recapitulated canonical circuitry between lateral PFC and dorsal caudate (t = 3.234, p = 0.008) that was positively correlated with delta-beta coupling (r = 0.718, p = 0.029), but not theta-gamma coupling (r = -0.358, p = 0.344). In addition, we found evidence for the canonical circuitry between medial PFC and nucleus accumbens (t = 5.494, p < 0.001), although the strength of this circuit was not correlated with the strength of theta-gamma coupling (r = -0.489, p = 0.182). While the strength of functional connectivity in both circuits was predictive of goal-directed behavior (r > 0.48), only medial PFC to nucleus accumbens was predictive of reward-evaluation (r = 0.362). Conclusions: These experiments provide a novel neurobehavioral model for the biological basis of symptoms of depression. The underlying cause of anhedonia may be reduced integrity of circuitry for goal-directed behavior, wherein lateral PFC displays reduced connectivity to the dorsal caudate nucleus and decreased delta-beta coupling to the motor cortex. Additionally, symptoms of anxiety might drive increased engagement of reward-evaluation circuitry, such that medial PFC displays increased connectivity with nucleus accumbens and elevated theta-gamma coupling to perceptual regions. The discovery that frontal-striatal circuitry exhibits distinct oscillatory control signals that are altered by specific symptoms of depression will provide novel targets for individually precise interventions using non-invasive brain stimulation. Keywords: Research Domain Criteria (RDoC), Major Depression Disorder, Cognitive Neuroscience, Functional MRI (fMRI), EEG Disclosure: Nothing to disclose. P263. Sex Differences in Sleep Slow-Wave Activity in Major Depression May Reflect Fundamental Differences in Regulation of Neuroplasticity and Disease Pathophysiology Jennifer Goldschmied, Elena Goldstein, Samantha Costello, Emma Palermo, Margaux Games, Philip Gehrman University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Males with major depressive disorder (MDD) have been shown to exhibit less slow-wave activity (SWA) than females with MDD or their healthy counterparts. SWA has been implicated in the homeostatic regulation of neuroplasticity; thus it is possible that due to these SWA deficiencies, males with MDD may have impairments in the modulation of cortical excitability, and by extension learning and memory. This could also suggest that manipulating SWA in males with MDD could serve to reduce these deficiencies and improve performance. Therefore, the aim of the present study was to examine sex differences in short-term memory performance and cortical excitability at baseline and in response to selective slow-wave disruption. Methods: 17 individuals (7 M) with MDD were recruited for an ongoing clinical trial. Auditory stimulation was utilized to disrupt slow-wave sleep on one of two overnight sleep laboratory visits (Baseline, SWD) separated by one week. Following each overnight visit, a computer-based N-back task was administered to assess short-term memory, and motor evoked potentials (MEP) generated from transcranial magnetic stimulation (TMS) were measured as a proxy measure of cortical excitability. Repeated measures ANOVA was used to evaluate sex differences in memory performance at baseline and change in MEP amplitude following SWD with condition (Baseline, SWD) as the within-subject factor and sex (F, M) as the between-subjects factor. Results: Results from repeated measures ANOVA revealed a significant main effect of sex for accuracy on the N-Back, (F(1,16) =7.42, p = .02). At baseline, males with MDD showed significantly worse accuracy on the N-Back than females (F(1,16) =11.49, p < .01). However, following SWD, this discrepancy was reduced, and males showed significantly improved performance, t(6)=-2.10, p = .04, that was no longer significantly different from females. With regard to cortical excitability, results from repeated measures ANOVA revealed a significant Condition x Sex interaction, (F(1,15) =4.42, p = .05) indicating that while males with MDD exhibited a trend towards increased MEP following SWD, t(6)=-1.85, p = .057), females demonstrated no change. Conclusions: Our results demonstrate that SWD may differentially impact males and females with MDD. While females did not exhibit any modulation of cortical excitability following SWD, males with MDD showed evidence of increasing cortical excitability, providing preliminary evidence that SWA is directly associated with the regulation of neuroplasticity in males with MDD. Relatedly, this increase in cortical excitability was also paralleled by an improvement in memory performance on the N-back, suggesting that SWA manipulation can potentially be used as a future intervention target. These results may also suggest that the observed sex differences in SWA in those with MDD reflect a fundamental neurobiological difference in disease mechanisms in males and females. Keywords: Neuroplasticity, Slow-Wave Activity, Sleep, Major Depressive Disorder Disclosure: Nothing to disclose. P264. Stress Levels, Psychological Symptoms, and C-Reactive Protein Levels in COVID-19: A Cross-Sectional Study Gislaine Zilli Reus, Taiane de A. Cardoso, Ritele H. Silva, Jessica L. Fernandes, Camila O. Arent, Graziela Amboni, Laura A. Borba, Alex Paulo Z. Padilha, Maria Eduarda M. Botelho, Amanda L. Maciel, Silvio José B. Soares, Margarete D. Bagatini, Claudia Dallagnol, Marta Elisa Brighenti, Zuleide Maria Ignacio, Luciane B. Ceretta, Joao de Quevedo University of Southern Santa Catarina, Criciuma, Brazil Background: Although many studies have pointed out a possible relationship between COVID-19 and the presence of psychiatric and neurological disorders, the majority of the studies have important limitations, such as not having a control group of individuals without COVID-19 or reporting the presence of psychiatric conditions through self-reported tools. This study investigates the role of COVID-19 infection on stress, anxiety, depression, insomnia, and C-reactive protein (PCR) levels. Methods: This cross-sectional study included a matched sample of adult individuals with COVID-19 (cases; n = 114) or without COVID-19 (controls; n = 236). We used Mini International Neuropsychiatric Interview – Plus for the diagnosis of psychiatric disorders. The severity of depressive or insomnia and anxiety symptoms were assessed using the Hamilton Rating Scale for Depression and for Anxiety, respectively. The stress level was evaluated by an inventory of stress symptoms adapted from Checklist-90-R. Blood samples were used to assess the CRP levels. The comparison between groups was tested using Man-Whitney U test. The data showed as absolute and relative frequency were compared using the chi-squared test. Spearman correlation was used to test the association between two continuous variables. Results: Findings showed greater severity of depressive symptoms (p = 0.034), higher levels of stress (p = 0.020), and greater CRP levels (p = 0.014) in individuals with COVID-19 when compared to controls. The severity of depressive (p = 0.005) and insomnia (p = 0.006) symptoms, as well as the CRP levels (p = 0.013) were more remarkable in individuals with moderate/severe COVID-19 symptoms than in asymptomatic or individuals presenting mild symptoms. We found a positive correlation between stress and severity of anxiety, depression, or insomnia in individuals with or without COVID-19 (p < 0.001). There was a positive correlation between PCR levels and severity of anxiety, depression, or levels of stress in individuals with COVID-19. Interestingly, we found that individuals with COVID-19 and depression had greater CRP levels as compared to individuals with COVID-19 without current MDD (p = 0.023). Conclusions: Individuals with COVID-19 showed greater severity of psychological symptoms, which may impact the development of psychiatric disorders in the future. CPR levels seem to be a promising biomarker to help on the earlier detection of the post-COVID depression. Longitudinal studies are needed to understand the clinical trajectories of these individuals. Keywords: Emotional Stress, Depression and Anxiety, Inflammation, c-Reactive Protein, COVID-19 Disclosure: Nothing to disclose. P265. The Relationship Between Free Triiodothyronine (FT3) and Performance on the Iowa Gambling Task in Bipolar Disorder: Sex Differences Explored Caitlin Millett, Megan Shanahan, Katherine Burdick Feinstein Institute for Medical Research, Glen Oaks, New York, United States Background: Thyroid dysfunction has been implicated in the pathophysiology of bipolar disorder (BD) because of the known effects on cognition and mood. Here we aimed to examine the effect of thyroid hormones and behavior on a reward learning paradigm, the Iowa Gambling Task (IGT). Methods: The study included 101 males and 94 females with a diagnosis of BD. BD diagnosis was confirmed by the structured clinical interview based on the DSM-IV (SCID-IV). For both males and females, we performed a repeated-measures analysis of covariance (RM ANCOVA) with five blocks of the IGT as the within-subjects factor, free triiodothyronine (FT3) high-versus-low median split as the between-subjects factor, and age in years as a covariate. To assess the associations between three expectancy valence (EV) measures (consistency, attention to losses, and recency) and FT3, we performed partial Pearson correlations controlled for age. Results: We observed a significant interaction between FT3 and IGT blocks in males with BD (F(4,95)=2.9, p = 0.02), but not in females with BD (F(4,88)=0.2, p = 0.9). For males, those in the low FT3 group appeared to have a more gradual and normal learning curve compared to those in the high FT3 group. In terms of learning strategies, we observed a significant negative correlation between FT3 and consistency in males (R = -0.3, p = 0.001), indicating that they employ a more exploratory approach in association with higher FT3. We did not observe this relationship in the females (R = -0.03, p = 0.7). We did not find significant associations in any of the other EV measures. Conclusions: We observed differential reward learning styles in males with high versus low FT3 in the IGT. These results suggest a role of the thyroid in emotional learning and exploratory behaviors in males with BD. Keywords: Bipolar Disorder, Monetary Reward, Thyroid Hormone Disclosure: Nothing to disclose. P266. Different Brain Functional Connectome Changes Following 12-Week Treatment With Mixed Amphetamine Salts in ADHD Youth With and Without Familial Risk for Bipolar I Disorder Kun Qin, Du Lei, Ziyu Zhu, Maxwell Tallman, Thomas Blom, Jeffrey Welge, L. Rodrigo Patino, John Sweeney, Robert McNamara, Melissa DelBello University of Cincinnati College of Medicine, Cincinnati, Ohio, United States Background: Prevalence rates of attention deficit/hyperactivity disorder (ADHD) in youth with bipolar I disorder (BD) are substantially higher than the general population, and prospective studies have found that prodromal ADHD commonly precedes the onset of BD and significantly increases risk for developing BD. Additionally, having a first-degree relative with BD robustly increases the risk of developing BD, and youth with a first-degree BD relative exhibit higher rates of ADHD and more severe ADHD symptoms. Although the pathoetiological mechanisms associated with the risk of developing BD in youth with ADHD remain poorly understood, retrospective studies suggest that antecedent psychostimulant exposure for the treatment of ADHD may precipitate or exacerbate manic symptoms in a subset of vulnerable individuals. The initial onset of BD frequently occurs during the peripubertal period which is associated with dynamic changes in frontal cortex connectivity with limbic regions implicated in emotional regulation. Although cross-sectional resting-state MRI studies indicate that both BD and ADHD are associated with hypo- and hyper-connectivity in different cortical networks, the majority of these studies did not control for BD familial risk, ADHD comorbidity, and/or psychostimulant exposure. In the present study, we used graph theoretical analysis to investigate the effects of 12-week psychostimulant treatment on brain network organization and connectivity in ADHD youth with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD. Methods: ADHD youth (ages 10-18 years) with (‘high-risk’ for BD, HR) and without (‘low-risk’, LR) a first-degree relative with BD and typically developing healthy controls (HC) were enrolled. LR youth received 12-week open-label mixed amphetamine salts-extended release (MAS-XR), and HR youth were randomized to MAS-XR or placebo (PBO). All ADHD patients met DSM-5 criteria for ADHD (any type), had no exposure to psychostimulants for at least 3 months prior to enrollment, and had no comorbid mood, conduct, eating, or psychotic disorders. Resting-state fMRI scans were acquired on a Philips 3.0 T MR scanner at baseline and week 12. The Automated Anatomical Labeling (AAL90) template included 90 regions/nodes, some of which are implicated in attention and emotion regulation. Whole-brain global- and nodal-level network topological properties were evaluated using graph theoretical analysis. Global topological metrics included global efficiency (Eglob), local efficiency (Eloc), clustering coefficient (Cp), characteristic path length (Lp), normalized clustering coefficient (γ), normalized characteristic path length (λ) and small-worldness scalar (σ). Nodal topological metrics reflecting centrality include degree, betweenness, and efficiency. We first compared the three patient groups and the HC group at baseline and endpoint using MANCOVA. Group x time interaction effects were then investigated using linear mixed models with age and sex as covariates. FDR correction was applied to control for multiple comparisons. Results: A total of n = 135 youth were included in the analysis (HC, n = 45; LR-MAS, n = 46; HR-MAS, n = 28; HR-PBO, n = 16). No significant group differences were observed for age, sex, handedness, or prior psychostimulant exposure in youth with ADHD. At baseline, LR-MAS youth exhibited reduced global efficiency Eglob and increased characteristic path length Lp, as well as topological abnormalities in the superior parietal cortex and orbital and medial prefrontal cortex (mPFC) compared with HC. Both HR-MAS and HR-PBO exhibited reduced nodal efficiency in the mPFC, and HR-MAS additionally exhibited reduced degree in the cingulum and increased caudate efficiency, compared with HC. Following 12-week MAS-XR treatment, significant group x time interactions were observed between LR-MAS and HR-MAS for global efficiency (p = 0.012), which increased in LR-MAS but not HR-MAS, and characteristic path length (p = 0.0045), which decreased in LR-MAS and increased in HR-MAS. Additionally, group x time interactions were observed for nodal efficiency of the right amygdala (p = 0.0001), which decreased in HR-MAS but not LR-MAS, and nodal degree in the left superior parietal gyrus (p = 0.0002), which increased in LR-MAS but not HR-MAS. For HR-PBO vs. HR-MAS, a significant group x time interaction was observed for betweenness in the right inferior frontal gyrus (p = 0.0005), which increased in HR-MAS but not HR-PBO. Consistent with a normalizing effect, LR-MAS did not exhibit any global or nodal differences compared with HC at week 12, whereas HR-MAS exhibited significant residual reductions in left mPFC and right caudate nodal efficiency and new reductions in the right amygdala nodal efficiency. Conclusions: Psychostimulant-free ADHD youth with and without a BD family history exhibit different global and nodal network connectivity abnormalities compared with healthy youth at baseline, and their functional brain connectomes change differently following 12-week MAS-XR treatment. These differences in network connectivity and plasticity suggest that ADHD in conjunction with BD family history is associated with different neuropathophysiologic processes that are less amenable to modification with psychostimulant treatment. Keywords: Bipolar Disorder, ADHD, Resting State Brain Imaging, Prodrome Disclosure: Nothing to disclose. P267. Differential Changes in Left Uncinate Fasciculus Microstructure following 12-Week Mixed Amphetamine Salts in ADHD Youth With and Without Familial Risk for Bipolar I Disorder: A Prospective DTI Study Du Lei, Kun Qin, Ziyu Zhu, Maxwell Tallman, Thomas Blom, Jeffrey Welge, L. Rodrigo Patino, John A. Sweeney, Melissa DelBello, Robert McNamara University of Cincinnati College of Medicine, Cincinnati, Ohio, United States Background: Prevalence rates of attention deficit/hyperactivity disorder (ADHD) in youth with bipolar I disorder (BD) are substantially higher than the general population, and prospective studies have found that prodromal ADHD commonly precedes the onset of BD and significantly increases risk for developing BD. Additionally, having a first-degree relative with BD robustly increases the risk of developing BD, and youth with a first-degree BD relative exhibit higher rates of ADHD and more severe ADHD symptoms compared with ADHD youth with healthy parents. Although the pathoetiological mechanisms associated with the risk of developing BD in youth with ADHD remain poorly understood, retrospective studies suggest that antecedent psychostimulant exposure for the treatment of ADHD may precipitate or exacerbate manic symptoms in a subset of vulnerable individuals. Furthermore, the initial onset of BD frequently occurs during the peripubertal period which is associated with dynamic changes in frontal cortex connectivity with limbic regions implicated in emotional regulation. The uncinate fasciculus (UF) is the main fiber tract connecting the ventral prefrontal cortex with the amygdala, and meta-analyses of cross-sectional diffusion tensor imaging (DTI) studies indicate that BD patients exhibit lower bilateral UF fractional anisotropy (FA). Although prior evidence suggests that ADHD is not associated with alterations in UF FA, the majority of these studies did not control for BD familial risk and/or psychostimulant exposure. The present DTI study investigated the effects of 12-week psychostimulant treatment on bilateral UF FA in ADHD youth with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD. It was hypothesized that HR ADHD youth would exhibit lower bilateral UF FA at baseline, and greater reductions in bilateral UF FA following 12-week psychostimulant treatment, compared with LR ADHD youth and healthy controls. Exploratory analyses investigated associations between UF FA and relevant symptoms including mania and dysregulation. Methods: ADHD youth (ages 10-18 years) with (‘high-risk’, HR) and without (‘low-risk’, LR) a first-degree relative with BD and typically developing healthy controls (HC) were enrolled. LR youth received 12-week open-label mixed amphetamine salts-extended release (MAS-XR), and HR youth were randomized to MAS-XR or placebo (PBO). All ADHD patients met DSM-5 criteria for ADHD (any type), had no exposure to psychostimulants for at least 3 months prior to enrollment, and had no comorbid mood, conduct, eating, or psychotic disorders. Clinician ratings of ADHD (ADHD-RS), mania (YMRS), depression (CDRS-R), global functioning (CGAS), and global symptom severity (CGI-S), and parent-rated Child Behavior Checklist (CBCL), were obtained at baseline and week 12. DTI scans were acquired on a Philips 3.0 T MR scanner at baseline and week 12. Tract-based analysis (Tract-Based Spatial Statistic, TBSS) were applied to evaluate large fiber tracts, and FA and mean diffusivity (MD) maps were calculated. FA and MD were extracted from the bilateral UF, and group x time interaction effects were assessed using linear mixed effect models with age and sex as covariates. Linear correlation analyses evaluated associations between baseline-endpoint changes in DTI metrics and clinical ratings. Results: A total of n = 145 (HR, n = 45; LR, n = 51; HC, n = 49) youth (mean age: 14.0 ± 2.5 years) were enrolled. There were no significant baseline group differences in demographics including sex, race, pubertal status, hand dominance, body mass index, and in the ADHD groups prior exposure to psychostimulants and ADHD age at onset. At baseline, there were no significant main effects of group (4) for left (p = 0.09) or right (p = 0.85) UF FA or left (p = 0.23) and right (p = 0.16) UF MD, and neither UF FA nor UF MD correlated with symptom ratings. Following 12-week MAS-XR treatment (median MAS-XR dose: LR-MAS: 17.7 ± 5.2 vs HR-MAS: 15.8 ± 5.7, p = 0.12), the LR-MAS group exhibited significantly greater reductions in ADHD-RS total score (p = 0.015) and inattentive subscale scores (p = 0.0001) compared with HR-MAS, and a larger percentage of LR-MAS youth achieved remission (ADHD-RS endpoint score of <18) compared with HR-MAS (92% vs. 61%, p = 0.0001). For all ratings, there were no significant baseline-endpoint change differences between HR-MAS and HR-PBO. For left UF FA, a significant group by time interaction was found (p = 0.005), and the baseline-endpoint change in the HR-MAS group differed significantly from HR-PBO (p = 0.003), LR-MAS (p = 0.015) and HC (p = 0.007). Specifically, left UF FA decreased significantly in HR-MAS (-5.3%, p = 0.029) but not HR-PBO ( + 3.4%, p = 0.37), LR-MAS ( + 1.7%, p = 0.41), or HC ( + 2.6%, p = 0.27). Changes in left UF FA did not correlate significantly with changes in any symptom rating in either HR-MAS or LR-MAS. Conclusions: Contrary to our first hypothesis, psychostimulant-free ADHD youth with a BD family history did not exhibit lower bilateral UF FA compared with ADHD youth without a BD family history or healthy controls. In support of our second hypothesis, ADHD youth with, but not without, a BD family history exhibited reductions in left UF FA following 12-week MAS-XR treatment. This differential response to MAS-XR in ADHD youth with a BD family history may be relevant to lower UF FA observed in youth with BD, and additional studies are warranted to evaluate the long-term impact of psychostimulant exposure. Keywords: Bipolar Disorder, ADHD, Diffusion Tensor Imaging (DTI), Prodrome Disclosure: Nothing to disclose. P268. A Neural Signature for Emotion Regulation in Depressed Adults: Associations With Real-World Management of Stress and Suicidal Ideation Sarah Herzog, Barbara H. Stanley, Maria A. Oquendo, Hanga Galfalvy, Tse Hwei Choo, Kevin Ochsner, J. John Mann, Noam Schneck Columbia University Irving Medical Center, New York, New York, United States Background: Emotion regulation (ER), the ability to modulate emotional responses to meet situational demands and personal goals, is essential to psychological wellbeing. Across age groups, individuals who report ER difficulties also experience higher levels of suicidal ideation (SI). ER is often assessed by explicitly prompting participants to regulate; however, this may not approximate how people regulate in their daily lives. In the current study, we aimed to clarify how directed and non-directed ER in the context of an fMRI task relates to management of stress and SI in real-world settings, in a sample of unmedicated depressed adults. Based on prior work linking mood- and anxiety-related psychopathology to non-directed ER, we hypothesized that only non-directed ER would predict lower stress-responsive increases in SI. Methods: To quantify ER, we applied a two-step multivariate pattern analysis (MVPA) to train and test a signature that identifies ER-related neural activity. The training sample consisted of 77 unmedicated adults, including depressed and healthy individuals, who completed two fMRI tasks. In Task 1, participants were prompted to simply view negative images (“look) or use an ER strategy of reappraisal (“reappraise”) while viewing. In Task 2, participants were cued to recall negative autobiographical memories while using either an “immersed” (i.e., first-person) or “distanced” (i.e., third person) perspective, for 12s. The distanced perspective represents a regulated approach to recall. All memory trials began with a 10s non-directed recall period in which participants were not instructed to immerse or distance. A binary classifier was first trained on Task 1 data to identify a spatially distributed neural pattern (i.e., signature) associated with reappraisal trials (AUC = 0.63, 1,000-permutation p-value<0.001). In a subset of 33 depressed adults, the signature was used to track non-directed ER expressed during the Task 2 recall period and directed ER during the "distanced" period. Neural signature output in Task 2 reflects the degree to which participants’ neural activity resembles the putative neural systems underlying reappraisal in Task 1. Participants also completed a 7-day ecological momentary assessment (EMA) period during which they reported on SI and stressors six times daily, within 2-hour epochs. Stress-responsive increases in SI were used as an indication of difficulty regulating negative mood states. Results: Signature output during Task 2 was higher for distance trials compared to immerse trials (b[5925]=-0.030, SE = 0.070, p < .001). We examined whether signature output for non-directed ER (recall period) and directed ER (“distanced” period) predicted changes in SI following negative stressors. A time-varying stress indicator was used to denote EMA epochs with and without stressors. Separate mixed effects regression models were fit with stress-reactive SI change as the outcome variable; signature output, the time-varying stress indicator (stressor vs. no stressor), and their interaction, as the predictors; subject-specific random intercepts, and an AR1 covariance matrix. There was a significant interaction between the stress indicator and signature output for the recall period, with higher signature output predicting greater SI increases after a stressor (b[920]=5.70; SE = 2.73; t = 2.08; p = 0.037). The interaction for signature output during the “distanced” period (i.e., directed ER) and stress-responsive SI was not significant (b(857)<0.00; SE < 0.00; t = 1.07; p = 0.284). Conclusions: In validation of the neural signature, Task 2 distance trials were associated with the same neural systems underlying Task 1 reappraisal trials. Signature output for the directed ER period was not associated with stress-responsive SI, as hypothesized. However, contrary to our expectation, signature output for the non-directed ER period predicted greater (rather than lower) stress-responsive SI. While it may appear paradoxical that greater engagement of neural systems supporting ER is related to increased stress-reactive SI, in the context of depression, engagement of a neural pattern associated with dampening initial affective responses may be a compensatory response in anticipation of difficulty managing negative affective states. These findings have important implications for mood- and suicide-related clinical interventions in depressed populations. Keywords: Emotion Regulation, Functional MRI (fMRI), Ecological Momentary Assessment, Suicidal Ideation, Depression Disclosure: Nothing to disclose. P269. Hopelessness as a Key Indicator of Suicide Risk: Clinical and Magnetoencephalographic Results Elizabeth Ballard, Roshni Nischal, Dede Greenstein, Courtney Burton, Grace Anderson, Carlos Zarate, Jessica Gilbert National Institute of Mental Health, Bethesda, Maryland, United States Background: A suicide crisis is a medical emergency. Clinical and neurobiological targets for the acute suicide risk state are critically needed for better identification and treatment. However, patients often minimize suicidal ideation (SI) due to concerns of hospitalization and stigma; additionally, SI may be particularly reactive to dramatic fluctuations over time, necessitating the identification of non-SI indicators of active suicide risk. There is a longstanding research literature linking hopelessness, or pessimism about the future, with risk for suicidal thoughts, behavior and death. However, less is known about whether hopelessness can be a proximal sign for imminent suicide risk as distinct from a chronic risk factor which places an individual for increased suicide risk over their lifetime. Additionally, understanding the neural correlates of hopelessness in the context of suicide risk could be useful for future intervention development and evaluation of response to treatment in at-risk individuals. In this analysis, we examined hopelessness as a potential indicator of active suicide risk by comparing patients who had experienced a suicide crisis in the last two weeks to those who had experienced a suicide crisis more than a year previously. Additionally, in a sample of participants across the suicide risk spectrum, we examined electrophysiological correlates of hopelessness using magnetoencephalography (MEG) to evaluate hopelessness as a potential biomarker for acute suicide risk. Methods: Participants: Participants were recruited as members on one of five groups across the continuum of suicide risk, ranging from individuals who recently attempted or seriously considered suicide in the last two weeks (“acute crisis”, n = 14), individuals who had a history of suicide attempt, but no suicidal ideation in the last year (“chronic suicide risk”, n = 39), patients with chronic suicidal ideation (n = 12), patient controls (n = 38), and healthy volunteers (n = 27). Clinical Measures: Participants were assessed for hopelessness (Beck Hopelessness Scale (BHS)), suicidal ideation (Scale for Suicidal Ideation (SSI) and Columbia Suicide Severity Rating Scale (C-SSRS)), depression (Montgomery-Asberg Rating Scale (MADRS)) and psychological pain (Mee-Bunney Psychological Pain Scale (MBP)). Effect sizes were used to quantify differences between active crisis and chronic suicide risk groups. MEG: Participants across all groups underwent two resting state MEG scans with their eyes closed within two days of clinical ratings. MEG power was source-localized in the theta (4-8 Hz), alpha (9-14 Hz), beta (15-29 Hz), gamma (30-58 Hz), and high gamma (62-118 Hz) frequencies using synthetic aperture magnetometry. Linear mixed effects models examined effects of hopelessness, group, and group-by-hopelessness interactions on whole-brain estimates of power. Results: Clinical: When comparing the “active crisis” and “chronic suicide risk” groups, hopelessness was greater in the “active crisis” group (Effect Size= 1.26 95%CI: 0.58-1.90), similar to depression ratings (MADRS total score: 1.49, 95%CI: 0.79-2.18) and psychological pain (MBP: 1.22 95%CI: 0.56-1.87). In contrast, effect sizes for SI ranged from 0.93 (SSI, 95%CI: 0.22-1.63) to 0.26 (C-SSRS, -.37-0.89). MEG: There were no significant group or group-by-hopelessness effects for any of the MEG measures. However, there was a significant main effect of hopelessness (p < 0.05 cluster-level correction) across all frequency bands (theta, alpha, beta, gamma, and high gamma), where negative associations were found between total BHS score and source-localized power in a right frontal region centered in the anterior insula, a key node of the salience network. Conclusions: Hopelessness may be a key indicator of acute suicide risk and is linked with brain areas implicated in the salience network, which has been linked to the transition from suicidal thoughts to suicidal behaviors. Future evaluations of hopelessness as a potential indicator of suicide risk and biomarker of response to suicide-focused treatments are indicated. Limitations include the small sample size of recently suicidal individuals; further analyses are needed to disentangle the relationship between hopelessness, SI and depression at both the clinical and electrophysiological levels. Keywords: Suicide, Hopelessness, MEG Disclosure: Nothing to disclose. P270. Chronic Escitalopram in Healthy Volunteers Has Specific Effects on Reinforcement Sensitivity Christelle Langley*, Sophia Armand, Brice Ozenne, Annette Johnson, Camilla Borgsted, George Savulich, Qiang Luo, Rudolf Cardinal, Trevor Robbins, Dea Stenbæk, Gitte Knudsen, Barbara Sahakian University of Cambridge, Cambridge, United Kingdom Background: There have been several studies of the effects of acute or sub-chronic escitalopram on cognition in healthy volunteers, which have found changes in learning and reinforcement outcomes (Kanen et al., 2021; Seymour et al., 2012; Skandali et al., 2018). In contrast, to our knowledge, there have been no studies of the chronic effects of escitalopram on cognition in healthy volunteers. The interpretation of acute effects is complicated by the possible pre- and post-synaptic actions of the drug. Therefore, it is important both to understand the steady state effects of escitalopram in healthy people, but also because it is given chronically to patients with Major Depressive Disorder (MDD), as well as other psychiatric conditions, such as Obsessive-Compulsive Disorder (OCD). We aimed to investigate the effect of the chronic selective serotonin reuptake inhibitors (SSRI), escitalopram, on ‘hot’ and ‘cold’ cognition, and particularly on reinforcement learning. Methods: The study was a double-blind placebo controlled trial of 66 participants of which 32 received 20mg of escitalopram (mean age (s.d.) 24.25 (5.56)) and 34 received placebo for 21 days (mean age (s.d.) 25.76 (6.64)). Participants were semi-randomised into the two groups and matched for age, sex and IQ. The study was pre-registered on clinicaltrials.gov (NCT04239339). Comprehensive questionnaires and neuropsychological tests were conducted and serum escitalopram measures were taken. Questionnaire measures included demographics (e.g. ethnicity, education, and marital status), personality, life experiences, medical history, mood, stress and sleep quality. Neuropsychological tests included both ‘hot’ and ‘cold’ cognition, including domains such as learning, memory, attention, executive functioning, emotion recognition and reinforcement learning. Statistical analyses were conducted in R, version 4.1.1 (R Foundation for Statistical Computing). All questionnaire group comparisons were conducted using two-tailed t-tests in R. The Benjamini-Hochberg (Benjamini and Hochberg, 1995) false discovery rate (FDR) with q = .05 correction was used for the questionnaires. Group comparisons for cognitive measures were conducted using linear models allowing for unequal variances with the gls function from the R package nlme. The Sequential Model-Based/Model-Free task analysis was conducted according to Decker et al. (2016), where a generalized linear mixed-effects regression analysis of group behaviour data was performed using the lme4 R package. Age, sex, IQ and reaction time were included as covariates in the analyses. The corrections for multiple comparisons were conducted according to the pre-registered cognitive domains using the Benjamini-Hochberg FDR with q = .05. In addition the Probabilistic Reversal Learning task was analysed using hierarchical Bayesian methods, incorporating parameters that have been studied previously in the reinforcement learning literature(Kanen et al., 2019; Marzuki et al., 2021). Results: The analysis revealed that the escitalopram group had lower reinforcement sensitivity compared to placebo controls on both the Sequential Model-based/Model-Free (estimated coefficient = -.34, p = .004, 95% CI[-.575 to -.105]) and the Probabilistic Reversal Learning tasks (MD = -2.676 [90% HDI, -5.285 to -.396]). There were no significant differences between the two groups for any of the other cognitive measures. There were no significant group differences at baseline on any of the questionnaires. Examining the change scores between the baseline visit and the 3 week visit there was a significant difference between the two groups on 2 dimensions of the CSFQ-14 Sexual Functioning Questionnaire (Orgasm/Ejaculation (t(42.25)=2.68, p = .01) and Orgasm/Completion (t(42.25)=2.68, p = .01)), where the escitalopram group had significantly lower responses. Using the serum escitalopram levels we confirmed that all participants in the escitalopram group complied with the drug administration. Conclusions: Unlike previous reports on the acute effects of serotonin3, we did not find any significant effects on ‘cold’ cognitive measures after 3 week SSRI administration. In addition, there were no effects on emotion recognition or moral judgements. Importantly, we did find significant effects specific to reinforcement learning, where the escitalopram group had lower reinforcement sensitivity. These findings have key implications for the role of serotonin in reinforcement learning. In addition, the results have clinical implications as they may reflect the ‘blunting’ effect often reported by MDD patients receiving chronic SSRI treatments (Marazziti et al., 2019). Keywords: Cognition, Reward Sensitivity, Selective Serotonin Reuptake Inhibitors (SSRIs) Disclosure: Nothing to disclose.	36456693	PMC9714397	NO-CC CODE	2022-12-06 23:15:03	no	Neuropsychopharmacology. 2022 Dec 1; 47(Suppl 1):63-219	utf-8	Neuropsychopharmacology	2,022	10.1038/s41386-022-01484-1	oa_other
==== Front Neuropsychopharmacology Neuropsychopharmacology Neuropsychopharmacology 0893-133X 1740-634X Springer International Publishing Cham 36456694 1485 10.1038/s41386-022-01485-0 Abstracts Collection ACNP 61st Annual Meeting: Poster Abstracts P271-P540 1 12 2022 12 2022 47 Suppl 1 220370 © The Author(s), under exclusive licence to American College of Neuropsychopharmacology 2022 issue-copyright-statement© American College of Neuropsychopharmacology 2022 ==== Body pmc December 4-7, 2022 Phoenix Arizona Sponsorship Statement: Publication of this supplement is sponsored by the ACNP. All content was reviewed and selected by the Program Committee, which held full responsibility for the abstract selections. Only disclosures for presenting authors are listed. Asterisks in the author lists indicate presenter of the abstract at the annual meeting. Abstract numbers do not correlate to poster number assigned for presentation at the Annual Meeting. P271. Negative and Positive Urgency-Related Left Dorsolateral Prefrontal Cortex Activity During Emotion Processing Predicts Manic Symptom Changes One Year Later in Distressed Young Adults Maya C. Schumer, Michele A. Bertocci, Haris A. Aslam, Simona Graur, Genna Bebko, Richelle S. Stiffler, Alexander S. Skeba, Tyler J. Brady, E. Kale Edmiston, Henry W. Chase, Sheri L. Johnson, Mary L. Phillips University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: There is a critical need for identifying robust biomarkers of objective risk factors associated with Bipolar Disorder (BD), particularly those predictive of mania/hypomania and mixed states, as these are pathognomonic features of BD. Moreover, identifying biomarkers of future BD risk informs not only early risk detection and targets for treatment developments, but also aids our understanding of pathophysiological processes underlying BD. Impulsivity is a widely studied characteristic of BD that is elevated during manic and mixed episodes and persistent during euthymic periods. While impulsivity is a multi-faceted trait, emotion-triggered impulsivity, or emotion-based rash action, is perhaps the most important form, as it is associated with a broad range of key outcomes in BD, including and notably suicidality. Negative and positive urgency (NU/PU), defined as the tendencies to act impulsively in response to negative and positive affect respectively, are evident in adults with BD and have been identified as risk factors for future BD, yet neuroimaging studies examining neural correlates of urgency in the context of predicting BD risk are sparse. We previously reported negative relationships between NU and PU-related blood oxygen level dependent (BOLD) activity in the left dorsolateral prefrontal cortex (L DLPFC) during approach emotion processing and lifetime mania risk in a transdiagnostic cohort (excluding young adults with BD diagnosis) of 106 young adults who were seeking treatment for psychological distress. However, it has not yet been tested whether urgency-related L DLPFC activity is a predictor of manic symptoms over time, and if the relationship is specific to mania or if it also extends to depressive symptoms. Methods: Twenty-one distressed adults ages 18-23 (mean age 20.84 ± 1.49, 17 female), from the initial aforementioned transdiagnostic cohort that were scanned on a 3 T fMRI while performing an implicit facial emotion processing task, completed baseline and 12-month follow-up assessments of hypo/mania and depression measured by the Moods Spectrum Scale (MOODS) Mood Manic and Depressive Domains. Angry and happy face-related activity at baseline scan was examined using an anatomical mask of regions supporting emotion processing and executive function, which included the L DLPFC. Parameter estimates were extracted from separate multiple regression models of NU and PU covarying for age and gender, p < 0.001, uncorrected, k = 20. We performed two parallel, separate multiple linear regression models with NU and PU-related L DLPFC BOLD activity, baseline MOODS Mood Manic Domain score, age, and gender as predictors and Mood Manic Domain score at 12-month follow-up as the dependent variable. To test whether the L DLPFC was specific to predicting mania, we performed two additional multiple regression models (one for NU, one for PU) controlling for baseline depression, with 12-month MOODS Mood Depressive Domain score as the dependent variable. Results for both the primary analysis with mania and the specificity analysis with depression were each considered significant at p < 0.025, Bonferroni-corrected for two models. Results: The initial models with baseline demographic variables explained 18% of the variance in 12-month MOODS Mood Manic Domain score. The full model including NU-related L DLPFC activity was significant, F(2,16)=4.88, p = 0.009, with an adjusted R2 of 0.44, such that the L DLPFC explained 26% of the variance and was significantly associated with mania one year later (β = -8.07, t = -2.95, p = 0.009). The full model including PU-related L DLPFC activity was also significant, F(2,16)=4.2, p = 0.016, with an adjusted R2 of 0.39, such that the L DLPFC explained 21% of the variance and was significantly associated with mania one year later (β = -7.24, t = -2.61, p = 0.019). In the specificity analysis with 12-month MOODS Mood Depressive Domain score, the full model including NU-related L DLPFC activity was not significant, F(2,16)=3.56, p = 0.029, with an adjusted R2 of 0.338. L DLPFC activity was not significantly associated with depression one year later (β = -6.24, t = -2.23, p = 0.041). The full model including PU-related L DLPFC activity was not significant, F(2,16)=3.08, p = 0.047, with an adjusted R2 of 0.294. L DLPFC activity was not significantly associated with depression one year later (β = -5.53, t = -1.91, p = 0.074). Conclusions: Attenuated activation of NU- and PU-related L DLPFC at baseline both predicted changes in mania one year later, and their significant associations were indeed specific to mania. These preliminary findings underscore the importance of urgency-related central executive network activity during emotion processing in predicting manic symptom changes over time, indicative of greater risk for future BD. Future research should further extend these findings to a larger sample and assess if these markers can additionally predict conversion to BD in at-risk individuals, as well as investigate the potential for targeting the L DLPFC for therapeutic interventions. Keywords: Bipolar Disorder, Mania, Dorsolateral Prefrontal Cortex, Negative Urgency, Emotion Processing Disclosure: Nothing to disclose. P272. Systemic Inflammation Response Index (SIRI) Correlates to Inflammatory-Metabolic Markers in Treatment-Resistant Bipolar Depression (TRBDD) and May Predict Treatment Response Nausheen Baig, Kyle Decker, Stephen Murata, Debra Hoppensteadt, Jawed Fareed, Angelos Halaris Loyola University Chicago Stritch School of Medicine, Oak Brook, Illinois, United States Background: Inflammation has been associated with depressive illness and treatment resistance (Haroon and Miller, 2017; Miller et al., 2009), prompting a search for predictive biomarkers of treatment response. In a randomized, double-blind, placebo-controlled trial we demonstrated efficacy of escitalopram (ESC) + Celecoxib (CBX) compared to ESC + placebo (PBO) in TRBDD ( In this secondary analysis, we characterized treatment response (HAMD17) in relation to SIRI (monocytes x neutrophils/lymphocytes). We hypothesize that treatment response to ESC + CBX is associated with baseline of and/or changes in SIRI levels. Methods: The sample (N = 79) included healthy controls (n = 32) and TRBDD subjects (n = 47). TRBDD cohort included an ESC + CBX arm (n = 26) and ESC + PBO arm (n = 21). SIRI was calculated from complete blood count (CBC) drawn at baseline and end of treatment (week 8). Inflammatory biomarkers were measured using ELISA assays. Kynurenine pathway (KP) metabolites were measured using HLPC. HAMD17 scores were obtained weekly. Treatment remission was defined as HAMD17 < 7 by treatment week 8. Univariate correlations were explored between SIRI and inflammatory and KP biomarkers. Post-treatment HAMD17 was modeled according to treatment arm, baseline HAMD17, baseline SIRI, and relevant interactions using multivariate linear regression. Statistical analysis was conducted using R-3.6.3. Results: In the TRBDD sample (n = 47) there were n = 14 (38%) remitters by week 8. Remission was significantly associated with ESC + CBX treatment (X2 = 8.3, p = 0.004). Group comparison by treatment group revealed no differences in sex, BMI, or blood counts; however, patients in the CBX + ESC arm were older (p = 0.032). On univariate analysis of baseline SIRI, there were no significant correlations with treatment response at week 8 (whether measured as a continuous or categorical outcome), although baseline SIRI was correlated with baseline HAMD17 (p = 0.008). Baseline SIRI was significantly associated with higher TNF-alpha at week 8 (p = 0.007) and IL-2 at baseline (p = 0.053), elevated picolinic acid at both baseline (p = 0.034) and week 8 (p = 0.037), and lower 3-hydroxykynurenine at week 4 (p = 0.030). Notably, baseline SIRI also trended towards significant correlation with lower baseline IL-4 (p = 0.057) and higher baseline anthranilic acid (p = 0.071). Multivariate analysis showed HAMD17 was significantly associated with SIRI at week 8, adjusting for treatment arm (beta=5.74, p = 0.007, R2/R2 adjusted=0.553/0.531). Finally, HAMD17 at week 8 was significantly associated with ESC + CBX treatment (p < 0.002) and an interaction with baseline SIRI and baseline HAMD17 (β-estimate =8.3 [95% CI (0.23, 1.44)], p = 0.008) (R2/R2 adjusted = 0.354/0.286). Conclusions: Post-treatment HAMD17 was independently predicted by treatment arm and an interaction of baseline SIRI with HAMD17. This suggests that, although baseline SIRI may not be an independent predictor of treatment response, it may be indicative of poor prognosis amongst patients with elevated pre-treatment depressive severity. It is notable that this relationship is not dependent on treatment arm. Considering the associations with pro-inflammatory markers and KP metabolites reported here, it is plausible that the biological significance of SIRI may implicate these pathways. Future studies on SIRI with a larger sample size are warranted to clarify its potential role as a readily obtained/accessible predictive biomarker of treatment response in TRBDD. References: Halaris, A., Cantos, A., Johnson, K., Hakimi, M., and amp; Sinacore, J. (2020). Modulation of the inflammatory response benefits treatment-resistant bipolar depression: A randomized clinical trial. Journal of Affective Disorders, 261, 145–152. 10.1016/j.jad.2019.10.021 Haroon, E., and amp; Miller, A. H. (2017). Inflammation Effects on Glutamate as a Pathway to Neuroprogression in Mood Disorders. Modern Trends in Pharmacopsychiatry, 31, 37–55. 10.1159/000470805 Mazza, M. G., De Lorenzo, R., Conte, C., Poletti, S., Vai, B., Bollettini, I., Melloni, E. M. T., Furlan, R., Ciceri, F., Rovere-Querini, P., and amp; Benedetti, F. (2020). Anxiety and depression in COVID-19 survivors: Role of inflammatory and clinical predictors. Brain, Behavior, and Immunity, 89, 594–600. 10.1016/j.bbi.2020.07.037 Mazza, M. G., Palladini, M., De Lorenzo, R., Magnaghi, C., Poletti, S., Furlan, R., Ciceri, F., COVID-19 BioB Outpatient Clinic Study group, Rovere-Querini, P., and amp; Benedetti, F. (2021). Persistent psychopathology and neurocognitive impairment in COVID-19 survivors: Effect ofinflammatory biomarkers at three-month follow-up. Brain, Behavior, and Immunity, 94,138–147. 10.1016/j.bbi.2021.02.021 Miller, A. H., Maletic, V., and amp; Raison, C. L. (2009). Inflammation and its discontents: The role of cytokines in the pathophysiology of major depression. Biological Psychiatry, 65(9), 732–741. 10.1016/j.biopsych.2008.11.029 Zahorec, R. (2021). Neutrophil-to-lymphocyte ratio, past, present and future perspectives. Bratislavske Lekarske Listy, 122(7), 474–488. 10.4149/BLL_2021_078 Zhang, Y., Xing, Z., Zhou, K., and amp; Jiang, S. (2021). The Predictive Role of Systemic Inflammation Response Index (SIRI) in the Prognosis of Stroke Patients. Clinical Interventions in Aging, 16, 1997–2007. 10.2147/CIA.S339221 Keywords: Bipolar I Depression, Systemic Inflammation, Neuroprotection, Neuropharmacology Disclosure: Nothing to disclose. P273. The Continuous-Performance Emotion-Regulation (CERT) Task: A Novel fMRI Paradigm to Investigate Inter-Neural Network Connectivity Shifts During Affect-Regulation in Depressed and Healthy Subjects Jungwon Cha, Amit Anand* Harvard Medical School, Boston, Massachusetts, United States Background: Inter-network connectivity shifts are part of dynamic changes in brain configuration while it interacts with the environment. Hence, a continuous design corresponds more to real-life conditions. Furthermore, shifts in internetwork connectivity corresponds more accurately to brain function as it reconfigures to different emotion regulation conditions rather than static measurement during a single state. We have designed a novel resting state fMRI paradigm – the continuous-performance emotion-regulation task (CERT) to capture brain network re-configuration during emotion regulation. This study used CERT and Group-level Independent Components Analysis (ICA) to investigate differences in inter-neural network connectivity shifts during maintenance and regulation of negative emotions to distinguish between (i) Bipolar Depressed (BDD) and Major Depression (MDD), (ii) Patients (PA = BDD + MDD) and Healthy Controls (HC), and (iii) MDD patients who at high risk (HRMDD) and at low risk (LRMDD) for bipolar disorder. Methods: Subjects: Data is included from a large sample of N = 298 medication-free participants recruited from the outpatient psychiatry clinics at the Indiana University School of Medicine and Cleveland Clinic, and by advertisement. All BDD and MDD participants were required to be medication-free for at least 2 weeks. The final analyses included 249 participants: 50 BDD (18 BDI, 32 BDII), 116 MDD (35 HRMDD, 45 LRMDD, 36 unspecified); and 83 HC subjects. CERT Imaging: Functional MRI data were acquired in two different conditions: during separate runs of 5.25 minutes each, subjects were continuously shown negatively valences pictures while they either tried to maintain emotional response to the pictures or in the following set tried to suppress their emotional response using reappraisal techniques. Data collected from each of the runs was corrected for motion and physiological noise and band-pass filtered to retain frequencies between 0.008 - 0.08 Hz. Internetwork Connectivity: Analysis: Group-level Independent Components Analysis (ICA) was performed using GIFT toolbox. The preprocessed resting state functional MR images of all subjects, both for maintenance condition (249 images) and regulation condition (249 images), were decomposed into independent components. Fourteen independent components were selected for Resting State Network (RSN) including dorsal and ventral Default Mode network (dDMN and vDMN), anterior and posterior Salience Networks (ASN and VSN), right and left Executive Control Network (ECN), Sensorimotor network (SMN), Precuneus network (PN), Visual and Auditory Networks (VN and AN), and the Language Network (LN). With the selected set of components, univariate tests were performed to investigate the inter-network connectivity difference between each individual group (BD, MDD, HRMDD, LRMDD, PA and HC) in the two different conditions (Maintain vs. Regulate) through Repeated-measure ANOVA (RMANOVA). Results: Condition Effect: Within group - HC, MDD, and LRMMD exhibited differences in internetwork connectivity shifts during Maintain and Regulation Conditions for several pairs of networks including the DMN and SN. However, BDD and HRMDD groups did not show significant connectivity shift between the two conditions. Group x Condition Effect:BDD vs. MDD The interaction between group and condition was significant (FDR corrected p < .01) for inter-network connectivity between SMN-AN, SN-ECN and SN-VN. PA vs. HC: The interaction between group and condition showed a significant difference (FDR corrected p < .01) in several pairs of FNC including vDMN-AN, dDMN-AN, and dDMN-ECN correlations. HRMDD vs. LRMDD: significant interaction between the group and condition was seen for AN-VSN internetwork connectivity (p < 0.01, uncorrected). Group Effect: No differences between groups were seen individually within Maintain and Suppress conditions. Conclusions: Using a novel continuous emotion maintenance and regulation paradigm, differential internetwork connectivity-shifts distinguished between depressed subjects and healthy controls, MDD and BDD groups, and high and low-risk for bipolar disorder depressed subjects. Hence, internetwork connectivity-shifts across emotion regulation conditions rather than static measurement of internetwork connectivity in a single condition may serve as a more efficient biomarker for depression and depression sub-types. Keywords: Brain Networks, Dynamic Functional Connectivity, Continuous Performance Task, Affect Regulation, Depression Disclosure: Nothing to disclose. P274. The Heinz C Prechter Bipolar Research Program: A Comprehensive Ontological Approach to the Study of Bipolar Disorder Sarah Sperry, Paul Jenkins, Kelly Ryan, Ivy Tso, Melvin McInnis University of Michigan, Ann Arbor, Michigan, United States Background: Bipolar disorder (BD) is a complex multidimensional illness characterized by pathological swings in moods and emotions that vary over time. The etiology of BD is not known, but causality is considered to be plural. The HC Prechter Bipolar Research Program at the University of Michigan is a collaborative network of research projects focused the discovery of mechanisms underlying the etiology of BD as well as the study of predictive patterns of outcomes of disease. The core features of the program include deep phenotyping and ongoing longitudinal collection of clinical data. The data are organized into 7 ontological platforms: 1) Neurocognitive functioning; 2) Personality, 3) Motivated behaviors; 4) Sleep and Circadian patterns; 5) Life Story and experiences; 6) Treatment and outcomes patterns; 7) Disease states. Methods: There are currently 1,385 participants in the Prechter Cohort (mean age at enrollment is 39.2 years, 62% female) including 874 individuals with a BD and 282 controls. The median longitudinal follow-up is 9 years (range: 0 – 16). Deep clinical phenotyping gathers clinical data from each of the 7 ontological platforms. Standardized interviews, assessment batteries, and self-report questionnaires are used to assess neurocognitive functioning (e.g., visual memory, fine motor dexterity), personality (e.g., NEO PI-R), motivated behaviors (e.g., Alcohol Use Disorder Identification Test), sleep and circadian rhythms (e.g., PSQI, Munich Chronotype Questionnaire), life story (e.g., Life Events Checklist, Childhood Trauma Questionnaire), treatment and outcomes patterns (e.g., medication review, mood measures). Biological modelling of disease states uses induced pluripotent stem cell (iPSC) methods. To identify patterns between baseline characteristics and longitudinal course of illness, we employ multilevel modeling, dynamic structural equation modeling, path analysis, and mathematical modeling approaches. Results: We present primary findings from each of the ontological platforms herein. Neurocognitive Functioning: Individuals with BD have poorer cognitive performance compared to controls on visual memory (t = 3.31, p = .001) and fine motor dexterity (t = 4.21, p < .001) at baseline, one- and five-year follow ups. However, there is no overall difference in the rate of decline in neurocognition between BD and controls. Personality: Individuals with BD have higher neuroticism which tends to remain stable over time. Stability of neuroticism has a moderately strong association with depression over time (effects range from 0.21 to 0.52, ps < .001). Motivated Behaviors: Individuals with BD tend to increase their alcohol use over time (Est = .33, 95% credibility interval [.30,.35]). Increases in alcohol use appear to increase risk of depression 6 months later (Est = .04, 95% CI [.02, .07]) and greater mood instability in the following year (Est = .12, 95% CI [.05, .18]). Even mild levels of alcohol use predicted greater impairment to friendships (β = .01, p = .02) and work-related functioning ((β = .01, p = .04). Sleep and Circadian Patterns: Circadian timing, as estimated from self-report chronotype appears to affect longitudinal outcomes, with those identifying as late chronotype experiencing a greater frequency of depressive symptoms over time (Est=1.29, 95% CI [1.14,1.47]). Life Story and experiences: Disruptions in childhood maternal attachment and current romantic attachment in the context of a history of childhood trauma were associated with greater depression severity (accounting for 12% of the total effect of childhood trauma on depression severity). Treatment and outcomes patterns: Mathematical modeling of longitudinal mood data suggests that individuals with BD are characterized by significant affective instability, wherein mood states do not follow a rhythmic process – BD episodes arise in the context of persistent instability with failure to return to normal states once perturbed. Disease States: Synaptic activity within iPSC cells differ significantly between BD and controls (p < 0.05); exosomal source / cargo significantly influences synaptic activity. The addition of control derived exosomes significantly increases the synaptic density in bipolar (iPSC) derived neurons (p < 0.01). Conclusions: We have identified 7 ontological platforms that are critical to the understanding of longitudinal course and heterogeneity in BD. Baseline characteristics across these platforms seem to robustly predict severity and course of depressive symptoms. The organizational structure of the data benefits from consideration of an ontological approach in order to study static, categorical, and dynamic longitudinal features of the illness. There are biological, neurocognitive, personality, behavioral, sleep, life story, and outcome differences in comparing BD with controls and between individuals with bipolar that provides the basis for the study of underlying mechanistic differences in subtypes of illness. Future research with the HC Prechter Bipolar Research Program aims to use data-driven approaches to identify strata of individuals based on these 7 ontological platforms to implement personalized, and precision-medicine based interventions. Keywords: Bipolar Disorder, Longitudinal Study, Deep Phenotyping, Mood and Cognition Disclosure: Nothing to disclose. P275. Behavioral and Electrophysiological Profiles along the Continuum of Suicide Risk: A Potential Role for Ketamine in Suicide Prevention Steven Lamontagne, Jessica Gilbert, Carlos Zarate, Elizabeth Ballard National Institute of Mental Health, Bethesda, Maryland, United States Background: Suicide is a leading cause of death worldwide, accounting for at least one million fatalities per year (World Health Organization, 2021). Despite increasing rates of suicide, risk mitigation strategies have been largely unsuccessful. Two major barriers hinder these efforts: (1) a paucity of objective markers that probe suicidal states, constraining suicide research to self-report and (2) limited understanding of the neurobiology of suicide, precluding the development of biological interventions. Although progress has been made on these fronts, there is considerable heterogeneity in the characterization of suicide risk across clinical trials and neuroimaging studies. Thus, there is an urgent need to identify neural correlates associated with varying levels of suicide risk, as well as develop novel fast-acting therapeutics to modulate activity within these neural networks. Methods: Seventy-five male and female adults (Mage=39.89, range 19-65) were recruited through a suicide-focused research protocol (NCT02543983). Whereas most studies broadly define suicide risk based on lifetime history of attempt(s), we examined parameters along a continuum of risk: (1) those with a suicide attempt in the past two weeks and/or lifetime suicidal ideation with intent (High Risk; HR) (n = 15), (2) those with a history of attempt, but no suicidal behavior or ideation with intent in the past year (Moderate Risk; MoR) (n = 18) (3) those with anxiety or mood symptoms, but no suicide history (Low Risk; LR) (n = 19), and (4) those without psychiatric or suicide history (Minimal Risk; MinR) (n = 23). We used a CTF 275-channel whole-head magnetoencephalography (MEG) scanner to examine electrophysiological correlates of suicide. During MEG scanning, participants completed a modified Life-Death Implicit Association Task, which is a computer task measuring associations of oneself to either life or death based on reaction times to words that represent each category (faster reaction time denotes stronger implicit bias) (Nock et al., 2010). The behavioral outcome of interest was the D-score, defined as the difference in mean reaction times between self-death and self-life trials divided by the standard deviation of all trials (positive D-scores reflect a stronger self-death association). MEG data were source-localized in the gamma (30-58 Hz) frequency, a proxy measure of excitation-inhibition balance, using a 1 s window from word onset and a linearly constrained minimum variance beamforming algorithm. A linear mixed-effects model implemented in AFNI was used to evaluate differences in gamma power between self-life and self-death word pairings. As a proof-of-concept open-label pilot study, we examined the effects of subanesthetic-dose ketamine, a N-methyl-D-aspartate (NMDA) receptor antagonist, known to have rapid anti-suicidal ideation properties, on gamma power in a small group of HR participants (N = 5). Results: Behavioral results showed that D-scores in the HR group did not differ from zero (p = 0.78), but were significantly higher compared to the MoR, LR, and MinR groups (ps<0.01). D-scores for the latter three groups did not differ from each other (ps>0.43) and were significantly lower than zero (ps<0.001), denoting a self-life bias. Across groups, a linear mixed effects model showed enhanced gamma power for self-death compared to self-life conditions in the posterior cingulate cortex (PCC) (p < 0.05), a region associated with self-referential processing. A significant group-by-condition interaction (p < 0.05) revealed group differences in gamma power within the PCC, right insular cortex, and orbitofrontal cortex (OBF). Extracting gamma power estimates in these regions showed higher gamma power for self-death compared to self-life trials in the OBF for the HR group (p < 0.01) and the insula and PCC for the MinR group (ps<0.05). No differences in gamma power between self-life and self-death trials emerged for the LR group (ps>0.05). In the ketamine pilot study, D-scores were not affected by ketamine administration (p = 0.57); however, a session-by-condition interaction (p < 0.05) revealed enhanced gamma power for self-death trials in the left insula after ketamine administration compared to baseline (p < 0.001). Post-ketamine insular gamma power for self-death trials inversely correlated with D-score (r = -0.89, p < 0.05), suggesting the insula might be an important biomarker for implicit cognitions about death in HR individuals. Conclusions: These findings point to differential implicit cognitive processing of life and death depending on suicide risk, highlighting the need for patient-centered risk assessment in suicide prevention. Our findings also implicate a role for pharmacotherapies that modulate gamma activity, particularly in the PCC and insula, in risk mitigation. Toward this end, our preliminary data show promising effects of ketamine in modulating neural correlates of suicide-related cognitive processing. Keywords: Suicide Risk Factors, Implicit Association Test, Ketamine, Magnetoencephalography Disclosure: Nothing to disclose. P276. The COVID19 Pandemic Disrupts Associations Between Neural Reward Connectivity and Affective and Anxiety Symptoms in Young Adults Kristen Eckstrand, Erika Forbes, Michele Bertocci, Henry Chase, Jeanette Lockovich, Richelle Stiffler, Haris Aslam, Alex Skeba, Simona Graur, Genna Bebko, Mary Phillips University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: The COVID19 pandemic has imposed significant morbidity and mortality, including the worsened mental health outcomes of higher depression and anxiety severity. Experiences of young adults during the pandemic - such as social isolation and increased stress - limit exposure to rewarding experiences and are associated with poorer mental health. Altered connectivity between neural reward regions, such as the ventral striatum (VS), dorsal anterior cingulate cortex (dACC), and ventrolateral prefrontal cortex (vlPFC), with other neural regions involved in salience detection and executive control has been implicated in depression and anxiety and may be a mechanism by which the COVID19 pandemic has worsened affective and anxiety symptoms in young adults. The purpose of this cross-sectional study was to examine whether the COVID19 pandemic altered relationships between neural reward connectivity and affective and anxiety symptoms. Methods: 98 young adults (23.5 ± 2.9 years; 63 F/35 M sex) were included in this cross-sectional sub-study derived from a larger longitudinal study examining the development of mood and anxiety symptoms in young adulthood. Participants were divided into two pandemic groups, those who completed the study prior to the COVID19 pandemic (n = 39) and during the pandemic (n = 59), using the national lockdown date (March 13, 2020). Participants completed self-report measures of depression (Mood and Anxiety Symptom Questionnaire (MASQ) – Anhedonia Depression), anxiety (MASQ-Anxious Arousal), anhedonia (Snaith Hamilton Pleasure Scale), lifetime trauma exposure (Trauma History Questionnaire) and underwent fMRI scanning during a standardized monetary reward task. fMRI data were preprocessed using fMRIPrep 20.2.6 and 1st level analyses were completed in SPM12. The 1st level GLM included reward expectancy (RE), reward prediction error (RPE), and uncertainty expectancy (UE) as regressors. Wholebrain functional connectivity during RE and RPE was assessed using bilateral VS, dACC (Brodmann area 32), and vlPFC seeds. Six two-group 2nd level models (3 seeds x 2 connectivity conditions) were performed in SPM12 comparing neural connectivity between COVID19 pandemic groups. Each model included independent variables of self-reported depression, anhedonia, and anxiety, interacting with pandemic group. Age, sex, IQ, psychotropic medication load, bipolar diagnosis, and lifetime trauma exposure were included as covariates. Significance was defined in SPM12 as punc<0.001, cluster corrected at pFWE<0.05. Results: Participants who completed the study during the COVID19 pandemic reported higher anhedonia (t = -2.112, p = 0.019) and higher anxiety (t = -2.789, p < 0.003) severity, but not depression severity, compared to those who completed the study before the COVID19 pandemic. The COVID19 pandemic impacted associations between affective symptoms and both dACC and vlPFC connectivity during RE and RPE. During RE, lower dACC-mPFC (kE=108 voxels, pFWE=0.015) connectivity was associated with higher anhedonia severity during the COVID19 pandemic compared to lower anhedonia severity before the COVID19 pandemic. Lower vlPFC connectivity with the left cerebellum (kE=131 voxels, pFWE=0.009), bilateral thalamus (kE=165 voxels, pFWE=0.002), right parietal lobule (kE=171 voxels, pFWE=0.002), middle cingulate cortex (kE=94 voxels, pFWE=0.044), and right precentral gyrus (kE=133 voxels, pFWE=0.008) during RE was associated with higher anxiety severity during the COVID19 pandemic compared to lower anxiety severity before the COVID19 pandemic. During RPE, higher vlPFC connectivity with the right temporoparietal junction (kE=119 voxels, pFWE=0.022), right dorsomedial prefrontal cortex (kE=176 voxels, pFWE=0.003), dACC (kE=98 voxels, pFWE=0.002) and middle cingulate cortex (kE=181 voxels, pFWE=0.002) during RPE was associated with higher anhedonia severity during the COVID19 pandemic compared to lower anhedonia severity before the pandemic. Higher dACC connectivity with the left vlPFC (kE=169 voxels, pFWE=0.001) and right ventral postcentral gyrus (kE=144 voxels, pFWE=0.003) was associated with higher anxiety severity during the COVID19 pandemic compared with lower anxiety severity before the COVID19 pandemic. Conclusions: The COVID19 pandemic changed the direction of associations between anhedonia and anxiety severity and reward circuitry connectivity. Higher anhedonia severity was associated with lower dACC connectivity with salience regions during RE and higher vlPFC connectivity with the central executive network during RPE, suggesting a diminished ability to anticipate rewards and overregulation of reward response may have contributed to higher anhedonia severity during the pandemic. The opposite pattern emerged for anxiety, where higher anxiety severity was associated with a combination lower vlPFC connectivity with the central executive network during RE and higher dACC connectivity with salience regions during RPE. This indicates a lesser ability to evaluate and attend to potential rewards, combined with a heightened sensitivity to rewards, impacted anxiety severity during the pandemic. Combined, opposing and ineffective compensatory relationships between reward circuitry and both salience and central executive networks during the pandemic may be a mechanism for the elevated anxiety and depression severity observed during the pandemic. Keywords: The COVID-19 Pandemic, Reward Circuitry, Depression and/or Anxiety, Young Adults Disclosure: Nothing to disclose. P277. Altered Patterns of Central Executive, Default Mode and Salience Network Activity and Connectivity are Associated With Concurrent and Future Depression Risk in Two Independent Young Adult Samples Michele Bertocci, Yvette Afriyre-Agyemang, Satish Iyengar, Renata Rozovsky, Richelle Stiffler, Haris Aslam, Genna Bebko, Mary Phillips University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: A range of subsyndromal-syndromal affective symptoms underlie emotional distress and are often early predictors of future depression in young adults. Greater functional connectivity (FC) among the central executive network (CEN), supporting emotional regulation (ER) subcomponent processes such as working memory (WM), the default mode network (DMN), supporting self-related information processing, and the salience network (SN), is thought to interfere with cognitive functioning and predispose to depressive disorders. These patterns of neural network function are therefore potential early, objective neural markers of depressive disorders; yet, few studies have examined how activity within, and FC among, these neural networks during WM and ER tasks are associated with depression severity in young adults. We aimed to: 1. elucidate relationships among activity and FC in these networks and concurrent depression, using a paradigm designed to examine WM and ER capacity; 2. examine the extent to which these relationships were specific to depression versus mania/hypomania; 3. test whether findings in a first, “discovery” sample could be replicated in a second, “test” sample of young adults; and 4. test whether such relationships also predicted future depression and/or mania/hypomania severity. Methods: We examined relationships among concurrent depression and mania/hypomania severity and neural activity and FC during the above paradigm in two independent samples of young adults: a discovery sample: n = 90, 60 female, age=21.7 (2.0) and a test sample: n = 96, 65 female, age=21.6(2.1). Depression severity was assessed using the Hamilton Depression Rating Scale, and mania/hypomania severity, using the Young Mania Rating Scale. We examined activity and FC using an emotional regulation neural mask comprising key regions in each network: the dorsolateral prefrontal cortex and caudate in the CEN; the precuneus in the DMN; and the dorsal anterior cingulate cortex and amygdala in the SN (FWE p = .001, k > 10). Elastic net variable selection was performed using GLMNET followed by negative binomial regression to determine relationships among clusters of significant activity and FC in this mask and concurrent depression and mania/hypomania severity in the discovery sample. Negative binomial regression analyses determined whether the significant neural activity and FC-affective symptom relationships in the discovery sample were replicated in the test sample. We then examined relationships among neural activity and FC and future depression and mania/hypomania severity in a subsample derived from both samples (n = 61, 45 female, age =21.6 (2.1)) who were clinically assessed for up to 12 months after the scan. Results: In the discovery sample during working memory: dlPFC activity (Odds Ratio (OR):1.80, CI:1.42-2.28, qFDR < .001), precuneus activity (OR:1.43, CI:1.12-1.82, p = .004, qFDR=0.004), were positively associated with concurrent depression severity. These relationships were replicated in the test sample: dlPFC activity (OR:2.90, CI:2.31-3.64, qFDR < .001) and precuneus activity (OR:1.88, CI:1.54-2.29, qFDR < .001). These measures were also positively associated with future depression severity: dlPFC activity (OR:2.81, CI:2.21-3.56, qFDR<0.001), and precuneus activity (OR:1.49, CI:1.21-1.85, qFDR<0.001). In the discovery sample during emotional regulation: dlPFC activity (OR:4.27, CI:2.09-8.72, qFDR < .001), precuneus-dACC FC (OR:1.64, CI:1.06-2.53, qFDR = .046), precuneus-dlPFC FC (OR:1.67, CI:1.22-2.29, p = .002), and dlPFC-dACC FC (OR:1.49, CI:1.09-2.04, qFDR = .028) were positively associated with concurrent depression severity. These relationships were replicated in the test sample: dlPFC activity (OR:2.65, CI:1.94-3.63, qFDR < .001), precuneus-dACC FC (OR:11.22, CI:7.26-17.34, qFDR<0.001) and dlPFC-dACC positive FC (OR:2.61, CI:1.89-3.60, qFDR<0.001). These measures were also positively associated with future depression severity: dlPFC activity (OR:3.55, CI:2.52-5.0, qFDR < .001) precuneus-dACC FC (OR:6.26, CI:3.62-10.83, qFDR < .001) and dlPFC-dACC FC (OR:3.65, CI:2.34-5.68, qFDR < .001). There were no relationships among any neural measures and concurrent mania/hypomania severity in the discovery sample. Conclusions: Identifying and replicating largescale neural network predictors of concurrent and future depression severity during working memory and emotional regulation paradigms is a step toward identifying objective markers of risk for future depressive disorders, and can provide neural targets to better guide and monitor early interventions in at-risk young adults. Keywords: Mood Disorders, Emotional Regulation, Working Memory fMRI, Default Mode Network (DMN), Salience Network Disclosure: Nothing to disclose. P278. Neonatal Maternal Separation-Induced Alternation of MiRNAs and Their Possibility as Biomarkers of Major Depressive Disorder Shuken Boku, Hiroyuki Toda, Minori Koga, Naoto Kajitani, Minoru Takebayashi Kumamoto University, Kumamoto, Japan Background: As major depressive disorder (MDD) impairs a patient’s life over a long period, early detection of MDD is essential for minimization of loss and suffering due to MDD. However, early detection of MDD remains difficult because of the lack of clinically useful biomarkers of MDD. A lot of studies have shown that early-life stress (ELS) is involved in the vulnerability and treatment-resistance of major depressive disorder in adults. In addition, recent studies have reported that miRNAs may be involved in the biological effects of ELS. These suggest that ELS-associated miRNA may be a potential biomarker of MDD. Therefore, here we examined the possibility of ELS-associated miRNAs as biomarkers of MDD. Methods: The possible candidates of ELS-associated miRNAs were identified with neonatal maternal separation (NMS) in rats, a common animal model of ELS. The levels of miRNAs in peripheral blood of MDD patients (N = 64) and healthy controls (N = 75) were estimated with quantitative RT-PCR. Receiver operating characteristic (ROC) curve analyses were performed to estimate the possibility of the possible candidates of ELS-associated miRNAs. Results: we performed the microarray analysis of miRNAs derived from peripheral blood of NMS rats and identified four miRNAs which were significantly altered in NMS rats and expressed in both rats and human. ROC analyses showed that area under the curve (AUC) of each miRNA was under 0.7000, which means that each miRNA has only limited diagnostic power of MDD. However, AUC of the combination of these four miRNAs was 0.9444 with sensitivity of 0.8750 and specificity of 0.9200, which means that the combination of these four miRNAs has the high diagnostic power of MDD. Conclusions: The combination of the four ELS-associated miRNAs is expected as a clinically useful biomarker of MDD. Keywords: Major Depressive Disorder (MDD), Biomarker, miRNA, Early life stress (ELS) Disclosure: Nothing to disclose. P279. Anhedonia is Associated With Paraventricular Nucleus of Thalamus to Nucleus Accumbens Resting-State Functional Connectivity Bianca Leonard, Steven Granger, Joren Adams, Liv McMillan, Michael Yassa University of California Irvine, Irvine, California, United States Background: The paraventricular nucleus of the thalamus (PVT) is gaining attention for its roles in integrating salient experiences, arbitration of motivational conflict, retrieval of long-term fear memories, and behavioral regulation. The PVT responds to salient stimuli of both positive and negative valence, likely due to its reciprocal connections with the nucleus accumbens (NAc) and amygdala (AMY). Recently, our lab defined the resting state functional connectivity (RSFC) patterns of the PVT in humans using resting state fMRI and established that a similar PVT network to that found in rodents exists in humans. Interruption of the PVT-AMY-NAc circuitry in rodents is associated with depressive- and anxiety-like behavior in rodents - notably anhedonia as a function of sex. Here, we investigated if anhedonia was associated with changes in PVT-NAc and PVT-AMY RSFC. Methods: We collected self-reported depression and anxiety scores (Beck Depression Inventory-II and Beck Anxiety Inventory), and resting-state functional magnetic resonance imaging (fMRI) scans in a human sample (n = 63, 48 females) that endorsed a range of depression and anxiety symptom severity. Latent factor analysis applied to the depression and anxiety symptom scores empirically differentiated symptom classes in this sample. We derived four latent factors from this analysis, including an anhedonia factor. RSFC was calculated as the time-series correlations between the activity of the PVT with the AMY and NAc. PVT-NAc and PVT-AMY FC were correlated with the anhedonia factor scores using Spearman’s rank correlation coefficient. Results: PVT-NAc FC was positively associated with anhedonia factor scores in participants with psychiatric symptoms (PS, R = 0.34, p = 0.018), but not in participants without psych symptoms (NPS, R = 0.23, p = 04). Males had a significant positive relationship between PVT-NAc FC and anhedonia (R = 0.65, p = 0.0082), but females did not (R = 0.13, p = 0.38). PS males had the strongest correlation between PVT-NAc FC and anhedonia factor scores (0.77, p = 0.0054) where females did not quite reach significance (R = 0.3, p = 0.075). PVT-AMY and anhedonia factor scores did not correlate significantly among any of these analyses. Conclusions: These findings suggest that changes in PVT-NAc circuitry may relate to severity of reported anhedonia and support the use of latent factor analysis to better understand patient symptom reporting and categorization. Future work will include analysis to investigate if this is occurring in a sex-specific way and test remaining factors collected in the study with PVT FC. Keywords: Paraventricular Nucleus of the Thalamus, Resting State Functional Connectivity, Anhedonia, Sex-specific Effects, Latent Factor Analysis Disclosure: Nothing to disclose. P280. Dorsal Attention Network Neural Activity During Explore-Exploit Decision Making is Linked to Mood-Dependent Impulsivity in Older Adults Angela Ianni, Beatrice Langer, Michael Hallquist, Alexandre Dombrovski University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: The decision to attempt suicide may result from an impaired ability to select among alternative options in a state of intense negative emotions. Personality traits such as neuroticism and impulsivity have been implicated suicidal behavior, and mood-dependent impulsivity (urgency) may play an important role in the suicidal crisis, however little is known about how trait predispositions impact decision-making. Reinforcement learning provides a framework for behavioral and neural studies of the interface between personality factors and decision-making. Explore-exploit decisions are one example, where the decision is to utilize known information to take advantage of (exploit) an option known to be good or explore other lesser-known options. Optimal behavior requires adequate learning from sampled rewards and appropriate generation and comparison of alternative options. While prior work has linked suicidal behavior to impaired reward-guided learning, the neural basis of aberrant decision making in the acute suicidal crisis is not well understood. The dorsal attention network (DAN) is involved in the voluntary orienting of visuospatial attention, allowing for generation of a set of actions that can be taken in a particular situation. The DAN is comprised of several functionally connected brain regions including the intraparietal sulcus, superior pre-central sulcus, inferior pre-central sulcus, and motion-sensitive area MT complex (MT + ). Prior work in the lab has shown that these subregions play distinct roles in explore-exploit decision making behavior. Furthermore, computational models suggest importance of effective information compression to limit cognitive burden and optimize behavior in explore-exploit decision-making. Parameters to characterize reward-guided learning and decision-making in this type of task include entropy (measure of information content), change in entropy, and reward prediction error. We hypothesized that the UPPS personality dimensions negative urgency and lack of premeditation would be linked with aberrant behavior and DAN neural activity during explore-exploit decision making. Methods: 146 cognitively intact individuals, aged 49-80 years, were recruited from four groups: healthy volunteers and three groups with depression (previously attempted suicide, suicidal ideation but no suicide attempts, and no suicidal ideation or suicide attempts). Individuals completed several cognitive and personality assessments including the UPPS-P impulsive behavior scale. Subscale dimensions of negative urgency (tendency to act rashly under extreme negative emotions) and lack of premeditation (tendency to act without thinking) were used for imaging analyses due to hypothesized association with suicidal behavior. Individuals also completed an explore-exploit “clock task” during an fMRI scan, where action values varied along a continuous interval marked by visuospatial and time cues. Participants were instructed to explore the interval extensively to discover the most rewarding options. We utilized the SCEPTIC selective maintenance reinforcement learning model, previously developed in the lab, to calculate trial-by-trial action values across discretized intervals of time, reward prediction errors, entropy (measure of information content), and change in entropy. We used the Matlab VBA toolbox for model fitting and extracted trial-by-trial parameter values for model-based fMRI analyses. fMRI images were pre-processed and then extracted signal from four DAN subregion ROIs (premotor, caudal posterior parietal cortex, rostral posterior parietal cortex, and MT + ) was deconvolved using a leading hemodynamic deconvolution algorithm to estimate neural activity, which was stimulus locked and averaged across trials. Multilevel modeling implemented in R was used to test for significance between the model-derived behavioral parameters and neural data, with significance thresholding of p < 0.05, FDR corrected. Results: We found a positive effect of UPPS negative urgency on the neural response to increases in the number of potentially useful options in all regions of the DAN (all p < 0.05, FDR corrected) with the greatest effect in the caudal posterior parietal cortex. This effect remained significant in all regions when controlling for age, education, total UPPS, and anxiety diagnosis. Furthermore, we found the negative urgency was also positively correlated with responses to unsigned prediction error (surprise) in the caudal posterior parietal cortex and MT + but not in other DAN regions when controlling for age, education, total UPPS, and anxiety diagnosis. Mood-independent impulsivity measured by UPPS lack of premeditation did not modulate DAN encoding of reinforcement. Conclusions: Mood-dependent impulsivity is associated with exaggerated responses to reinforcement when the best option is harder to identify. This enhanced sensitivity to global uncertainty could represent a neural mechanism underlying aberrant decision-making in the suicidal crisis. Keywords: Explore-exploit Dilemma, Computational Models of Decision-making, Impulsivity, Suicide Disclosure: Nothing to disclose. P281. Dimensional Antidepressant Response to Repetitive Transcranial Magnetic Stimulation Can Be Predicted Using Pretreatment Resting-State Functional Connectivity Benjamin Wade, Tracy Barbour, Kristen Ellard, Joan Camprodon Massachusetts General Hospital, Charlestown, Massachusetts, United States Background: Repetitive Transcranial Magnetic Stimulation (rTMS) is an effective treatment for depression. To date, however, few studies have evaluated whether antidepressant response to rTMS is predictable from pretreatment neuroimaging measures. Depression is, moreover, a symptomatically heterogenous disorder. Previous work from our group has demonstrated that prospective prediction of antidepressant response to other antidepressant treatments (i.e., ketamine and electroconvulsive therapy) is improved by predicting symptom changes along latent dimensions of depression rather than changes in the overall severity of depressive symptoms. Here, we compared the performance of machine learning models using pretreatment patterns of resting-state functional connectivity (RSFC) to predict rTMS-related symptom changes along the total score of the 17-item Hamilton Depression Rating Scale (HDRS), the HDRS-6 subscale, and three previously identified latent dimensions of the HDRS: core mood and anhedonia (CMA), somatic disturbances (SoD), and insomnia. We hypothesized that predictions made for changes in the CMA dimension and the HDRS-6 would be more accurate than those for the HDRS-17 total score. Methods: Patients (n = 26; mean age [SD] = 41.1 [14.1] years; percent male=50%) received rTMS to the left DLPFC. Stimuli were delivered at 120% resting motor threshold at 10 Hz (3000 stimuli per session, 36 sessions total). All patients underwent resting-state fMRI (rs-fMRI) scans within 1 week of their first rTMS session and depressive symptoms were assessed with the HDRS-17 every 2 weeks, including the first and last day of treatment. Pretreatment rs-fMRI scans were parcellated into 200 regions using the Schaefer atlas. For each subject, regional global connectivity values were computed using the graph theoretic node degree, calculated as the sum of pairwise correlations surviving a Fisher Z-transformed correlation threshold (Z ≥ 0.4). Regional global connectivity values were used as predictive features in random forest regression (RFR) models to predict changes along the HDRS-17, HDRS-6, CMA, SoD, and insomnia outcome measures. All RFR models were trained and validated using 10-repeated 10-fold cross validation with a nested grid search for parameter optimization. The performance of models was evaluated based on the coefficient of determination (i.e., the fraction of explained variance) in the out-of-sample test data. The significance of each model’s fit was assessed using permutation tests (B = 1000 permutations) and multiple comparisons were adjusted across the set of all outcome measures using a Bonferroni correction, yielding a critical value of 0.05/5 = 0.01. Results: The mean coefficient of determination (i.e., fraction of explained variance) in the prediction of change in the CMA dimension was significantly above zero (R^2 = 0.19, q = 0.005) and was significantly higher than the HDRS-17 total score (R^2 = -0.07). Elevated global connectivity of a division of the right somatomotor network (SMN), right dorsal prefrontal cortex division of the default mode network (DMN), and right precuneus/posterior cingulate DMN division all predicted poorer symptom reduction while elevated connectivity components of the right dorsal attention network (DATTN) predicted more reduced CMA symptoms following rTMS. Changes along other HDRS subscales were not predicted above chance levels (all q > 0.05). Conclusions: Our findings support that the pretreatment global connectivity of components of the DMN, SMN, and DATTN are predictive of dimensional antidepressant response to rTMS. Previous reported on DMN connectivity as predictive of antidepressant response in other pharmacological and behavioral treatments. Interestingly, changes in core mood symptoms (depressed mood, interests, psychomotor retardation, and weight loss) were predicted more accurately than those for somatic or sleep disturbance symptom clusters. These findings align with related work showing improved prediction of treatment outcomes using latent dimensional outcomes in ketamine and electroconvulsive therapy. Future work will seek to replicate these findings in a larger sample. Keywords: Repetitive Transcranial Magnetic Stimulation, Depression, Resting-state Functional Connectivity, Machine Learning, Symptom Dimensions Disclosure: Nothing to disclose. P282. Deriving Candidate TMS Targets for Bipolar Disorder With Brain Lesions Causing Mania or Depression Joseph Taylor, Summer Frandsen, Amit Anand, Faith Gunning, David Silbersweig, Katherine Burdick, Amy Brodtmann, Maurizio Corbetta, Gonçalo Cotovio, Natalia Egorova, Sophia Gozzi, Jordan Grafman, Andrew Naidech, Albino Oliveira-Maia, Thanh Phan, Joel Voss, Michael Fox, Shan Siddiqi Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Patients with bipolar disorder need better treatments. Transcranial magnetic stimulation (TMS) shows promise, but the optimal brain regions to stimulate are unknown. In this study, we derived a map that is causally implicated in mood valence based on the functional connectivity of brain lesions causing mania versus depression. Methods: We combined seven independent lesion datasets for mania and depression into a single model. First, we estimated the connectivity of each lesion location using a normative connectome (n = 1000). Second, we created a mood valence map from the differential functional connectivity patterns of mania versus depression lesions. Third, we used permutation testing to determine whether the absolute mean voxel value in the mood valence map was stronger than chance (10,000 permutations). Fourth, we assessed specificity of the mood valence map with control lesions not associated with mood disturbance. Finally, we assessed whether the average coordinates of common TMS targets (i.e., bilateral 5 cm, anti-subgenual, dorsomedial) were preferentially connected to the mood valence map. Results: Mania is more likely to be associated with lesions functionally connected to right prefrontal cortex, and depression is more likely to be associated with lesions functionally connected to left prefrontal cortex. This mood valence map was stronger than expected by chance (p < 0.05) and did not change when multiple control lesions were added into the model (spatial correlation r > 0.99). The left anti-subgenual TMS target was preferentially connected to the negative valence map (p < 0.01), and the right dorsomedial prefrontal cortex was preferentially connected to the positive valence map (p < 0.005). Conclusions: Mood valence shows left-right frontal lobe asymmetry, consistent with existing literature. However, the topography of this asymmetry could guide optimization of TMS treatment targets for bipolar depression versus mania. Future analyses will search for a candidate treatment target functionally connected to both mania and depression lesions, which may be relevant for mood stabilization. Keywords: Bipolar Disorder, Brain Networks, Lesion, TMS Targeting, Human Connectome Disclosure: Nothing to disclose. P283. Decrease in Calculated Brain Age Following ECT Michael Henry, Tracy Barbour, Kristen Ellard, James Luccarelli, Isabella Conner, Amanda Yang, Joan Camprodon Massachusetts General Hospital, Boston, Massachusetts, United States Background: The cognitive side effects of electroconvulsive therapy (ECT) have long caused concerns that it causes damage to the brain. Instead, structural imaging studies have found that brain regions that exhibit plasticity show an increase in tissue volume following a course of ECT. Recently, machine learning based algorithms have been developed that use regional brain volumes to estimate the person’s physiologic brain age. We hypothesized that the increase in volume seen following ECT in depressed individuals would be sufficient to decrease the brain age calculated by the BrainAgeR machine learning based algorithm. We report the results of this pilot study below. Methods: Structural magnetic resonance imaging (MRI) scans from 44 subjects with major depressive disorder (MDD) who had undergone imaging as part of other studies on the imaging changes induced by ECT were assessed for inclusion in this analysis. 37 subjects, aged 19-65, 20 males, were found to have completed a pretreatment and post treatment scan suitable for analysis. Depression rating scales obtained prospectively included the Quick Inventory of Depressive Symptomology (QIDS) and the 28-item Hamilton Depression Rating Scale (HAMD-28). Physiologic brain age was estimated for each scan using the BrainAgeR algorithm. Since we had a priori hypothesized that calculated brain age would decrease, the results were analyzed using a one-tailed paired t-test. Given the exploratory nature of this study, the results were not corrected for multiple comparisons. Results: Across participants, there was a significant decrease in estimated physiologic brain age after a course of ECT (mean change = -0.79 years, p = 0.037). When stratified according to clinical response, the within-group change in the responder and non-responder groups did not reach significance. When stratified by remitters versus non-remitters, for both the QIDS and the HAMD-28, the non-remitter group showed a significant decrease in calculated age (QIDS: mean change = -0.99 years, p = 0.018; HAMD-28: mean change = -0.95 years, p = 0.033), while the remitters did not. Conclusions: These data suggest that the BrainAgeR algorithm for calculating physiologic brain age is sufficiently sensitive to detect the effects of an acute treatment for depression. Also, consistent with our hypothesis, the mean calculated physiologic age decreased following ECT in a cohort of depressed subjects. When analyzed according to clinical response, the subjects who did not remit showed a statistically significant decrease in calculated age while the remitters did not. Given the small sample size and the preliminary nature of these findings, replication in a larger study sample is indicated. Keywords: Brain Age, Electroconvulsive Therapy, MRI Disclosure: Roche Pharmaceuticals: Employee (Spouse) P284. Depression as a Disease of White Matter Network Disruption: Characterizing the Relationship Between White Matter Lesions and Depression in Patients With Multiple Sclerosis Erica Baller, Matthew Cieslak, Timothy Robert-Fitzgerald, Sydney Covitz, Melissa Martin, Matthew Schindler, Amit Bar-Or, Ameena Elahi, Michael Fox, Abigail Manning, Clyde Markowitz, Nebojsa Mirkovic, Christopher Perrone, Victoria Rautman, Donovan Reid, Guy Schultz, Shan Siddiqi, Sunil Thomas, John Detre, Russell Shinohara, Theodore Satterthwaite University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Multiple sclerosis (MS) is an immune-mediated neurological disorder that affects one million people in the United States, with up to 50% of patients experiencing a lifetime depression. However, mechanisms of depression in MS remain under-investigated. Previous research using lesion network mapping has demonstrated that strokes in gray matter associated with depression disrupt a reproducible depression network. However, such methods have not been used to investigate how white matter lesions (WMLs) relate to depression in MS. This study aims to define how depression in adults with MS is associated with white matter lesion (WML) location and burden in a retrospective sample. Methods: Participants with MS were identified from the electronic medical record. The depressed individuals (DI) included persons with evidence of depression as indicated by a ICD-10 depression diagnosis (F32-F34.), a prescription for antidepressant medication, or screening positive via PHQ2/9 (n = 232, age (SD) = 49 (12), % females = 86). The age- and sex-matched non-depressed comparators (NDC) included persons with no prior depression diagnosis, psychiatric medications, and were asymptomatic on PHQ2/9 (n = 148, age (SD) = 47 (13); % females=79). Structural MRI was obtained as part of routine care at 3 T using a research-quality protocol. WMLs were automatically segmented using the algorithm Method for Inter-Modal Segmentation Analysis and projected onto a standard template. Seventy-seven white matter tracts (WMT) were evaluated. The volume of WMTs intersecting each lesion was computed via streamline filtering in DSI Studio. Total volume of lesions irrespective of tract was also calculated and compared between diagnostic groups. Age and diagnostic effects were assessed with general linear models and T-tests. Enrichment of effects in a previously described depression network by Siddiqi et al., 2021, was also evaluated. Multiple comparisons were controlled using the false discovery rate (Q < 0.05). Results: Streamline filtering recapitulated previously known patterns of MS disease, with high proportions of streamlines impacted in the optic radiations, inferior fronto-occipital fasciculi, medial longitudinal fasciculi, and corticopontine tracts. Greater age was associated with higher disease burden in 54/87 WMTs (PFDR < 0.05). Total lesion volume was not significantly different between the two groups (P = 0.07, NS). However, when using streamline filtering, DIs had a higher mean disease burden across all WMTs (P < 0.05, Cohen’s d = 0.17), which was driven by a greater burden of disease within the depression network. Conclusions: We present a novel approach for calculating the relationship of WML to depression disease burden. We demonstrate that overall lesion burden, irrespective of location, does not differ between diagnostic groups. Rather, when looking at disease burden in streamlines that intersect with the lesions, we show that DIs have greater overall disease burden as compared NDCs, which is driven by a higher burden of disease in fibers that connect areas of the depression network. Future work using white matter lesion network mapping in MS could also advance our understanding of the mechanisms of depression more broadly. Keywords: Depression, Multiple Sclerosis, Electronic Medical Record, Brain MRI Disclosure: Nothing to disclose. P285. Tianeptine Improves Anticipation for Reward in a Mouse Model of Early Developmental Exposure to Fluoxetine Emily Cambre, Elena Christenfeld, Gabriella Sahyoun, Jonathan Javitch, Sarah Canetta* Columbia University, New York, New York, United States Background: Deficits in reward processing, commonly referred to as anhedonia, are a hallmark of multiple psychiatric disorders. In the context of depression, anhedonia is one of two core symptoms needed for diagnosis, and often predicts more severe outcomes. Although selective serotonin reuptake inhibitors (SSRIs) are the frontline treatment for depression, they are ineffective for many individuals, and in certain cases can even exacerbate symptoms including anhedonia. Therefore, there is a clear gap in our understanding of how antidepressants affect reward processing, and why they may be variably effective across individuals. Methods: Male and female pups on a C57BL/6 J (C57) background were injected with either FLX (10 mg/kg, i.p) or saline from postnatal (PN) day 2 to 11. Motivation and hedonic perception were measured in adult PN FLX or vehicle (PN VEH) exposed mice using a progressive ratio (PR) and lickometer task, respectively. During PR testing, mice were food restricted to 90% of their baseline weight and during lickometer testing, mice were maintained on ad libitum citric acid water with food removed 12 hours prior to testing. Cages with co-housed PN FLX and PN VEH mice were subsequently randomized to receive either chronic FLX or vehicle control (water) as adults. FLX (18 mg/kg) was administered through the drinking water for three weeks and then continuously throughout behavioral testing. A two-week washout period followed completion of post-FLX behavioral testing before beginning TIA administration. Mice that had previously received FLX, received TIA and the mice receiving control remained the same. A 30 mg/kg solution of TIA NaCl dissolved in 0.9% sterile saline, or saline as a control, was injected intraperitoneally twice per day for 14 days. TIA injections continued throughout behavioral testing. Results: We found that as adults, PN FLX animals show decreased motivation to pursue rewards, manifest as a decrease in total presses (Mann-Whitney, p = 0.0017, n = 31 PN VEH, 29 PN FLX), session time (t-test, p = 0.0011, n = 31 PN VEH, 29 PN FLX), break point (Mann-Whitney, p = 0.0018, n = 31 PN VEH, 29 PN FLX) and rewards retrieved (t-test, p = 0.0475, n = 31 PN VEH, 29 PN FLX). These mice also show decreased latency to approach the lever and begin pressing (Mann-Whitney, p = 0.0005, n = 31 PN VEH, 29 PN FLX), which may reflect decreased reward anticipation. Strikingly, in the lickometer test, there were no differences in the number of times the PN FLX and PN VEH mice licked the freely available reward (Mann-Whitney, p = 0.9796, n = 31 PN VEH, 29 PN FLX) suggesting no change in hedonic perception. Interestingly, we found that adult FLX administration did not alter any of these variables in PN FLX mice. By contrast, adult TIA administration increased reward anticipation (Mann-Whitney, p < 0.0001, n = 14 PN FLX-SAL, 15 PN FLX-TIA) but did not consistently alter variables associated with motivation including total presses (Mann-Whitney, p = 0.1456, n = 14 PN FLX-SAL, 15 PN FLX-TIA), session time (t-test, p = 0.8056, n = 14 PN FLX-SAL, 15 PN FLX-TIA), break point (Mann-Whitney, p = 0.1486, n = 14 PN FLX-SAL, 15 PN FLX-TIA) and rewards retrieved (t-test, p = 0.8641, n = 14 PN FLX-SAL, 15 PN FLX-TIA). Conclusions: Our results confirm prior work demonstrating that early developmental exposure to FLX in mice, during a period of brain development occurring during the third trimester in a human, produces adult animals that show decreased motivation in adulthood. We expand on this phenotype to show these effects also include reward anticipation, while reward learning and hedonic perception remains intact. Our results also demonstrate that these deficits in motivation and reward anticipation in PN FLX animals are resistant to subsequent FLX administration in adulthood. By contrast, the atypical antidepressant TIA improves reward anticipation, although motivation remains unaltered. Overall, these findings suggest that PN FLX mice may represent a model of SSRI-resistant affective behavioral changes and that TIA may be a promising alternative treatment from SSRIs for humans with suspected in utero early developmental exposure to SSRIs, and more broadly in a subset of people with SSRI-resistant depression. Keywords: Reward Processing, SSRI, Tianeptine Disclosure: Nothing to disclose. P286. Early and Late Predictors of Depression Treatment Response in the Clinic: An Observational Study Kathryn Ridout, Mubarika Alavi, Samuel Ridout, Constance Weisner, Esti Iturralde The Permanente Medical Group, Santa Rosa, California, United States Background: Major depressive disorder (MDD) is a chronic, recurrent illness impacting 20.6%1 of the U.S. population, causing significant disability, and costing $326.2 billion annually. Patient risk for subsequent depressive episodes increases from 50% to 90% after the first compared to the third episode. Incomplete depressive episode treatment response is the strongest predictor of depression relapse and reoccurrence and increases the likelihood of work productivity loss and disability. As such, improving treatment response during a patient’s first clinical presentation for a depressive episode would have the strongest impact on decreasing depressive disorder prevalence, chronicity, and associated disability. Evidence suggests that MDD treatment response is improved with early detection, intervention, and appropriate treatment. However, treatment response varies between patients, and, to date, there are no tools to predict which patients will respond to treatment. This study aimed to examine clinical predictors of depression treatment response early and later in the course of treatment. Methods: Retrospective, observational study in a large, integrated health system. Participant inclusion criteria include age ≥ 18-years-old, a Patient Health Questionnaire-9 score of ≥ 5, and seeking depression outpatient care between 3/2020-12/2021. Exclusion criteria include acute suicide risk, diagnoses of bipolar, psychotic, dementia, or active substance use disorders, current hospice, or home-based palliative care, residing in a skilled nursing or assisted living facility, or non-health plan member. Study variables were extracted from the electronic health record at 6 weeks and 6 months after starting depression treatment. Predictors of depression treatment response were chosen based on prior literature. Based on the previous literature and our sample size, we estimated power to be able to detect small effect sizes (f2 of 0.02). Results: Of the 27,858 patients, 68% were female, 48% were White, 19% were LatinX, 14% were Asian, 8% Black, and 11% other race. The majority of patients were age 18-39 (61%), followed by 40-59 (27%), 60-69 (8%), then 70 or older (4%); neighborhood deprivation index was equally distributed across categories (23% to 26%). Most patients had a Charleson comorbidity index of 0 (84%) and a body mass index of ≤24.9 (22%). The mean baseline PHQ-9 was 10.7 ± 3.9; the average wait time to first treatment appointment was 2.4 ± 5.2 days. Most patients have no history of outpatient mental health treatment (63%) or antidepressant therapy (79%) in the year prior. EHR predictors significant at both timepoints included baseline PHQ-9 score; previous psychiatry referral; patient outreach to psychiatry; outpatient psychiatric encounter for condition other than depression; previous antidepressants for disorders other than depression; gender; age; and BMI (P < .0001 for all). EHR predictors that differed by timepoint included race (lower treatment response at 6 months for LatinX populations; p < .0001); Charlson comorbidity index (lower response at 6 months with higher CCI; p < .0001); neighborhood deprivation index (lower response at 6 weeks for higher deprivation; p < .0001); and antidepressant start during depression treatment (higher response at 6 months with antidepressant; p < .0001). Conclusions: While many clinical variables predicted depression treatment outcomes both early and later in the course of treatment, some variables appeared to differentiate treatment response at each time point. This study provides the foundation for future work examining utilization of treatment course predictors in prospective treatment decision making. Keywords: Treatment Outcome Prediction, Personalized Medicine, Depression Treatment Response Disclosure: Nothing to disclose. P287. Melancholic Versus Non-Melancholic Features in Bipolar I Disorder Jake Stenzel, Tim Bigdeli, Michele Pato, Carlos Pato, Ayman Fanous University of Arizona College of Medicine-Phoenix, Phoenix, Arizona, United States Background: Melancholic features, as a specifier for Major Depressive Episode in both major depressive disorder and bipolar I disorder (BD-I), is characterized by depressive episodes with marked psychomotor disturbances, anhedonia, weight loss, and feelings of guilt as defined in the Diagnostic and Statistical Manual of Mental Disorders, 5th ed. (DSM-V) (American Psychiatric Association, 2013). While the validity of melancholia as a diagnostic concept has been widely debated, there is evidence to suggest that melancholia exhibits differences in pathophysiology and severity of disorder. Such distinctions include increase hypothalamic-pituitary-adrenal axis dysregulation, pro-inflammatory cytokines IL-5 and IL-4, suicide risk, and psychotic features. While the majority of investigations have focused on melancholia in the context of depression, our study investigated melancholic features amongst a patient population with BD-I from the Genomic Psychiatry Cohort (GPC) (Pato et al., 2013). The aims our study were to identify the prevalence of melancholic features in a large population of patients with BD-I (n = 4025) and compare clinical features and socio-demographic characteristics between melancholic and non-melancholic BD-I patients. Methods: The GPC has gathered a large cohort of patients with schizophrenia, bipolar disorder, and healthy controls from across the United States and select sites abroad. All enrolled participants were asked to complete a screening questionnaire to assess their psychiatric history. Participants enrolled as probable cases were interviewed by mental health professionals using the Diagnostic Interview for Psychosis and Affective Disorders (DI-PAD). Information on ascertainment and diagnosis of the study population are provided in a previous publication (Bigdeli et al., 2020). Diagnosis of psychiatric disorders was obtained from the DI-PAD. Patients were included in this study if they completed all necessary DI-PAD and screener material needed for each analysis and had a diagnosis of bipolar I disorder (with or without psychosis). DSM-V criteria for melancholic features were assessed using the DI-PAD and screening questionnaire. Socio-demographic factors and severity of illness were derived from the DI-PAD and screening questionnaire. Results: Participants with a diagnosis of BD-I who completed the DI-PAD and screener questionnaire for all measured variables resulted in a N of 4025). Those fulfilling criteria for melancholia comprised 74.04% (n = 2980). Compared to non-melancholic BD-I, melancholic BD-I was more prevalent in females (77.05% vs 70.52%, OR = 1.40; p < 0.0001). Having a family history of psychiatric disorders was associated with melancholic BD-I as compared to non-melancholic BD-I (67.15% vs 61.05%, OR = 1.30; p < 0.001). In addition, melancholic BD-I was significantly associated with having suicidal ideation lasting at least one week (79.73% vs 60.86%, OR = 2.55; p < 0.0001) and suicidal ideation lasting greater than one month (69.62% vs 23.12%, OR = 7.62; p < 0.0001) when compared to non-melancholic BD-I. No significant association was found with the presence of psychosis, nicotine use (defined by having smoked over 100 cigarettes in their lifetime), or history of recreational drug use. Conclusions: When compared to non-melancholic BD-I, patients with melancholic BD-I were more likely to be female and to have a family history of psychiatric disorders. Melancholic BD-I patients were also found to have a marked increase in periods of suicidal ideation lasting greater than one week and greater than one month. This observation is in accordance with previous studies on melancholic features in major depression (Dold et al., 2021; Tondo et al., 2020). Our study relied on DSM-V criteria for determination of melancholic features, whereas alternative instruments may have resulted in varied prevalence. Additional exploration of comorbidities and both GWAS and NGS data are planned and may provide further insight into melancholic features in BD-I. Keywords: Bipolar I Disorder, Psychiatric Disorders, DSM-5, Clinical Psychiatry Disclosure: Nothing to disclose. P288. Single Nucleus RNA Sequencing of Mouse Ventral Tegmental Area Neurons Following Input-Specific Stimulation Reveals Differential Activation Patterns Rhiana Simon, Joshua Yee, Koichi Hashikawa, Garret Stuber, Larry Zweifel, Marta Soden* University of Washington, Seattle, Washington, United States Background: The ventral tegmental area (VTA) is home to a heterogeneous population of dopamine neurons as well as significant populations of glutamatergic and GABAergic cells. The VTA receives inputs from over two dozen brain regions. We found previously that specific inputs differentially innervate VTA subregions, and stimulation of select inputs leads to varying behavioral outcomes and distinct spatial patterns of cellular activation, as measured by Fos protein expression. This implies that different subsets of VTA neurons are activated by distinct inputs to drive different behavioral modalities, but the specific genetic identity of these activated neurons is not known. Methods: To address this question, we used Cre-dependent viral expression of channelrhodopsin (ChR2) to stimulate three distinct VTA inputs in the mouse: GABAergic inputs from the lateral hypothalamus (LH), GABAergic inputs from the nucleus accumbens (NAc), and glutamatergic inputs from the prefrontal cortex (PFC). Male and female Vgat-Cre (Slc32a1) or Vglut1-Cre (Slc17a7) mice were injected with AAV1-FLEX-ChR2-YFP (or AAV1-FLEX-YFP as a control) in one of the indicated regions and fiber optics were implanted above the VTA. Following 20 Hz blue light stimulation we collected tissue and performed single-nucleus RNA sequencing (snRNAseq). We integrated the sequencing data from all four groups to generate a comprehensive analysis of VTA cell types based on differential gene expression and examined expression of immediate early genes (IEGs) to determine which cell clusters were preferentially activated by stimulation of each input. Spatial expression of a selection of marker genes was validated using multi-plex in situ hybridization. Results: From approximately 40,000 cells sequenced we identified over 9,000 neurons with high quality gene expression data. These neurons segregated into 3 clusters of primarily dopaminergic cells, identified by expression of canonical markers such as Th, Slc6a3 (DAT), and Ddc. We also identified multiple clusters of primarily GABAergic cells, clusters of primarily glutamatergic cells, and one major cluster that expressed a mix of GABAergic, dopaminergic, and glutamatergic markers. These clusters showed differential expression patterns of critical genes important for determining cellular physiology and connectivity, including ion channels and neurotransmitter and neuropeptide receptors. We confirmed spatially distinct expression patterns of a selection of marker genes using multi-plex in situ hybridization and found that the three dopaminergic clusters tended to segregate along the lateral to medial axis, consistent with established differences in function between known dopaminergic subpopulations. Stimulation of LH-GABA, NAc-GABA, or PFC-glutamate inputs all led to increased IEG levels compared to control mice. Analysis of the percentage of cells in each cluster that showed IEG expression revealed unique patterns of cluster activation for each stimulation group compared to control (Fisher’s exact test, P < 0.05). Notably, the patterns of dopamine cell cluster activation were consistent with the spatial innervation patterns of each input (i.e., only LH-GABA inputs, which innervate the lateral VTA, induced significant activation in the most lateral dopamine cluster). We also observed variability in IEG expression in VTA glutamatergic clusters, with NAc-GABA inputs inducing the most activation. Conclusions: This data set represents a large and comprehensive single-nuclear sequencing analysis of the mouse VTA. Analysis of differentially expressed genes between identified cell clusters provides valuable information regarding the organization of this critical brain region and the cellular properties and functions of specific neuronal subsets. We also provide proof-of-concept of a novel approach to analyzing activation of cell clusters following optogenetic stimulation and demonstrate differential activation of VTA cell types by distinct inputs. These findings advance our understanding of the circuit architecture governing inputs to the midbrain dopamine system and the regulation of dopamine-dependent behaviors. Keywords: Dopamine, Ventral Tegmental Area (VTA), RNAseq Disclosure: Nothing to disclose. P289. Analysis of Mitochondrial DNA Variants and Their Association With Brain Structural Measures in Bipolar Disorder Ana Mendes-Silva, Suyi Shao, Lucas Taniguti, Mikaela Dimick, Clement Zai, James Kennedy, Benjamin Goldstein, Vanessa Gonçalves Centre for Addiction and Mental Health, Toronto, Canada Background: Mitochondria are the main energy source for neurons and other brain cells and play crucial roles in various neural processes, such as neurogenesis, neuroplasticity, and neurotransmission. The dysfunction in those organelles may alter critical neuronal processes underlying abnormal brain development and cognitive impairment in psychosis because of a lack of energy and higher levels of inflammatory molecules. Several clinical, genetic, and neuroimaging studies implicate mitochondrial dysfunction to play a crucial role in bipolar disorder (BD) pathophysiology. The present study aimed to characterize the mtDNA variants in youth diagnosed with BD and investigate the association between two common mitochondrial DNA (mtDNA) variants with structural brain changes. Methods: We included 96 European-Caucasian adolescents (54 BD and 42 healthy controls (HC)), within the age range 13-20, from both sexes. Psychiatric diagnoses were determined based on semi-structured diagnostic interviews. Mitochondrial DNA was extracted from saliva and the MiSeq platform was used to sequence the samples. mtDNA variants were identified using the mtDNA-Server pipeline and common variants were selected with a minor allele frequency (MAF) of at least 5%. Functional analysis was performed by Mutserve to identify mtDNA variants potentially harmful. The MutPred, Selection Score and Mito tool algorithms assigned for each potentially harmful variant identified by functional analysis were summed to build a pathogenicity score. Further, the pathogenicity score and individual variants were tested with the phenotypes using logistic regression in R. Subcortical volumes, cortical thickness and cortical surface area were determined for a-priori determined regions of interest (ROIs) (anterior cingulate cortex (ACC) and amygdala) and associations with each mtDNA variants and pathogenic score were tested using general linear model in SPSS, adjusting for age, sex, and intracranial volume (ICV) when needed. To account for multiple tests, a Bonferroni-corrected significance threshold of 0.0125 was applied. Results: A total of 1382 homoplasmic variants were identified in our sample, of which 67 were common variants (MAF > 0.05). Functional analysis revealed that 9 of the 67 common variants were non-synonymous, and the variants (MT-ND2):m.4917 A > G, and (MT-ND1):m.4216 T > C were classified as potentially harmful. Logistic regression analysis showed the association of mtDNA pathogenicity score and 4216 T > C variant with BD (OR: 1.57 [95% Cl: 1.04, 2.38], p = 0.031; and OR: 1.99 [95% Cl: 1.04, 3.81], p = 0.037, respectively). Regression analysis, adjusted for sex and age, showed that both pathogenicity score and the individual variant (MT-ND1):m.4216 T > C were nominally associated with amygdala volume in the whole cohort (ß43.07, p0.037, and ß82.78, p0.024, respectively). No effect of pathogenicity score or individual variants were found for cortical thickness, surface area or volume in ACC. None of these associations were significant after adjustment for multiple testing. Conclusions: Our findings indicate that youth with BD have a greater presence of the two reported pathogenic complex I variants m.4917 A > G and m.4216 T > C. Moreover, higher pathogenicity score and the variant m.4216 T > C were associated with greater amygdala volume, which could reflect neuroinflammation caused by mitochondrial dysfunction. Further studies in other populations are required to support these findings. Keywords: Mitochondria, Mitochondrial DNA, Bipolar Disorder, Youth, Amygdala Disclosure: Nothing to disclose. P290. RNA Sequencing of the Limbic System in Major Depressive Disorder Fernando Goes, Leonardo Collado-Torres, Peter Zandi, Joel Kleinman, Daniel R. Weinberger, Ran Tao, Daniel Weinberger, Thomas Hyde Johns Hopkins School of Medicine, Baltimore, Maryland, United States Background: Major Depressive Disorder (MDD) is the second leading cause of disability worldwide and a significant risk factor for suicide. Novel treatments are urgently needed, yet the development of new types of antidepressants has been hindered by limited understanding of the pathophysiology of MDD. In the current study, we perform the largest and most comprehensive molecular study of MDD in post-mortem brain samples Methods: We have performed expression profiling on a large cohort of post-mortem brains with MDD and healthy controls. RNA sequencing using a ribozero protocol (median depth 132 million reads) was performed comprising a total of 432 males and female samples from the Anterior Cingulate Cortex (ACC) and 429 samples from the Amygdala. Differential expression analyses were conducted using limma voom, while accounting for typical post-mortem confounds, including a measure of differential susceptibility to degradation. Control for multiple testing was performed using a false-discovery rate of 5%. Results: A total of 630 and 106 genes were differentially expressed in the ACC and Amygdala respectively. The mean fold change was modest (OR ~ 1.1) and the most highly differentially expressed gene in both the ACC and Amygdala was FUS, an RNA-binding protein previously associated with neurodegeneration. Moreover, using a transcriptional-wide association study approach in the Anterior Cingulate and Amygdala, we have found 88 genes meeting Bonferroni levels of significance and 309 genes meeting an FDR < 5% level of significance. Among the Bonferroni corrected genes are several previously associated at GWAS significance levels (for example, SORCS3, ZKSCAN7, LIN28B, RMT61A, RANGAP1, FADS1, TMEM258, FNIP2, NRG1, among many others) as well as numerous novel genes, such as SIRT1, which was identified in a GWAS of severe MDD in China and now finds convergence evidence for association in a European Ancestry sample Conclusions: Major Depressive Disorder is associated with several hundred differentially expressed genes, consistent with a large polygenic contribution across molecular features. Defining the molecular landscape of gene expression in brain regions associated with the pathophysiology of MDD may lead to the identification of novel therapeutic targets, particularly in conjunction with risk loci identified in large-scale genetic studies Keywords: Transcriptome, Postmortem Human Brain Tissue, Major Depressive Disorder (MDD), Anterior Cingulate Cortex (ACC), Amygdala Disclosure: Nothing to disclose. P291. Immunometabolic Gene Pathways are Associated With Anhedonia and Altered Following a TNF-Alpha Antagonist in Patients With Depression and High Inflammation Mandakh Bekhbat, Ebrahim Haroon, Sarah Etuk, Jennifer Felger, Andrew Miller, Michael Treadway Emory University, Atlanta, Georgia, United States Background: Inflammation and altered glucose metabolism are two pathways implicated in the pathophysiology of anhedonia in major depressive disorder (MDD). These pathways are hypothesized to synergize within circulating immune cells, whereby inflammatory activation can shift metabolic demand and reprogram cellular energy sources to fuel pro-inflammatory activities, including systemic inflammation and its effects on the brain and reward processing to contribute to symptoms of anhedonia. We have previously shown relationships between symptoms of anhedonia and a whole-blood gene expression pattern consistent with increased reliance on glycolysis (as opposed to oxidative phosphorylation [OXPHOS]), a hallmark of metabolic change in activated cells, but only among MDD patients with high inflammation (plasma C-reactive protein [CRP] > 3 mg/L). We have also reported associations between combined plasma inflammatory and glucose metabolism biomarkers and reduced willingness to expend effort, a key index of motivational anhedonia, in MDD patients with CRP > 3 mg/L undergoing anti-inflammatory challenge. These findings suggest that immunometabolic shifts may contribute to, and serve as intervention targets for, anhedonia symptoms in depressed patients with high inflammation. The tumor necrosis factor (TNF)-alpha antagonist infliximab was previously found to improve symptoms of anhedonia, including effort-based motivation, selectively among MDD patients with higher inflammation. Here, we analyzed microarray data form a cohort of MDD patients with high inflammation before and after a single anti-inflammatory challenge with infliximab versus placebo in order to discover novel immunometabolic signatures for infliximab’s effects on anhedonia. Methods: N = 42 medically stable, medication-free depressed patients aged 21-65 with high inflammation (CRP > 3 mg/L) were studied before and two weeks after a single infusion of infliximab (5 mg/kg body weight) or placebo. Anhedonia was assessed using the subscale from the Inventory of Depressive Symptoms-Self Report (IDS-SR) and whole blood gene expression was profiled via the Clariom S (ThermoFisher) gene array platform at baseline and Week 2 post-infusion. Microarray data were SST-RMA-normalized and batch-corrected. Differential expression analysis was conducted using linear models for microarray data (limma) to assess treatment x time interaction effects while controlling for clinical covariates (age, sex, and race). Functional pathway enrichment was queried using the Kyoto Encyclopedia of Genes and Genomes (KEGG), and WikiPathways databases. Results: At baseline, anhedonia severity was negatively associated with 117 gene probes (R < -0.30, p < 0.01) which were enriched for the Mitochondrial Complex I of the OXPHOS system pathway (p < 0.05, q < 0.1). Among those who received infliximab, patients who responded with >50% reduction of anhedonia had greater baseline expression of 82 gene probes which enriched IL-17 and NF-kappa B signaling pathways (p < 0.05, q < 0.1) compared to non-responders. Longitudinal gene expression and behavioral data were available from fifteen infliximab- and fourteen placebo-treated patients. At two weeks following a single infusion of infliximab, there was a significant decrease in the expression of 44 gene probes that enriched PI3K/AKT/mTOR as well as Glycolysis and gluconeogenesis pathways (p < 0.05, q < 0.1). In contrast, only 36 genes were significantly changed following placebo, which enriched broad, non-specific pathways. Conclusions: Our results indicate that anhedonia in MDD patients with high inflammation is associated with altered energy metabolism pathways, including lower OXPHOS at baseline, which may reflect a pro-glycolytic shift characteristic of activated immune cells. In addition, increased expression of inflammatory pathways at baseline predicted reduced anhedonia scores following infliximab, consistent with our previous findings. Finally, the effects of infliximab involved reductions in glycolysis and upstream mTOR signaling pathways. These findings highlight the metabolic reprograming in circulating immune cells that are associated with systemic inflammation in MDD, which may lead to identification of new metabolic and anti-inflammatory targets for treatment of anhedonia and/or MDD with increased inflammation. Keywords: Immune Modulation, Immunometabolism, Anhedonia, Precision Medicine for Depression, Microarray Disclosure: Nothing to disclose. P292. Pharmacogenomic Testing and Symptom Remission in Mood Disorders: Systematic Review and Meta-Analysis Lisa Brown, Joseph Stanton, Kanika Barthi, Abdullah Al Maruf, Daniel Mueller, Chad Bousman Great Scott! Consulting, Brooklyn, New York, United States Background: Pharmacogenomic (PGx) testing has emerged as a compelling strategy that clinicians can use to inform medication selection and dosing, but the clinical efficacy of this strategy has been questioned. Here, we sought to systematically review and meta-analyze prospective, controlled clinical trials for an association between PGx testing and depressive symptom remission in patients with major depressive disorder (MDD). Methods: We reviewed PubMed and the bibliographies of systematic reviews published up to July 12, 2022. We included prospective, controlled clinical trials examining the association between PGx testing and depressive symptom remission in adults and were available in English. The study adhered to the 2020 PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) reporting guidelines and was registered in PROSPERO (CRD314807). Extracted information included trial publication year, trial design, sample size, trial eligibility criteria, trial duration, participant’s characteristics (i.e., age, gender, ancestry), remission measure used, and description of the genes included in the PGx test performed. Data was extracted by two independent reviewers and a third reviewer determined discrepancies. Each trial was assessed for risk of bias and a random-effects model was used to estimate pooled risk ratios. Depressive symptom remission was defined as a Hamilton Depression Rating Scale-17 score ≤ 7, Clinical Global Impression scale ≤ 2, or Patient Health Questionnaire ≤ 5. Results: Thirteen trials including 4767 patients were analyzed, including 10 randomized controlled trials (RCTs) and three open label trials. Across all included trials, those that received PGx-guided antidepressant therapy (n = 2395) were 1.41 (95% CI: 1.15 - 1.74, p = 0.001) more likely to achieve remission compared to those that received unguided antidepressant therapy (n = 2372). Pooled risk ratios for randomized controlled trials and open label trials were 1.46 (95% CI: 1.13 - 1.88) and 1.26 (95% CI: 0.84-1.88), respectively Conclusions: PGx testing is associated with a modest but significant increase in depressive symptom remission in adults with MDD. Heterogeneity in PGx test composition and accompanying prescribing recommendations across trials are likely contributing to uncertainty about the efficacy of PGx testing in the literature. Keywords: Pharmacogenomics, Depression, Pharmacogenetics Disclosure: Great Scott Consulting: Founder (Self), Tempus Labs: Employee (Self), Myriad Genetics: Stock / Equity (Self) P293. Whole and Single Cell Human Cortical Spheroid Transcriptomics in Response to Low-Dose Ketamine Mark Niciu, Natalia Schmidt, William Guiler, Amanda Decker, Brian Kinnaird, Kornel Schuebel, Carlos Zarate, David Goldman University of Iowa Health Care, Iowa City, Iowa, United States Background: Major depressive disorder (MDD) has a ~16% global lifetime prevalence and is associated with extensive morbidity and mortality including suicide. Standard first-line antidepressant medications are effective for some but not all MDD patients, as exemplified by numerous real-world effectiveness trials including the Sequenced Treatment Alternatives to Relieve Depression (STARD) and the International Study to Predict Optimized Treatment for Depression (iSPOT-D). As also exemplified in these trials, first- and second-line antidepressant medications have monoaminergic mechanisms of action, so it is not surprising that MDD patients who fail a first trial are at greater risk of failing subsequent medication trials of medications due to their pharmacodynamic similarities. On the other hand, the N-methyl-D-aspartate receptor antagonist and glutamate modulator ketamine at subanesthetic doses, has rapid-acting antidepressant and anti-suicidal efficacy in such patients. The mechanisms of action of ketamine, e.g., molecular, and cellular response, have been studied in the brains of preclinical model organisms and peripheral human tissue but have yet to be reported in human central nervous system-like tissue. In this study, we report whole and single cell transcriptomic data from human stem-cell derived cortex-like spheroid/organoid cultures exposed to low-dose ketamine and its bioactive metabolites in vitro. Methods: For stem cell lines created in the laboratory, whole blood samples were collected from subjects participating in clinical studies at the National Institutes of Health Experimental Therapeutics and Pathophysiology Branch. Peripheral blood mononuclear cells (PBMCs) were then isolated and reprogrammed into human induced pluripotent stem cells (hiPSCs) via Sendai virus-mediated transient transfection of plasmids containing Yamanaka factors, e.g., Klf4, c-Myc, Oct-3/4, and Sox2. Other validated stem cell lines, e.g., PENN025i-71-58 and STAN062i-164-2, were purchased from WiCell (Madison, WI, U.S.A.). Human pluripotent stem cells (hPSCs) were then differentiated into human cortical spheroids (hCSs), closely adhering to the methodologies pioneered by Sergiu Pasca’s laboratory (Stanford University, Palo Alto, CA, U.S.A.). After reaching maturity (>40 days in vitro), hCSs were exposed to low-dose ketamine and other bioactive ketamine metabolites, e.g., 2 R,6R-hydroxynorketamine (HNK). Briefly, we followed the next-generation Ion Torrent RNA Sequencing Pipeline (ThermoFisher Scientific, Waltham, MA, U.S.A.) and the 10x Genomics Chromium Single-Cell System (10x Genomics, Pleasanton, CA, U.S.A.) for the whole and single cell RNA-Seq analyses, respectively. Results: For the whole cell transcriptomics, no genes were ≥1.5x up-regulated by brief (1 hour) exposure to low-dose (10 mcM) racemic ketamine, relative to vehicle control. 10 genes were ≥1.5x down-regulated (p < 0.05) but did not survive False Discovery Rate (FDR) correction. In response to (2 R,6 R)-hydroxynorketamine (1 hour, 5 mcM), 13 genes were ≥1.5x up-regulated with FDR corrected p ≤ 0.05, and 4 genes were ≥1.5x down-regulated with FDR-corrected p ≤ 0.05. For the up-regulated genes, pathway analyses revealed 3 clusters: axon guidance (microtubule-mediated), transcriptional regulation (histones), and translational regulation (ribosomal proteins). In subsequent experiments, hCSs were briefly exposed to multiple low doses of racemic ketamine. Single cell RNA-Seq was performed in collaboration with the Iowa Institute of Human Genetics Genomics Division. At the time of abstract submission, this data is being processed with anticipated presentation for the first time at the 2022 American College of Neuropsychopharmacology’s Annual Meeting (Phoenix, AZ, U.S.A.) Conclusions: We have identified several genes/clusters altered by low-dose ketamine and/or its bioactive metabolites in hPSC-derived cortical-like organoid cultures. In the whole cell transcriptomic analyses, 2 R,6R-hydroxynorketamine, which is hypothesized to have non-NMDA receptor antagonist properties [Zanos et al. (2016) Nature 533(7604):481-6, PMID: 27144355], robustly upregulated several genes involved in cytoskeletal reorganization, e.g. stathmin-like 2 (STMN2), beta-2B tubulin and microtubule-associated proteins 1B and 2. Due to the known effects of low-dose ketamine on synaptic plasticity, it appears logical that cytoskeletal genes appear to be rapidly up-regulated. One major future direction is to determine if these such transcriptional alterations correlate with antidepressant efficacy. This may be assessed by stratifying at “efficacy bookends,” e.g., hCSs derived from ketamine non-responders and ketamine remitters, in response to brief, low-dose incubation with racemic ketamine and/or its bioactive metabolites. Keywords: Transcriptomics, Ketamine, (2 R,6 R)-Hydroxynorketamine, Stem Cells, Brain Organoids Disclosure: Johnson and Johnson-Janssen: Contracted Research (Self) P294. Neurodevelopmental Signature of a Transcriptome-Based Polygenic Risk Score for Depression Yuliya Nikolova, Amy Miles, Fernanda Dos Santos Centre for Addiction and Mental Health, Toronto, Canada Background: Transcriptomic studies in post-mortem human brain tissue have begun to shed light onto the molecular mechanisms implicated in Major Depressive Disorder (MDD). Yet, it remains unclear how these transcriptomic shifts may impact brain structure or function in vivo. We recently leveraged common cis-eQTL single nucleotide polymorphisms (SNPs) to create a transcriptome-based polygenic risk score (tPRS) reflecting developmental shifts towards depression-like corticolimbic gene expression patterns, based on 76 genes differentially expressed in MDD. We identified distinct sex-specific neurofunctional and neurostructural signatures associated with higher tPRS and MDD risk in young adults, independent of the effects of traditional MDD PRS emerging from genome-wide association studies (GWAS). To elucidate the neurodevelopmental impact of tPRS and provide insight into when these risk phenotypes may first emerge, we set out to map an expanded version of tPRS, comprising 332 genes differentially expressed in MDD, onto individual differences in brain structure and depressive symptoms in children participating in the Adolescent Brain Cognitive Development (ABCD) study. Methods: This study used tabulated neuroimaging data, collected at baseline, from 5124 non-Hispanic white participants in the ABCD study (2737 male, 2387 female; 9.9 ± 0.6 years of age (range: 8.9 – 11.0)). We used the tool MetaXcan and the CommonMind Consortium reference transcriptome to impute relative gene expression in the dorsolateral prefrontal cortex (dlPFC) at the individual level. Individual SNP contributions were determined based on weighting in a tissue-specific elastic net prediction model (DLPFC_newMetax.db). Using this model, we were able to impute 9347 genes, which included 332 out of the 566 genes previously identified in a meta-analysis of case-control post-mortem brain corticolimbic transcriptome datasets (‘metaA-MDD genes’). tPRS was computed as the sum of the imputed expression values of the 332 imputed genes, each weighted by the direction of effect in the original post-mortem meta-analysis. Separate linear mixed effects models were used to test main effects of tPRS and tPRS-by-sex interaction effects on volume in each subcortical segmentation (n = 14), and cortical thickness and surface area in each Desikan-Killiany atlas-based cortical parcellation (n = 68). Within each analysis, FDR-correction was applied to account for testing in multiple regions. In each case, tPRS, sex, age, estimated total intracranial volume (eTIV, not included in cortical thickness analyses) and 10 genetic components were modeled as fixed effects, while site was modeled as a random effect. Depressive symptoms were indexed using the t-score from the depressive syndrome scale of the parent-reported Child Behavior Checklist (CBCL). Results: We identified a significant tPRS-by-sex interaction effect on volume in the right hippocampus (HPC.R: t = -3.087, pFDR = 0.028) and right pallidum (PAL.R: t = -2.808, pFDR = 0.035), wherein higher tPRS was associated with lower volumes of both structures in females (HPC.R: t = -3.058, pFDR = 0.002; PAL.R: t = -3.112, pFDR = 0.002) but not in males (HPC.R: t = 1.379, pFDR = 0.227; PAL.R: t = 1.208, pFDR = 0.227). Higher tPRS was further associated with greater cortical thickness in the left posterior cingulate cortex when tested in the full sample (t = 3.739, pFDR<0.001). No other neurostructural effects emerged. Lower right hippocampal and pallidum volume further showed trend-level associations with higher depressive symptoms in the female subsample (HPC.R: t = -1.942, p = 0.052, pFDR = 0.052; PAL.R: t = -2.121, p = 0.034, pFDR = 0.052), where the right pallidum volume was also a significant mediator of an indirect effect of tPRS on elevated depressive symptoms (PAL.R, p = 0.016). In contrast, posterior cingulate cortical thickness was not associated with depressive symptoms when tested in the full sample (t = 0.009, p = 0.993). All effects’ significance remained unchanged when a more conventional GWAS-based measure of MDD PRS was included as an additional covariate. Conclusions: Our results suggest that genetic variants associated with shifts towards a depression-like corticolimbic transcriptome may have a distinct sex-specific neurodevelopmental signature affecting the morphology of corticolimbic regions, which may indirectly increase risk of depression, particularly in female participants. Future studies will examine the effects of tPRS on brain maturation and depressive symptom change across adolescence to identify trajectories of risk amenable to early prevention efforts. Keywords: Depression, Polygenic Scores, MRI, ABCD Study, Brain Structure Disclosure: Nothing to disclose. P295. Correlation Between Gene Expression of SIRT2 - SIRT7 and Cognitive Function in Recurrent Major Depressive Disorder Angelos Halaris, Maria Filip, Piotr Galecki Loyola University School of Medicine, Maywood, Illinois, United States Background: Sirtuins are proteins found in all aerobic organisms. They are enzymes that regulate important biological functions and are involved in several pathways. There are seven known sirtuins in humans (Sirt1-Sirt7). They are present in all organisms, including bacteria. Sirtuins have been associated with caloric restriction, aging, metabolism, cancer, transcriptional silencing, chromosomal stability, cell differentiation, stress response, inflammation, apoptosis, DNA repair, and prevention of age-related ocular diseases. Sirt1 is involved in gene silencing, cell cycle, fat and glucose metabolism and cellular oxidative stress. A genetic study has received considerable attention for association with depression and determined that Sirt1 is a potential gene target. The study conducted by the CONVERGE Study Cconsortium (China, Oxford and Virginia Commonwealth University Experimental Research on Genetic Epidemiology) identified two loci that contributed to the risk of MDD on chromosome 10: one is close to the Sirt1 gene (P52.53310210) and the other is localized in one of the introns of the LHPP gene (P56.45310212). A case-control investigation in Japan showed that one SNP (rs10997875) in the Sirt1 gene could play a role in MDD pathophysiology. It was also found that there is a link between the Sirt1 gene (rs3758391) and depressive disorders. Further, it has been shown that Sirt1 expression in the peripheral blood from individuals with depression is significantly less than healthy subjects. Sirt1 expression is markedly downregulated in the blood of MDD patients when compared with control subjects and those with remitted MDD. Given the relative paucity of studies in depressive disorders, we conducted the present study. Methods: Seventy-two newly admitted hospitalized patients who met diagnostic criteria for recurrent major depressive disorder (MDD) and inclusion/exclusion criteria, were enrolled in the study after signing informed consent. A comparative group consisted of 74 healthy volunteers with a negative history of any mental disorder. Complete clinical and laboratory data about these subjects were retrieved from the Department’s database. The following data were collected: sirtuin 2-7 mRNA expression levels, results of 21 cognitive assessment tests, clinical parameters describing the patient’s depressive disorder. The data were analyzed in an effort to detect possible differences in sirtuin 2-7 mRNA expression levels between the group of patients and the healthy controls, correlations between levels of expression of sirtuin 2-7 mRNA, and the results of cognitive assessment in the group of MDD patients, correlation between levels of sirtuin 2- 7 mRNA expression and clinical data describing the course of the disease in the group of patients. The study was approved by the Bioethics Committee No. RNN/137/17/ KE and No. RNN/303/18/ KE, and the collection of biological research material was approved by the Bioethics Committee No. RNN /566/08/ KB. The specific aims of the study included: 1. Determine whether gene expression of sirtuins 2-7 differed between patients and healthy controls. 2. Establish whether there were correlations between gene expression levels for sirtuins 2-7 and the severity of cognitive impairment in these patients. 3. Establish whether there any correlations between gene expression levels of these genes and clinical variables in these patients. Results: The levels of gene expression of sirtuins 2-7 differed statistically significantly between the two study groups. The levels of expression of the sirtuin 2, sirtuin 3, sirtuin 4, sirtuin 5 and sirtuin 7 genes were significantly higher in patients compared to healthy controls. Conversely, the level of expression of the sirtuin 6 gene was statistically significantly higher in healthy controls compared to the patient group. There were statistically significant correlations between the level of expression of the sirtuin 4 gene and cognitive function on the Stroop B scale for time, and the level of expression of the sirtuin 7 gene and cognitive function in the California Language Learning Test CVLT2 and in scale SIET raw. Lastly, there were statistically significant correlations between the level of gene expression of two sirtuins, 3 and 6, and the Hamilton Depression score on the day of admission to the hospital. Conclusions: Expression of genes for sirtuins 2-7 in patients with recurrent MDD is significantly different from the expression of these genes in healthy subjects. There were three significant correlations between gene expression for sirtuins 2-7 and the severity of cognitive impairment in patients versus healthy controls. The gene expression of sirtuins may prove to be a biomarker for cognitive impairment in this patient population if confirmed in future studies. Keywords: Major Depressive Disorder (MDD), Biomarker, Genes, SNP, Sirtuins Disclosure: Nothing to disclose. P296. Genome-Wide Methylation Study of Suicide Attempt in Bipolar Disorder Suggests Epigenetic Pathway to Peripheral Immune Alterations Salahudeen Mirza, Camila De Carvalho-Lima, Alexandra Del Favero-Campbell, Alexandre Rubinstein, Brenda Cabrera-Mendoza, Consuelo Walss-Bass, Joao Quevedo, Jair Soares, Gabriel Fries Institute of Child Development, University of Minnesota, Minneapolis, Minnesota, United States Background: Suicide is a leading cause of death worldwide and has shown extensive comorbidity as well as genetic correlation with bipolar disorder (BD). BD patients show a 10-30-fold increase in risk for suicide. DNA methylation (DNAm) patterns involve both genetic and experiential contributions and may be promising biological substrates for suicide attempt (SA) risk. Previous studies have considered DNAm in SA, but often lack matched diagnosis reference groups for better disentanglement between the pathophysiologies of SA and psychiatric disorder. We conducted an extensive epigenome-wide association study of SA in BD. Methods: DNA from seventy-nine BD patients with a lifetime history of SA (BD/SA) and eighty-four BD patients without a lifetime history of SA (BD/non-SA) was isolated from the buffy coat fraction of whole blood. 500 ng of DNA were bisulfite converted and hybridized to the Illumina Infinium EPIC BeadChip, which queries methylation at over 850,000 cytosine-preceding-guanine (CpG) sites. Analyses considered genome-wide methylation differences between BD/SA and BD/non-SA. False discovery rate (FDR) significant (q < 0.05) differentially methylated positions (DMPs) were detected using linear models with the limma package in R, and Šidák correction significant (Šidák p < 0.05) differentially methylated regions (DMRs) were detected using the comb-p algorithm within the ENmix package in R. Area under the curve (AUC) of the receiver operating characteristic (ROC) curve was used to evaluate the prediction capability of a binomial generalized linear model predicting BD/SA from the beta-values for the FDR significant DMPs. Gene ontology (GO) pathway enrichment analysis using the DMPs at p < 0.001 was performed using the gometh function in the missMethyl R package. Weighted gene co-methylation network analysis (WGCNA) using the DMPs at p < 0.05 and located at transcriptional start sites was performed in the R package WGCNA, and modules were evaluated for relation to BD/SA with age and sex adjusted binomial generalized linear models, as well as subjected to GO pathway enrichment analysis. GWAS enrichment analysis was performed using a gene set consisting of the DMPs at p < 0.05 and the summary statistics for recent GWAS of SA. DMP and DMR analyses were adjusted for age, sex, the first three genomic ancestral principal components, DNAm-based white blood cell count proportion estimates, and DNAm-based smoking score estimates. Results: Six FDR-significant DMPs were related to BD/SA, with the leading site nearest to the IL8 gene (hypomethylated in BD/SA; logFC = -0.33, pFDR= 0.03). AUC for the ROC curve predicting BD/SA from the six FDR significant DMPs was 83.5% (CI = 77.4-89.59%). Nine significant DMRs, ranging from 3 to 19 CpG sites, were identified, with the leading DMR nearest to the CLDN9 gene (Šidák p = 7.09 ×10-9). GO pathway enrichment analysis from DMPs implicated biological processes modulated by calcium. In the WGCNA analysis, six co-methylated modules, ranging from 188 to 7237 CpG sites, were identified. All six modules were significantly related to BD/SA, with one module (β = 7.15, pFDR = 0.004) being enriched for genes related to immune processes in GO analysis. GWAS enrichment analysis revealed no gene set enrichment in summary statistics for SA in BD, SA in psychiatric disorders, and SA in the general population. Conclusions: Results suggest an epigenetic signature associated with SA in BD. These biological factors may be useful to delineate a more severe subgroup of BD patients prone to suicidal behaviour. DMP and WGCNA analyses converge on immune-related processes, which parallels existing literature documenting peripheral immune alterations in suicidal behaviour. The divergence between genetic and epigenetic correlates of suicidal behaviour suggests that risk for suicidal behaviour may involve different biological mechanisms at each of these levels. Therefore, integrating genetic and epigenetic information together may offer further clarity in understanding the pathophysiology of suicidal behaviour. On both fronts, replication studies are needed to understand the reliability of identified (epi)genetic markers, if they are to be incorporated into (preventive) intervention efforts. Keywords: Epigenome Wide Association Studies, Suicide Attempt, Bipolar Disorder (BD), Peripheral Blood Marker, DNA Methylation Disclosure: Nothing to disclose. P297. Differential MicroRNA Expression Profiling of Neural-Derived Extracellular Vesicles in Bipolar Disorder Patients: A Preliminary Analysis Gabriel R. Fries, Salahudeen Mirza, Camila Nayane de Carvalho Lima, Wei Zhang, Giselli Scaini, Amit Srivastava, Zhongming Zhao, Benson Mwangi, Jair C. Soares, Joao de Quevedo University of Texas Health Science Center at Houston, Houston, Texas, United States Background: Bipolar disorder (BD) is a globally prevalent psychiatric disorder associated with functional impairment and elevated risk for suicide. There is an urgent need to identify biological mechanisms underlying BD to facilitate risk assessment, prevention, and intervention efforts. Epigenetic processes may be one pathway by which experiential risk and genetic liability interface to drive risk for BD. In particular, microRNAs (miRNAs) have recently been highlighted as important candidates in the study of psychiatric disorders due to their dynamic expression and ability to regulate the expression of many target genes. However, peripherally sampled miRNAs do not directly reflect the expression of miRNAs in the brain. We isolated extracellular vesicles (EVs) from plasma which were tagged for neural origin to identify a peripheral marker of brain gene expression. We then conducted a differential miRNA expression analysis between BD patients and healthy controls (HC) to identify miRNAs which may be differentially expressed (DE) in the brain of living BD patients. Methods: This preliminary analysis included 20 patients with BD and 20 age- and sex-matched HC. EVs were isolated from plasma with the ExoQuick® ULTRA EV Isolation Kit (Systems Biosciences) and further immunoprecipitated for the L1 cell adhesion molecule (L1CAM) neural adhesion protein, a validated marker for neural-derived EVs. Total RNA was isolated from the neural–derived EVs with the exoRNeasy Midi Kit (Qiagen) and prepared for RNA sequencing on the Illumina NextSeq. Sequencing reads were submitted to the ERCC exceRpt small RNA-seq pipeline (v.4.6.2) with default settings (adapter trimming, alignment to miRBase hg38). After filtering steps, 157 miRNAs remained for differential expression analysis in DESeq2 (v.1.36). The design was adjusted for age (median split) and sex, with HC as reference. Benjamini-Hochberg false discovery rate (FDR) procedure corrected for multiple comparisons. FDR significant DE miRNAs were inputted to the miEAA 2.0 web tool to identify implicated gene ontology (GO) processes (miRTarBase, miRWalk), diseases (MNDR), pathways (KEGG, miRWalk), and target genes (miRTarBase). We focused on the pathways, processes, and target genes which implicated the largest number of FDR significant DE miRNAs. Results: The differential expression analysis revealed 27 FDR significantly differentially expressed miRNAs in patients, led by hsa-miR-301a-3p (log2FC = 3.88, pFDR = 1.32 ×10-6). Variance stabilized hsa-miR-301a-3p expression classified BD patients with an AUC of the ROC curve of 70% (CI = 54-86%). Sixteen miRNAs were upregulated and 11 miRNAs were downregulated at the FDR significance threshold. Implicated GO processes included enzyme binding, cell proliferation, neuron projection, and positive regulation of protein phosphorylation (miRTarBase); positive regulation of transcription from RNA polymerase II promoter, extracellular region, blood coagulation, and endoplasmic reticulum membrane (miRWalk). Diseases included carcinoma, ductal, breast; hepatocellular carcinoma; breast cancer; and colorectal cancer. Pathways included yersinia infection, regulation of actin cytoskeleton, growth hormone synthesis, secretion, and action, AGE-RAGE signaling pathway in diabetic complications (KEGG); cancer, focal adhesion, integrated pancreatic cancer pathway, and angiogenesis (miRWalk). Target genes included NUFIP2, PTEN, WASL, and MBNL1. Conclusions: Our preliminary results support an epigenetic biosignature to BD and validate the potential for neural-derived EVs to characterize miRNA expression patterns in BD and other psychiatric disorders. Established relevance of target genes to nervous system function as well as psychiatric disorders is promising. Further investigation will be required to better understand the functional implications of the observed miRNA alterations, as well as their concordance to studies conducted in post-mortem brain tissue. In particular, given higher burden for certain diseases in BD patients, miRNA alterations may be one possible pathway to disease risk, although this hypothesis is preliminary. Regardless, findings offer a promising first step to incorporating brain-specific miRNAs to monitoring as well as intervention work. Keywords: Bipolar Disorder, MicroRNA, Extracellular Vesicles, Epigenetics Disclosure: Nothing to disclose. P298. Functional Magnetic Resonance Spectroscopy of Emotional Processing at 7 T Alexander Lin, Katherine Breedlove, Jessica Busler, Eduardo Coello, Huijun Liao, Pamela Mahon, Pamela Mahon Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Functional MR Spectroscopy (fMRS) at 7 T provides increased sensitivity and spectral dispersion that enables the detection of dynamic changes in metabolite concentrations in the brain under specific functional tasks. Recently, fMRS experiments performed at 7 T have successfully detected changes in metabolite concentrations. To maximize the sensitivity of these experiments, high stability of the system and the patient are required (i.e., B0-homogeneity, negligible B0-drifts, effective water suppression, reduced eddy currents, and negligible patient motion). Moreover, special processing is required to account for different external instabilities of the experiment. This work presents a novel fMRS methodology to investigate metabolic changes related to emotional processing with improved sensitivity at 7 T. An acquisition protocol for the evaluation of a visual go-no-go task was designed. Furthermore, a processing pipeline that corrects measurement instabilities is presented. Methods: Data acquisition: All data was acquired on a 7 T scanner (Terra; Siemens, Erlangen, Germany). Patients were scanned in a single channel transmit 32-channel receive head coil with additional dielectric padding to improve for B0 inhomogeneity. High-resolution MRI images were acquired using magnetization-prepared rapid gradient echo with different inversion times (MP2RAGE) using TR = 2.530 ms, TE1 = 1.64 ms, TE2 = 3.5 ms, TE3 = 5.36 ms, TE4 = 7.22 ms, Flip=7°, FOV = 256 cm, 0.7×0.7×0.7 mm resolution, acceleration factor of 2, for a scan time of 6 minutes. A 20x40x20mm3 volume was acquired using a semi-LASER localization sequence optimized for 7T28 with the gradient-modulated FOCI pulses to reduce maximum B1 (TE = 28 ms, TR = 5 s, 128 scans, spectral width 6 kHz). VAPOR water suppression will be interleaved with the outer-volume saturation. Unsuppressed water signal was acquired using 4 averages for eddy current correction and as an internal reference for metabolite quantification. 768 signal averages were acquired during the full experiment for a total acquisition time of 24 minutes. Visual stimuli and fMRS paradigm: The fMRS paradigm consisted of an affective go/no-go task to assess emotional regulation. Participants were instructed to silently read each word presented and then to button-press for words in normal font (go trial) and to inhibit this response for words in an italicized font (no-go trial). Stimuli (words) had negative, positive, and neutral valence. Performance during the task and post-task assessment of stimuli recognition and valence were used to determine participant compliance with task instructions. Data Processing and Quantification: The pipeline used for reconstruction of the spectra was implemented in python using OpenMRSLab and included the following steps: (1) coil combination, (2) frequency alignment to water, (3) zero-order phase removal, (4) combination of 4 signal averages (sub-blocks) to increase SNR for processing, (5) water removal at each sub-block using HSVD, (6) frequency alignment to NAA, (7) zero-order phase removal, (8) filtering of the dynamic temporal signal (e.g. sliding window average, Fourier thresholding), and (9) combination of sub-blocks to obtain the desired temporal resolution to match the paradigm. Frequency/phase corrections eliminated distortions caused by external effects that affect the measurement, such as B0-drifts, eddy currents, temperature, or the BOLD effect. The reconstructed spectra, corresponding to consecutive time points, were fitted using LCModel with a simulated basis set. Results: The analysis of the auditory fMRS data showed the presence of BOLD signal when subtracting each STIM block with its nearest REST block. Variations in metabolite concentrations were observed and enhanced using dynamic filtering. The quantified metabolite concentrations processed with the different methods superimposed with the visual paradigm are shown. The visual experiment served as a validation for the methodology as it was able to reproduce previously published experiments. Conclusions: This is a promising development for the application of functional MRS to the study of mood disorders such as bipolar disorder. Altered performance in neural activation and neurometabolites levels has previously been correlated with this go-no-go tasks in patients with bipolar disorder. Therefore, the ability to measure dysfunction in the glutamatergic systems that may be indicative of network dysfunction observed in mood disorders is a promising application of functional MRS. Keywords: Functional Magnetic Resonance Spectroscopy, Emotional Regulation, Ultra-high Field MRI, GoNoGo Disclosure: Agios Pharmaceuticals, Biomarin Pharmaceuticals, Design Therapeutics, Moncton MRI: Consultant (Self), BrainSpec, Inc: Founder (Self) P299. Fronto-Limbic Functional Connectivity as a Predictor of Emotional Arousal in Youth at Risk for Bipolar Disorder Samantha Hu, Aniruddha Shekara, Maxwell Tallman, Rodrigo Patino, Thomas Blom, Jeffrey Welge, Jeffrey Strawn, Kaitlyn Bruns, David Fleck, Manpreet Singh, Caleb Adler, Melissa DelBello University of Cincinnati College of Medicine, Cincinnati, Ohio, United States Background: Antidepressants are a first-line treatment for youth with anxiety and/or depression; however, their use can carry significant risk of adverse psychiatric events (e.g., increased irritability, aggression, impulsivity, psychosis) associated with increased emotional hyperarousal (EH). This may be particularly important in individuals with a family history of bipolar disorder (BD). Previous studies have shown that selective serotonin re-uptake inhibitors (SSRIs) may disrupt fronto-limbic neural pathways critical for emotion regulation; however, the neural mechanisms of EH remain largely unknown. We used functional neuroimaging to study youth with familial risk for BD receiving treatment for anxiety and/or depression to determine whether changes in emotion-associated fronto-limbic networks are predictive of dysfunctional emotional arousal. Methods: Seventy-five youth with a family history of BD and a current depressive or anxiety disorder were recruited at the University of Cincinnati and Stanford University. All participants received escitalopram and during the treatment period of up to 16 weeks, 14 youth developed EH. All participants completed a functional magnetic resonance imaging (Fmri) scan at baseline. A second scan was obtained at the time of EH occurrence. Ten youth on escitalopram matched by time on treatment, age, sex, and site who did not develop EH during the 16-week period were also scanned for comparison. EH was defined by clinician assessment, which prioritized observation of arousal-like behaviors in a clinical setting, but also incorporated parental and child self-reports, such as the Treatment-Emergent Activation and Suicidality Assessment Profile (TEASAP). During fMRI scans, participants viewed a series of stimuli and were asked to press a button when presented with an infrequent target (circles) in the context of infrequent distractors and more frequent, standard items (squares). Distractors were either emotional or neutral images. Analysis of fMRI data was performed using the CONN toolbox 21a in MATLAB. ROI-to-ROI functional connectivity analyses were performed using the Harvard-Oxford Atlas-based insular cortex (INS), orbitofrontal cortex (OFC), amygdala (AMY), inferior frontal gyrus (IFG) pars opercularis and pars triangularis, and anterior cingulate cortex (ACC) seed regions. Differences in functional connectivity between emotional and neutral distractors were compared at baseline and between baseline and EH scans. Contrasts were analyzed with repeated measures ANOVA and random-effects mixed ANOVA. Significant ROI-ROI connectivity differences were reported with cluster-level threshold p-FDR < 0.05. Results: Functional connectivity changes in participants who developed EH after treatment: At baseline, participants who developed EH while receiving escitalopram displayed no statistically significant differences in ROI-ROI functional connectivity when comparing emotional and neutral stimuli. However, over time, participants with EH showed increased connectivity between left IFG pars opercularis and left insula (p-FDR = 0.0105) and between left IFG pars opercularis and right insula (p-FDR = 0.0105) when comparing emotional with neutral stimuli. Functional connectivity changes in participants who developed EH and matched patients after treatment: Compared with matched subjects, participants who developed EH did not have significant baseline differences in functional connectivity when contrasting emotional and neutral stimuli. Over time, participants who developed EH exhibited increased connectivity between ACC and left IFG pars triangularis (p-FDR = 0.0075) when compared to matched subjects. Conclusions: Youth with family history of BD who developed EH during escitalopram treatment did not demonstrate any differences in fronto-limbic functional connectivity at baseline. However, there are longitudinal changes in emotional processing circuitry over the course of treatment with escitalopram, implying that SSRIs may play a role in mediating disruptions in these fronto-limbic neural circuits. Due to the small sample size of 14 EH participants, further study is necessary to confirm these findings. Our results suggest that a larger sample of at-risk youth is necessary to elucidate the mechanisms underlying treatment-induced EH and uncover potential targets for pharmacotherapy in this patient population. Keywords: Bipolar Disorder, fMRI, Task-based Functional Connectivity, Emotional Arousal Disclosure: Janssen Pharmaceuticals: Speakers Bureau (Self), Janssen Pharmaceuticals: Advisory Board (Self), Otsuka: Speakers Bureau (Self) P300. Examining Resting State Functional Connectivity in Suicidal Ideation and Attempts Patricia Burhunduli, Zhuo Fang, Katie Vandeloo, Pierre Blier, Jennifer Phillips* University of Ottawa Institute of Mental Health Research, Ottawa, Canada Background: Suicide remains one of the most common preventable causes of death, although predicting and reducing suicide-related behaviours remains challenging. Studies have shown that suicide cannot be solely explained as a catastrophic outcome of major depressive disorder (MDD), but risk factors are multifaceted and neurobiological processes play an important role. Studies have shown that aberrant neural networks may be involved in suicide-related thoughts and behaviours. This study examined resting-state functional connectivity (FC) differences between suicidal ideation (SI) and suicide attempts (SA) in patients with treatment-resistant MDD. Methods: The sample consisted of N = 40 patients with treatment-resistant MDD (n = 21 suicide ideators, n = 19 suicide attempters). Resting state functional magnetic resonance imaging (fMRI) data were acquired at 3 T and pre-processed using the default CONN Functional Connectivity pipeline. Group-level differences in resting-state functional connectivity between patients with SI and SA were completed using a region of interest (ROI)-to-ROI approach. SI and SA were clinically characterized using the Columbia Suicide Severity Rating Scale (C-SSRS); comparisons were statistically significant at pFDR≤0.05. Results: Resting-state functional connectivity of the right hippocampus and the default mode network (DMN) (bilateral lateral-parietal lobe) were increased in patients with SI compared to SA (pFDR= 0.02). The connectivity of the DMN (right lateral-parietal lobe) and regions within the salience network, such as the anterior cingulate cortex (ACC) and the bilateral supra marginal gyrus (SMG), were significantly decreased in the SI group (pFDR=0.03). Functional connectivity between the caudate and the hippocampus/posterior parahippocampus was negatively correlated with current SI severity in the full patient sample (N = 40; pFDR=0.05), and in the attempter group specifically (n = 19; pFDR=0.008). Conclusions: Our preliminary resting state fMRI analyses shows significant differences in DMN functional connectivity in patients with a suicide attempt history compared to those with suicidal ideation only. There were significant associations between limbic network functional connectivity and current suicidal ideation severity. These findings support potential neural circuits involved in the neurobiology of suicidal ideation and suicide attempt history in patients with treatment resistant MDD. Keywords: Resting State Functional Connectivity, Suicide Attempt, Suicidal Ideation, Treatment-resistant Depression, Magnetic Resonance Imaging Disclosure: Nothing to disclose. P301. Regional Grey Matter Volume Correlates With Anxiety, Apathy and Resilience in Geriatric Depression Beatrix Krause, Prabha Siddarth, Hanadi Ajam Oughli, Lisa Kilpatrick, Katherine Narr, Helen Lavretsky* UCLA Semel Institute for Neuroscience and Human Behavior, Los Angeles, California, United States Background: Geriatric depression (GD) is associated with significant medical comorbidity, cognitive impairment and brain atrophy, premature mortality, and suboptimal treatment response. While apathy and anxiety are common comorbid symptoms in GD, resilience has been found to be a protective factor. Understanding the relationship between brain morphometry and symptoms and resilience in GD could inform clinical treatment targets. Gray matter volume (GMV) is altered in GD but correlations with mood symptoms and particularly resilience are less available. Methods: Forty-nine older adults over 60 y.o. (38 women) diagnosed with major depressive disorder undergoing concurrent antidepressant treatment participated in the study. Depressive symptoms, as well as apathy, anxiety and resilience were assessed along with an anatomical T1-weighted MRI scan. We used a general linear model (GLM) to identify clusters in which GMV correlated with each measure. Freesurfer 6.0 was used to process T1-weighted images and perform voxel-wise whole-brain GLMs using age and sex as covariates. Partial Spearman correlations controlling for age and sex were used to investigate the association between clinical variables. Results: Greater depression severity was, as expected, associated with greater anxiety (r = .53, p = .0001). Greater resilience was associated with lower depression (r = -.33, p = .03) and reduced apathy (r = .39, p = .01). Greater GMV in widespread, partially overlapping clusters across the brain was associated with reduced anxiety and apathy, as well as increased resilience Conclusions: Our results suggest that GMV in extended brain regions is a potential marker for resilience in GD and overlaps in specific smaller regions with GMV markers for depression and anxiety. Since resilience is a protective factor against depression, future studies should examine GMV changes in these regions with treatment. Keywords: Depression, MRI, Resilience, Apathy, Anxiety Disclosure: Nothing to disclose. P302. Reduced Prefrontal γ-Aminobutyric Acid and Glutamate Levels in Major Depression: Results of a Proton Magnetic Resonance Spectroscopy Study Chris Ritter, Andreas Buchmann, Sabrina Müller, Martin Volleberg, Melanie Haynes, Carmen Ghisleni, Ralph Noeske, Ruth Tuura, Gregor Hasler* University of Fribourg, Villars-sur-Glâne, Switzerland Background: In MDD, perturbations of the major inhibitory and excitatory neurotransmitters, γ-aminobutyric acid and glutamate respectively, as well as Glx (glutamate + glutamine) have been extensively reported in a multitude of brain areas, but few studies have examined changes in glutamine, the metabolic counterpart of synaptic glutamate. In this study we investigated changes in glutamate, GABA, Glx, as well as glutamine levels in a voxel in the left dorsolateral prefrontal cortex of participants with no, past and current major depressive disorder in a large, unmedicated sample, recruited from the general population. Methods: Cross-sectional design using 3-T 1H-MRS in participants recruited from the community. Our sample consisted of 251 healthy controls, 98 subjects with a history of past major depressive disorder, as well as 47 subjects who met the diagnostic criteria for current major depressive disorder. Diagnostic groups were comparable regarding age, education, income and diet. Our main outcome measures were GABA, glutamate and glutamine concentrations in left dorsolateral prefrontal cortex. Results: Participants with past major depressive disorder had lower glutamate (r = .162, p = .010, n(Healthy; Past MDD) = 234, 93) and GABA (r = .184, p = .002, n(Healthy; Past MDD) = 236, 92), as well as higher glutamine (r = .165, p = .043, n(Healthy; Past MDD) = 153, 66) compared to participants without major depressive disorder. GABA concentrations were negatively associated with acute depressive symptoms (HAMD: rho = -.117, p = .022, n = 322, MADRS: rho = -.125, p = .018, n = 362, BDI: rho = -.150, p = .005, n = 363), while glutamine was positively associated with neuroticism (rho = .202, p = .002, n = 240). Conclusions: In a large, unmedicated community sample, reduced prefrontal GABA concentrations were associated with past and current major depressive disorder, consistent with histopathologic studies reporting reduced glial cell and GABA cell density in the prefrontal cortex in depression. Patients with major depressive disorder also demonstrated increased glutamine levels, indicative of increased synaptic glutamate release, adding to previous evidence for the glutamate hypothesis of major depressive disorder. Keywords: Gutamate, Glutamine, GABA, Depression Disclosure: Nothing to disclose. P303. Impaired Coronary Microvascular Reactivity in Youth Bipolar Disorder Benjamin Goldstein, Nilesh Ghugre, Idan Roifman, Brian McCrindle, Bradley MacIntosh, Kody Kennedy Centre for Addiction and Mental Health, Toronto, Canada Background: Cardiovascular disease (CVD) is excessively prevalent and premature in bipolar disorder (BD), even after controlling for traditional cardiovascular risk factors. The increased risk of CVD in BD may be subserved by microvascular dysfunction. We set out to extend the prior literature on coronary microvascular dysfunction in psychiatric populations by focusing on BD in youth. Methods: Participants were 86 youth, ages 13-20 years (n = 39 BD, n = 47 controls). Coronary microvascular reactivity (CMVR) was assessed using blood-oxygen-level-dependent T2-weighted magnetic resonance imaging including an established breath-holding paradigm. Images were acquired during normal breathing (baseline), following 60-seconds of hyperventilation, and every 10-seconds during a 40-second breath-hold. Measure of left ventricular structure (e.g. mass, volume) and function (e.g. ejection fraction) were evaluated based on 12-15 short- and long-axis cardiac-gated cine images. A linear mixed-effects model controlling for age, sex, and body mass index assessed for between-group differences in CMVR (i.e., a time-by-group interaction). Results: The breathing paradigm induced a significant change in T2-relaxation time in the overall sample (i.e., CMVR; β = 0.36, p < 0.001). CMVR was significantly lower in BD vs HC (β = -0.11, p = 0.004). Post-hoc pairwise analyses revealed that significant between-group differences were evident after 30- and 40-seconds of breath-holding (p = 0.003 and p < 0.001, respectively). Measures of left ventricular structure and function were within normal ranges and did not significantly differ between groups. Conclusions: There was evidence of coronary microvascular dysfunction, despite normal gross cardiac structure and function, in youth with BD. These findings converge with prior findings of adults with major depressive disorder and post-traumatic stress disorder. Future studies integrating larger samples, prospective follow-up, and blood-based biomarkers are warranted. Keywords: Bipolar Disorder, Cardiac Reactivity, BOLD fMRI Signal, Adolescent Disclosure: Nothing to disclose. P304. Connectome-Based Prediction of Mood Lability in Youth at Familial Risk for Bipolar Disorder Danella Hafeman, Feldman Jamie, Mary Phillips University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Mood lability, defined as frequent and marked changes in mood, is a predictor of mood disorder onset, poor psychosocial functioning, and suicidal thoughts and behaviors. Given the clinical relevance, it is critical to better characterize this phenotype, including the neural underpinnings. In recent years, whole-brain predictive approaches (e.g., Connectome Predictive Modeling) have emerged as useful tools for assessing the degree that edge strengths predict a given phenotype (e.g., anxiety or irritability), using a cross-validation approach. However, the interpretability of the networks that emerge from these analyses are limited. Network-Based Statistic (NBS) is an approach developed over a decade ago that identifies networks associated with a phenotype; recently, this approach has been adapted to a predictive context, using k-fold cross-validation (NBS-Predict). The combined use of NBS and NBS-Predict have the potential to identify reproducible and interpretable networks associated with a mood lability phenotype. Methods: We recruited 69 youth 10-15 years old with family history of bipolar disorder; 33 of which were selected to have elevated mood lability (defined as an average of >10 on the parent- and self-reported Child and Adolescent Lability Scale, CALS). We also scanned 42 age- and sex-matched healthy controls. Family history was confirmed via either the Structured Clinical Interview for DSM-IV (mania and depression sections) and/or medical records. Youth were assessed using Kiddie Schedule for Affective Disorders and Schizophrenia, present and lifetime; they were also administered several questionnaires, including the CALS. Youth had an MRI scan on a 3 T scanner (Verio or Prisma), which included a 6-minute resting state protocol. Data were analyzed using fmriprep (20.2.6 LTS) and xcp-d (stable version), adjusting for motion, global signal, and derivatives (36p model). Participants with excessive motion (mean framewise displacement > .5 mm or max motion > 3 mm; n = 14) or artifact observed on visual inspection (n = 1) were excluded, yielding 96 participants with acceptable data (58 at-risk, 38 healthy controls). Using nilearn, we extracted timeseries from the Shen parcellation (268 parcels) and generated connectivity matrices. We next used NBS-Predict to assess predictive models for risk status (at-risk vs. healthy) and mood lability; all models were adjusted for scanner, mean framewise displacement, age, and sex. Models were tested using 5-fold cross validation (10 repetitions), using the optimized prediction model (established in the training set) and thresholds of p < .01 and p < .05, with 5000 permutations. NBS (v1.2) was used for additional exploratory models, including testing signed networks separately and testing CALS as a dichotomous predictor. Results: Using an individual edge threshold of p < .05, we identified a network that predicted mood lability (correlation: 0.335 (0.299, 0.371), p = 0.017), explaining 10.3% of the variance. Limiting to the most influential edges (weight threshold=1), we found 52 edges (15 positively correlated; 37 negatively correlated). Visual networks (esp. Visual II) were highly represented in selected edges. Visual II connectivity with motor, salience, and medial frontal networks was correlated with lower mood lability, while Visual II-frontoparietal network connectivity was correlated with greater mood lability. Testing networks in NBS, we found a significant network negatively correlated with mood lability (5000 permutations, p = .004); a positively correlated network approached significance (5000 permutations, p = .055). These networks were largely overlapping with those identified by NBS-Predict, but with more nodes in medial frontal and frontoparietal networks. Dichotomizing mood lability at a median split (10), we found a significant network negatively predicting mood lability. We were not able to classify at-risk versus healthy controls using NBS-Predict; similarly, NBS did not identify any significant networks between these two groups. Conclusions: We find that a connectome-based approach identified a network (consisting of both negative and positive edges) that predicts mood lability in a sample of healthy controls and at-risk youth. While we hypothesized that subcortical and medial frontal networks would be most correlated with mood lability, we were surprised to find that primary visual cortex was heavily represented in the predictive network. Most strikingly, more connectivity between visual and motor context (anatomically consistent with the visual dorsal stream) was associated with less mood lability. Interestingly, previous work has shown that the visual dorsal stream is activated by naturalistic emotional stimuli and may be important for action preparation; in this way, higher connectivity within the dorsal stream may enhance emotion regulation strategies, and thus less mood lability. While NBS and NBS-Predict generated similar edges, one important difference is that networks prominent in NBS (e.g. medial frontal) did not feature so prominently in NBS-Predict results; this may be related to their out-of-sample performance. While k-fold cross-validation enhances reproducibility of findings, the gold standard approach is out-of-sample replication; in the future, we will plan to test this model in an independent sample of at-risk youth. Keywords: Mood Dysregulation, Resting-state fMRI, Familial Risk of Bipolar Disorder Disclosure: Nothing to disclose. P305. Estrogen Receptor Beta Mediates Stress-Susceptibility in the Male Brain Polymnia Georgiou, Ta-Chung M. Mou, Liam E. Potter, Xiaoxian An, Panos Zanos, Michael S. Patton, Katherine J. Pultorak, Sarah M. Clark, Vien Ngyuyen, Chris F. Powels, Katalin Prokai-Tatrai, Istvan Merchenthaler, Laszlo Prokai, Margaret M. McCarthy, Brian N. Mathur, Todd D. Gould University of Wisconsin Milwaukee, Milwaukee, Wisconsin, United States Background: Depression is the leading cause of disability worldwide afflicting ~16% of the population. Although, women are diagnosed with depression twice as often as men, there are approximately 109 million men worldwide suffering from the disease. In addition, men suffering from depression are at higher risk to lose their life from suicide, one of the most common symptoms of depression, with suicide rates being four times higher in men than women. In susceptible populations, stress is a major risk factor for the development of mental disorders, including depression. While the role of estrogen receptors in the pathophysiology of depression and as treatment targets in females are widely elucidated, their role in males is not well understood. Methods: We used a subthreshold social defeat stress model which consists of three cycles of two-min physical stress and fifteen min of sensory stress, in combination with immunohistochemistry, neuronal tracing, RNAscope, in vitro electrophysiology, in vivo optogenetics, in vivo chemogenetics, in vivo fiber photometry and surgical/pharmacological hormonal manipulations to investigate the role of estradiol and its receptors in the development of social avoidance and anhedonia in male and/or female mice (n = 10-15/experimental group). Statistical analysis was performed with ANOVAs followed by Holm-Sidak post-hoc analysis. If criteria for parametric analysis were not met, Kruskal-Wallis test was performed followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Results: We found that absence of estrogen receptor-β (ERβ) is associated with stress susceptibility in male but not female mice following exposure to a mild stressor and infusion of an ERβ-specific agonist in the basolateral amygdala (BLA) rescues this stress susceptibility in male mice. We identified a dense ERβ-expressing projection from BLA to the nucleus accumbens (NAc) and optogenetic stimulation of this projection resulted in the development of real-time place preference for the light-paired compartment in male mice while female mice did not show any preference between the light-paired and unpaired compartments suggesting a sex-dependent effect of this circuit. In addition, we observed that activity of these neurons is reduced in male mice lacking gonadal hormones subjected to mild stress and this is associated with stress susceptibility. Moreover, we demonstrated that optogenetic activation of this circuit reverses stress-induced maladaptive behaviors and induces stress resilience in hypogonadal mice, while chemogenetic inhibition of this circuit induces a stress susceptible phenotype following mild stress in intact mice. Estradiol (E2), often considered a female specific hormone, is distributed in the male brain via aromatization of testosterone. We identified that absence of E2, but not testosterone per se, underlies susceptibility to develop maladaptive behaviors following exposure to mild stress in males. Using brain-selective delivery of E2 through administration of a prodrug, which offers a viable treatment option in males, we demonstrated that E2 prevents the development of depression-related behaviors following acute/mild stress in hypogonadal male mice. Conclusions: Overall, our findings provide evidence for an estrogen-based mechanism underlying stress susceptibility and suggest a novel therapeutic strategy utilizing brain selective estradiol for treating depression in males Keywords: Estradiol, Depression, Neural Circuit and Animal Behavior, Estrogen Receptor Disclosure: A patent was filed for the development of brain selective estradiol compounds: Patent (Self) P306. Luteal Phase Epigenetic Biomarkers Identify Premenstrual Dysphoric Disorder (PMDD) and Selective Serotonin Reuptake Inhibitor (SSRI) Response in PMDD Liisa Hantsoo, Zachary Kaminsky, Jennifer Payne Johns Hopkins University, Baltimore, Maryland, United States Background: Premenstrual dysphoric disorder (PMDD) is considered a type of reproductive affective disorder and is characterized by affective symptoms that emerge in the luteal phase of the menstrual cycle and remit in the follicular phase. PMDD’s pathophysiology is poorly understood, although genetic vulnerabilities, including epigenetic changes, may contribute. Previous research has found DNA methylation variations that predict postpartum depression (another reproductive affective disorder) with up to 80% accuracy. This study explored these epigenetic markers in those with PMDD, compared with healthy controls, in the follicular and luteal phases in order to determine if these biomarkers were specific for postpartum depression or were more general biomarkers of reproductive affective disorders. Methods: Female control and PMDD participants were recruited from the community and completed two months of daily prospective symptom ratings (Daily Record of Severity of Problems (DRSP), a standard measure for premenstrual symptoms) to assess control or PMDD status, confirmed with SCID interview. Blood was collected during the follicular (days 5-11 of the menstrual cycle) or luteal phase (days -7 to -1 of the menstrual cycle, confirmed with urine lutenizing hormone test and serum progesterone levels ≥3 ng/ml). Additionally, women with PMDD received treatment with 50 mg of the selective serotonin reuptake inhibitor (SSRI) sertraline during a subsequent luteal phase and were characterized as either “responders” or “nonresponders” based on a minimum 30% reduction in DRSP score. Blood samples underwent sodium bisulfite pyrosequencing at the TTC9B and HPIBP3 genes and our published, established PPD biomarker linear model was used to evaluate whether our postpartum depression model was able to predict PMDD status compared to controls, and, in addition, predict SSRI response in the PMDD participants. Results: Blood samples were available for fifty-five participants (n = 26 control, n = 29 PMDD); 23 in the follicular phase and 32 in the luteal phase. Follicular samples failed to distinguish PMDD cases (N = 13) from controls (N = 9). However, luteal phase samples were able to distinguish PMDD cases (N = 10) from controls (N = 18) with an AUC of 0.71 (95% CI: 0.49-0.93). Our methylation biomarkers were also able to distinguish between SSRI responders (N = 5) and SSRI nonresponders (N = 5) using luteal phase blood with an AUC of 0.84 (95% CI: 0.57-1). Conclusions: This study examined CpG methylation levels at two loci within the HP1BP3 and TTC9B genes. We found that methylation patterns at these two genes were able to distinguish between PMDD cases and control participants but only when luteal phase blood was used indicating that the hormonal changes that occur during the luteal phase may be important. Our methylation biomarkers were also able to discriminate between SSRI responders and nonresponders in PMDD cases when luteal phase blood was used. Research on postpartum depression, another reproductive affective disorder, suggests that these loci may mediate sensitivity to changes in estrogen that occur during and after pregnancy which suggests that they may also be identifying women who are sensitive to the hormonal changes that occur during the luteal phase of the menstrual cycle. Our results also indicate that there may be biological differences between SSRI responders and nonresponders. We hope to replicate these findings in a large sample of PMDD cases and controls as well as examine our biomarker’s ability to predict case status in other types of reproductive affective disorders. Keywords: Epigenetic Biomarkers, Reproductive Affective Disorder, Premenstrual Dysphoric Disorder, Allopregnanolone, Estradiol Disclosure: Nothing to disclose. P307. Differential Cellular Response to Allopregnanolone in Postpartum Depression Sarah Rudzinskas, Maria Mazzu, Allison Goff, Crystal Schiller, Samantha Meltzer-Brody, David Rubinow, David Goldman, Peter Schmidt National Institute of Mental Health, NIH, Rockville, Maryland, United States Background: Given the health consequences, societal burden, and prevalence of postpartum depression (PPD), the recent FDA-approval of Brexanolone was an encouraging advance in PPD treatment. Allopregnanolone (ALLO), the naturally occurring steroid metabolite of progesterone that is chemically identical to Brexanolone, is thought to exert its anxiolytic- and antidepressant-like effects via positive allosteric modulation of GABAA receptors. However, ALLO’s mechanism of action at the molecular level, and whether that mechanism is specific to PPD, remains unclear. Methods: We examined the consequences of ALLO exposure using transcriptomics on lymphoblastoid cell lines (LCLs) derived from women with past PPD (n = 9) and women with no history of PPD or other psychiatric illness (n = 10, i.e., Controls). All LCLs were treated for 60 hours total with either ALLO (three spikes, 100 nM/spike) or DMSO vehicle, creating four different experimental groups: Control:DMSO, Control:ALLO, PPD:DMSO, and PPD:ALLO. LCLs were then collected for AmpliSeq RNA-sequencing and analyzed for differential gene expression 1) between Control and PPD LCLs, at baseline (Control:DMSO vs. PPD:DMSO) and after ALLO treatment (Control:ALLO vs. PPD:ALLO) and 2) within either Control (Control:DMSO vs. Control:ALLO) or PPD LCLs (PPD:DMSO vs. PPD:ALLO). Quality control, unsupervised clustering, and expression analyses were performed in Transcriptome Analysis Console (TAC) 4.0, and Weighted Gene Correlation Network Analysis (WGCNA) was performed in R. Results: Differentially expressed genes (DEGs, p(nom)<0.05, log(Fold Change)>=\|1.25 \| ) were detected after ALLO treatment within, as well as between, PPD and Control LCLs. Between Control:ALLO vs. PPD:ALLO, 269 DEGs were observed, with Enrichr revealing many that were related to synaptic activity. Amongst these DEGs was Glutamate Decarboxylase 1 (GAD1), which was significantly (p(nom)<0.019) decreased in PPD:ALLO compared to Control:ALLO LCLs. Technical replication via qRT-PCR confirmed that GAD1 expression was decreased in PPD LCLs compared to Controls (Diagnosis: F(1,34)=5.25, p = 0.0283), but ALLO did not significantly effect expression (Treatment: F(1,34)=0.006121; Interaction: F(1,34)=0.0108). Overall patterns of gene expression also demonstrated that regardless of ALLO treatment, diagnosis was the primary driver of expression differences, suggesting a robust effect of PPD on gene expression, in line with previous data (Rudzinskas et. al, under review). Surprisingly, substantially more DEGs were induced with ALLO treatment in control LCLs (Control:DMSO vs. Control:ALLO, 265 DEGs), as compared to in PPD LCLs (PPD:DMSO vs. PPD:ALLO, 98 DEGs), with only 11 of these 363 total ALLO-related DEGs overlapping. Correspondingly, Gene Set Enrichment Analysis using MSigDB revealed networks linked to PPD:DMSO vs. PPD:ALLO DEGs were unique from, and occasionally opposite of, Control:DMSO vs. Control:ALLO DEGs. Similarly, WGCNA revealed statistically significant modules related to either treatment or diagnosis; no modules significant for both PPD and ALLO were observed. Conclusions: Together, these data highlight both ALLO-independent and -dependent molecular responses in PPD. While the Control:ALLO vs. PPD:ALLO comparisons of the transcriptional response reflect literature supporting ALLO’s modulation of synaptic signaling and GABA-related activity, the ultimately weak response of PPD LCLs to ALLO-treatment, especially compared to control LCLs, warrants further investigation. Furthermore, the striking lack of overlap in DEGs between diagnostic groups after ALLO-treatment suggests that ALLO ultimately activates unique and potentially divergent cellular pathways in women with PPD compared to controls. Thus, it may be beneficial to recognize ALLO’s ability to play distinct, diagnosis-dependent cellular roles when considering its therapeutic potential. Keywords: Postpartum Depression, Allopregnanolone, Transcriptomics Disclosure: Nothing to disclose. P308. Menstrual Cycle Brain Plasticity: Ultra-High Field 7 T MRI Reveals Changes in Human Medial Temporal Lobe Volume in Female Adults Rachel Zsido, Angharad Williams, Claudia Barth, Bianca Serio, Luisa Kurth, Frauke Beyer, Veronica Witte, Arno Villringer, Julia Sacher Emotion NeuroimaGinG (EGG) Lab, Max Planck Institute for Human Cognitive and Brain Sciences, Clinic for Cognitive Neurology, University of Leipzig, Leipzig, Germany Background: Female-specific variables, such as pregnancy, menopause, and the menstrual cycle, influence the brain and the risk for mental health disorders, with women at higher risk for cognitive and affective disorders when ovarian hormones rapidly fluctuate and decline. Only 3 percent of studies in the neurosciences, however, are conducted exclusively in females, creating a huge data gap. Here, we provide the first longitudinal high-field MRI dataset of the hippocampus across the menstrual cycle, demonstrating the natural variance of female brain structural plasticity in a region instrumental to memory and affect regulation. Methods: We performed longitudinal mapping of medial temporal lobe subregion morphology at 6 timepoints across the menstrual cycle in vivo using a dense-sampling protocol, ultra-high field neuroimaging and individually derived segmentation analysis in 27 healthy female participants (19-34 years). We acquired a total of 138 high resolution images for volumetric calculations of the hippocampus and medial temporal lobe. Using this well-powered within-subject design, we performed novel segmentation analysis of high resolution 7Tesla MRI scans to investigate whether ovarian hormone-modulated volumetric changes manifest differently across subregions of the medial temporal lobe complex. We also quantified cerebral spinal fluid and cerebral blood flow to confirm any volumetric changes were not driven by hormone-related water shifts or blood flow changes. Results: As hypothesized, linear mixed-effects modeling showed positive associations between estradiol levels and whole hippocampus volume (β = 108.26, 95% CI = 27 to 190, random effects SD = 174.47, p = 0.009). In MTL subregions, the addition of the estradiolprogesterone interaction to the model significantly improved model fit only for the CA1 (χ2(1) = 7.691, p = 0.006). Estradiol was positively associated with CA1 volume (β = 42.87, 95% CI = 21 to 65, p < 0.001), progesterone was negatively associated with CA1 volume (β = -150.02, 95% CI = -249 to -51, p = 0.003), and we observed a significant interaction of estradiol and progesterone with CA1 volume (β = 53.06, 95% CI = 16 to 90, random effects SD = 44.03, p = 0.005), such that at higher progesterone levels, the positive effect of estradiol on CA1 volume was attenuated. Progesterone was positively associated with subiculum volume (β = 13.12, 95% CI = 4 to 22, random effects SD = 43.29, p = 0.006) and with Area 35 volume (β = 11.98, 95% CI = 2 to 21, random effects SD = 44.01, p = 0.014). Finally, estradiol was positively associated with parahippocampal cortex volume (β = 24.33, 95% CI = 10 to 39, random effects SD = 32.48, p = 0.001). Conclusions: We found unique associations between ovarian hormones and CA1, perirhinal Area 35, subiculum, and parahippocampal cortex volumes across the menstrual cycle. These findings suggest that ovarian hormones alter structural brain plasticity in brain subregions that are differentially sensitive to hormones. Recognizing female brain health as more than just how female brains differ from male brains requires shining a spotlight on female-specific variables, such as the menstrual cycle, as a primary variable of interest. By providing detailed neural phenotyping of brain areas substantially implicated in cognitive and neurodegenerative disease, we establish an important reference: an integrative benchmark for evaluating female MRI biomarkers in health and disease and a prerequisite to ultimately assess depression and dementia risk later in life, pathologies which affect females twice as often as males and more frequently during times of a changing ovarian hormone environment. Keywords: Human Neuroimaging, Hippocampus, Menopause, Mood, Childhood Stress, Neuroendocrinology, Women’s Health, Sex Differences Disclosure: Nothing to disclose. P309. Mast Cell Activity Induces Fosb as a Negative Feedback Mechanism to Limit Neuroinflammation and Anaphylactic Response Natalia Duque-Wilckens, Dimitri Joseph, Maradiaga Nidia, Srinivasan Vidhula, Szu-ying Yeh, Hari Subramanian, Eric Nestler, Adam Moeser, Alfred Robison Michigan State University, Bath, Michigan, United States Background: Brain inflammation plays a central role in neurodegenerative and psychiatric disorders, but the mechanisms by which events such as peripheral infection or environmental stressors result in persistent central neuroimmune activation remain unclear. Mast cells, innate immune cells mostly known for their role in allergy, are uniquely positioned to play a key role in amplifying inflammatory responses leading to chronic brain diseases. In addition to multiple peripheral tissues, mast cells reside in the brain and meninges, are highly sensitive to immune challenges and stress (which can permanently change their activity) and can release an impressive variety of bioactive molecules ranging from pro- and anti-inflammatory cytokines to proteases and neurotransmitters, directly affecting brain physiology. Surprisingly, the transcriptional mechanisms controlling acute-and long-term mast cell responses remain largely unexplored. Based on initial findings that stress and antibody-mediated activation of mast cells dramatically increase the expression of FosB, which encodes the FosB and ΔFosB transcription factors critically involved in long-term modulation of neuronal activity, we used transgenic mice in combination with in vitro and in vivo experiments to test the hypothesis that FosB plays a fundamental role in regulating stimulus-induced mast cell activation and mediator release. Methods: 1) To create the first mice in which FosB expression is ablated specifically in mast cells (MCFosB-), we crossed the Mcpt5-Cre with the Cre-dependent floxed FosB mouse lines. 2) For in vitro experiments, bone marrow progenitor cells were harvested from femurs of WT and MCFosB-mice and cultured in media supplemented with stem cell factor and Il-3 for 6 weeks to generate bone marrow derived mast cells (BMMC). Next, several approaches were used to assess BMMCs responses to IGE-antigen or lipopolysaccharide (LPS) stimulation: electron microscopy, intracellular Ca2+ measurements, and mediator release. Finally, to confirm the role of FosB in mast cell activity, we overexpressed ΔFosB or its dominant negative binding partner ΔJunD into WT and MCFosB- BMMCs 3) For in vivo experiments, WT and MCFosB- male and female mice were exposed to passive systemic anaphylaxis or LPS injections and rectal temperature, peripheral and central release of inflammatory mediators, and activation levels of mesenteric mast cells were assessed. 4) Finally, to uncover regions of ΔFosB binding in mast cell chromatin, we performed CUT and RUN using baseline and IGE-antigen activated BMMCs, and further combined these data with RNAseq analysis to find potential genes directly regulated by ΔFosB after stimulation. 5) Statistical analyses: samples sizes were n = 3-6 for in vitro studies and 8-10 for in vivo studies. T-test, two-way ANOVAS, or repeated measures ANOVAS were used depending on experimental design. Results: 1) BMMCs derived from MCFosB- do not express FosB or ΔFosB and, compared to WT, show increased activated appearance at baseline and after stimulation as well as heightened stimulus-induced Ca2+ mobilization and release of proinflammatory mediators (p < 0.001). Preliminary data suggest that overexpression of ΔFosB inhibits stimulus-induced mediator release in both WT and MCFosB- BMMCs while ΔJunD exerts the opposite effect. 2) MCFosB- mice show exaggerated hypothermic responses (p < 0.01), increased plasma and hypothalamic content of inflammatory cytokines (both p < 0.05), and heightened activation of mesenteric mast cells (p < 0.05) The overlap of CUT and RUN and RNAseq data suggest that ΔFosB functional targets include dual specificity phosphatase Dusp4 and thymic stromal lymphopoietin TSLP, known regulators of mast cell activity. Currently, we are validating these findings using gene and protein expression analyses. Conclusions: Taken together, these data show that FosB products exert an inhibitory effect on mast cell activation and proinflammatory mediator release and suggest that this effect could be partly mediated by DUSP1 mediated dephosphorylation of mitogen-activated protein kinases (MAPKs). These results provide a novel negative feedback mechanism of mast cell regulation relevant to brain and peripheral disorders associated with exacerbated inflammation. Keywords: Neuroinflamation, ΔFosB, Mast Cells Disclosure: Nothing to disclose. P310. Gestational Stress Exposure Effects on Microglia- Synaptic Interactions in the Rat Prefrontal Cortex and Nucleus Accumbens Across the Peripartum Period Kathryn Lenz, Courtney Dye, Dominic Franceschelli, Amanda Ringland, Benedetta Leuner The Ohio State University, Columbus, Ohio, United States Background: Pregnancy confers a period of heightened vulnerability to mood disorders, with an estimated 20% of new mothers experiencing Postpartum Depression (PPD). The mechanisms contributing to mood dysregulation and impaired maternal care in PPD are not well understood, but stress during pregnancy is a strong risk factor. We previously discovered pregnancy drives microglial changes in brain regions regulating mood and maternal care, including the medial prefrontal cortex (mPFC) and nucleus acccumbens (NAc). Pregnancy also induces widespread neuroplasticity to facilitate motivation and maternal care, and because microglia have been linked to synaptic remodeling in both stressed and non-stressed states, we hypothesize that aberrant microglia-mediated synaptic remodeling may contribute to PPD. Methods: We used a rodent model of gestational stress that induces a PPD-like phenotype (i.e., behavioral despair, anhedonia, impaired maternal care). Rats underwent chronic variable stress or control handling (n = 8-10/group/endpoint) from gestational days (GD)7-20 and were sacrificed on GD21 or postpartum day (PD) 8 and brains collected. In one cohort, qPCR was performed on mPFC and NAc tissue to measure a select panel of synaptic transcripts. In another cohort, brains were sectioned, and immunofluorescent staining performed against a postsynaptic glutamatergic synaptic marker (PSD-95), a marker extracellular matrix protein marker for perineuronal nets (WFA), a pan-microglia marker (Iba1), and a lysosomal marker (CD68) to identify phagocytic microglia. Staining density and 3D co-localization were quantified with ImageJ and IMARIS software. Data were analyzed via 2 way ANOVA and Tukey post-hocs if significant main effects found. All experimental protocols in animal studies were approved by the Institutional Animal Care and Use Committee of The Ohio State University and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Results: Gestational stress led to increased microglia and phagolysosome staining in the mPFC at PD8 (p < 0.05) and NAc at both GD21 (p < 0.05) and PD8 (p < 0.0001). In NAc, gestational stress led to decreased expression of the pre-synaptic glutamatergic marker Vgat1 at GD21 and the general pre-synaptic marker synaptophysin at PD8 (p < 0.05). Stress also led to a decrease in PSD-95 immunostaining in the NAc (p < 0.05), but no stress-induced differences in microglia engulfment of PSD-95 were observed with Imaris 3D rendering analysis. In mPFC, neither synaptic mRNA expression nor PSD95 immunostaining were impacted by stress exposure. However, we did find changes in perineuronal nets (PNNs), which are extracellular proteins responsible for synaptic stability. We found that PNNs increased from GD21 to PD8 in control animals (p < 0.0001), and that gestational stress led to a significant decrease in PNN levels relative to controls at GD21 (p < 0.001) and PD8 (p < 0.01). However, there were no significant stress effects on microglia engulfment of PNNs in the Imaris 3D rendering analysis. The NAc had few to no WFA-stained PNNs across any group. Conclusions: In the PFC, PNNs, but not synaptic targets, were decreased by gestational stress exposure. In the NAc, synaptic targets were decreased by gestational stress exposure. Thus gestational stress has region-specific impacts on the synaptic elements it affects, even though both regions showed similar microglial changes. Since PNN and synaptic loss were not mediated by microglia phagocytosis, future work will assess degradation of these synaptic and stabilizing by microglia-derived secreted factors. Microglial-mediated remodeling of perineuronal nets is a novel mechanism for maternal neuroplasticity. Understanding how remodeling of perineuronal following gestational stress may contribute to the underlying pathophysiology of PPD is an exciting avenue for future research and interventional strategies. Keywords: Postpartum Depression, Microglia, Medial Prefrontal Cortex, Stress, Synapse Disclosure: Nothing to disclose. P311. Serotonergic IL-1R1 Modulates Transporter and Neuronal Activity in a Sex-Dependent Manner in Response to Peripheral Inflammation Paula Gajewski-Kurdziel, Alaina Tillman, Hideki Iwamoto, Nicole Baganz, Matthew Robson, Ning Quan, Randy Blakely Florida Atlantic University, Jupiter, Florida, United States Background: Multiple neuropsychiatric disorders have been found to display immune system alterations (Kerr et al., 2005). The high incidence of comorbidity of neuropsychiatric disorders with chronic inflammatory conditions, such as rheumatoid arthritis, suggests a commonality in the manifestation of these conditions (Evans et al., 2005). Inflammatory cytokines have been found to impact multiple dimensions of neural signaling, from neurotransmitter release and transport, to neural excitability and plasticity (Capuron and Miller, 2011). Forebrain-projecting serotonin (5-HT) neurons of the dorsal raphe (DR) play a key role in regulating behaviors related to mood, fear, sleep, feeding and social interactions. Dysfunction in 5-HT neurotransmission is believed to contribute to multiple neuropsychiatric disorders. Our work is focused on identifying molecular and circuit-level mechanisms that can translate peripheral innate immune system activation into changes in behavior. We have reported that peripheral activation of the innate immune system rapidly increases the activity of the serotonin transporter (SERT), a main determinant of 5-HT neurotransmission (Zhu et al., 2010). Multiple groups, including our own, have demonstrated high level expression of IL-1R1 on 5-HT neurons (Okaty et al., 2015; Liu et al., 2019). Identifying projections and physiological consequences of 5-HT expressing IL-1R1 neurons may clarify how the immune system can influence behavior through discrete circuits and lead to refined treatments for behavioral alterations arising in neuropsychiatric disorders. Methods: We used adult male and female transgenic mice that allow for conditional elimination of IL-1R1 (IL-1R1loxP/loxP) or restoration of IL-1R1 on an otherwise IL-1R1 knockout background (IL-1R1r/r). Mice were administered LPS (0.2 mg/kg i.p.) or saline and sacrificed by transcardial perfusion three hours after treatment (n = 8-12). Brain slices containing DR were immunolabeled with 5-HT and cFos and counts for colocalization were obtained by a blind observer. 5-HT projection sites were stained for cFos (DAB) and cell counts were obtained through automated counting (Nikon NES Elements software). 5-HT uptake assays on midbrain synaptosomes were performed after peripheral LPS with or without the presence of serotonergic IL-1R1. Candidate projection sites were targeted for stereotaxic surgery to inject the retrograde tracer, Fluorogold (FG), in ePet1:Cre;IL-1R1r/r mice to visualize colocalization of serotonergic IL-1R1 (via a transcriptional reporter) with FG. In vivo chronoamperometry was utilized to determine the effect of local IL-1β (2 ng) on 5-HT clearance in the dorsal hippocampus (CA3) of wild type male mice. Acute brain slice recordings of DR 5-HT neurons were obtained using whole cell patch clamp with current injection used to produced tonic firing. IL-1β was perfused (10 ng/ml) onto the slice and recordings were taken. Results: We demonstrate nonuniform expression IL-1R1 throughout the DR subregions containing 5-HT neurons. Serotonergic IL-1R1 shows enrichment evident in the dorsal and dorsolateral subregions of the DR. Upon peripheral LPS treatment, males showed an IL-1R1-dependent decrease in serotonergic cFos throughout the DR, whereas females only demonstrated reduced cFos levels in a subset of DR regions. Examination of the median raphe revealed female-specific decreases in serotonergic neuronal activity dependent on the expression of IL-1R1. The lateral habenula (LHb) appears to be a direct target of serotoninergic IL-1R1 neurons, and we observed a female-specific serotonergic IL-1R1-dependent decrease in LHb neuronal activity after LPS. Other brain regions revealed a dependence of serotonergic IL-1R1 expression to maintain baseline neuronal activity. Additionally, we show that the ability of peripheral LPS treatment to stimulate SERT in midbrain synaptosomes is lost in our serotonergic IL-1R1 knockout. Local application of IL-1β in the CA3 subregion of the dorsal hippocampus resulted in increased 5-HT clearance, whereas ex vivo DR slice electrophysiology revealed an inhibition of 5-HT neuron firing in response to IL-1β. Reversal potential analysis suggests a role for membrane K + channel activation as underlying IL-1R1 actions. Conclusions: Our findings support a growing appreciation that 5-HT neurons contribute to changes in CNS physiology following peripheral immune activation. Our studies are among the first to reveal a sex specificity of serotonergic IL-1R1 on the regulation of 5-HT neuron activity. Since the subregions of DR 5-HT neurons have unique projection patterns, the differences in 5-HT neuron activity post-LPS in males and females suggest a basis for sex-specific differences in projection targets that can drive distinct behavioral responses. Our findings indicate that IL-1β modulates 5-HT neurotransmission both via regulation of SERT and by suppression of 5-HT firing, and thereby 5-HT release. Our current efforts are aimed at investigating the behaviors dependent on serotonergic IL-1R1 activation, as well as elucidating the intracellular signaling cascade of serotonergic IL-1R1 in hopes of revealing targets important for modulating 5-HT intrinsic activity. Our ongoing work will further clarify links between elevated inflammatory signaling and how, by acting through 5-HT pathways, these signals can provide for both normal, health promoting behavioral responses as well as impact risk for neuropsychiatric disorders. Keywords: Serotonin, Dorsal Raphe, Interleukin 1 Receptor, Interleukin 1beta, LPS Disclosure: Nothing to disclose. P312. Type I Interferon Signaling Mediates Chronic Stress-Induced Synapse Loss and Behavioral Deficits Ashutosh Tripathi, Alona Bartosh, Danny Perez Sierra, Anilkumar Pillai The University of Texas Health Science Center, Houston, Texas, United States Background: Inflammation and synaptic deficits have been associated with pathophysiology of many neuropsychiatric disorders. Chronic stress is a major risk factor for many neuropsychiatric conditions and is known to induce inflammation and behavior deficits. Type I interferons (IFN-I) are key players in peripheral inflammatory response and are responsible for mood and behavior deficits. Findings from our published study identified a crucial role of complement component 3 (C3) in IFN-I-mediated changes in neuroinflammation and behavior under chronic stress conditions. C3 is the central hub of complement activation pathways and is known to tag synapses to be eliminated. The receptor of C3 (C3ar1) is highly expressed on microglia and infiltrating monocytes/macrophages. In the present study, we hypothesized that C3ar1 activation is involved in IFN-I signaling-mediated chronic stress-induced synapse loss and behavior deficits. Methods: To test our hypothesis, we conducted experiments using C3ar1 knockout mice. To block IFNAR signaling, mice were administered intraperitoneally with anti-IFNAR antibody. We used the chronic unpredictable stress (CUS) paradigm in mice. Three chambers social behavior test, Golgi staining for spine density, immunohistochemistry to study microglial activation and synapse pruning, flow cytometry for monocyte infiltration, and qRT-PCR for cytokine measurements were conducted in our experiments. Data were analyzed using two-tailed Student’s t-tests (for two-group comparisons) or Analysis of Variance (ANOVA; for multiple-group comparisons). p < 0.05 was considered significant. Bonferroni’s posthoc test was performed within the comparison. Results: Our findings show that CUS significantly increased serum IFNβ levels and social behavior deficits. Increased microglial activation and synapse loss were found in the prefrontal cortex (PFC) following CUS. In addition, decreased sociability index was correlated with increased inflammation and decreased synapse numbers in CUS-exposed mice. Peripheral blockade of IFN-I signaling attenuated the above CUS-induced neuro-immune alterations and behavioral deficits. Furthermore, C3ar1 mediates systemic IFNβ-induced neuroinflammation and social behavior deficits. Conclusions: Our findings identify a key role of C3ar1 in IFN-I-mediated changes in neuro-immune alterations under chronic stress. Collectively, these results support the rationale that targeting peripheral IFN-I pathway represents a promising therapeutic option especially for patients with an elevated immune profile as seen in many depressed subjects. Additional studies are warranted to investigate the brain cell types responsible for the C3ar1-mediated effects. Keywords: Interferon, Neuroinflammation, Complement Component 3, Synapse Loss, Behavior Deficits Disclosure: Nothing to disclose. P313. Chronic Stress Exposure Alters the Gut Barrier Integrity: Sex-Specific Effects on Microbiota and Jejunum Tight Junctions Ellen Doney, Laurence Dion-Albert, Francois Coulombe-Rozon, Natasha Osbourne, Renaud Bernatchez, Sam Paton, Fernanda Neutzling Kaufmann, Roseline Olory Agomma, Jose Luis Solano Lopez, Raphael Gaumond, Katarzyna Dudek, Joanna Kasia Szyszkowicz, Signature Consortium, Manon Lebel, Alain Doyen, Audrey Durand, Flavie Lavoie-Cardinal, Marie-Claude Audet, Caroline Menard Université Laval, Quebec City, Canada Background: Major depressive disorder (MDD) is currently the most prevalent mood disorder and a leading cause of disability worldwide. However, 30-50% of patients are unresponsive to commonly prescribed antidepressants, highlighting untapped causal biological mechanisms. Dysfunction in the microbiota-gut-brain axis, the bidirectional communication between the central nervous system and gastrointestinal tract, has been implicated in MDD pathogenesis. Aligning with the neuroimmune hypothesis of depression, MDD has a high comorbidity with inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, suggesting that inflammation-driven gut barrier dysfunction may affect emotion regulation and vice versa. Exposure to chronic stress disrupts blood-brain barrier integrity in a sex-specific manner through loss of the tight junction protein Claudin-5, leading to the development of anxiety- and depression-like behaviors. Still, little is known about intestinal barrier function in these conditions particularly for the small intestine where most food and drug absorption takes place. Methods: Thus, here we investigate how chronic social (n = 12-20/group/sex) or variable stress (n = 5-10/group/sex), two mouse models of depression, impact the jejunum (JEJ) intestinal barrier in males and females. As chronic stress is the main environmental risk factor for MDD, it is commonly used in animals to alter behaviors and investigate underlying biology. Chronic social defeat stress is a mouse model of depression based on social dominance which produces two distinct phenotypes of stress response: stress-susceptible and resilient mice. The susceptible subgroup display distinct behavioral changes reminiscent of depressive symptoms in humans with increased social avoidance, anxiety, anhedonia, despair, body weight changes, metabolic disturbances, and corticosterone reactivity. Furthermore, loss of BBB integrity occurs only in the brain of susceptible, but not resilient, mice. Another leading stress paradigm is the variable stress model, during which mice are exposed to a repetitive sequence of three stressors, namely tube restraint, tail suspension, and foot shocks. In this paradigm, females and males develop depression-like symptoms at different time points making it a strong model for investigating sex differences. Mice were subjected to stress paradigms followed by analysis of gene expression profiles of intestinal barrier-related targets by quantitative PCR, fecal microbial composition with sequencing studies, and blood-based markers using ELISAs. We also took advantage of machine learning and developed algorithms to characterize in detail tight junction morphological changes. Translation value of a potential gut health biomarker was validated on blood human samples from the Montreal Signature biobank (N = 15-29 individuals/group/sex). Results: Altered microbial populations as well as changes in gene expression of JEJ tight junctions were observed depending on the type and duration of stress, with sex-specific effects (Claudin-3: social stress for males, two-tailed unpaired t-test p = 0.0002 vs p = 0.288 for females; variable stress for males, two-tailed unpaired t-test p = 0.4234 vs p = 0.0079 for females). We confirmed that stress-induced alterations in tight junction gene expression are also reflected at protein level. Thin 6-um slices were double stained with Cldn3 (red) and F-actin (green), and morphological analysis performed using the Imaris software (Claudin-3; two-tailed unpaired t-test p = 0.0357). Intriguingly, with unsupervised k-mean clustering of four features of tight junctions - ruffles, width, fragmentation, and diffusion - we identified a cluster of ruffled junctions in stressed animals. Ruffling is associated with inflammation, so we evaluated if LPS injection recapitulates stress-induced changes in the JEJ and observed profound sex differences (Claudin-3: LPS treatment for males, two-tailed unpaired t-test p = 0.0001 vs p = 0.2799 for females). Finally, LPS-binding protein (LBP), a marker of gut barrier leakiness, was associated with stress vulnerability in mice (for males p = 0.0033 after social stress vs for females p = 0.0188 after variable stress) and translational value was confirmed on blood samples from women with MDD (two-tailed unpaired t-test p = 0.7285 for men vs p = 0.0434 for women). Conclusions: Our results provide evidence that chronic stress disrupts intestinal barrier homeostasis in conjunction with the manifestation of depressive-like behaviours in a sex-specific manner in mice and possibly, human depression. We developed tools and algorithms to analyze in detail tight junction morphological changes and identified circulating LBP as a gut leakiness potential biomarker that could help better diagnose and inform treatment strategies for mood disorders. Keywords: Social Defeat Stress, Sex Differences, Gut Microbiome, Brain Immune Gut Axis, Cytokines Disclosure: Nothing to disclose. P314. Psilocybin Alters Behavior and the Intestinal Microbiota in a Wild Type Mouse Model by Mechanisms That Are Not Fully Dependent on 5HT2A and 5HT2C Receptors Zachary Cordner, Emese Prandovszky, Megan Pedicini, Hua Liu, Lindsey Macias, Mikhail Pletnikov, Kellie Tamashiro, Robert Yolken Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Much enthusiasm has emerged for the therapeutic potential of psilocybin, with growing evidence of remarkable benefit for depression as well as several other psychiatric disorders. However, despite promising clinical data, our understanding of psilocybin’s therapeutic mechanisms remains limited. The lack of mechanistic studies appears to be driven, in part, by the assumption that psilocybin’s agonism of serotonin receptors in the brain – known to be responsible for the drug’s psychedelic effects – also explains its diverse therapeutic benefits, though several recent studies suggest that other mechanisms are likely involved. The microbiome-gut-brain axis, which is increasingly recognized as a driver of behavior and modulator of psychotropic drug effects, is a plausible but largely unexplored target of psilocybin. Here, we begin to assess the effects of psilocybin on behavior, the intestinal microbiota, and the dependence of psilocybin-induced changes on 5HT2A and 5HT2C receptors. Methods: In the first study, adult male and female C57BL/6 J mice were exposed to a single dose of saline, psilocybin, the 5HT2A and 5HT2C receptor antagonist ketanserin, or psilocybin co-administered with ketanserin. The head twitch response, a validated behavioral measure of central 5HT2A receptor agonism, was measured 30 minutes after treatment. Elevated plus maze, social interaction, and forced swim behaviors were measured between 3 and 6 days after treatment. In a second cohort, male mice were again treated with saline, psilocybin, ketanserin, or psilocybin co-administered with ketanserin. Then, 3 days after treatment, behavior was assessed, and intestinal contents were collected for 16 S rRNA sequencing to determine bacterial composition and diversity. Finally, intestinal contents were collected from mice treated with saline or psilocybin then transplanted by gavage to naive male mice, followed by behavioral analysis. Results: Psilocybin induced a robust head twitch response, increased exploratory behavior in the elevated plus maze, increased social behavior in the social interaction test, and decreased immobility in the forced swim test. Co-administration of ketanserin fully blocked the head twitch response without significantly altering psilocybin’s effects on other behavioral outcomes. In a separate cohort, treatment with psilocybin produced broad alteration of the intestinal microbiome, with particularly marked changes in the large intestine that were only partially blocked by pre-treatment with ketanserin. Finally, transplantation of intestinal contents from psilocybin-treated mice to naive untreated mice resulted in behavioral changes consistent with the effects of psilocybin treatment. Conclusions: Our findings demonstrate that a single dose of psilocybin leads to behavioral changes in mice that are relevant for studies of resilience and affective disorders. Our results further indicate that the behavioral changes may not be fully dependent on psilocybin’s agonism of 5HT2A and 5HT2C receptors. Further, psilocybin appears to broadly alter the intestinal microbiome and transplantation of intestinal contents reproduces behavioral change associated with psilocybin treatment, suggesting a previously unknown microbiome-gut-brain mechanism of action. Keywords: Psilocybin, Gut Microbiome, Mood and Anxiety Disorders, Translational Animal Models Disclosure: Nothing to disclose. P315. Transcriptional Machinery of Microglial Stress Response for Mental Illness Pathology Masayuki Taniguchi, Kazutoshi Matsushita, Rei Mishima, Mitsutaka Kadota, Shigehiro Kuraku, Tomoyuki Furuyashiki Kobe University Graduate School of Medicine, Kobe, Japan Background: Clinical studies have suggested the presence of neuroinflammation in depressive patients. Rodent studies have demonstrated that chronic stress activates microglia, thereby leading to depression-related behaviors via the induction of proinflammatory molecules. Microglia appear to utilize different yet overlapping molecules in multiple brain areas to cause behavioral alterations. However, histological markers for microglial activation can only capture a limited facet of microglial responses. Thus, microglial responses to chronic stress and their functions remain poorly understood. Methods: We subjected male C57BL/6 N mice to acute or chronic social defeat stress. We categorized the mice after chronic social defeat stress into susceptible and resilient mice according to the level of social avoidance, a typical depression-related behavior. Then, we isolated microglia from multiple brain areas of these mice, including the medial prefrontal cortex (mPFC), primary sensory and motor cortices, hippocampus, nucleus accumbens (NAc), and hypothalamus, and subjected these cells to single-cell RNA-seq. In addition, we more deeply analyzed the transcriptome of microglia from the mPFC and NAc, two representative brain areas with distinct responses to social defeat stress, with bulk RNA seq. We then predicted transcription factors responsible for stress-induced changes in the microglial transcriptome and examined their functional significance using genetic and surgical manipulations. Results: Using single-cell RNA-seq of microglia isolated from multiple brain areas in mice, we found that microglial transcriptomes manifested the brain region-specificity and individual variability of stress susceptibility after chronic social stress. Deeper transcriptome analyses of microglia from the mPFC and NAc segregated gene clusters of brain region-specific (local) and non-specific (global) responses to chronic social stress, and the global responses encoded individual variability of stress susceptibility. Epigenomic analyses with H3K27ac ChIP-seq and ATAC-seq predicted distinct transcription factors involved in the respective responses, and surgical and genetic manipulations identified a peripheral stress signal that induced the global transcriptional responses to confer microglia with stress susceptibility. Conclusions: These findings suggest that microglia integrate local and global stress signals via transcriptional machinery to determine their activation state, promoting mental illness pathology. Keywords: Social Defeat Stress, Depression, Microglia, Neuro-Inflammation, Medial Prefrontal Cortex Disclosure: Nothing to disclose. P316. From Blue to Gray: Inflammation, Aging and Neurodegeneration in Depression Ebrahim Haroon, Diana Beltran, Xiangchuan Chen, Evanthia Wommack, Blaine Roberts, Deqiang Qiu, Felicia Goldstein, Michael Treadway, Jennifer Felger, Andrew Miller Emory University, Atlanta, Georgia, United States Background: Depressed patients experience a 2-5 times higher risk of neurodegenerative disorders such as dementia (Alexopoulos, 2019; Byers and Yaffe, 2011). However, predisposing risk factors that enable clinicians to stratify risk and initiate preventive measures are unclear. We propose that chronic inflammatory activation in depression promotes and sustains this risk. Our previous data have demonstrated that increased inflammation in depression increases the risk of glutamate toxicity and leads to toxic disorganization of neural systems linked to emotional and cognitive functions (Haroon et al., 2016). Herein, we examined if increased inflammation in the brain as measured in cerebrospinal fluid (CSF) was associated with increases in CSF makers of neurodegeneration in depressed versus controls. Methods: 54 subjects (35 depressed and 19 non-depressed control subjects) participated in the study and provided CSF samples and clinical and demographic information. Study participants were aged 35-65 and unmedicated with psychotropic medications. No patient was taken off medications for the sake of the study. CSF immune markers were assessed using methods described previously (Felger et al., 2020). CSF neurodegeneration markers were assayed using SIMOA assay on the Quanterix platform (Rissin et al., 2010). The immune marker panel included c-reactive protein (CRP), tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1beta, and their circulating receptors [type 2 TNF (TNFR2), IL-6 (IL6sr), and IL-1 receptor antagonist (IL1ra). The neurodegeneration panel included neurofilament light chain protein (NFL), glial fibrillary acidic protein (GFAP), hyperphosphorylated tau-181 (Tau), abeta-(ab)42 and ab40. Ab42:40 ratio was used to differentiate neurodegenerative from immune effects on amyloid metabolism. Extended regression analyses were used to examine the relationship between CSF immune and neurodegeneration markers across groups, considering subject characteristics as covariates, including age, sex, body mass index, 10-year vascular risk, race, and educational, marital, and occupational status. In addition, structural equation modeling (SEM) was used for mediation analysis. Results: Of the inflammatory markers, CSF TNFR2 was differentially associated with neurodegeneration markers as a function of depressed group status. Indeed, there was a significant CSF TNFR2 by depressed group interaction that was positively associated with CSF NFL (cf=0.51, p = 0.015), CSF GFAP (cf=0.90, p < 0.001), CSF Tau (cf=0.50, p = 0.013) and negatively associated with CSF ab42:40 ratio (cf = -0.55, p = 0.005). No CSF TNFR2 by control group interactions were significantly associated with neurodegeneration markers. Mediation models indicated that CSF TNFR2 indirectly mediated the negative effect of age on CSF Abeta42:40 (cf = -0.25, p = 0.014); and the positive effect of age on CSF Tau (cf=0.28, p = 0.009), CSF GFAP(cf=0.22, p = 0.02), and CSF NFL (cf=0.42, p < 0.001). Conclusions: Ours is among the first studies to concurrently examine CSF markers of neuroinflammation and neurodegeneration in depressed and control subjects. We focused on depressed patients aged between 35 and 65 due to the confluence and build-up of neurodegenerative risk factors in midlife (Marsland et al., 2015). CSF increases in TNFR2 were associated with increases in markers of neurodegeneration in depressed but not control subjects. Profiling proinflammatory activity compounded by the combined risk of aging and depression may better predict the risk of neurodegeneration. While sex was included as a covariate, this study was not powered to detect sex differences. Treating the risk of neurodegeneration among aging depressed patients is of great interest. Treatment with immune-modulating or neuroprotective agents in addition to well-known antidepressants may be useful in this group. Keywords: Neuroinflammation, Neurodegeneration, Major Depression, Alzheimers, Late-Life Depression Disclosure: Nothing to disclose. P317. Relationship Between Interleukin-6 (IL-6) Blood Levels and Treatment Outcome in Patients With Treatment-Resistant Bipolar Disorder Depression (TRBDD) Evan Sitar, James Sinacore, Angelos Halaris Loyola University School of Medicine, Maywood, Illinois, United States Background: Immune system activation and inflammatory response have been increasingly linked in the pathophysiology of psychiatric disorders. It has been proposed that stress is associated with sustained activation of the innate immune system, leading to the release of pro-inflammatory cytokines in the nervous system (Haapakoski et al., 2015). Recent literature has identified a potential relationship between alterations in the cytokine network and depression in bipolar disorder (BD) (Halaris et al., 2021). Elevated expression of pro-inflammatory cytokine IL-6 has been positively associated with the subject’s experience of depressive and anxiety symptoms, particularly in BD depression (Muneer 2016), and has been proposed to reflect symptom severity and likelihood of treatment response. Furthermore, specific single nucleotide polymorphisms (SNPs) have been shown to influence IL-6 expression and may predispose individuals to depression (Sundaresh et al., 2019). Understanding depressive illness in the context of inflammation triggered by stress may aid in intervention. For the present study, we hypothesized that depression in BD will be accompanied by an elevated blood level of IL-6, and that this elevation correlates positively with the illness severity in BD. Finally, we predicted that the addition of the cyclooxygenase-2 inhibitor and anti-inflammatory celecoxib (CBX), in conjunction with escitalopram (ESC), a selective serotonin reuptake inhibitor, will reduce IL-6 blood levels and improve treatment outcomes in BD patients with depression. We also identified carriers of a particular SNP (rs4553185) among the study’s BD cohort and anticipate this SNP will influence IL-6 expression. Methods: Data was derived from forty patients that completed a 10-week, randomized, double-blind, two-arm, placebo-controlled trial examining the efficacy of CBX combined with ESC in treating TRBDD (Halaris et al., 2020). Both sexes were included. Patients were selected after they met criteria for TRBDD, which included failure to reach remission following 8 weeks of two or more adequate trials of antidepressant drug treatment (Hidalgo-Mazzei et al., 2019). Subjects that met criteria underwent a physical exam, medical history, laboratory tests, and completed several assessment scales. The severity of depressive symptomatology was assessed via the Hamilton Rating Scale for Depression (HAM-D) and Hamilton Anxiety Rating Scales (HAM-A), Beck Depression Inventory, and Beck Anxiety Inventory at Baseline, as well as at Weeks 1, 2, 4, and 8. Patients scoring 18 or higher on the HAM-D 17 scale were randomized and treated with either CBX and ESC, or ESC alone for 8 weeks. Blood samples were obtained via venipuncture for IL-6 at Baseline, Week 4, and Week 8. Patients were either excluded by an inability to adhere to visit schedule or due to having incomplete data sets. An Independent Sample t-test was used to compare IL-6 values between BD patients and Healthy Controls. Depression and anxiety were extracted from assessment scales, and both Pearson correlations and partial correlations were used to examine the relationship between scores and IL-6 levels. Comparison of IL-6 values between CBX + ESC and ESC treatment groups, as well as with respect to treatment response or remission status, were conducted using an analysis of covariance. Results: There was a statistically significant difference (p = 0.007) between mean Baseline IL-6 values in the BD group (Mean ± standard deviation) (1.51 ± 1.30) (n = 43) and the Healthy Control group (0.94 ± 0.69) (n = 53). No clinical associations were found between IL-6 levels and HAM-D, HAM-A, or other assessment scores. After controlling for Baseline IL-6, comparing the change in IL-6 values from Week 4 to Week 8, as well as the difference between Week 8 IL-6 values, rendered no statistically significant difference between BD patients treated with ESC (n = 15) and BD patients treated with ESC and CBX (n = 25). Outcome of treatment was categorized into treatment response when patients had a 50% or less reduction in HAM-D 17 score, or higher score than baseline, and into treatment remission when patients had a HAM-D 17 score ≤7. When BD patients were identified in terms of treatment outcome, the adjusted mean Week 8 IL-6 values steadily decreased (p = 0.074) when looking from no response (Adjusted Mean ± standard deviation) (1.65 ± 0.20) to treatment response (1.54 ± 0.25), and finally to treatment outcome (1.06 ± 0.17). We also assessed carrier status of the SNP (rs4553185) and distinguished the BD patient cohort between carriers (n = 35) and non-carriers (n = 6) of the SNP. Results will be presented in the poster. Conclusions: The significantly elevated Baseline blood levels of IL-6 in BD patients suggests an elevated expression of pro-inflammatory cytokines in patients with depression. This finding supports the hypothesis that immunological dysregulation may play a role in depressive illness. The observation that Week 8 IL-6 values decreased as treatment outcomes improved suggests that IL-6 blood levels may have the potential to predict treatment response. While there was no correlation between IL-6 levels and severity of depression or anxiety, nor was there a difference between IL-6 values in either treatment group, recruiting a larger sample size and extending the duration of treatment may impact these findings and accentuate the role that IL-6 plays in depression. Keywords: Bipolar Disorder, Treatment-Resistance, Depression Inflammation Cytokine, Celecoxib, Escitalopram Disclosure: Nothing to disclose. P318. Assessment of Complement Cascade Components and Ketamine’s Mechanism of Action in Patients With Treatment Resistant Depression Brandi Quintanilla, Ashutosh Tripathi, Alona Bartosh, Peixiong Yuan, Dede Greenstein, Carlos Zarate, Anilkumar Pillai University of Texas Health Science Center, Houston, Texas, United States Background: Over 300 million people world-wide suffer from major depressive disorder (MDD). Unfortunately, only 30-40% of patients with MDD receive remission after conventional monoamine antidepressant therapy. In recent years, ketamine has revolutionized the treatment of MDD, with its rapid antidepressant effects manifesting within a few hours as opposed to weeks with conventional antidepressants. Many research endeavors have sought out to identify Ketamine’s mechanism of action in mood disorders, with several studies implicating ketamine’s role in neuroinflammation regulation. The complement system is an important component of the innate immune response vital for the regeneration processes, including neurogenesis. The complement pathway has been implicated in the pathophysiology of depression and studies have shown significant increases in Complement component 3 (C3) expression in the PFCs of depressed suicide subjects. Given complement’s role in depression, ketamine’s/complement’s ability to modulate glutamatergic transmission, and the fund of research highlighting ketamine’s anti-inflammatory properties; there is reason to suspect an overlay between the complement system pathway and ketamine’s mechanism of action. To investigate this, we hypothesized an increase in baseline complement system levels that are subsequently attenuated by ketamine administration at varying time points. Methods: Thirty-nine unmedicated individuals with MDD (23 F) and 25 healthy volunteers (HVs, 16 F) participated in a randomized, double-blind trial comparing intravenous ketamine (0.5 mg/kg) to placebo. Blood was obtained at baseline and at three post-infusion timepoints (230 minutes, Day 1, and Day 3). Plasma was then aliquoted into cryotubes and stored at -80 °C until thawed for assay. In this secondary analysis from a placebo-controlled double-blind inpatient crossover ketamine trial, C3a levels were determined by ELISA. Due to notable skew in our data, we log transformed C3 values. We used a linear mixed model with C3 (log ng/ML) as our outcome and included fixed effects of drug (KET, PBO) and drugtime to test our hypotheses. Models also included time as a main effect, sex, age, and baseline C3 as covariates, and a random intercept per person. Results: We did not detect overall drug differences or differences on C3a levels at any of the time points. The model adjusted overall drug effect was (collapsed over time) (Ket - Pbo = -0.008 (SE = 0.04) t = -0.203 (df=286), p = 0.84). The Ketamine/Placebo difference at each time point was 230 min: Ket - Pbo = -0.0517 (SE = 0.065) t = -0.795(df=282), p = 0.4271; Day 1: Ket – Pbo = -0.0250 (SE = 0.0652), t = -0.384 (df=279), p = 0.7014; and Day 3: Ket - Pbo 0.0531 (SE = 0.0695), t = 0.764 (df=278), p = 0.4458. Conclusions: Our findings did not show a significant effect of ketamine on plasma C3a levels. Additional analyses on other complement proteins and their association with inflammatory markers are in progress. Keywords: Treatment Resistant Depression, Ketamine, Complement Pathway Disclosure: Nothing to disclose. P319. Serum MCP-1 Protein Levels Correlate With Depression Symptom Severity and Predict Relapse Status in Major Depressive Disorder Qingqin Li, Randall Morrison, Maggie Fedgchin, Vanina Popova, Wayne Drevets Johnson and Johnson Pharmaceutical, Titusville, New Jersey, United States Background: Major depressive disorder (MDD) is an episodic condition with a relapsing and remitting disease course. Neuroimmune mechanisms have been associated with the pathophysiology of depressive symptoms in subgroups of patients with MDD. The current study investigated the correlation between immune-related protein levels and depression symptom severity and their association with future relapse/recurrence episodes. Methods: Serum samples were collected from participants with MDD drawn from an observational clinical study OBSERVEMDD0001 (ClinicalTrials.gov Identifier: NCT02489305, n = 422) and three interventional clinical studies SUSTAIN-1 (NCT02493868, n = 462), TRANSFORM-1 (NCT02417064, n = 320) and TRANSFORM-2 (NCT02418585, n = 420). Immune-related proteins were assayed using MRBM InflammationMap protein panel v1.0 consisting of 46 protein analytes. Samples from multiple time points were included in the analysis correlating protein levels with depression symptom severity as measured by Montgomery Asberg Depression Rating Scale (MADRS). Protein analyte level was subject to BoxCox transformation to approximate a normal distribution and Protein analytes with > 80% data missing rate were removed from the analysis. Samples with protein analyte below the lowest limit of detection (LLOD) were set to missing data. A linear mixed model adjusting sample storage age, assay batch (where applicable), subject age, sex, and symptom severity score was fit to assess the relationship between protein levels and depression symptom severity. For the relapse/recurrence analysis, only the “baseline” stable samples from OBSERVEMDD0001 and SUSTAIN-1 were used. For OBSERVEMDD0001, the “baseline” samples when participants were clinically stable (MADRS ≤ 14) were included in the analysis. For SUSTAIN-1, the week 16 samples after subjects achieved stable remission or stable response (MADRS ≤ 12) at the end of the optimization phase but before randomizing to either continuing esketamine plus antidepressant or switch to placebo plus antidepressant for the maintenance phase were used. In the OBSERVEMDD0001 study, subjects were followed up for up to 2.8 years during which patients continued antidepressant treatment. Clinical evaluations were performed at regular time intervals to detect relapse/recurrence events. Proteins associated with relapse/recurrence in each study/treatment arm were analyzed separately. Meta-analyses were also performed by type of antidepressant or disease population (treatment-resistant depression (TRD) and non-TRD). Results: ~28 protein analytes were detectable in >80% of the samples. Interleukin 12 subunit p40 (IL12p40) and MCP-1 were associated with MADRS symptom severity score in OBSERVEMDD0001 (β = -6.56×10-3, p = 0.0001, adjusted p-value = 0.003 for IL12p40, and β = 2.38×10-2, p = 0.005, adjusted p-value = 0.07 for MCP-1). The MCP-1 correlation with MADRS score was also observed in both TRANSFORM-1 (β = 5.10×10-3, p = 0.03) and TRANSFORM-2 (β = 4.54×10-3, p = 0.06) studies, but not in SUSTAIN-1. IL12p40 finding was not significant however in other studies. For the relapse analysis, stable “baseline” samples from 63 relapsers and 154 non-relapsers were retained for OBSERVEMDD0001. Nine proteins were nominally associated with relapse status during follow-up (p < 0.1). For SUSTAIN-1, samples from in the esketamine arm (21 relapsers and 34 non-relapsers) and in the placebo arm (33 relapsers and 48 non-relapsers) were retained in the analysis. Five proteins and 1 protein from esketamine and placebo arm, respectively, were nominally associated with relapse status during the maintenance phase (p < 0.1). Meta-analysis across the two studies (three separate analyses) identified monocyte chemotactic protein 1 (MCP-1) to be consistently predicting relapse status among the three analyses (p = 0.003, adjusted p-value = 0.09). Conclusions: These results are noteworthy given evidence that chemokines play major roles in mediating interactions between immune cells in the periphery and those within the CNS. We provide evidence that higher MCP-1 levels are associated with worse depression symptom severity scores, which is consistent with a recent meta-analysis indicating that MCP-1 is elevated in depressive subjects compared to healthy controls. Furthermore, baseline MCP-1 is associated with the relapse disease trajectory. Keywords: MDD, MCP-1/CCL2, Relapse, Depressive Symptoms Disclosure: Janssen Research and Development, LLC: Employee (Self), Johnson and Johnson: Stock / Equity (Self) P320. Improvement in Depression and Fluid Cognition Following Accelerated Theta Burst Stimulation for Treatment-Resistant Major Depressive Disorder in Autism Spectrum Disorders Elizabeth Blank, Steve Wu, Donald Gilbert, John Sweeney, Elizabeth Smith, Lauren Schmitt, Rebecca Shaffer, Meredith Will, Travis Larsh, Paul Horn, Kelli Dominick, Craig Erickson, Ernest Pedapati Cincinnati Children’s Hospital, Cincinnati, Ohio, United States Background: Major depressive disorder (MDD) disproportionately affects individuals with autism spectrum disorders (ASD). MDD is a leading cause of functional impairment, social withdrawal, and suicide. Tragically, it is estimated that individuals with ASD have a 4X greater risk of developing MDD and a 9X greater risk of completed suicide. Despite this urgent need, intervention research specifically targeting MDD in ASD is scarce. No medication trials have focused on MDD in ASD. Standard-of-care medications for mood disorders, such as selective serotonin reupdate inhibitors, may be unpredictable or ineffective in individuals with ASD. Repetitive transcranial magnetic stimulation (TMS) is an efficacious, non-pharmaceutical treatment for MDD with strong meta-analysis support, including over 1,000 patients from randomized controlled trials (RCT), and is covered by most insurers. However, repetitive TMS therapy involves 40-minute sessions of high-intensity pulses over a period of 4 to 6 weeks. The lack of evidence in ASD, duration of therapy, and sensory concerns have severely limited the use and scalability of TMS in the ASD population. The study team is highly experienced in using TMS and has piloted an innovative accelerated low-intensity TMS protocol optimal for MDD in ASD individuals. Methods: This is an interim analysis of an in-progress intervention trial of accelerated theta burst stimulation (aTBS) on MDD in ASD (see NCT05271357). The primary outcome was Hamilton Depression Rating Scale (17-item; HDRS), and the secondary outcome was the NIH Cognitive Toolbox. Exploratory outcomes involved EEG connectivity. The study design is a randomized, active-comparator study of unilateral or bilateral dorsolateral prefrontal cortex (DLPFC) aTBS stimulation (including mandatory two-week lead-in and waitlist control groups). ATBS sessions are administered 3X daily for ten days (30 sessions). The intervention was delivered using a Magstim Horizon (Magstim/EGI, Whitland, UK) using a 70 mm figure-eight EZ cool coil. Resting motor threshold was determined with single-pulse TMS and electromyography. Theta Burst Stimulation: Participants received intermittent theta burst stimulation (50 Hz in 5 Hz bursts) for a total of 600 pulses total at 90% of RMT for each session. We used the Beam F3 localization method. EEG recordings are acquired using Netamp 400 amplifier (MagstimEGI, Eugene, OR) and 128-channel Hydrocel nets in sound-attenuated booths. We performed source estimation using Brainstorm to compute L2-normed, depth-weighted minimum norm estimation source model to generate a current source density (CSD) map to reconstruct time series activations. Standard weighted alpha phase lag index connectivity measures of left prefrontal to right anterior cingulate cortex were estimated for each subject. Statistics: As this is an interim analysis, we examined the effect of aTBS (unilateral and bilateral) on outcome measures. For our primary outcome, we examined HDRS change from baseline immediately, and 4-weeks post-treatment using FDR-corrected paired t-tests. Secondary variables were compared using t-tests. The relationship between changes in frontocingulate connectivity and change in HDRS score was explored using Pearson correlation. Results: The analysis below reflects the seven subjects who have completed the aTBS treatment and have had at least a 4-week follow-up visit at the time of abstract submission. Since November of 2021, we have screened 14 subjects (2 females), two subjects on the waiting list, one subject scheduled for treatment, and one subject who withdrew before treatment. Our pilot demographics include 57% Caucasian and 14% African American, Hispanic, and Asian. Primary outcome: Mean baseline HDRS (M = 20.3, SD = 3.5) was significantly reduced immediately following treatment (M = 7.0, SD = 2.8, t = 10.5, FDR p = 4.4×10-5, Cohen’s d = 3.9, n = 7) and sustained at Week 4 (M = 5.4; SD = 2.7; t = 9.7, FDR p = 6.9×10-5, Cohen’s d = 3.7, n = 7). Secondary outcome: Baseline Fluid Cognition composite score (M = 43.1, SD = 4.7) significantly improved at Week 4 following aTBS intervention (M = 51.4, SD = 6.6; t = -3.1, p = .02, Cohen’s d = 1.18, n = 7). Exploratory: EEG source analysis was only available on five subjects at the time of the abstract. Reduction of prefrontal to cingulate connectivity following aTBS was predictive of improved HDRS score (R = .82, p = .09, n = 5). Conclusions: We present early but promising results of using accelerated TBS in a diverse sample of individuals with ASD and MDD. We observed significant improvement in MDD symptoms leading to remission but also improvements in objectively measured Fluid Cognition. We have identified a potential neural biomarker suggesting that left prefrontal (stimulation site) and cingulate connectivity are associated with depression symptoms. The availability of a 10-day non-pharmaceutical intervention for rapid remission of MDD would be highly impactful. The negative sequelae of MDD are particularly devastating, exacerbated by the lack of evidence-based treatments and atypical responses in ASD. A TMS protocol optimized for MDD in ASD, which can be used with commercially available TMS machines, would greatly improve access to care, reduce the need for specialists, and provide rapid recovery as a mid-level option between medications and electroconvulsive therapy. Keywords: Autism and Depression, Theta Burst Transcranial Magnetic Stimulation, Electroencephalography Disclosure: Nothing to disclose. P321. Acute rTMS Effects on EEG-Based Functional Cortical Networks Camila Cosmo, Amin Zandvakili, Thaise Toutain, José Garcia Vivas Miranda, Noah Philip Brown University, Center for Neurorestoration and Neurotechnology, Providence VA Medical Center, Providence, Rhode Island, United States Background: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that generates electrical current in the cortex through magnetic pulses, modifying brain networks. Several studies have shown the effectiveness of rTMS in neuropsychiatric disorders, including pharmacoresistant depression. However, these previous trials have mostly indicated therapeutic and neurophysiological effects following a long-term treatment course. Identification of brain-based biomarkers of early therapeutic response remains an important unanswered question in the field. Here we used a novel graph-based analysis method, called Functional Cortical Networks (FCN), to examine electroencephalograpy (EEG) data, hypothesizing that changes would occur early in the rTMS treatment course. Methods: Resting-EEG activity was measured in fifteen patients with pharmacoresistant depression following five rTMS sessions (5 Hz, 120%MT, 3,000 pulses/session, left dorsolateral prefrontal cortex). Ten minutes of eyes closed; 64-channel EEG was recorded at baseline (pretreatment; T0) and following five rTMS sessions (T1). An FCN model was constructed by applying time-varying graphs and motif synchronization. Each EEG electrode position was identified as a node, and the synchronization between electrodes is called edge. The main outcome was the weighted-node degree; this parameter reflects how many times the nodes (i.e., EEG electrodes) were synchronized during the EEG acquisition period. Analyses were performed using the EEGLAB/MATLAB software system (Mathworks, Inc.). Results: A paired t-test showed a significant acute effect of rTMS over the left posterior area, with an increase of 37.825 in the weighted-node degree after five rTMS sessions (95% CI, 468 to 75.181); and marginally significant at the left frontal region (95% CI, -1.663 to 111.981). No other significant changes were observed when hemispheres and additional regions were analyzed (p ≥ .05). Conclusions: FCN models might work as a sensitive measure of acute changes in neural mechanisms underlying therapeutic rTMS, as indicated by these pilot findings. Five rTMS sessions were enough to evoke higher synchronization between electrodes in the left posterior and frontal areas, with a statistically significant increase in the first. Our data are accordant with prior trials that have shown that increased left frontal activity might be linked to decreased negative affect, while increased central and posterior activity is thought to reflect increased arousal, which may be involved in reduced fatigue and increased motivation. Based on our findings, FCN models may contribute to further understanding of acute mechanisms underlying rTMS treatments. Future examination will investigate whether early EEG changes can serve as a potential predictor of therapeutic rTMS response. Keywords: Repetitive Transcranial Magnetic Stimulation (rTMS), Functional Brain Network, Treatment Resistant Depression, Non-invasive Neuromodulation Disclosure: Nothing to disclose. P322. Subcallosal Cingulate Deep Brain Stimulation for Treatment-Resistant Depression: Findings From a Double-Blind Randomized Controlled Trial and Open-Label Extension Phase Sidney Kennedy, Peter Giacobbe, Sakina Rizvi, Amanda Ceniti, Rima Styra, Andres Lozano St. Michael’s Hospital, Toronto, Canada Background: Deep brain stimulation (DBS) to subcallosal cingulate (SCG) white matter has been evaluated in ‘Treatment-Resistant Depression’ (TRD) with mixed findings. Despite a large number of positive findings in open-label studies, randomized controlled trials (RCTs) have so far not supported these results. The primary objective of this study was to compare the efficacy of active vs. sham DBS stimulation to the SCG in reducing depressive symptoms over a 6-month randomized, double-blinded phase, followed by open-label evaluation of outcomes for a further 18 months. Methods: Thirty-three male and female patients with TRD were enrolled at Toronto Western Hospital, University of Toronto between 2010 and 2016 in this randomized, placebo-controlled double-blind crossover trial. Participants had a current Major Depressive Episode of more than 12 months duration and had documented resistance to at least 4 adequate depression treatments from a minimum of 3 treatment categories in the current episode. Thirty-one of these patients subsequently underwent DBS implantation. Using a Balaam crossover design, participants were randomized to one of four stimulation sequences during two consecutive 3-month treatment phases: ON-ON, ON-OFF, OFF-ON, or OFF-OFF. The primary RCT endpoint was change in Hamilton Depression Rating Scale (HDRS-17) from baseline to 3 and 6 months, with a reduction of 5 points representing a minimum clinically important difference. Response rate was calculated as ≥50% reduction in HDRS-17 score from baseline. At the end of the 6-month randomized phase, all participants received active stimulation for an additional 18 months in an open-label extension phase. Results: In the double-blind phase, no statistically significant treatment effect was observed for either intent-to-treat (p = 0.4725) or as-treated (p = 0.7397) analyses, using a linear random-effects model. All four treatment groups demonstrated a reduction in HDRS-17 scores at 3 and 6 months, but this did not significantly differ by stimulation status. No unanticipated adverse device effects or deaths were reported. In the subsequent open-label extension phase, significant reductions in HDRS-17 scores from baseline were observed at 12-, 18-, and 24-months post-implantation (all p < 0.0001) across the full sample. Response rates at 12, 18, and 24 months were 64.5% 63.6%, and 71.0%, respectively. Significant and progressive improvements in functional impairment, as measured by the Sheehan Disability Scale, were also observed at 12 months (p = 0.002), 18 months (p = 0.0003), and 24 months (p < 0.0001) post-implantation. Conclusions: The double-blind RCT phase failed to demonstrate a statistically significant difference between active and sham stimulation. In the open-label phase, the majority of participants responded to DBS, a particularly noteworthy finding in this TRD population. There are several potential explanations for these results: first, the Balaam crossover design may have resulted in an inadequate duration to detect a treatment effect. Second, this design led to a reduced sample size for each treatment condition. Third, the improvements observed with sham stimulation may be related to microlesion (insertion) effects, or finally, due to frequent visits with the study psychiatrist. This study also supports the safety of DBS to SCG for TRD, with reported adverse events consistent with those previously reported in DBS trials. Keywords: DeepBrain Stimulation, Treatment-Resistant Depression, Randomized Balaam Crossover Design, Open-Label Follow-up Disclosure: Abbvie: Consultant (Self), Boehringer-Ingelheim: Advisory Board (Self), Janssen: Consultant (Self), Janssen: Contracted Research (Self), Lundbeck: Consultant (Self), Lundbeck: Contracted Research (Self), Lundbeck Institute: Consultant (Self), Merck: Consultant (Self), Otsuka: Other Financial or Material Support (Self), Otsuka: Consultant (Self), Pfizer: Contracted Research (Self), Sunovion: Consultant (Self), Servier: Consultant (Self), Brain Canada: Grant (Self), CIHR: Grant (Self), Ontario Brain Institute: Grant (Self), Strategy for Patient-Oriented Research (SPOR): Grant (Self), Field Trip Health: Stock / Equity (Self), Abbott: Other Financial or Material Support (Self) P323. The Astrocyte Protein, S100B’s Potential Role as a Predictor of Treatment Outcome of Transcranial Magnetic Stimulation for Depression Andrew Fukuda, Lauren Hindley, Eric Tirrell, Linda Carpenter Brown University, Butler Hospital, Providence, Rhode Island, United States Background: Transcranial Magnetic Stimulation (TMS) is effective for Major Depressive Disorder (MDD) but the therapeutic mechanism of action is not fully elucidated and there is no biomarker used to inform clinicians and patients of the likelihood of a favorable clinical outcome. Astrocytes have been implicated to play role in depression pathophysiology but have not been explored in TMS. S100 Calcium Binding Protein (S100B) is ubiquitously and selectively expressed in the astrocytes and plays a variety of functions including neuroplasticity and neuroinflammation and has been shown to be increased in the serum of depressed patients compared to non-depressed controls and holds promise as a clinical biomarker in TMS. We provide preliminary data showing association of peripheral levels of S100B and clinical outcome. Methods: A prospective observational study at an outpatient TMS clinic from consenting patients consisting of a collection of behavioral measures and serum at pre and post TMS treatment (standard clinical treatment targeting left DLPFC daily for 6 weeks) were carried out. The Inventory of Depressive Symptomatology (IDS-SR) as a measure of overall depressive symptom severity. Serum enzyme linked immunosorbent assay (ELISA) for S100B was performed. A repeated measures general linear model (GLM) was performed to determine whether change in S100B concentration differed significantly between those who achieved clinical remission and those who did not with time (pre vs post) as the within-subjects factor and clinical outcome (remission vs non-remission) as a between-subjects factor, with gender and age as covariates. A binary logistic regression was performed to ascertain the effects of age, gender, baseline depression score, and baseline S100B concentration on the likelihood that participants will achieve remission. Results: 66 patients (26 Males, 44 Females) had pre and post IDS-SR and S100B concentrations. The remission rate was 44%. GLM showed no statistically significant effect observed with time but there was a statistically significant difference between remitters and non-remitters (F = 4.641, p < 0.05), with a post hoc t-test showing remitters to have lower S100B concentration at baseline (p < 0.05, cohen’s d = 1.213) and post-treatment (p < 0.05, d = 0.445) compared to non-remitters. The logistic regression model was statistically significant, χ2(4)=11.031, p < .05. The model explained 20.6% (Nagelkerke R2) of the variance in achieving clinical remission and correctly classified 65.2% of the cases. Lower S100B concentration prior to treatment course was associated with increased likelihood of achieving remission. Conclusions: Although S100B did not show a significant change with TMS treatment, those who achieved clinical remission had a lower concentration compared to non-remitters at both timepoints. Furthermore, lower S100B level at baseline was associated with increased likelihood of achieving clinical remission and holds promise as a predictive biomarker of clinical outcome. Studies are underway to look at specific symptomatic domains that S100B may be associated with in depression. Keywords: Astrocytes, Biomarker, Repetitive Transcranial Magnetic Stimulation (rTMS), Natural Setting, Treatment Resistant Depression Disclosure: Nothing to disclose. P324. TMS-EEG Indices as High Potential Predictive Markers of iTBS Response in MDD Rebecca Strafella, Reza Zomorrodi, Jennifer Lissemore, Yoshihiro Noda, Zafiris Daskalakis, Daniel M. Blumberger, Daphne Voineskos Centre for Addiction and Mental Health, Toronto, Canada Background: Intermittent theta-burst stimulation (iTBS) to the L-DLPFC is effective for treatment-resistant depression (TRD). We indexed transcranial magnetic stimulation-electroencephalography (TMS-EEG) markers, the N45 and N100 amplitudes as well as overall TMS evoked potential activity (GMFA-AUC) at baseline and post-iTBS, comparing separated and contiguous iTBS schedules. TMS-EEG markers were also compared between iTBS responders and non-responders. Methods: TMS-EEG was analyzed from a triple-blind 1:1 randomized trial for TRD, comparing a separated (54 min interval) and contiguous (0 min interval) schedule of 2×600 pulse iTBS for 30 treatments. Participants underwent TMS-EEG over the L-DLPFC at baseline and post-treatment. 114 participants had usable TMS-EEG at baseline, and 98 at post-treatment. TMS-evoked potential (TEP) components (N45, N100) were examined via global mean-field analysis. Results: The N100 amplitude decreased from baseline to post-treatment regardless of treatment group (F(1, 106.02 = 5.20, p = 0.02). There were no changes in N45 amplitude in either treatment group. In responders, the N100 amplitude decreased after iTBS (F(1,102.13) = 11.30, p = 0.001, pcorrected= 0.0004). Responders showed higher post-treatment N45 amplitude than non-responders (F(1, 94.14) = 4.11, p = 0.045, pcorrected= 0.016). Higher baseline N100 amplitude predicted lower post-iTBS depression scores (F(4, 106) = 6.28, p = 0.00014). Conclusions: These results further the evidence for an association between neurophysiological effects of iTBS and treatment efficacy in TRD in the largest collected pre/post rTMS TMS-EEG sample to date. Future studies are needed to solidify the predictive potential for clinical applications of TMS-EEG markers. Keywords: Theta-Burst Stimulation, TMS-EEG, Treatment Resistant Depression Disclosure: Nothing to disclose. P325. Dose Dependent Effects of Transcranial Photobiomodulation on Brain Temperature in Major Depression Akila Weerasekera, Eva Ratai, Jacek Dmochowski, Katherine A. Collins, Aura M. Hurtado, Luis De Taboada, Maia B. Gersten, Julie A. Clancy, Matthew J. Hoptman, Molly K. Irvin, Allison Sparpana, Elizabeth Sullivan, Xiaotong Song, Arwa Adib, Paolo Cassano, Dan V. Iosifescu Massachusetts General Hospital, Boston, Massachusetts, United States Background: Transcranial photobiomodulation (t-PBM) with near-infrared (NIR) light penetrates the cerebral cortex and is absorbed by the mitochondrial enzyme cytochrome c oxidase (CCO), stimulating the mitochondrial respiratory chain. t-PBM also significantly increases cerebral blood flow (CBF) and oxygenation. Small-scale studies have reported that t-PBM may be an effective treatment in major depressive disorder (MDD). However, t-PBM may affect brain temperature, and those effects are unclear. Therefore, possible excessive brain warming during t-PBM is a concern and must be investigated. In this pilot study we evaluated the dose-dependent brain temperature effects of t-PBM in MDD subjects. Methods: We enrolled 30 adult subjects (age 18-65 years) meeting DSM-5 criteria for MDD, not treatment-resistant (0-2 failed antidepressants in the current episode), either unmedicated or on stable doses of antidepressants, with no other significant medical or psychiatric comorbidities. All subjects underwent three t-PBM sessions with distinct doses (peak irradiance; low: 50, medium: 300, high: 850 mW/cm2, low and medium doses were administered in continuous wave [CW] mode, high dose in pulsed wave [PW] mode) and sham treatment. 1H-MRS data: using a 3 T Siemens Trio MRI scanner, single-voxel (SV) MRS was performed with a PRESS sequence (TE/TR = 30 ms/2 s, dynamic averages = 32×4). A voxel with a volume of 30 mm × 30 mm × 15 mm was placed on the left prefrontal region. Brain temperature (°C) was derived by analyzing the 1H-MRS spectrum chemical shift differences between the water (~4.7 ppm) and NAA (~2.01 ppm) peaks, using jMRUI software’s HLSVD method and Zhu et al formula: Tbrain (°C) = 36 - [103.8 × (ΔH2O − NAA - 2.6759)]. Results: After quality control procedures, the following group numbers were available for both pre- and post- temperature estimations: Sham (n = 10), Low (n = 11), Medium (n = 10), High (n = 8). We did not detect significant post-irradiation temperature differences between any of the tPBM-active or sham groups (unpaired t-test; p value range 0.105-0.781). We also tested for potential differences in the pre-post variability of brain temperature in each group. As for t-PBM active groups; lowest fluctuation (variance) was observed for the medium-dose (σ2 = 0.29), next for the low-dose (σ2 = 0.47) and the highest fluctuation was for the high-dose (σ2 = 0.67). The sham condition showed the overall lowest fluctuation (σ2 = 0.11). Conclusions: Overall our MRS thermometry results show that no significant brain temperature elevations occur during the t-PBM application procedure. The brain temperature variations observed pre- and post- t-PBM sessions were not statistically significant. The lowest temperature variation was observed for medium-dose while highest was for the high-dose. These preliminary results indicate a favorable safety profile for t-PBM treatment with NIR in regard to changes in brain temperature. Keywords: Neuromodulation, Photobiomodulation, Near-Infrared Light, Depression, MR Spectroscopy Disclosure: Niraxx Light Therapeutics Inc: Advisory Board (Self), Niraxx Light Therapeutics Inc: Board Member (Self), Niraxx Light Therapeutics Inc: Consultant (Self), Niraxx Light Therapeutics Inc: Founder (Self), Niraxx Light Therapeutics Inc: Stock / Equity (Self), Niraxx Light Therapeutics Inc: Patent (Self) P326. A Novel Cortical Target for Neuromodulation of Reward Network Activity and Affect Regulation Merage Ghane, Simona Graur, Michele Bertocci, Henry Chase, Tyler Conrad, Alexander Skeba, Rachel Kaskie, Sabine Janssen, Lisa Bonar, Tyler J. Brady, Fabio Ferrarelli, Mary Phillips University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Recurring hypo/manic episodes are a characteristic symptom of Bipolar Disorder (BD) and associated with interfering impulsivity, reward sensitivity, and sensation-seeking. Existing treatments come with severe side effects and high relapse rates, highlighting a need for more effective and mechanistically targeted treatments. Elevated left ventrolateral prefrontal (vlPFC) and ventral striatum (VStr) activity during reward expectancy (RE) has shown associations with hypo/mania risk and dysregulated affective states. While noninvasive neuromodulation of subcortical structures is not yet robustly possible, there are cortical targets, such as the vlPFC, that have direct and indirect projections to RE-relevant subcortical circuitry. We aim to further understand the mechanistic role of the vlPFC in RE-related activity, affect regulation, and hypo/mania to identify novel targets for neuromodulatory intervention. To do this, we are examining how inhibitory continuous (c)TBS to the left vlPFC, compared to left somatosensory (control), and sham differentially impacts RE-related activity and acute affect in BD and healthy adults (18-35 years). We hypothesized that one of the three cTBS conditions would be associated with greater decrease in left vlPFC and left ventral striatum (vStr), and that change in activity would be associated with change in affective state. Methods: To date, 11 adults (73% female) completed all sessions and pre and post-TBS fMRI assessments of RE-related neural activity and affect, using an uncertain probabilistic reward task (Card Guessing Task). We used a priori defined seeds in the left vlPFC and left vStr to test whether one of the three stimulation (stim) conditions showed a greater decrease in RE-related activity from pre-to-post stimulation. Additionally, we used linear regression within stim condition and tested whether change in vlPFC and vStr activity accounted for significant variance in positive and negative affect change from pre-to-post stim. Given that this data is part of an ongoing clinical trial, group and cTBS condition targets are blinded until the study end. Results: Due to continued data collection, we are primarily focusing results on effect sizes to maintain best practices until sufficiently powered for robust statistical tests. One cTBS condition led to greater decreases in RE-related left vlPFC (partial EtaSq = .11) and left vStr (partial EtaSq = .27) activity vs. the other cTBS conditions. For this condition only, change in left vlPFC activity, but not vStr accounted for a significant portion of the variance in negative (B = .763, p = .048) and positive affect change (B = -.657, p = .01). Change in vlPFC activity accounted for 29.9% of the variance (r2 = .299) in negative affect change (after accounting for positive affect change, full model r2 = .559) and 41.6% of the variance in positive affect change (after accounting for negative affect change; full model r2 = .677). Conclusions: The left vlPFC is a promising neural target for neuromodulatory intervention in adults with BD. Preliminary results provide proof of concept and suggest that targeted cTBS reduces RE-related activity in both the cortical left vlPFC and subcortical vStr. However, only reductions in RE-related activity in the vlPFC appear to contribute to the regulation of positive and negative affect. Given that data collection is ongoing, we plan to extend these methods to a larger sample and additionally test for a causal role of the vlPFC in hypo/mania related symptoms and reward sensitivity. Future work should test the transdiagnostic relevance of this approach in conditions impacted by affect dysregulation and/or elevated approach motivation/impulsivity more broadly. Keywords: Theta-Burst Stimulation, Reward Expectancy, Affective Instability, vlPFC, Bipolar Disorder, fMRI Disclosure: Nothing to disclose. P327. Imaging Brain SV2A Density Measured With [11 C]UCB-J μPET as a Readout Of Depression and Antidepressant Response in a Mouse Model of Depression Claire Leroy, Sebastien Goutal, Denis David, Wadad Saba, Maud Goislard, Michel Bottlaender, Fabien Caillé, Phi T Nguyen, Alain Gardier, Rene Hen, Emmanuelle Corruble, Romain Colle, Nicolas Tournier, Indira Mendez-David* Université Paris-Saclay, UVSQ, Centre de Recherche en Epidémiologie et Santé des Populations (CESP), UMR 1018, CESP-Inserm, ORSAY, France Background: The high prevalence of major depressive disorder (MDD) and the varying degrees of antidepressant effectiveness justify investigating the molecular determinants of pathogenesis and therapeutics. Several studies provided evidence of reduced synaptic density and cell loss in rodent models of depression. Antidepressant therapies can rapidly induce synaptogenesis and reverse these neuronal deficits. Estimating synaptic density in the living brain is now possible in positron emission tomography (PET) using novel radiotracers targeting the synaptic vesicle glycoprotein-2-A (SV2A). Recently, synaptic density deficiency has been proven in a small number of high severely depressed patients. However, the effect of an antidepressant drug on synaptogenesis in the human brain remains to be evaluated in vivo. This preclinical study aims to profile using SV2A PET imaging the synaptic density with depression-like phenotype and antidepressant drug responses in a well-validated mouse model based on the elevation of glucocorticoids. Methods: Adult male C57BL/6JRj mice were subjected to chronic corticosterone treatment (CORT, 35 ug/ml) in the drinking water to induce a depression-like phenotype. Mice were thereafter subjected to 4-weeks of selective serotonin reuptake inhibitors (SSRI) antidepressant treatment (fluoxetine, 18 mg/kg/day er) or Vehicle (VEH). Synaptic density was compared between CORT-Fluoxetine (N = 6), CORT-VEH (N = 6) and VEH-VEH (N = 5) using a 90-min PET acquisition after injection of [11 C]UCB-J (3.32 Mbq ± 1.43), a SV2A radioligand. Depression score and antidepressant response were assessed by calculating the latency to feed at the Novelty Suppressed Feeding test, before (at 4 weeks) and after (at 8 weeks) antidepressant treatment. Standardized Uptake values ratio between 60-90 min of PET acquisition (SUVr) of [11 C]UCB-J binding were extracted in cortex and hippocampus and normalized to heart blood using Pmod. Results: [11 C]UCB-J SUVr was decreased in the cortex and hippocampus of CORT-VEH mice compared to VEH-VEH and CORT-Fluoxetine group. [11 C]UCB-J SUV in CORT-Fluoxetine and VEH-VEH groups were not significantly different. Chronic corticosterone (CORT-VEH)-induced increase in depression score was significantly reduced after a 4-week fluoxetine treatment (CORT-Fluoxetine mice) (p = 0.014), confirming antidepressant-like efficacy. Interestingly, we observed an inverse correlation between the depression score and [11 C]UCB-J SUVr in the cortex (p = 0.026). Conclusions: This study suggests that the antidepressant effects of SSRIs may be related to presynaptic terminal restoration. Synaptic plasticity might contribute to the neurobiological substrate of depression and may be a target for the management of therapy efficacy. Further investigations using an immunohistochemical study of synaptogenesis is currently ongoing for a better characterization of the biological meaning of this change in [11 C]UCB-J binding. Keywords: Synaptogenesis, Biomarker, Positron Emission Tomography (PET), Depression, Antidepressant Disclosure: Scientific/Medical Advisory Board Member: Advisory Board (Spouse) P328. Variability in Sleep and Activity While Hospitalized Relate to Post-Discharge Outcomes in Youth Anna Van Meter, Wei Guo, Sun Jung Kang, Kathleen Merikangas NYU School of Medicine, New York, New York, United States Background: Discharge from inpatient psychiatric care is typically based on observed reductions in patients’ externalizing (e.g., manic, psychotic) symptoms and/or resolution of safety concerns related to suicidal or homicidal ideation. However, these markers of discharge readiness are flawed, given high rates of readmission1. Other markers of patients’ mental health, including sleep quality and activity patterns, may provide a more valid assessment of stability and discharge readiness. Importantly, measurement of sleep and activity can be made using wearable devices and is not subject to the same biases as self-report (including motivation to feign wellness in order to go home). We used a wrist-based actigraph, the GENEActiv, to assess stability and quality of sleep and physical activity in adolescent inpatients over the course of hospitalization to determine whether there is an association between these metrics and post-discharge outcomes. We hypothesized that patients who had disturbed sleep and/or inconsistent patterns of physical activity would be more likely to experience clinically significant symptoms one month post-discharge than patients whose sleep and activity are consistent. Additionally, we hypothesized that collecting data with the GENEactiv will prove to be both safe and feasible in the inpatient environment. Methods: All new admissions to an adolescent inpatient unit who were capable of providing informed consent were invited to participate. Participants completed self-report measures of depression (PHQ-9), mania (GBI-10M), and anxiety (GAD-7) at admission, discharge, and one-month post-discharge. Participants wore a GENEactiv for the duration of their hospitalization. Linear regression, controlling for age and self-reported sex (assessed separately from gender), tested associations between change in symptoms (baseline-to-discharge and baseline-to-follow-up) with actigraph-based sleep and activity metrics while hospitalized. Results: There were 108 participants with valid actigraph data. Mean age was 15.0 years old (SD = 1.4), 80% were female. Higher average activity during periods of rest were associated with higher average GAD (β = -1.99, p = 0.007; β = -1.59, p = 0.044) and PHQ (β = -3.25, p < 0.001; β = -2.05, p = 0.030) scores at discharge and follow-up, respectively. Variability in activity during rest periods was also associated with less improvement in GAD (β = -2.82, p = 0.002) and PHQ (β = -4.14, p < 0.001) scores at discharge. Similarly, participants who woke up more frequently during their sleep period had higher GAD (β = -0.33, p = 0.027) scores at follow-up and variability in the number of nighttime awakenings was associated with higher GAD (β = -0.65, p = 0.019) and PHQ (β = -0.92, p = 0.005) scores at discharge. Variability in sleep duration was also associated with less improvement in PHQ (β = -2.44, p = 0.018) from baseline to discharge. Participants with high variability in the difference in their level of activity across the day had less improvement in their PHQ scores (β = -39.91, p = 0.029) from baseline to follow-up. Participants who were more sedentary showed less improvement in their GBI-10M scores (β = -0.02, p = 0.030) at follow-up. No other variables were associated with change in manic symptoms. Surprisingly, greater variability in sleep onset time was associated with improvement in GAD (β = 2.32, p = 0.027) and PHQ (β = 6.40, p < 0.001) scores from baseline to discharge. Only 13 participants required urgent care (ED or hospitalization) post-discharge; this outcome was not associated with sleep or activity. Conclusions: Nighttime agitation and wakefulness were associated with less improvement in depression and anxiety at both discharge and follow-up. Surprisingly, there was no meaningful association between manic symptoms and activity or sleep. The result that sleep onset variability was associated with better outcomes may be related to circadian shifting (i.e., moving from being up all night to sleeping at night) over the course of the hospitalization, rather than day-to-day changes. Future research must evaluate whether stabilization of patients’ sleep/activity over the course of hospitalization, rather than averages and standard deviations, relate to outcomes. If so, these metrics have potential to help determine discharge readiness for patients hospitalized for internalizing concerns. Keywords: Actigraphy, Adolescent, Psychiatric Hospitalization Disclosure: Nothing to disclose. P329. Real-Time Assessment of Positive and Negative Affective Fluctuations and Mood Lability in a Transdiagnostic Sample of Youth Reut Naim, Shannon Shaughnessy, Ashley Smith, Sarah L. Karalunas, Katharina Kircanski, Melissa A. Brotman National Institutes of Health, NIMH, Bethesda, Maryland, United States Background: Emotional lability, or rapid and intense fluctuations in affect, is associated with increased psychopathology and impairment in youth. Although not a diagnostic criterion, emotional lability has been documented in depression, anxiety, and attention-deficit/hyperactivity disorder (ADHD) and is a transdiagnostic risk factor for general psychopathology. However, prior research has primarily relied on retrospective report, focused on negative valanced emotions, and has yet to include participants with Disruptive Mood Dysregulation Disorder (DMDD), a diagnosis which manifests with emotional lability. The present study leverages real-time ecological momentary assessment (EMA) to examine naturally occurring aberrant shifts in affective states in a transdiagnostic pediatric sample. This is the first EMA study that includes participants with DMDD that also explores both negative and positive affective fluctuations. We had two main aims. First, we wanted to compare emotional lability between different diagnostic groups. We hypothesized that youth with psychopathology would have elevated levels of emotional lability when compared to healthy volunteer (HV) children. We also anticipated that DMDD and ADHD participants would experience greater emotional lability than participants with anxiety disorders (ANX), due to emotional lability being an inherent aspect of these disorders. Our second aim was to compare momentary positive and negative affective fluctuations between diagnostic groups. Based on the previous literature, we hypothesized that patients would have more daily affective fluctuations compared to HV participants. In an exploratory manner, we wanted to assess whether fluctuations in negative and positive fluctuations would be associated with functional impairment across the different diagnostic groups. Methods: 130 participants (M = 12.55 years, SD = 2.51 years, 70% males, 65.40% White/Caucasian) with primary diagnoses of DMDD, ADHD, ANX, or HV completed a previously validated one-week EMA protocol for irritability (Naim et al., 2021). Participants rated mood change and affective symptoms three times per day for one week. Items of interest included ratings of positive affect, specifically momentary happiness and giddiness, and negative affect, specifically momentary anxiety, anger, and unhappiness. A composite score for each positive and negative component of emotional lability was generated using an unweighted average of all items in each category. Before compositing, we assessed within- and between-person reliability of the EMA-items using multilevel confirmatory factor analyses (MCFA) which yielded overall medium-to high reliability (range: 0.41- 0.89). To capture within-person variations in affective states over time, a computation of root mean successive squared difference scores (RMSSD) for these composite scores was applied. ANOVAs were conducted to compare affect fluctuations among diagnostic groups and linear regressions were conducted to test for their association with functional impairment, which was assessed by clinician-reported measure (Clinical Global Impressions Severity Scale, CGI-S). Results: Compliance rate with the EMA protocol was high (M = 78.54%, SD = 16.38%), with all groups presenting similar rate. As expected, differences in emotional lability across diagnostic groups were found, with clinical groups exhibiting higher levels of emotional lability compared to HV (all βs > 0.34, ps<0.046). Within the patient groups, DMDD youth demonstrated the most labile and fluctuating negative and positive affect (βs > 0.41, ps<0.049). Emotional lability was associated with global impairment in the whole sample (βs > 0.11, ps< 0.015). Conclusions: Findings provide evidence that emotional lability is a salient and important mechanism to understand in the context of childhood mood disorders, particularly DMDD. Findings also provide evidence that aberrant fluctuations in positive affect may be just as important as aberrant fluctuations in negative affect, and both are related to psychopathology in youth. Additionally, this study supports added advantages of using real-time in-vivo measures to augment classic, retrospective parent/child or clinician clinical assessments. Targeting labile mood in-vivo may also be a potential treatment for DMDD, which is essential as few treatments for the disorder have been developed. As our sample was largely composed of White/Caucasian males, future work should increase sample diversity, to further examine generalizability. Keywords: Emotional Dysregulation, Mood Disorders, Ecological Momentary Assessment, Youth, Pediatric Disclosure: Nothing to disclose. P330. Predicting the Likelihood of Remission With Antidepressant Medication in Depression: A Practical Patient-Level Machine Learning Approach Adina Fischer, Scott Fleming, Kelsey Hagan, Bailey Holt-Gosselin, Alan Schatzberg, Leanne Williams Columbia University Irving Medical Center, New York, New York, United States Background: Less than half of patients with major depressive disorder (MDD) experience symptom remission from their first antidepressant medication (ADM) trial. There is limited empirical data to guide ADM selection for an individual patient. As such, clinicians rely on trial-and-error and evidence from what works for the average patient when treating MDD with first-line ADM, which results in symptom persistence and extended morbidity and mortality for those who do not remit. Using state-of-the-art machine-learning methods, we developed a model that identified patient-specific predictors of likelihood remission (and non-remission) with first-line ADM. Methods: Recursive feature elimination was used to develop a parsimonious (25 feature) gradient-boosted decision-tree model that predicted binary remission (yes/no) from ADM. Features included pre-treatment clinical, demographic, cognitive, and behavioral variables collected from participants (N = 1008) during the baseline visit of the International Study to Predict Optimised Treatment in Depression (iSPOT-D): a randomized, parallel-model, open-label, repeated-measure, longitudinal 8-week trial assessing response to three commonly prescribed first-line ADMs (sertraline, escitalopram, venlafaxine). Remission was defined as a score < 7 on the 17-item Hamilton Depression Rating Scale (HDRS) at a 12-week follow-up. Models were evaluated based on their accuracy and AUROC for predicting remission on a held-out test set using only measurements collected at baseline. Shapley values were used to ascertain which variables most impacted model estimates at the group and individual (patient) levels. Results: The trained model exhibited performance significantly exceeding random chance on a held-out test set (AUROC = .64, 95% CI = .55 to .71, p < .001; Accuracy = .63, 95% CI = .56 to .70, p = .010). Of participants who were identified as being at high risk of non-remission by our model (i.e., predicted probability of remission < 20%), model accuracy was .71. Features identified by recursive feature elimination to be of greatest predictive value include measures of pre-treatment: depression symptom severity, anxiety symptom severity, impaired cognitive function (verbal memory and information processing speed), impaired identification of facial emotions (fear and disgust), and agitation (facial and motor). This model was compared to models using common linear modeling approaches with the same number of features (25). Elastic net regularization yielded poorer performance on the test set relative to our approach (AUROC = .59, Accuracy = .55), as did use of simpler downstream models, such as canonical logistic regression (AUROC = .54, Accuracy = .55) and logistic regression with elastic net regularization (AUROC = .59, Accuracy = .61). Conclusions: Using sophisticated machine-learning methods, we developed a model that identified patient-specific predictors of the likelihood of remission from first-line ADM, which could have powerful clinical implications if implemented in outpatient psychiatry or primary care settings. Moreover, findings illustrate the benefit of using sophisticated machine-learning models (gradient-boosted decision trees) over simple logistic regression, as this method improved both model accuracy and interpretability. Results highlight how variables obtained from standard clinical evaluations may be subjected to machine-learning models to assist in developing individually tailored treatment plans, thereby optimizing outcomes and reducing morbidity and mortality for patients with MDD. Keywords: Antidepressants, Major Depressive Disorder (MDD), Machine Learning, Prediction, Remission Disclosure: Nothing to disclose. P331. TMS Doses Based on Motor Threshold Differ Between DLPFC, OFC, and Motor Cortex: A Case for Electric Field Dosimetry in Clinical Studies Helmet Karim, Ahmad Mayeli, Francesco Donati, Brian Coffman, Daniel M. Blumberger, Noah Philip, Mary Phillips, Carmen Andreescu, Rebecca Price, Fabio Ferrarelli University of Pittsburgh Western Psychiatric Institute and Clinic, Pittsburgh, Pennsylvania, United States Background: Transcranial magnetic stimulation (TMS) is utilized for treating several neuropsychiatric disorders, but outcomes remain variable. The motor threshold (MT) is used to standardize doses but does not account for structural variability in other cortical areas, such as dorsolateral prefrontal cortex (DLPFC) or orbitofrontal cortex (OFC). Electrical field modeling can help estimate those differences. We aimed to compare E-fields of motor cortex, DLPFC, and OFC at MT and 120% of MT, which is the standard dose in most TMS studies. Methods: We collected T1-weighted images and MT from compulsive behavior disorder patients (n = 53) and healthy controls (n = 12). We estimated E-fields over motor cortex, DLPFC, and OFC at 100% and 120% of MT. Separate two-way repeated measures ANOVA with post-hoc t-tests were performed. Results: In the compulsive behavior sample, E-fields at MT in the motor cortex and DLPFC were not statistically different but were greater than OFC. Motor cortex E-fields at MT were less than DLPFC E-fields at 120% MT but was not statistically different when compared to OFC E-fields at 120% MT. A similar pattern was identified in controls. Conclusions: MT may not be an effective standard for dosimetry in TMS with variable dosing depending on the region of interest. E-field modeling can be used to deliver similar doses of TMS across participants or adjust for differences in dosing, thus allowing personalized TMS treatments. Keywords: TMS, Electrical Field Modeling, Depression, Neuromodulation, OCD Disclosure: Nothing to disclose. P332. Deep Phenotyping in Routine Inpatient Psychiatric Care: Methods, Feasibility, Early Results, Potential Applications Agustin Yip, Savannah Layfield, Lucie Duffy, Samantha Wong, Joann Chen, Abdirahman Osman, Fernando Rodriguez-Villa, Steven Gelda, Eliot Gelwan, Philippe Beauchamp, Habiballah Rahimi Eichi, Joshua Salvi, Justin T. Baker, Alisa Busch, Jane Eisen, Kerry Ressler McLean Hospital, Belmont, Massachusetts, United States Background: There is increasing acknowledgment of the need for the integration of ‘continuous telemetry’ and ‘deep phenotyping’ into routine psychiatric care as a way of generating real-world evidence and quantitative objective measures for the comparative effectiveness and assessing heterogeneity of treatment effects of established and novel interventions. Here we report, to our knowledge, the first instance of concurrently deploying electronic health records (EHR), patient-reported outcome measures (PROMs), actigraphy, ecological momentary assessments (EMA), and biomarker and genomic data collection from individuals admitted for depression/suicidality and anxiety to an academic psychiatric inpatient unit. Methods: Participants admitted to depression and anxiety inpatient psychiatric units at McLean Hospital completed a battery of self-assessments (depression: QIDS-SR-16, anxiety: GAD-7, functional status: BASIS-24) and screening instruments (borderline personality disorder [BPD]: MSI-BPD, posttraumatic stress disorder [PTSD]: PCL-5, substance use: DAST-10, alcohol use: AUDIT) within 48 hours of admission and within 24 hours of discharge as part of the hospital’s Clinical Measurement Initiative (CMI), complementing electronic health records (EHR) data. In addition, patients were offered an opportunity to opt into our genomics (Biobank), wearable device (actigraphy), real-time active and passive assessments of suicidality, and context-adaptive multimodal informatics (CAMI -- audiovisual recording of clinician-patient interactions) initiatives as these modalities were rolled out on to the units. This project received IRB approval. Results: Over a two-year period, 753 inpatients completed CMI on admission, 552 (73.3%) of whom also reported depression (QIDS-SR-16) and anxiety (GAD-7) symptom severity, and functional status (BASIS-24), upon discharge from the inpatient hospital stay. A large subset (n = 434, 57.6%) consented to add-on phenotyping, comprising Biobank (n = 301), actigraphy (n = 134), CAMI (n = 24), and suicidality-related assessments (n = 28). Most patients who met inclusion criteria consented to CMI, but uptake was variable for the other phenotyping modalities, reflecting – in part – differences in participant burden and rollout timing. Participants showed robust improvement in depressive (QIDS-SR-16 mean [SD] admission vs discharge 15.6 [5.6] vs 8.7 [5.0]; t(530) = 29.27, p < 0.001) and anxiety (GAD-7 mean [SD] admission vs discharge 13.5 [5.9] vs 6.3 [5.1]; t(529) = 29.46, p < 0.001) symptoms and functional status (BASIS-24 mean [SD] admission vs discharge 37.6 [14.1] vs 21.2 [11.9]; t(542) = 29.42, p < 0.001) during a relatively brief (mean [SD] = 12.6 days [14.9]) acute inpatient admission. There was a similar improvement in suicidality (BASIS-24 thoughts of ending life χ2(10, n = 542) = 300.5, p < 0.001; QIDS-SR-16 thoughts of death and suicide χ2(6, n = 545) = 266.0, p < 0.001) and BASIS-24 self-harm thoughts (χ2(10, n = 548) = 291.7, p < 0.001). Screening positive for BPD on the MSI-BPD was associated with worse depressive-symptom and suicidality severity at both admission (18.6 [4.4] vs 14.3 [5.6] p < 0.001 and 52.2% vs 32.1% severe to very severe suicidality p < 0.001, respectively) and discharge (9.8 [5.4] vs 8.3 [4.8] p = 0.002 and 10.0% vs 7.8% severe to very severe suicidality p < 0.001, respectively), but not with length of stay (13.4 days [16.1] vs 12.2 days [14.5] p = 0.4). Screening positive for PTSD on the PCL-5 was associated with worse depressive-symptom and suicidality severity at admission (19.1 [3.7] vs 13.9 [5.7] p < 0.001 and 50.5% vs 33.7% severe to very severe suicidality p < 0.001, respectively), length of stay (14.7 days [16.1] vs 11.4 days [13.9] p = 0.04), and with depressive symptom severity (10.9 [5.3] vs 7.8 [4.6] p < 0.001) but not suicidality severity on discharge (10.1% vs 8.8% severe to very severe suicidality p = 0.2). QIDS-SR-16 composite sleep disturbance at admission was related to increased reports of ‘thoughts of ending life’ at admission (p = 0.001), and ‘sleeping too much’ on admission was related to ‘thoughts of ending life’ at discharge. QIDS-SR-16 ‘sleeping too much’ was not associated with actigraphy-derived sleep duration measured up to first 5 nights of wear (F(3,58)=0.93, p = 0.4). Sleep duration was not associated with QIDS-SR-16 ‘thoughts of ending life’ at admission (F(4,57)=1.20, p = 0.3) but was inversely associated with ‘thoughts of ending life’ at discharge (F(3,58)=6.57, p = <0.001). Conclusions: The addition of a rotating set of deep phenotyping modalities to EHR data and routine PROM collection is eminently feasible, if challenging, in a well-resourced inpatient setting. Demographic, comorbidity, and actigraphy (sleep) factors modify the effect of inpatient care among those admitted for depression/suicidality and anxiety. We show robust improvements in a broad range of transdiagnostic, standardized and validated outcome measures during a relatively brief inpatient stay. Early data suggest that quantitative sleep actigraphy may complement self-report measures as a robust predictor of outcomes, in particular suicidal thinking, upon discharge. Overall, these data demonstrate the feasibility of deep phenotyping and quantitative assessments, integrated with routine inpatient psychiatric care, to provide real-world evidence for comparative effectiveness and assessing heterogeneity of treatment outcomes. Keywords: Digital Phenotyping, Mood Disorders, Suicidality Disclosure: Nothing to disclose. P333. High-Throughput Platform for the Discovery of Novel Structural Neuroplasticity Modulators Pushpa Kumari, Vasin Dumrongprechachan, Yevgenia Kozorovitskiy Northwestern University, Evanston, Illinois, United States Background: Structural plasticity at the level of dendrites and dendritic spines, mediated by neuronal activity, critically shapes the wiring of mammalian brain circuits crucial for development and behavior. Deficits in dendritic spine plasticity are central to neurological impairments and mental health disorders. Recent studies characterize role of pharmacological neuroactive agents such as ketamine, psilocybin and entactogens in amelioration of these deficits. Despite the extensive research focused on activity-dependent synaptic plasticity mechanisms, the field lacks screening tools to perform accurate quantitation of synaptic plasticity in a high-throughput format. Methods: Here we develop, characterize, scale down, and validate a high-throughput screening platform for dynamic measurements of structural plasticity and overcome the bottlenecks in neuroscience drug discovery. The strength of our platform is based on genetically encoded biosensor designs, where the translation of a high-throughput compatible reporter, Luciferase or Nanoluciferase, fused with PSD95(Δ1.2) is driven under the control of synaptic activity-dependent promoters (Arc, cfos). The readout of these sensors—change in luminescence signal—is a direct, quantitative measurement of local translation and potentiation of dendritic spines. The best performing of 3 distinct sensor designs is further developed the study. Cortical and hippocampal mouse neuronal cultures from males and females are used, although the platform is compatible with neurons derived from human pluripotent stem cells. Each experiment generating images, Western Blot data, or neuroplasticity results includes a minimum of 3 biological replicates, with multiple technical replicates. Repeated measures ANOVA are used for most statistical analyses. Results: Our activity dependent sensors localize in dendritic spine of primary cortical neurons and provide a luciferase readout in response to known positive neuronal activity inducers such as Forskolin (FSK), gabazine and ketamine, in dose dependent manner. Following NIH assay guidelines, the z score (z‘> 0.4, range 0.884-0.925) at two different concentrations of FSK and DMSO control ensures the quality and reproducibility of our HTS platform. Screening ~1280 FDA-approved small-molecules followed by unsupervised cut off analysis provided us with a preliminary list of top hits that may positively affect structural plasticity, including previously known and novel modulators. We are validating new hits further using optically-evoked de novo spinogenesis platform to identify novel plasticity enhancers. Conclusions: Altogether, our platform provides an unbiased high-throughput screening approach that causally associates neuronal activity with an accurate quantitative luciferase readout and allows rapid screening of large numbers of novel neuroactive compounds. This approach should help gain mechanistic insights into signaling pathways for druggable target development for basic neuroscience applications and therapeutics for numerous neurological and mental health disorders. Keywords: Neuroplasticity, Dendritic Spine, High-Throughput Disclosure: Nothing to disclose. P334. Prediction of Antidepressant Response Using Machine Learning and Resting-State EEG Chao Wang, Wei Wu, Akshay Ravindran, Vinit Shah, Maimon Rose, Adam Savitz, Amit Etkin Alto Neuroscience Inc., Los Altos, California, United States Background: Antidepressant medications such as the selective serotonin reuptake inhibitors (SSRIs) are the first line treatment for major depressive disorder (MDD), yet the response rates remain low, and the current practice still relies on trial-and-error. Here, we sought to develop and prospectively validate a machine-learning (ML) model for predicting individual response to SSRI treatment, using 19-channel resting-state electroencephalography (rsEEG) data from two MDD clinical trials (total N = 346). Methods: Data from 93 MDD patients in the SSRI arm of an open-label clinical trial were used for model development. Features extracted from the pre-treatment rsEEG were used to build an ML model to predict change in Hamilton Depression Rating Scale (HAMD17) scores. The model was evaluated with 10-fold nested cross-validation, and additionally prospectively tested on 42 unseen holdout open-label SSRI patient samples. We also assessed the model’s capacity for cross-study prediction by applying the model to the sertraline (N = 99) and placebo (N = 112) arms of a double-blind randomized clinical trial (RCT), respectively. Results: We identified a regularized linear regression model that was significantly predictive of the observed HAMD17 score change in the open label study (r = 0.35, p < 0.001). Applying the model to the holdout samples yielded similar results (r = 0.32, p = 0.019). The model was also generalizable across studies, being predictive of the outcome in the sertraline arm of the RCT (r = 0.28, p = 0.003). Importantly, when applying the model to the placebo arm of the RCT, it failed to predict outcome (r = 0.03, p = 0.375), suggesting that the model was specific to SSRIs (versus placebo). Conclusions: Our findings demonstrated that individual response to antidepressants can be robustly predicted in a specific manner by using EEG and machine learning. The successful prospective replication on the holdout set, and generalization across studies, demonstrates the promise of precision psychiatry approaches. Keywords: Depression, EEG Biomarkers, Machine Learning, Treatment Prediction Disclosure: Alto Neuroscience Inc.: Founder (Self), Alto Neuroscience Inc.: Employee (Self) P335. Toll-Like Receptor 4 Agonist Elicits an Inflammatory Response After in Vivo Immune Challenge in Major Depressive Disorder Leandra Figueroa-Hall, Kaiping Burrows, Jennifer L. Stewart, Ahlam M. Alarm, Chibing Tan, Rayus Kuplicki, T. Kent Teague, Rajagopal Ramesh, Victoria B. Risbrough, Martin P. Paulus, Jonathan Savitz Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Approximately 1/3 of individuals with major depressive disorder (MDD) display inflammation, but the precise mechanisms are not understood. While experimental lipopolysaccharide (LPS; endotoxin) studies have shown behavioral, immunological, and physiological changes in healthy individuals, these studies cannot reveal putative, aberrant inflammatory and regulatory mechanisms in MDD, or distinguish between peripheral and central mechanisms. Hence, a mechanistic approach is required to pinpoint which immunoregulatory mechanisms are defective in MDD. One plausible, mechanistic pathway is the toll-like receptor 4 (TLR4) pathway that upon activation with LPS, produces inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF), which are elevated in some individuals with MDD. Here, we examined immune serum markers to identify peripheral changes, and whether these markers are correlated with TLR4-responsive miRNAs isolated from astrocyte-enriched extracellular vesicles (AEEV). Methods: This preliminary work leveraged a randomized clinical trial (NCT# NCT03142919) involving acute administration of LPS or saline to individuals with MDD and healthy controls (HC). This study was approved by the Western Institutional Review Board, participants’ written informed consent obtained, and carried out in accordance with the principles expressed in the Declaration of Helsinki. MDD (n = 53) and HC (n = 18) were randomized to LPS (0.8 ng/kg) or placebo (saline) and serum samples immunotyped for IL-6 and TNF from baseline (T0), 2 hours (T2), and 24 hours (T24) post LPS/saline infusion using mesoscale discovery. For AEEV experiments, serum was used to isolate total EV, and a GLAST biotin-conjugated antibody used for astrocyte enrichment. AEEV miRNAs from previously published exploratory sequencing data [MDD (n = 8); HC (n = 5)] were examined for correlations with IL-6 and TNF. Analyses were performed in R Studio, version 1.4.1717. Results: Preliminary results revealed statistically significant main effects of visit for IL-6 (F(2,203)= 53.2, p < 0.001, η2g = 0.344) and TNF (F(2,205)= 44.9, p < 0.001, η2g = 0.305), with pairwise comparisons indicating T2 was significantly different from baseline and T24. There was no significant interaction between the effects of diagnosis and visit for IL-6 and TNF, but there was a significant interaction between the effects of drug and visit for IL-6 (F(2,200)= 88.0, p < 0.001, η2g = 0.468) and TNF (F(2,202)= 99.7, p < 0.001, η2g = 0.497). There was also a significant three-way interaction between visit, drug, and diagnosis for TNF (F(2,196)= 3.28, p = 0.04, η2g = 0.032), but not IL-6. Previously published exploratory sequencing data showed statistically significant changes in several AEEV miRNAs including hsa.let.7 f.5p and hsa.miR.374a.5p, which were decreased at T2 and then resolved back to baseline at T24. Here, for the first time, we show that at T2, hsa.let.7 f.5p has a trending positive relationship with IL-6 (r2 = 0.196) and TNF (r2 = 0.183) and hsa.miR.374a.5p with IL-6 (r2 = 0.321) and TNF (r2 = 0.253). Statistics: 1. Immune serum markers were analyzed with general linear models with diagnosis [MDD (n = 53) vs. HC (n = 18)] and drug [LPS (n = 12, HC); (n = 28, MDD) vs. saline (n = 6, HC); (n = 25, MDD)] as factors, visitdrug as the primary interaction, and IL-6 as the primary outcome. The Shapiro-Wilk normality test revealed non-normal data for IL-6 and TNF, therefore data were log transformed. A one-way ANOVA revealed significant main effects of visit on IL-6 (F(2,203) = 53.2, p < 0.001, η2g = 0.344) and TNF (F(2,205)= 44.9, p < 0.001, η2g = 0.305). A two-way ANOVA indicated significant interaction effects between drug and visit for IL-6 (F(2,200)= 88.0, p < 0.001, η2g = 0.468) and TNF (F(2,202) = 99.7, p < 0.001, η2g = 0.497). A three-way ANOVA revealed significant three-way interaction between visit, drug, and diagnosis for TNF (F(2,196) = 3.28, p = 0.04, η2g = 0.032). 2. Correlations were determined for immune markers and AEEV miRNAs, hsa.let.7 f.5p and hsa.miR.374a.5p; [MDD (n = 8); HC (n = 5)]. Here, we show that at T2, hsa.let.7 f.5p has a trending positive relationship with IL-6 (r2 = 0.196) and TNF (r2 = 0.183) and hsa.miR.374a.5p with IL-6 (r2 = 0.321) and TNF (r2 = 0.253). Conclusions: This is the first experimental LPS administration study in depressed individuals. These preliminary findings provide novel insight into LPS-induced inflammatory responses after in vivo immune challenge in MDD and its association to brain-enriched extracellular vesicle miRNAs. In vivo immune challenge elicits temporal-specific inflammatory responses in depressed individuals that trend positively with AEEV miRNAs, which may help elucidate the relationship between peripheral and central mechanisms in MDD. Keywords: Lipopolysaccharide, miRNAs, Astrocyte-Derived Exosomes (ADE), Major Depressive Disorder (MDD) Disclosure: Nothing to disclose. P336. Irritability and Suicidal Ideation in Depressed Individual Across the Lifespan: Clinical Significance and Potential Neurocircuit Mechanisms Manish Jha, Cherise Chin-Fatt, Abu Minhajuddin, Madhukar Trivedi UT Southwestern Medical Center, Dallas, Texas, United States Background: Recent reports have linked irritability to suicidal ideation (SI) in adults with major depression. Here, we seek to (Study 1) evaluate whether the association between irritability and SI differs based on age, and (Study 2) identify the neurocircuit mechanisms of irritability that mediate its association with SI. Methods: Study 1: Individuals in VitalSign6 quality improvement project who had irritability [Concise Associated Symptom Tracking irritability domain (CAST-IRR)]and SI [Concise Health Risk Tracking suicidal thoughts factor (CHRT-SUI)] measures were included N = 2248). Linear regression analysis with an age-by-CAST-IRR interaction was used to evaluate whether association between irritability and SI differed based on age. In this analysis, CHRT-SUI was the dependent variable, age-by-CAST-IRR was the independent variable of interest, and sex, race, and ethnicity were covariates. To interpret the significant interaction, post hoc analyses were conducted where separate linear regression analysis for pediatric (aged 12 to 17 years) and adult (aged 18 to 64 years) groups were conducted with CHRT-SUI as the dependent variable, CAST-IRR as the independent variable of interest and sex, race, and ethnicity as covariates. Study 2: Participants of the Establishing Moderators and Biosignatures of Antidepressant Response in Clinical Care (EMBARC) study with magnetic resonance imaging (MRI), CAST-IRR and CHRT-SUI data available were included (N = 274). Resting-state functional connectivity (FC) among 121 cortical and subcortical regions were computed (n = 7260 FC pairs). Age, sex, race, ethnicity and site were covariates in all regression analyses. Separate linear regression analyses for each of the 7260 FC pair were used to evaluate their association with CAST-IRR, and those with unadjusted p value <0.0005 were then used in mediation analysis. Baron and Kenny approach used to evaluate whether FC pairs accounts for the effect of irritability on SI with age, sex, race, ethnicity and site as covariates. This approach uses three separate linear regression models to evaluate how much of the predictive ability of a variable (irritability) for an outcome (SI) is accounted for by a third variable (FC pair). Results: Study 1: N = 1677 and N = 571 individuals were between ages of 12-17 (pediatric) and 18-64 (adult) years, respectively. The age-by-CAST-IRR interaction was significant (p = 0.0003) where the association between CAST-IRR and CHRT-SUI was higher in pediatric (β = 0.25, SE = 0.03) versus adult (β = 0.13, SE = 0.03) group. Study 2: Fifteen FC pairs were associated with irritability at p < 0.0005 threshold of which nine FC pairs included the striatum. Functional connectivity of dorsal striatum to lingual and superior temporal regions significantly mediated (p < 0.05) the association between symptoms of irritability and SI. Conclusions: Association between irritability and SI was stronger in youths as compared to adults. Dysfunctions within the striatum may mediate this association, and serve as targets for developing novel circuit-specific treatments. Future studies are needed to replicate and extend these findings, especially in youths with depression. Keywords: Irritability, Pediatric Irritability, Neurocircuitry, Depressive Disorders, Resting-state fMRI Disclosure: Acadia Pharmaceuticals: Contracted Research (Self), Janssen Research and Development: Contracted Research (Self), Neurocrine Biosciences: Contracted Research (Self), Navitor/Supernus: Contracted Research (Self), Eleusis: Advisory Board (Self), Eliem/Worldwide Clinical Trials: Consultant (Self), Guidepoint Global: Consultant (Self), Janssen Global Services: Consultant (Self), Janssen Scientific Affairs: Consultant (Self), Medscape/WebMD: Honoraria (Self), Clinical Care Options: Honoraria (Self), NACCME: Honoraria (Self), Global Medical Education: Honoraria (Self) P337. Hybrid Concept Elicitation and Cognitive Debriefing Patient Interviews to Establish Content Validity of the Dimensional Anhedonia Rating Scale (DARS): Anhedonia is a Core Symptom of MDD; PRO are Important to Assess the Patient Voice Stephanie Bean, Rahul Dhanda, Christina Graham, Deborah Hoffman, Mariam Rodriguez, Adrian Ionescu, Stella Karantzoulis, Sidney Kennedy, Sakina Rizvi Neurocrine Biosciences, Inc., San Diego, California, United States Background: Anhedonia is a core symptom of major depressive disorder (MDD) and is commonly defined as a loss of interest or pleasure. As a key diagnostic criterion for MDD, anhedonia contributes to patients’ health-related quality of life and poor outcomes with current treatments. With this, accurate measurement of anhedonia is crucial to reliably support research efforts and potential pharmacotherapy development in MDD. The 17-item Dimensional Anhedonia Rating Scale (DARS) was developed as a self-reported instrument to address the limitations of existing anhedonia measurements. Notably, the DARS was designed to be generalizable across patient cultures and experiences. To support the available evidence of measurement properties of the DARS, the purpose of this qualitative study was to evaluate and establish content validity of the DARS in adults with anhedonia in MDD through a targeted literature review (TLR), clinician interviews, and patient interviews. Methods: The TLR was conducted to identify patient-relevant concepts (i.e., signs/symptoms, impacts of signs/symptoms on the patient’s day-to-day life and functioning) of anhedonia in MDD and develop a preliminary conceptual model. The model was evaluated and revised through qualitative interviews with clinicians (N = 6). The insights derived through the TLR and clinician interviews informed the development of patient interview materials, including a concept elicitation (CE) / cognitive debriefing (CD) discussion guide. The CE exercise was designed to elicit patient-relevant concepts of anhedonia in MDD, while the CD exercise was designed to evaluate patient understanding and use of the DARS via a think-aloud method. One-on-one telephone interviews were conducted with N = 20 patients with clinician-confirmed anhedonia in MDD. Interviews were audio-recorded, transcribed, and subsequently coded using qualitative analysis software to identify reported patient-relevant concepts as well as interpretation, clarity, and relevance of the DARS instructions, items, and response options. Reported concepts were analyzed for salience (i.e., most relevant and important to patients), which were then used to finalize the conceptual model. With the final model, an item mapping exercise was conducted, with the goal of evaluating concept coverage of the DARS. Results: Twelve symptoms and 39 impacts were reported by patients, of which 10 symptoms and 24 impacts were deemed salient and used to finalize the conceptual model. The item mapping exercise revealed that the DARS provided suitable concept coverage in this patient population. CD results demonstrated that the DARS instructions, items, and response options were generally well understood and clear to patients. Additionally, most item concepts were shown to be relevant to patients’ experiences; some inconsistencies with specificity or relevance were identified with three items (i.e., two items in the foods / drinks domain, one item in the social activities domain). Conclusions: Content validity of the DARS was evaluated and confirmed through hybrid CE/CD interviews with patients with anhedonia in MDD. Further performance and other measurement properties of the DARS will be evaluated through planned psychometric analyses. Keywords: Clinical Outcome Assessments, Patient Reported Outcomes, Content Validity of a Scale, The Dimensional Anhedonia Rating Scale Disclosure: Neurocrine Biosciences: Employee (Self) P338. Using Multimodal Neuroimaging to Differentiate Depression in BD From MDD Ruth Asch, Ryan Cool, Sophie Holmes, Margaret Davis, Richard Carson, Patrick Worhunsky, Hilary Blumberg, Irina Esterlis Yale School of Medicine, New Haven, Connecticut, United States Background: Mania/hypomania are a defining diagnostic feature of bipolar disorder (BD), but depression is its most frequent clinical presentation, often leading to misdiagnosis of BD as major depressive disorder (MDD). The metabotropic glutamate receptor 5 (mGluR5) is implicated in cognitive function and mood regulation, thus representing a potential treatment target for disorders associated with glutamate dysfunction, such as BD and MDD. Here we investigate differences in mGluR5 between individuals with MDD, BD, and healthy controls (HC) and examine relationships between mGluR5, brain function, and clinical symptoms. Methods: Within each study, participants were well matched for demographic characteristics (age, sex, race/ethnicity, smoking status). For Study 1, 62 individuals (37% male, 17 MDD, 27 BD, 18 HC] participated in positron emission tomography (PET) imaging with radiotracer [18 F]FPEB to estimate mGluR5 availability (as estimated by the volume of distribution, VT) in three regions of interest: orbitofrontal (OFC), ventromedial (vmPFC), and dorsolateral prefrontal cortices (dlPFC). Group differences in VT were determined using a multivariate ANOVA with OFC, vmPFC, and dlPFC VT as the dependent variables, and diagnosis (HC, MDD, BD) as the independent variable, followed by Fishers-LSD pairwise comparisons. To evaluate associations between dlPFC mGluR5 availability and clinical variables, we computed Pearson’s r. All tests were 2-tailed, and findings were considered significant at p < 0.05 level. Statistical tests were conducted in SPSS (v28). In Study 2, we used functional MR imaging (fMRI) to examine BOLD responses of 48 individuals (48% male, 15 MDD, 14 BD, 19 HC) to fearful displays of facial affect (FEAR) and examined correlations between FEAR responses and dlPFC mGluR5 availability as measured by PET. Processing and analysis of fMRI were performed in SPM12. The three stimulus conditions (fear, happy, neutral) were modeled separately in an event-related design that included regressors for motion-correction parameters. Group differences in FEAR responses (FEAR > fixation), as well as relationships with dlPFC mGluR5 availability and mood symptoms were examined using standard linear modeling (i.e., one-way ANOVAs with/without covariates of interest). Whole-brain group differences and correlations were investigated at a voxel-level p < 0.01 with a cluster-level threshold of puncor<0.05 (kE>600). The mean FEAR BOLD response for significant clusters identified in the whole-brain analyses were extracted (MarsBaR), and ANOVAs with Bonferroni’s post hoc tests were used for pair-wise comparisons. Participants in both studies completed clinical scales (MADRS, BDI and POMS) to assess mood symptoms. Cognitive domains of working memory and psychomotor speed were evaluated using the Cogstate testing battery (Groton maze learning test and detection test, respectively). Results: In Study 1, there was a main effect of group in mGluR5 availability (F = 2.3, p = 0.04), with lower mGluR5 in BD relative to MDD (p < 0.01, -15.6%) and HC (p < 0.04, -13.8%). For participants with BD in a depressed mood state (n = 17), dlPFC mGluR5 availability was associated with psychomotor speed (r = -0.6, p = 0.03), and working memory (r = -0.5, p = 0.03); and depressed mood (r = -0.7, p = 0.005) and working memory (r = -0.6, p = 0.05) among subjects with MDD. In Study 2, BD displayed greater FEAR responses in the right temporal-parietal junction (p = 0.012, kE=830) relative to HC (padj=0.03) and MDD (padj <0.001). Across all subjects, there was a correlation between dlPFC mGluR5 and the FEAR response in dlPFC/anterior cingulate (ACC) (p = 0.012, kE=624). Medial PFC/ACC FEAR response was negatively correlated with mood symptoms in BD (p < 0.001, kE=2728), but not MDD. Conclusions: These data demonstrate differences between BD and MDD in PFC mGluR5 availability and relationships between mGluR5 availability and brain function related to emotion processing that may influence clinically meaningful indices of mood symptoms and cognitive function. Further, mGluR5 PET and fMRI may represent useful tools in the differential diagnosis of BD, and mGluR5 as a potential therapeutic target. Keywords: Metabotropic Glutamate Receptor 5 (mGluR5), Positron Emission Tomography (PET), Functional Magnetic Resonance Imaging (fMRI), Bipolar Disorder (BD), Major Depressive Disorder (MDD) Disclosure: Nothing to disclose. P339. Antidepressants That Increase Mitochondrial Energetics May Elevate Risk of Treatment-Emergent Mania Mark Frye, Manuel Gardea Resendez, Brandon Coombs, Marin Veldic, Susannah Tye, Francisco Romo Nava, Aysegul Ozerdem, Miguel Prieto, Alfredo Cuellar-Barboza, Nicolas Nunez, Balwinder Singh, Richard Pendegraft, Alessandro Miola, Susan McElroy, Joanna Biernacka, Eva Morava, Tamas Kozicz Mayo Clinic, Rochester, Minnesota, United States Background: Preclinical evidence suggests that antidepressants (ADs) may differentially influence mitochondrial energetics. This study was conducted to investigate the relationship between mitochondrial function and illness vulnerability in bipolar disorder (BD), specifically risk of treatment emergent mania (TEM). Methods: Participants with BD already clinically phenotyped as TEM + (n = 176) or TEM- (n = 516) were further classified whether the TEM associated AD, based on preclinical studies, increased (Mito + , n = 600) or decreased (Mito-, n = 289) mitochondrial electron transport chain (ETC) activity. Comparison of TEM + rates between Mito+ and Mito- ADs was performed using generalized estimating equations to account for participants exposed to multiple ADs. Results: Adjusting for sex and BD subtype, TEM + was more frequent with antidepressants that increased (24.7%), versus decreased mitochondrial energetics (13.5%, OR = 2.12, p = 0.00002). Conclusions: Our preliminary retrospective data suggests there may be merit in reconceptualizing AD classification, not solely based on monoaminergic conventional drug mechanism of action, but additionally based on mitochondrial energetics. Future prospective clinical studies on specific antidepressants and mitochondrial activity are encouraged. Recognizing pharmacogenomic investigation of drug response may extend or overlap to genomics of disease risk, future studies should investigate potential interactions between mitochondrial mechanisms of disease risk and drug response. Keywords: Bipolar Disorder, Mitochondria, Electron Transport, Mania, Genetic Variation Disclosure: Myriad: Grant (Self), Assurex Health: Grant (Self), Carnot Laboratories: Other Financial or Material Support (Self), Chymia: Other Financial or Material Support (Self) P340. The Association Between Cellular Senescence and Clinical Presentation in Late-Life Depression Johanna Seitz-Holland, Benoit H. Mulsant, Charles F. Reynolds III, Daniel M. Blumberger, Jordan F. Karp, Meryl A. Butters, Ana Paula Mendes-Silva, Erica L. Vieira, George Tseng, Eric J. Lenze, Breno S. Diniz Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Late-life depression (LLD) is common and associated with excessive age-related medical morbidity, cognitive decline, and increased mortality risk. Previous research has suggested that at least a subset of individuals with LLD experience accelerated aging. In addition, previous studies have implicated the role of cellular senescence, a hallmark of biological aging for accelerated aging in LLD. Senescence refers to a state of permanent cell cycle arrest and changes in the cellular secretome, referred to as senescence-associated secretory phenotype (SASP). Previous studies consistently demonstrated an increased expression of SASP proteins in individuals with LLD compared to healthy individuals. However, it is unclear how the increased expression of SASP proteins relates to clinical presentation, including mental and physical health, within individuals with LLD. Therefore, the present study aimed to examine which demographic, physical, and mental health variables are related to peripheral SASP proteins in LLD. Methods: We examined the relationship between the SASP and demographic, clinical, and cognitive variables in 426 individuals with LLD (mean age 68.9 years, 64% female). We obtained age, self-reported sex, and self-reported race for all individuals. Characteristics of major depressive disorder, including the length of the current depressive episode, age of onset of the first depressive episode, recurrence, and comorbid anxiety disorders, were obtained during the SCID-IV-TR interview. The severity of symptoms was evaluated by the total scores on the Montgomery-Asberg Depression Rating Scale (MADRS), 17-item Hamilton Depression Rating Scale (HAM-D), Brief Symptom Inventory (BSI), Penn State Worry Questionnaire (PSWQ), Anxiety Sensitivity Index (ASI), Scale of Suicidal Ideation (SSI), Medical Outcomes Survey- Mental. We recorded self-reported years of education, Mini-Mental Status Examination (MMSE), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), and the Delis-Kaplan Executive Function System (DKEFS). In addition, we collected anthropometric data, pulse and blood pressure, fasting glucose levels, ICD 10 diagnosis of cardiometabolic disorders, Cumulative Illness Rating Scale – Geriatrics (CIRS-G), and the Medical Outcomes Survey- Physical. We calculated a SASP index based on 22 SASP proteins identified by preclinical studies and our previous publications. Our statistical analyses consisted of three steps. First, we conducted three factor analyses to group clinical variables into domains related to 1) depression and anxiety characteristics, 2) cognitive functioning, and 3) physical health. Using regression analyses, we then explored the association between the SASP index and age and sex. Last, we examined the association between the SASP index and the factors determined in step one, computing correlation and regression analyses. Results: Factor analyses revealed two factors related to depression and anxiety (34.69% and 15.10% variance accounted for), one related to cognitive functioning (55.64% variance accounted for), and three related to physical health (27.08%, 17.33%, 13.21% variance accounted for). The physical health 1 comprised the CIRS- G and Medical Outcomes Survey- Physical scales, BMI, glucose, waist/hip ratio, and physical comorbidities. A regression analysis demonstrated a significant effect of age (T = 5.24, p < .001, standardized Beta = 0.24) and sex (T = -4.11, p < .001, standardized Beta = -0.19). Older and male participants presented with a higher SASP index. A higher SASP index correlated with worse cognitive functioning (r = -0.18, p = 0.001), and worse physical health 1 (r = 0.41, p < .001). Regression analyses with the SASP index as the dependent variable and age, sex, and the six factors as independent variables showed a significant effect of age (p < .001), sex (p = 0.0050), and physical health 1 (p < .001). Physical health 1 was also the most significant independent variable when splitting our sample into males (p < .001) and females (p < .001). Conclusions: Our findings highlighted the role of physical health in accelerated aging in LLD. In addition, the SASP index was associated with age and sex, and cognitive functioning. In contrast, the SASP index was not related to the severity or chronicity of depression and anxiety characteristics. This finding does not contradict our previous studies that have consistently demonstrated an increased SASP index in individuals with a major depressive disorder compared to non-depressed older adults. However, it suggests that the SASP index is more closely associated with physical health and cognitive functioning within individuals with LLD. Therefore, our findings are consonant with a viewpoint that depression is an unwanted co-traveler with co-occurring medical burden and associated disability. They further suggest that future treatment efforts should include interventions and lifestyle modifications that target general health, such as weight loss and exercise programs and optimized control of chronic medical conditions such as diabetes, hypertension, and hypercholesterolemia. Furthermore, longitudinal studies are needed to understand if the inhibition of cellular senescence is a promising treatment target in LLD. Keywords: Depression, Accelerated aging, Senescence Disclosure: Nothing to disclose. P341. An Exploratory Machine Learning-Based Approach to Predicting Outcomes With Intravenous Racemic Ketamine Treatment of Major Depressive Disorder Albert Arias, Richard Feinn, Patrick Oliver Virginia Commonwealth University, Richmond, Virginia, United States Background: We recently reported a large (424 patients) retrospective case series of depressed subjects (most of which had treatment-resistant depression) treated with intravenous ketamine in regular clinical practice (Oliver et al., in press). Good remission (38%) and response (72%) rates were achieved but logistic regression did not identify any convincing significant predictors of response based on available demographic and phenotypic variables. Similar to other recent efforts to predict treatment response in depression (Lin et al., 2021, Athreyu et al., 2019) we sought to use machine learning on our extensive database to identify a model for predicting ketamine response and important variables for the prediction. Methods: The patients were adults with Major Depression N = 400 (those of the 424 with complete outcomes data) and were recruited from the community as part of regular practice at private clinics. Ketamine infusions were administered at a starting dose of 0.5 mg/kg/40 minutes for six infusions within 21 days and continued as needed with titration for the effect of partial disassociation as per the clinical course, further details are available in the forthcoming publication. The PHQ-9 was the primary outcome measure and was completed at intake, prior to each infusion, and then every two weeks via electronic form. Response (>50% improvement from baseline) and remission (PHQ-9 score <5) were used as the categorical outcomes. The Random Forest (RF) package in R Studio was used to generate models of outcomes based on 25 available demographic and other phenotypic variables (e.g., sex, age, suicidal ideation, BMI, etc.). Results: The RF model for remission was fairly accurate in predicting outcomes with an area under the curve (AUC) = 75.1%, and accuracy of 73.5%, with the model for response somewhat less accurate; AUC = 71%, accuracy 68%. For remission, the most important variables in the model (most with > 5% mean decrease in Gini index) were; baseline PHQ-9, total number of infusions, age, dosage per kg average, BMI, baseline anxiety, current use of benzodiazepine, a history of an alcohol problem, and suicidal ideation, amongst others). The most important variables in the response model were quite similar. Conclusions: This preliminary exploratory analysis suggests that a set of variables that are easily and quickly obtained at intake can provide a good estimate of the probability of response and remission with ketamine treatment for prospective patients with MDD. It could also potentially be used to help estimate the ideal number of infusions to optimize the probability of response in a patient. These models could also potentially be improved upon by discovering additional important variables (e.g., neurobehavioral traits, transdiagnostic phenotypic features, genetic markers) in future studies. Though there is clear room for improvement, these findings suggest further study of these precision medicine methods may advance the field. Keywords: IV- Ketamine, Ketamine, Treatment Resistant Depression, Machine Learning, Precision Medicine for Depression Disclosure: Nothing to disclose. P342. Efficacy of Adjunctive Cariprazine in Patients With Major Depressive Disorder and Anxiety Symptoms: A Post Hoc Analysis Maurizio Fava, Vladimir Maletic, Chen Chen, Julie Adams, Ken Kramer, Majid Kerolous AbbVie, Madison, New Jersey, United States Background: Anxiety symptoms are common in patients with major depressive disorder (MDD). The presence of these symptoms makes diagnosis and treatment of MDD more difficult and can negatively impact treatment outcomes. Cariprazine is a dopamine D3-preferring D3/D2 and serotonin 5-HT1A receptor partial agonist that is approved to treat adults with schizophrenia or manic, mixed, and depressive episodes associated with bipolar I disorder. The efficacy of cariprazine as adjunctive treatment for patients with MDD and inadequate response to antidepressant treatment (ADT) alone has been evaluated in late-stage clinical studies. This post hoc analysis evaluated the effects of adjunctive cariprazine on symptoms of depression in patients with MDD and anxiety symptoms using data from a positive phase 3 fixed-dose, randomized, double-blind, placebo-controlled trial (NCT03738215). Methods: Patients with MDD and inadequate response to ongoing ADT were randomized to placebo + ADT, cariprazine 1.5 mg/d + ADT, or cariprazine 3 mg/d + ADT for 6 weeks of double-blind treatment; the primary efficacy outcome was change from baseline to week 6 in Montgomery-Åsberg Depression Rating Scale (MADRS) total score. Post hoc analyses evaluated change from baseline in MADRS total score in subgroups of patients with and without baseline anxiety symptoms. The anxiety subgroup included patients with a baseline score ≥7 on the Hamilton Depression Rating Scale (HAM-D) Anxiety/Somatization factor (sum of 6 items: psychic anxiety, somatic anxiety, gastrointestinal somatic symptoms, general somatic symptoms, hypochondriasis, and insight). Analyses used a mixed-effects model for repeated measures; statistical significance was determined using the 95% confidence interval (95% CI) associated with the least squares mean difference (LSMD) versus placebo. Results: A total of 751 patients were included in the intent-to-treat (ITT) population and post hoc analysis. In the overall ITT population, the difference in MADRS total score change from baseline to week 6 versus placebo + ADT was statistically significant after multiplicity adjustment for cariprazine 1.5 mg/d + ADT (-14.1 vs -11.5; adjusted P = .0050), but not for cariprazine 3 mg/d + ADT (-13.1; P = .0727). In post hoc analysis, 83% of patients (627/751) met criteria for anxiety at baseline. In patients with baseline anxiety, least squares (LS) mean change from baseline to week 6 in MADRS total score was significantly greater for cariprazine 1.5 mg/d + ADT versus placebo + ADT (-14.1 vs -11.7; LSMD [95% CI] = -2.4 [-4.2, -0.7]); for cariprazine 3 mg/d + ADT, LS mean change from baseline in MADRS total score was -13.1 (LSMD [95% CI] vs placebo + ADT = -1.5 [-3.2, 0.3]). In the smaller subgroup of patients without baseline anxiety (n = 124), LS mean changes from baseline in MADRS total score were numerically greater for cariprazine 1.5 mg/d + ADT (-12.8) and 3 mg/d + ADT (-10.1) compared with placebo + ADT (-9.8), but LSMDs (95% CIs) versus placebo did not reach statistical significance for either cariprazine dose (1.5 mg/d = -3.0 [-7.3, 1.3], 3 mg/d = -0.4 [-4.5, 3.7]). Conclusions: The high percentage of patients with anxiety symptoms in the positive fixed-dose study of cariprazine as adjunctive treatment in MDD supports previous findings that anxiety is a common feature of MDD. In post hoc analysis, cariprazine 1.5 mg/d + ADT compared with placebo + ADT was associated with significantly greater improvement in depressive symptoms in the subgroup of patients with MDD and baseline anxiety. In patients without baseline anxiety, the effect size versus placebo for cariprazine 1.5 mg/d + ADT was comparable to that observed in patients with baseline anxiety, though differences were not statistically significant, which was likely due to small sample size in this group. These outcomes suggest that adjunctive cariprazine was efficacious in reducing depressive symptoms in patients with MDD whether or not they had anxiety symptoms at baseline. Keywords: Cariprazine, Dopamine, Major Depressive Disorder, Anxiety Disclosure: AbbVie: Employee (Self) P343. Comparative Effectiveness of Medications in Bipolar Disorder in Real-World Settings Based on 60,045 Patients Markku Lähteenvuo, Tapio Paljärvi, Antti Tanskanen, Heidi Taipale, Jari Tiihonen Niuvanniemi Hospital, Kuopio, Finland Background: Bipolar disorder is a severe psychiatric disorder with a prevalence of 2-3% for the bipolar spectrum. The treatment patterns of bipolar disorder have changed during the recent years, with mood stabilizer usage decreasing and the usage of antipsychotics increasing. However, information of the comparative effectiveness of these different treatment options especially in real-world settings is still sparse. Methods: We identified anyone between the age of 16-65 years diagnosed with bipolar disorder in Finland (ICD-10: F30-F31) during the years 1987-2018 using Finnish nationwide registries and excluded anyone with a diagnosis of schizophrenia spectrum disorder (ICD-10: F2X) or dementia (F00-F03, G30) before their diagnosis of bipolar disorder (resulting cohort size: n = 60,045, 56.4% female, mean age 41.7 years (Standard Deviation (SD) 15.8 years). A sub-cohort of patients newly diagnosed with bipolar disorder and without use of antipsychotics or mood stabilizers one year prior to their first diagnosis of bipolar disorder was also identified (incident cohort, n = 26,395, 54.9% female, mean age 38.2 years (SD 13.0 years). Hospitalizations for psychiatric (ICD-10: FXX) and non-psychiatric (ICD-10: any other than F-diagnosis) reasons were used as proxy outcome measures for relapse and safety, respectively. Medication use was modelled using the Finnish Prescription registry and the established PRE2DUP method on a day-by-day basis. Exposures to medications were compared against non-exposure to the same medication group (non-exposure served as reference, for example: exposure to long-acting injectable antipsychotics (LAI) vs. non-use of antipsychotics or exposure to lithium vs. non-exposure to mood stabilizers). The hazard ratios (HR) with 95% confidence intervals (95% CI) for the outcome measures as per medication exposures were than calculated by using Cox hazard models during the follow-up years 1996-2018 using advanced within-individual models to eliminate selection bias. Exposures with less than 50 person-years of use were not reported. Separate analyses were run for the whole cohort and the incident cohort. The hazard ratios were adjusted (aHR) for the following covariates: other psychotropic medications used, order of treatment, time since diagnosis of bipolar disorder. Results: A total of 104,093 psychiatric hospitalization events were recorded during the follow-up dispersed between 26,159 individuals. The medications associated with lower risk of psychiatric hospitalizations were olanzapine LAI (aHR 0.54, 95% CI 0.37-0.80), haloperidol LAI (aHR 0.62, 95% CI 0.47-0.81), zuclopenthixol LAI (aHR 0.66, 95% CI 0.52-0.85), lithium (aHR 0.74, 95% CI 0.71-0.76), clozapine (aHR 0.75, 95% CI 0.64 – 0.87), carbamazepine (aHR 0.81, 95% CI 0.75-0.87), levomepromazine (aHR 0.88, 95% CI 0.83-0.93), lamotrigine (aHR 0.88, 95% CI 0.85-0.92), valproic acid (aHR 0.89, 95% CI 0.87-0.92), pregabalin (aHR 0.92, 95% CI 0.86-0.98) and chlorprothixene (aHR 0.93, 95% CI 0.86-0.99). Medications associated with a statistically higher risk were quetiapine (aHR 1.03, 95% CI 1.00 – 1.06) and ziprasidone (aHR 1.26, 95% CI 1.07-1.49). The results were mostly similar for the incident cohort. A total of 144,434 non-psychiatric (somatic) hospitalization events were recorded during the follow-up dispersed between 33,380 individuals. Of the studied medications, only lithium (aHR 0.77, 95% CI 0.74-0.81) and carbamazepine (aHR 0.91, 95% CI 0.85-0.97) were associated with significantly reduced risk of non-psychiatric hospitalizations, whereas risperidone (aHR 1.07, 95% CI 1.02-1.13), olanzapine (aHR 1.10, 95% CI 1.05-1.15), quetiapine (aHR 1.10, 95% CI 1.07-1.13), haloperidol (aHR 1.12, 95% CI 1.03-1.22), melperone (aHR 1.19, 95% CI 1.03-1.35), pregabalin (aHR 1.25, 95% CI 1.19-1.31), gabapentin (aHR 1.28, 95% CI 1.20-1.38), clozapine (aHR 1.29, 95% CI 1.07-1.55), ziprasidone (aHR 1.36, 95% CI 1.06-1.75) and haloperidol LAI (aHR 1.43, 95% CI 1.01-2.03) were associated with significantly increased risk of non-psychiatric hospitalizations. Results were similar for the incident cohort. Conclusions: Lithium and certain long-acting injectable antipsychotics were associated with best outcomes and should be favoured. Quetiapine and ziprasidone were associated with significantly increased risk for both psychiatric and non-psychiatric hospitalizations. Keywords: Bipolar Disorder, Antipsychotic Treatment Practice, Mood Stabilizers, Pharmacoepidemiology Disclosure: Janssen: Honoraria (Self), Janssen-Cilag: Honoraria (Self), Lundbeck: Honoraria (Self), Otsuka: Honoraria (Self), Recordati: Honoraria (Self) P344. Pharmacogenetic and Pharmacokinetic Modelling for Precision Prescribing of Venlafaxine in Late-Life Depression Daniel Mueller, Xiaoyu (Betsy) Men, Zachary Taylor, Victoria Marshe, James Kennedy, Eric Lenze, Benoit Mulsant, Charles Reynolds, Laura Ramsey University of Toronto, Toronto, Canada Background: Late-life depression (LLD) is characterized by major depressive disorder (MDD) in adults older than 50-65 years old. LLD is frequently associated with medical comorbidities (e.g., cardiovascular and cerebrovascular disease) and cognitive decline. More than 50% LLD patients fail to achieve remission or have a high risk of relapse once remitted. Therefore, predictive models using genetic biomarkers will be helpful in guiding and optimizing LLD treatment. Venlafaxine (VEN) is a commonly prescribed antidepressant for LLD treatment. VEN is primarily metabolized to its active metabolite o-desmethylvenlafaxine (ODV) by the cytochrome P450 (CYP) 2D6 enzyme. Given that both VEN and ODV are pharmacologically active, the sum of VEN and ODV is frequently referred to as the active moiety (AM) and analyzed as a single pharmacological entity. In this study, we aim to utilize a pharmacokinetic (PK) modelling method, which can estimate key PK parameters using sparse data across multiple time points to account for interindividual variability, to assess 1) whether CYP2D6 genotypes contribute to different VEN PK parameters, and 2) the relationship between VEN treatment responses and drug exposure. Methods: We investigated individuals that participated in the Incomplete Response in Late-Life Depression: Getting to Remission (IRL-GRey, NCT00892047). During the IRL-GRey study phase 1, participants received open-labeled VEN for 12 weeks, starting from 37.5 mg/d and titrated to 300 mg/d if tolerated. Remission was defined as a MADRS score ≤10 at both of the final two consecutive visits. The presence of any adverse effect was also recorded using UKU rating scale at the end of phase 1. We used the existing guideline by Caudle et al.to transfer CYP2D6 genotype to metabolizer status by summing the activity values of haplotypes. Individuals were identified as CYP2D6 poor metabolizers (PM), intermediate metabolizers (IM), normal metabolizers (NM), and ultra-rapid metabolizers (UM). We adapted and modified a pharmacokinetic (PK) model previously described by Lindauer et al. in NONMEM. Our PK model was adjusted for body weight and CYP2D6 metabolizer status. We analyzed whether different CYP2D6 metabolizers had different VEN clearance and exposure of VEN, ODV, and AM using one-way ANOVA. Further, we built regression models to assess the association between treatment responses and drug exposure. The significance level was p-value < 0.05. Results: The PK model (n = 304) demonstrated good predictive performance for VEN and ODV after adjusting for CYP2D6 metabolizer status and body weight. The four metabolizer groups had significantly different model-estimated VEN clearance, VEN exposure, and ODV exposure, while there was no difference across metabolizer status in AM exposure. Before dose adjustment, higher VEN exposure, higher ODV exposure, and higher AM exposure were all associated with poorer treatment efficacy (n = 295). After dose-adjustment, no association was found between treatment efficacy and dose-adjusted VEN exposure, or dose-adjusted AM exposure. The overall presence of adverse effect was associated with higher VEN exposure, higher ODV exposure, and higher AM exposure (n = 287). For specific adverse effects, orthostatic dizziness was associated with higher VEN exposure and higher AM exposure. Notably, PMs had the highest rate of orthostatic dizziness. Higher AM exposure was associated with the presence of nausea/vomiting. Conclusions: We adapted a transit PK model which fitted our data well. Our results showed significant impacts of CYP2D6 on estimated VEN PK parameters. By predicting PK parameters prior to treatment, extreme CYP2D6 metabolizers, especially CYP2D6 PMs, can benefit from genetic testing to avoid treatment-induced adverse effects. Keywords: Late-Life Depression, Pharmacogenetics, Pharmacokinetics, Venlafaxine Disclosure: Nothing to disclose. P345. Categorical Improvement Across Bipolar Depression Symptoms: Pooled Analyses of Cariprazine Randomized Phase II/III Trials Roger McIntyre, Lakshmi Yatham, Eduard Vieta, Lauren Aronin, Binh Nguyen* AbbVie, Madison, New Jersey, United States Background: Bipolar I disorder (BP-I) is a chronic mood disorder characterized by an admixture of manic, hypomanic, and depressive symptoms; depressive symptoms are the leading cause of morbidity and time spent unwell. Cariprazine is a dopamine D3-preferring D3/D2 and serotonin 5-HT1A receptor partial agonist that is approved to treat manic, mixed, and depressive episodes associated with BP-I. The efficacy of cariprazine in BP-I depression was established in 3 fixed-dose, randomized, double-blind, placebo-controlled phase 2/3 trials (NCT01396447, NCT02670538, NCT02670551). Data from these trials were pooled for post hoc analyses to evaluate clinically relevant symptom improvement using the 10 individual items of the Montgomery-Åsberg Depression Rating Scale (MADRS). Methods: Patients were randomized to fixed-dose cariprazine 0.75 mg/d (1 study only), 1.5 mg/d, or 3 mg/d; the primary endpoint in each study was MADRS total score change from baseline to week 6. For each individual MADRS item, post hoc analysis was conducted to determine the percentage of patients that shifted from mild or worse baseline symptoms (MADRS item score ≥2) to minimal or no symptoms (MADRS Item Score <2) at week 6; in a more rigorous analysis, the percentage of patients that shifted from moderate or worse baseline symptoms (MADRS Item score ≥4) to mild or better symptoms (MADRS Item Score ≤2) at week 6 was also evaluated. Individual MADRS item scores range from 0-6, with higher scores indicating greater symptom severity. Cariprazine 1.5 mg/d and 3 mg/d were pooled for post hoc analysis; cariprazine 0.75 mg/d was not included because it is not within the recommended dose range for cariprazine. Odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated for each comparison of cariprazine versus placebo. Results: The pooled intent-to-treat population included a total of 1383 patients (placebo=460; cariprazine=923 [1.5 mg=461, 3 mg/d = 462]). Across 9 of 10 MADRS items, 67.5% to 100% of patients had mild to severe baseline symptoms (score ≥2), and 25.2% to 82.3% of patients had moderate to severe baseline symptoms (score ≥4); a lower percentage of patients had baseline Suicidal Thoughts (score ≥2 = 14.2%; score ≥4 = 0%), which was likely due to exclusion criteria in the original studies. On 8 of 10 MADRS items, a significantly greater percentage of cariprazine- versus placebo-treated patients shifted from mild or worse severity to minimal or no symptoms at week 6 (OR [95% CI]): Apparent Sadness (1.6 [1.2, 2.0]; P = .0005]), Reported Sadness (1.6 [1.2, 2.0]; P = .0005), Reduced Sleep (1.5 [1.1,1.9]; P = .004), Reduced Appetite (1.4 [1.1, 1.9; P = .011), Concentration Difficulties (1.5 [1.1, 2.0]; P = .005), Lassitude (1.4 [1.1, 1.8]; P = .006), Inability to Feel (1.5 [1.2, 1.9]; P = .001), and Pessimistic Thoughts (1.5 [1.2, 2.0]; P = .001); no significant differences were observed on the Inner Tension and Suicidal Thoughts items. In patients with moderate or worse baseline symptoms, a significantly greater percentage of cariprazine- versus placebo-treated patients shifted to mild or better symptoms on 7 of 10 MADRS items (OR [95% CI]): Apparent Sadness (1.8 [1.4, 2.3]; P < .0001), Reported Sadness (1.5 [1.2, 2.0]; P = .008), Reduced Sleep (1.3 [1.0, 1.7]; P = .005), Reduced Appetite (1.8 [1.1, 2.9]; P = .002), Concentration Difficulties (1.6 [1.2, 2.1]; P = .002), Lassitude (1.6 [1.2, 2.1]; P = .001), and Inability to Feel (1.5 [1.1, 2.0]; P = .006); no significant differences were observed on the Inner Tension and Pessimistic Thoughts items and no patients had a score ≥4 on the baseline Suicidal Thoughts item. Conclusions: A significantly greater proportion of patients treated with cariprazine versus placebo shifted to a lower category of severity symptom across most individual MADRS items. This suggests that cariprazine was associated with clinically meaningful improvement across a broad range of depressive symptoms in patients with BP-I depression. Keywords: Bipolar I Depression, Bipolar I Disorder, Cariprazine, Depression, MADRS Disclosure: AbbVie: Employee (Self) P346. Precision Functional Brain Mapping to Understand the Mechanisms of Psilocybin Joshua Siegel, Dean Wong, Demetrius Perry, Timothy Laumann, Abraham Snyder, Marcus Raichle, Eric Lenze, Nico Dosenbach, Ginger Nicol Washington University, Saint Louis, Missouri, United States Background: Animal models suggest that limbic plasticity induced by psilocybin may be key to its antidepressant effects. To understand how the neurotrophic and psychological effects of psychedelics relate, it is necessary to measure psilocybin-induced plasticity in humans. Methods: In this cross-over study, we employed precision functional mapping (PFM) and diffusion basis spectrum imaging (DBSI) to measure changes in functional connectivity (FC) and inflammation, respectively, during and after psilocybin exposure in healthy adults (18-45 years). Participants received 25 mg of psilocybin (PSIL) and 40 mg of methylphenidate (MTH) 1-2 weeks apart. Participants underwent numerous imaging sessions prior to, during (60 minutes following injections), and in the weeks after drug sessions. Primary outcomes were imaging tolerability (defined as scan completion below a strict motion cutoff), subjective experience (e.g., Mystical Experiences Questionnaire), and limbic DBSI and FC. Results: Six adults (3 female) completed the study. Precision resting fMRI was acquired roughly every other day over approximately 1 month, for a mean of 34 scans (SD 5) per subject. All participants completed scans conducted during PSIL sessions, with high-quality obtained in 5/6 participants. Measurable differences between groups on the MEQ were observed across all four factors (t-test; p < 0.05). In the cortex, PSIL produced a decrease in BOLD signal power across regions that was reflected in reduced neurovascular response during task fMRI. In the limbic system, PSIL produced in increase in FC between structures (hippocampus, amygdala, Nacc, subgenual cingulate) that could not be attributed to neurovascular or respiratory changes. Persisting changes in hippocampal-frontal FC were observed 1 day after dosing. No persisting changes (after vs before) were observed in network modularity or signal power. Participants showed a trend towards decreased hippocampal DBSI restriction fraction (marker or inflammation) following PSIL exposure (linear mixed effects model, main effect of timexdrug; p = 0.08). Conclusions: These results demonstrate that treatment-related changes in FC and inflammation, measured via PFM and DBSI methods, respectively, occur following PSIL exposure and are observable and distinguishable from MTP in healthy younger adults. This study establishes the feasibility of precision imaging during acute PSIL exposure. Keywords: Psilocybin, Resting State Functional Connectivity, Precision Psychiatry, Hippocampus, Inflammation Disclosure: Nothing to disclose. P347. Both Levomilnacipran and Duloxetine Potently Inhibit Serotonin Reuptake Throughout Their Therapeutic Dose Range in Healthy Male Volunteers Stephen Daniels, Katerina Nikolitch, Jonathan S. James, Jennifer L. Phillips, Pierre Blier University of Ottawa Institute of Mental Health Research, Ottawa, Canada Background: Medications that potently inhibit serotonin (5-HT) reuptake and/or norepinephrine (NE) reuptake are effective in the treatment of major depressive disorder (MDD). Serotonin/NE reuptake inhibitors (SNRI) are first-line treatment for MDD, but for both venlafaxine and duloxetine, higher doses are required to inhibit the NE reuptake process than those necessary to potently inhibit 5-HT reuptake. In a previous analysis, it was shown that levomilnacipran, but not duloxetine, inhibits NE reuptake throughout its therapeutic range. Using positron emission tomography (PET), racemic milnacipran occupies only 60% of 5-HT transporters at its minimal effective dose in MDD (100 mg/day), whereas duloxetine occupies 84% administered in similar conditions. There is no such data for the occupancy of 5-HT transporters by levomilnacipran. An alternative approach to evaluate 5-HT reuptake activity has been to assess the depletion of whole blood 5-HT as 90% of 5-HT in the blood is in the platelets, which do not synthetize 5-HT, but have a 5-HT transporter that is identical to those on 5-HT neurons. Methods: Healthy male participants (18-40 years of age) were initially randomized to take either placebo for 21 days, levomilnacipran (40 mg/day for 7 days, increased to 80 mg/day for 7 days, and then 120 mg/day for 7 days), or duloxetine (60 mg/day for 7 days, 90 mg/day for 7 days, and 120 mg/day for 7 days). Participants could prolong administration periods to allow for adaptation to side effects. The concentration of whole blood 5-HT was determined using high performance liquid chromatography from samples collected at steady state levels of the drugs. Results: There were 10 completers in the placebo group, 10 for levomilnacipran, and 9 for duloxetine. Two participants withdrew due to side effects, one in each of the active treatment arms. Baseline levels of 5-HT ranged between 400 and 500 picomol/ml and remained unaltered in the subjects who received placebo. There was a robust depletion of 5-HT after 7 days with both duloxetine (75%) and levomilnacipran (85%), which were not significantly different from each other. Higher regimens of both drugs lowered 5-HT levels further and were below the detection level of our assay (F2, 6: 6.13; P < 0.001). Conclusions: These findings replicate our previous results using whole blood 5-HT depletion to assess the degree of 5-HT reuptake inhibition that duloxetine produces at 60 mg/day. Such results are also consistent being PET imaging in the brain. Levomilnacipran was equipotent with duloxetine in blocking 5-HT reuptake also at its minimal effective dose in MDD. Higher regimens of levomilnacipran and duloxetine led to further decreases of 5-HT levels consistent with prior results using larger doses of the SNRI venlafaxine. Taken together with prior results assessing NE reuptake inhibition in this group of healthy participants, these assays confirm that duloxetine is a SNRI, potently inhibiting 5-HT reuptake first and requiring a titration to 120 mg/day to inhibit NE reuptake. In contrast, levomilnacipran acts as a dual reuptake inhibitor from its minimal effective regimen in MDD. Keywords: Serotonin Transporter, Peripheral Blood Marker, Serotonin and Norepinephrine Reuptake Inhibitor Disclosure: Abbvie: Advisory Board, Grant, Honoraria (Self) P348. Brexanolone Therapeutics in Post-Partum Depression Involves Inhibition of Systemic Inflammatory Pathways A. Leslie Morrow, Irina Balan, Riah Patterson, Giorgia Boero, Holly Krohn, Todd O’Buckley, Samantha Meltzer-Brody University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States Background: Brexananolone (FDA approval, Feb 2019) has fast, long-lasting and remarkable efficacy (>60% HAM-D score improvement) in the treatment of post-partum depression (PPD). Treatment requires hospitalization and substantial patient expense. Understanding the therapeutic mechanism(s) offer insight to address the core pathology in PPD. We test the hypothesis that Brexanolone inhibits proinflammatory immune signaling and macrophage sensitivity in PPD patients to promote clinical recovery. Methods: PPD patients under treatment at UNC hospitals provided blood samples before and after Brexanolone infusion according to the FDA-approved protocol. All patients were under concurrent treatment with other psychotropic medications, but unresponsive prior to Brexanolone therapy. Serum was collected to determine neurosteroid levels and isolated cell lysates were examined for inflammatory markers at baseline as well as sensitivity to the inflammatory activators lipopolysacharide (LPS) and imiquimod (IMQ). Results: Brexanolone infusion alters multiple neuroactive steroid levels, inhibits inflammatory mediators, and desensitizes inflammatory response to promote clinical improvement. Brexanolone infusion increased allopregnanolone (+2443 pg/ml, 95%CI 2009-2711, Bonferroni’s p = 0.0001) and allotetrahydrodeoxycorticosterone (+63.4 pg/ml, 95%CI 48.3-78.4, Bonferroni’s p < 0.001), while decreasing 3α,5α-androsterone (-738 pg/ml, 95%CI -1243 to -232, Bonferroni’s p = 0.03) and 3α,5α-androstandiol (-23.1 pg/ml, 95%CI -33.9 to -12.4, Bonferroni’s p < 0.001) in a manner that was highly correlated with HAM-D score. Brexanolone infusion inhibited tumor necrosis factor-α (TNF-α, 58%, p = 0.03), and interleukin-6 (IL6, 61% p < 0.04), but not interferon gamma inducible protein-10, and these effects were correlated with HAM-D score improvement (TNF-α: Pearson r = 0.61, p < 0.06; IL6: Pearson r = 0.68, Bonferroni’s p < 0.03). Furthermore, Brexanolone infusion prevented the activation of TNF-α, IL1β and IL6 by both LPS and IMQ, suggesting a desensitized inflammatory response. Brexanolone infusion partially inhibited the LPS-induced elevation of IL-1β (-53.6%; Bonferroni’s p = 0.03), IL-6 (-50.4%; Bonferroni’s p = 0.03), and TNF-α (-31.3%; Bonferroni’s p = 0.02). Brexanolone infusion also partially inhibited the IMQ-induced elevation of IL-1β (-48.9%; Bonferroni’s p = 0.02), IL-6 (-47.0%; Bonferroni’s p = 0.02), and TNF-α (-32.4%; Bonferroni’s p = 0.048. Pearson correlation indicated that the inhibition of LPS- or IMQ-induced elevation of IL-1β, IL-6 or TNF-α was positively correlated with HAM-D score improvement. Conclusions: The data suggest that Brexanolone therapeutic actions involve the inhibition of inflammatory mediator production through desensitization of immune cells and changes in endogenous neurosteroid levels in blood. These data support the idea that inflammation plays a key role in post-partum depression and inhibition of specific inflammatory pathways underlies its therapeutic efficacy. Keywords: Allopregnanolone, Inflammatory Markers, Depression Disclosure: Sage Therapeutics: Grant (Self), UNC Chapel Hill: Patent (Self) P349. Investigation of the Pharmacological Properties of 2-Br-LSD, a Non-Hallucinogenic LSD Analog Adam Halberstadt, Yasmin Schmid, Emma Bonniwell, Janelle Lanham, Abdi Ghaffari, Andrew Cao, Hooshmand Sheshbaradaran, John McCorvy University of California, San Diego, La Jolla, California, United States Background: Psychedelic drugs such as lysergic acid diethylamide (LSD) and psilocybin induce hallucinogenic effects via 5-HT2A receptor activation. Over the last two decades, considerable clinical evidence has emerged indicating that LSD and psilocybin have therapeutic efficacy against a range of psychiatric disorders, including depression, anxiety, headache, pain, and substance abuse. Similar to LSD, the closely related derivative 2-bromo-LSD (2-Br-LSD, TD-0418A) is also being investigated as a potential treatment for depression and headache. The therapeutic efficacy of 2-Br-LSD is somewhat surprising, however, because 2-Br-LSD does not produce hallucinogenic effects in humans and reportedly acts as a 5-HT2A receptor antagonist. Methods: A combination of in vitro and in vivo pharmacological studies were conducted with 2-Br-LSD to assess its receptor interactions and potential mechanism-of-action. The interaction of 2-Br-LSD with 33 monoaminergic GPCR targets was assessed using G protein-dissociation and β-arrestin2 recruitment BRET assays. Head-twitch response (HTR) studies were conducted in male C57BL/6 J mice to determine whether 2-Br-LSD has an LSD-like behavioral profile (n = 5-8 mice/group). The HTR is rapid rotational head movement induced by psychedelic drugs in mice via 5-HT2A receptor activation. The HTR was detected in mice using an automated method based on artificial intelligence. Results: Compared to LSD, 2-Br-LSD shows considerably less off-target activity at GPCRs. The presence of a single bromine atom at the 2-position of LSD leads to partial agonist activity at several GPCRs, including the 5-HT2A subtype (Emax = 59% relative to serotonin); LSD, by contrast, has much higher agonist efficacy at 5-HT2A (Emax = 92%). Unlike LSD, 2-Br-LSD lacks activity at the 5-HT2B receptor, an interaction linked to valvulopathy and pulmonary hypertension. While LSD (0.1 mg/kg IP) induced the HTR in mice, administration of 2-Br-LSD at IP doses ranging from 0.1–10 mg/kg failed to induce the behavior. Furthermore, pretreatment with 2-Br-LSD (0.1–3 mg/kg IP) blocked the HTR induced by the 5-HT2A agonist/psychedelic drug 2,5-dimethoxy-4-iodoamphetamine (DOI) (F(4,26) = 17.96, p < 0.0001), indicating 2-Br-LSD is brain penetrant and interacts with the 5-HT2A receptor after in vivo administration. We also found that 2-Br-LSD produces weak recruitment of β-arrestin via 5-HT2A in vitro and has reduced potential to induce tolerance in vivo in the HTR paradigm. Conclusions: Overall, 2-Br-LSD has an improved pharmacological profile compared to LSD. 2-Br-LSD appears to act as a non-hallucinogenic 5-HT2A agonist, similar to the isolysergic acid derivative lisuride. Our studies provide insight into the non-hallucinogenic activity of 2-Br-LSD, which is probably a consequence of its weak partial agonist activity at the 5-HT2A receptor. Follow-up studies are necessary to assess whether 2-Br-LSD can mimic the therapeutic effects of LSD but with less potential to produce hallucinogenic side-effects. Keywords: Psychedelics, Psychedelic medicine, LSD, 5-HT2A Receptor, Head Twitch Response Disclosure: BetterLife Pharma Inc.: Contracted Research (Self) P350. GluN2B-NMDARs on Gabaergic Interneurons Mediate the Antidepressant-like, but not Psychotomimetic, Effects of Ketamine in Mice Santosh Pothula, Alexa-Nicole Sliby, Min Wu, Rong-Jian Liu, Ralph DiLeone, Ronald Duman Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States Background: A single subanesthetic dose of ketamine exerts rapid antidepressant-like effects via rapid glutamate efflux, activation of mTORC1 signaling, and enhanced synaptic transmission in the medial prefrontal cortex (mPFC); however, the initial cellular trigger for these synaptic and behavioral actions of ketamine remains unclear. Methods: Here, we used electrophysiology, biochemistry, molecular biology, genetic and behavioral approaches to determine the baseline sex differences in behavior after GluN2B conditional deletion from Sst- and Pvalb-interneurons, and effect of ketamine on chronic unpredictable stress (CUS)-induced behavioral deficits, activation of mTOR signaling cascade and changes in synaptic transmission in adult male and female GluN2Bfl/fl (WT) and Sst-creGluN2Bfl/- and Pvalb-creGluN2Bfl/- conditional knockout (cKO) mice. One-way or Two-way ANOVA post hoc Bonferroni or t-tests were used for data analysis and p < 0.05 was considered as statistically significant. Results: Acute treatment of ketamine significantly decreased NMDA-induced burst firing of Sst- and Pvalb-interneurons ex vivo in mPFC (p < 0.05). Deletion of GluN2B from Sst- or Pvalb-interneurons in the cKO produced changes in synaptic transmission in mPFC. Consistent with the disinhibition hypothesis, ketamine significantly decreased inhibitory, but enhanced excitatory transmission of layer II/III and V pyramidal neurons of mPFC (p < 0.05) and reverses CUS-induced behavioral deficits only in WT, but not Sst-creGluN2Bfl/- and Pvalb-creGluN2Bfl/- cKO, mice. In contrast, ketamine treatment-induced psychotomimetic effects (increased locomotion and sensory motor gating deficits) in both WT and Sst-creGluN2Bfl/- or Pvalb-creGluN2Bfl/- cKO mice in locomotor and prepulse inhibition tests. Preliminary data demonstrate that deletion of GluN2B from Sst-interneurons blocks ketamine-induced activation of mTOR signaling cascade. Ongoing studies are evaluating the effect of ketamine on mTOR signaling in Pvalb-creGluN2Bfl/- cKO mice. Conclusions: Our results demonstrate that Gabaergic interneurons are an initial cellular trigger for synaptic and antidepressant-like behavioral actions of ketamine. Consistent with the disinhibition hypothesis, ketamine actions are mediated by GluN2B-NMDARs on Gabaergic interneurons via disinhibition of pyramidal neurons, activation of mTOR signaling, and enhanced synaptic function in mPFC. Keywords: (R,S)-ketamine, NMDAR, GABAergic Interneurons, Antidepressant, Psychotomimetic Effects Disclosure: Nothing to disclose. P351. Anti-Anhedonic Profile of ENX-104, a Novel and Highly Potent Dopamine D2/3 Receptor Antagonist Krishna Vadodaria, Brian D. Kangas, David S. Garvey, William Brubaker, Diego A. Pizzagalli, Vikram Sudarsan, Kimberly E. Vanover, Jordi Serrats Engrail Therapeutics, San Diego, California, United States Background: Anhedonia, defined as the loss of pleasure derived from previously rewarding activities, is a behavioral phenotype present in many neuropsychiatric conditions, including major depression. Alterations in dopaminergic (DA) corticostriatal circuitry and dysfunction of mesolimbic DA pathways underlie reward processing deficits. At low doses, amisulpride, a D2/3 and 5-HT7 receptor antagonist, shows antidepressant effects and enhances reward-related neural responses, whereas at high doses it has been used as an antipsychotic. We hypothesized that pro-hedonic effects of amisulpride are mediated by its dopaminergic effects, specifically via blockade of presynaptic inhibitory D2/3 receptors at low receptor occupancy (RO), which in turn boosts DA neurotransmission. Based on this evidence, we developed a novel, selective D2/3 receptor antagonist, ENX-104 and predicted that it would enhance reward responsiveness at low doses (~40% D2/3 RO) without impairing motor function. We characterized the in vitro pharmacological profile, the pharmacokinetic (PK) profile, the corresponding receptor occupancies, and the behavioral impact of this investigational new drug, ENX-104. Methods: ENX-104 pharmacology was characterized using binding (radioligand competition) and functional (agonist or antagonist mode) assays in vitro with separate cell lines overexpressing recombinant human receptors: D2L, D3, D4.4, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT7. For PK characterization of ENX-104, rats (n = 3) were orally dosed (0.5 mg/kg [mpk] and 5 mpk) with plasma collection at eight timepoints post dose (5 min-24 h), with a group of rats (n = 5) for plasma and brain collection 1, 4, 8, 24 h post dose. In the D2/3 RO study, rats (n = 5) were orally dosed ENX-104 at 2.5 mpk and plasma and brains were collected at 1, 2, 4, 8, and 24 h post dose. In an ex-vivo competitive tracer binding assay, striatal tissue was incubated with radiolabled [3H]raclopride. Binding of labeled tracer was inversely proportional to ENX-104 occupancy at D2/3 receptors. Comparisons to vehicle were by Dunnetts’s test. PK and RO data were further utilized to build PK:pharmacodynamic (PD) models enabling the prediction of D2/3 RO at different doses and time points following ENX-104 treatment. To evaluate reward responsiveness, rats (n = 8) were trained on a touchscreen-based probabilistic reward task (PRT) analog using parameters based on the human task. To back-translate this into a preclinical model predictive of anti-anhedonic efficacy, we validated the PRT with amisulpride. Vehicle, amisulpride (1, 5, 50 mpk) or ENX-104 (0.5, 1, 2.5 mpk) were orally administered 4-5 h before the PRT session in separate studies with a Latin square design. For motor function, rats (n = 8) were orally dosed ENX-104 (1, 2.5, 10 mpk) and catalepsy was scored by an observer at 1.5, 3 and 4 h post dose. Rat paws were gently placed on a rubber bung and a score of 1 was given for each paw that remained in position for at least 15 seconds, and latency for the time each paw remained on the bung was calculated. Comparisons to vehicle were done by exact Wilcoxon rank sum tests. All animal study protocols were approved by IACUC and conduced in accordance with NIH guidelines. Results: ENX-104 is a potent dopaminergic antagonist, with Ki of 0.01 nM for D2L, 0.2 nM for D3, and 1.6 nM for D4 receptor subtypes. Among serotonergic receptors ENX-104 displayed binding affinity of 3.7 nM at the 5-HT2A receptor, acting as a partial antagonist (EC50 of 14 nM, Emax of ~40%). In comparison, ENX-104 displayed little or no functional activity at the 5-HT1A and 5-HT7 receptors. ENX-104 exhibited a short plasma half-life with significant brain enrichment and retention over the course of 24 h. The plasma half-life of ENX-104 (0.5mpk oral dose) is nearly 2 h with a tmax of ~30 mins. Although plasma concentrations reduced after 2 h, the maximum brain concentration is reached at ~4 h post dose. These data correspond with the D2/3 RO time course data, showing a maximum receptor occupancy at 4 h post dose. Based on these data, 4 h was chosen as a key time point for behavioral analyses. In the PRT, doses of amisulpride and ENX-104 targeting low but not high D2/3 RO significantly increased reward responsiveness. This was revealed by substantive increases in response bias (log b) without reductions in task discriminability (log d). General linear model tests of within-subjects contrast (quadradic term) yielded statistical significance across the dose-response function for ENX-104 (p = 0.048) and a trend for amisulpride (p = 0.065). Given the a priori hypotheses, we conducted paired t-tests of log b values. Low amisulpride doses were significantly different than vehicle (1 mpk, p = 0.03; 5 mpk, p = 0.04), and ENX-104 low doses were significantly different than vehicle (0.5 mpk, p = 0.006 and 1 mpk, p = 0.04). Notably, ENX-104 did not induce catalepsy at lower doses (1 and 2.5 mpk). Catalepsy was observed only in the 10 mg/kg group (D2/3 RO > 80%). Together with the PK:PD model, these data suggest that D2/3 RO of ~40-60% is the target range for anti-anhedonic effects of ENX-104, with catalepsy emerging only at RO greater than ~80%. Conclusions: These data predict ENX-104 could reduce anhedonia in humans at low doses without inducing extrapyramidal side effects. Our data support further investigation of ENX-104, a potent and novel D2/3 receptor antagonist, for clinical use in treating anhedonia in psychiatric disorders. Keywords: Anhedonia, Antidepressant, D2 Dopamine Antagonists, Reward, Depression Disclosure: Engrail Therapeutics: Employee (Self) P352. A Single Administration of a Psychedelic Can Persistently Change Synaptic Function Through 5-HT2A Receptor Activation in Rats Charles Nichols, Meghan Hibicke, Jason Middleton, Hannah Kramer, Jesper Kristensen LSU Health Sciences Center, New Orleans, Louisiana, United States Background: Certain psychedelics such as psilocybin have shown profound and long-lasting therapeutic benefit in several neuropsychiatric disorders including depression and substance use disorder. Clinical trials have shown that a single treatment can have therapeutic benefits lasting months to years. The molecular mechanisms underlying these effects remain unknown but are thought to involve changes in synaptic plasticity. Previously, we demonstrated that a single administration of psilocybin or lysergic acid diethylamide (LSD) to Wistar Kyoto (WKY) rats had long-lasting antidepressant-like effects. In the forced swim test (FST), immobility was dramatically reduced at five weeks (35 days) post treatment with a large effect size. Further, we also have demonstrated that a single treatment with psilocybin rescued deficits in object pattern recognition and decreased immobility in the FST when measured at five weeks in female Sprague-Dawley rats subject to adolescent chronic restraint stress. In these systems, we only tested the effects of psilocybin out to five weeks. In order to assess the durability of effect of a single administration of psychedelic, and to investigate the role of 5-HT2A receptors, we assessed the effects of a single treatment with psilocybin and 25CN-NBOH (a selective 5-HT2A receptor agonist) in the FST at 5 weeks, and 12 weeks in WKY rats. Further, we assessed synaptic function at 12+ weeks in the vmPFC. Methods: Male adult WKY rats were injected with either psilocybin or 25CN-NBOH (i.p., HCl salt, 1.5 mg/kg), or sterile saline. At five weeks, all rats were assessed in the FST. This involved placing rats into a plastic cylindrical tank (114 cm×30.5 cm) that contained 30 cm of water at 28-30°C for a five-minute swim, and video recording the sessions. Videos were scored for immobility, swimming, and climbing. 12 weeks after treatment, rats were again assessed in the FST to examine persistence of effect. After FST testing was complete, rats were transcardially perfused with a neuroprotective artificial cerebrospinal fluid (aCSF), and brains removed, sliced, and electrophysiological whole cell recordings were made from layer V pyramidal neurons of the vmPFC (~3-5 neurons per animal) to characterize synaptic and intrinsic properties of these neurons. Results: Both psilocybin and 25CN-NBOH produced a significant decrease in immobility and increase in swimming behaviors to an equivalent extent when measured at five weeks. These changes were stable, and present at the same effect size when measured at 12 weeks. Slice recordings showed that drug treatment depolarized resting membrane potential and decreased action potential firing thresholds in regular spiking neurons and had bidirectional effects in different classes of bursting neurons: psilocybin increased sEPSC amplitude in transient bursting neurons and decreased sEPSC frequency in rhythmic oscillatory bursting neurons. Conclusions: The effects of a single treatment with psilocybin or 25-CN-NBOH appear to be persistent, as no changes in effect sizes in the FST were measured between 5 weeks and 12 weeks. FST behaviors are mediated by vmPFC projections to the dorsal raphe nucleus. Our results indicate that both drugs have very long-lasting effects on synaptic function that facilitates the activity of neurons in the vmPFC. Because the dose of 25CN-NBOH used is predicted to be selective for 5-HT2A receptor activation, we conclude that 5-HT2A receptor activation is necessary and sufficient for the long-term effects of psychedelics. Keywords: Serotonin 5-HT2A Receptor, Psychedelics, Psilocybin, Depression, Model Systems Disclosure: Eleusis Therapeutics: Advisory Board (Self), Eleusis Therapeutics: Stock / Equity (Self), Eleusis Therapeutics: Royalties (Self), Palo Santo Ventures: Advisory Board (Self) P353. The NMDA Receptor Subunit GluN2D is a Target for Rapid Antidepressant Action Claus Normann, Stefan Vestring, Katharina Domschke, Josef Bischofberger, Tsvetan Serchov University of Freiburg, Freiburg, Germany Background: Major depressive disorder is a widespread and devastating condition that leads to enormous individual suffering and imposes a serious socioeconomic burden. Despite its vast impact, current medical treatment options are limited, and conventional antidepressants take weeks or even months to alleviate patient symptoms. Subanesthetic doses of ketamine effectively reduce depressive symptoms within hours but are often accompanied by distressing adverse effects limiting its broad clinical use. The identification of ketamine’s primary target and subsequent mechanisms further downstream relevant for its antidepressant efficacy could lead to more specific interventions with fewer side effects and better treatment outcomes. Methods: Young adult mice of both sexes were used for all experiments. Hippocampal brain slices were obtained after decapitation and patch-clamp recordings were performed according to previously published protocols to measure long-term synaptic plasticity (LTP), NMDAR currents and other cell parameters. Slices from mice expressing tdTomato in SOM-INs (SOM-Cre (SST tm2.1(cre)Zjh/J) were used to assess SOM-INs. Protein- and RNA levels were assessed by Western Blot and RT-PCR. For behavioral assments, the chronic despair model of depression, the Nosepoke Sucrose Preference Test in the IntelliCage and an Open Field Test were used. GRIN2D siRNA was applied by intrathecal injection together with in-vivo-jet-PEI solution. Results: We started by investigating the behavioral antidepressant properties of ketamine in an animal chronic despair model (CDM) of depression. In CDM, animals were subjected to ten-minute forced swim sessions on five consecutive days to induce a depression-like state, followed by a rest period of two days, and different readout assessments were performed thereafter. After a single intraperitoneal injection of ketamine (10 mg/kg body weight), immobility time was significantly reduced to baseline levels and sucrose preference was normalized. In ex-vivo hippocampal brain slices, LTP induction was completely abolished after swim stress and fully restored by ketamine treatment. In brain slices from naïve animals, ketamine facilitated LTP induction in a protocol designed to avoid ceiling effects. However, the underlying mechanism of this effect is not obvious, as hippocampal LTP is N-methyl-D-aspartate (NMDA)-dependent and ketamine is a noncompetitive NMDA receptor antagonist. The NMDAR is a heterotetramer composed of two GluN1 and two GluN2 subunits with widespread synaptic and extrasynaptic distribution on different neurons in the brain. A potential mediator of o positive modulation of LTP could be somatostatin-expressing interneurons (SOM-INs), which constitute a major subpopulation of GABAergic interneurons and provide feedback inhibition to distal dendrites of PCs in the hippocampus and the frontal cortex. As part of a feedback loop, these interneurons effectively control postsynaptic NMDA receptor activation and synaptic plasticity. In brain slices, NMDAR currents were inhibited to a larger degree in SOM-INs than in pyramidal cells. GluN2D subunits are preferentially expressed in SOM-INs, and ketamine has a higher affinity to NMDARs containing this subunit. Selective inhibition of GluN2D-containing NMDA receptors recovered stress-induced blockade of long-term synaptic potentiation, which is consolidated in later stages by structural synaptic modifications. The cellular and behavioral actions of ketamine could be fully mimicked in-vitro and in-vivo by the selective GluN2D antagonist NAB-14 and by small interfering RNA (siRNA)-mediated posttranscriptional silencing of its encoding gene GRIN2D in vivo. Conclusions: These findings identify the GluN2D subunit of the NMDA receptor on interneurons as novel and highly specific targets for drug treatment of major depression, including RNA-based therapies. Keywords: Depression, Synaptic Plasticity, NMDA Receptor, In-Vivo siRNA Treatment, Ketamine Disclosure: Janssen Pharmaceuticals: Honoraria (Self), GluN2D Antagonists to Treat Depression: Patent (Self) P354. Activation of the Rostral Agranular Insular Cortex is Involved in the Antidepressant Actions of Arketamine Yukio Ago, Rei Yokoyama, Atsushi Kasai, Masato Tanuma, Misuzu Hayashida, Yuto Shimazaki, Momoko Higuchi, Hisato Igarashi, Kaoru Seiriki, Shun Yamaguchi, Takanobu Nakazawa, Kenji Hashimoto, Hitoshi Hashimoto Hiroshima University, Hiroshima, Japan Background: Esketamine and arketamine are the S( + ) and R(-) enantiomers of ketamine, respectively, and esketamine nasal spray has been developed and recently approved for treatment-resistant depression. Accumulating evidence from rodent studies suggests that arketamine may show a more potent antidepressant-like effect and affect fewer perceptual disturbances than esketamine, but the antidepressant mechanism of ketamine enantiomers is not fully understood. In the present study, we aimed to explore and identify brain regions that contribute to the difference in antidepressant action between ketamine enantiomers by brain-wide mapping of immediate early gene expression. Methods: Experimental procedures involving animals and their care were conducted in compliance with the guidelines of the Guide for the Care and Use of Laboratory Animals, and all animal studies were approved by the Animal Care and Use Committees at Osaka University. We used post-weaning social isolation of mice as a model of depression. Male C57BL/6 J mice were weaned at postnatal day 21 and assigned to either group housing or isolation. At day 63, mice were subjected to a 6-min session of a forced swim test (FST). To visualize neuronal activation on a brain-wide scale with subcellular resolution, we used Arc-dVenus mice, which expresses a destabilized fluorescent reporter protein dVenus under the promoter of immediate-early gene Arc, and analyzed dVenus expression in the whole brains of mice in an unbiased manner by an automated imaging system FAST (block-face serial microscopy tomography), which we recently developed (Seiriki et al., Neuron 2017; Nat Protoc 2019). Results: Social isolation stress increased the immobility time of mice in the FST, and arketamine showed a greater potency of antidepressant-like effect than esketamine in isolation-reared mice. Both arketamine and esketamine increased neuronal activity in cortical and subcortical regions in isolation-reared Arc-dVenus mice, and then the machine learning classifiers with brain-wide activation mapping identified several candidates of brain areas including the rostral agranular insular cortex that may contribute to the antidepressant-like effect of arketamine as well as discrimination between the effects of arketamine and esketamine. Additionally, chemogenetics-mediated acute inhibition of excitatory neuronal activity in the rostral agranular insular cortex blocked the antidepressant-like effects of arketamine, but not of esketamine, in isolation-reared mice. Conversely, chemogenetic activation of the rostral agranular insular cortex neurons induced antidepressant-like effects in isolation-reared mice. Conclusions: These findings suggest that esketamine and arketamine have different neural mechanisms underlying their antidepressant actions. This study also implies that activation of the rostral agranular insular cortex is essential for exerting the antidepressant-like effects by arketamine. Keywords: Arketamine, Esketamine, Insular Cortex, Antidepressant Mechanisms, Neural Activity Disclosure: Nothing to disclose. P355. Ketamine Preservative Benzethonium Chloride Potentiates Hippocampal Synaptic Transmission and Inhibits Neurotransmitter Receptors and Transporters in Vitro Kyle Brown, Panos Zanos, Ruin Moaddel, Xi-Ping Huang, Chris Powels, Mike Michaelides, Edna Pereira, Todd Gould University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Benzethonium chloride (BZT) is an excipient antimicrobial used in numerous products including (R,S)-ketamine (ketamine) drug formulations. A 10 uM ketamine formulation, a concentration commonly used in preclinical studies, contains up to 12 nM BZT. Emerging evidence indicates BZT is functionally active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. Methods: We evaluated affinity and functional effects of BZT at neurotransmitter receptors and transporters. Radioligand binding assays determined the affinity of BZT for numerous targets whereas functional effects were established via assessment of neurotransmitter uptake, β-arrestin translocation, cAMP production, and/or Ca2+ mobilization. Plasma and brain distribution of BZT up to eight hours after systemic administration (i.e., intraperitoneal administration) was determined in male mice. The effect of BZT on in vitro synaptic transmission was assessed in the hippocampus of male mice via electrical stimulation of Shaffer collateral afferents and recording field excitatory postsynaptic potentials (fEPSPs) in the CA1 subfield. Results: BZT potently binds to numerous receptors at high to moderate affinity (e.g., σ2 Ki=7 nM; n = 3-5 independent experiments per receptor), inhibited transporter activity (e.g., dopamine transporter Ki=545 nM; n = 3 independent experiments per transporter), and did not exhibit agonist activity at any assessed metabotropic receptor (n = ≥3 independent experiments per receptor). BZT was detected in the periphery, but not in the brain, of mice following systemic administration (n = 4 at 10, 30, 60, 120, 240, and 480 min post-BZT injection). Bath application of BZT potentiated hippocampal Schaffer collateral-CA1 fEPSPs in mouse hippocampal slices (F(6,142)=6.60, p < 0.0001; EC50 = 2.03 nM). Specifically, bath application of 10 nM BZT (p < 0.0001; n = 14), 50 nM BZT (p = 0.028; n = 17), and 300 nM BZT (p = 0.021; n = 18), but not VEH, (n = 48), 0.3 nM BZT (p = 1.00; n = 19), 2 nM BZT (p = 0.72; n = 19), or 10000 nM BZT (p = 0.067; n = 14), significantly increased the fEPSP slope value. Separate experiments showed that the potentiating effect of 10 nM BZT persisted following BZT wash-out (t(5)=0.987, p = 0.37). Conclusions: BZT interacts with numerous membrane receptors and can induce synaptic potentiation measured in hippocampal slices. However, it does not readily cross the blood-brain barrier. Studies measuring peripheral endpoints following BZT-containing ketamine treatment or directly exposing systems to BZT-containing ketamine formulations (e.g., intracortical administration) should account for the effects of BZT. Our findings suggest that earlier data attributing physiological effects to ketamine may be impacted by BZT and that additional investigation into the functional impact of BZT is warranted. Keywords: (R,S)-ketamine, Synaptic Plasticity, Hippocampus, Slice Electrophysiology, Neurotransmission Disclosure: Nothing to disclose. P356. Transcriptomic Analysis of Rats Exposed to Chronic Mild Stress and Modulation by Prolonged Treatment With the Antipsychotic Drug Lurasidone Veronica Begni, Moira Marizzoni, Diana Morena Silipo, Kerstin Camile Creutzberg, Mariusz Papp, Annamaria Cattaneo, Marco Andrea Riva* University of Milan, Milan, Italy Background: Exposure of adult rats to chronic stress represents a valuable experimental paradigm to investigate and characterize the mechanisms that may contribute to the development of a pathologic phenotype. One consistent finding following stress exposure is the development of an anhedonic phenotype, which may be normalized through pharmacological intervention with different psychotropic drugs. Anhedonia is a key domain in mood disorder and originates from an array of alterations in different brain regions. In this respect, the prefrontal cortex may play a crucial role considering that it receives a number of functional inputs, and it has an important modulatory role on other structures, including the amygdala and nucleus accumbens. Here, we performed a transcriptomic analysis on the prefrontal cortex of rats exposed to the chronic mild stress (CMS) paradigm and we investigated the potential impact of chronic treatment with the antipsychotic drug lurasidone on such transcriptional alterations in order to identify genes that may contribute to stress susceptibility as well as those that could play a role in the therapeutic intervention with lurasidone. Methods: Adult male rats were initially exposed to CMS for 2-weeks and sucrose intake was measured as a proxy for anhedonia. Animals that showed reduced intake (vulnerable) were randomized to receive vehicle or the antipsychotic drug lurasidone (3.0 mg/kg, per os) for 5 more weeks while continuing CMS exposure. Blood and brain tissues were collected 24 h after the last manipulation for the molecular analyses. Whole-genome RNA sequencing analysis was conducted using the Illumina NextSeq500 platform in the Prefrontal cortex (PFC) of Control rats treated with Vehicle (CT/Veh), CMS rats treated with Vehicle (CMS/Veh), and CMS rats treated with Lurasidone (CMS/Lur). Differentially expressed genes (DEGs) identified by the Bioconductor package Deseq2 with FDR-corrected p-values (q < 0.05) were used to run pathway analyses and functional network prediction through Ingenuity Pathway Analysis (IPA). Results: In line with our previous studies, CMS exposure caused a significant reduction in sucrose intake, which was progressively normalized by lurasidone treatment (p < 0.001). Transcriptomic analysis in the prefrontal cortex allowed us to identify a high number of genes (>500) that were differentially expressed in CMS/Veh, as compared to CT/Veh, but that were also differentially expressed in CMS/Lur when compared to their vehicle-treated counterpart (CMS/Veh). Using Venn diagram analysis, we found that 107 genes in common were altered in CMS/Veh and CMS/Lur groups. By running pathway analysis, we found that most of these pathways show opposite modulation. Indeed, among the most significant changes, we found that netrin, CREB, and oxytocin signaling pathways were reduced in CMS/Veh and up regulated in CMS/Lur. Similarly, based on disease and function analysis, we observed an increase in cell and neuronal death in CMS/Veh, with opposite changes in rats treated with lurasidone. Conclusions: In summary, we provide new insights on how chronic lurasidone treatment may counteract the alterations produced upon prolonged exposure to stress, a key vulnerability factor for mental disorders. In particular, the prefrontal cortex appears to be particularly sensitive to stress exposure with a down-regulation of different pathways associated with neuronal guidance and synaptic plasticity. Our results suggest that pharmacological intervention with lurasidone can counteract such changes and may ultimately promote resilience. Keywords: Chronic Stress, Transcriptomics, Lurasidone, Plasticity Disclosure: Angelini: Speakers Bureau (Self), Lundbeck: Speakers Bureau (Self), Otzuka: Speakers Bureau (Self), Sumitomo Pharma: Honoraria (Self), Sumitomo Pharma: Grant (Self), Sunonion: Grant (Self) P357. The Formation of Stress Vulnerability is Controlled by 5-HTergic System in the Raphe Nucleus Through Striatal N-Acetyltransferase, Shati/Nat8l, in Mice Hajime Miyanishi, Shin-ichi Muramatsu, Atsumi Nitta Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama-shi, Japan Background: Depression is one of the most common mental diseases, with increasing numbers of patients in all over the world. Stress is closely related to the depression pathology. While some individuals show the vulnerable to stress, however, others are resilient. Revealing the underlying regulatory mechanisms of stress sensitivity could offer novel insights for understanding the pathogenesis of depression. Serotonin [5-hydroxytryptamine (5-HT)] is involved in depression pathogenesis, and 5-HT has been targeted for medical treatments of depression. The hereditary reduction of 5-HT in the brain induced the vulnerability to social stress in mice. Shati/Nat8l was identified from the brain of methamphetamine-induced psychosis model mice. Shati/Nat8l has N-acetyltransferase activity, and predominantly synthesizes N-acetyl-aspartate (NAA) from L-aspartate and acetyl-coenzyme. NAA expression in the brain was reported to be changed in the patients with depression. We previously found that Shati/Nat8l mRNA levels in the dorsal striatum were increased in the mice exposed repeated forced swimming stress. In the present study, we demonstrated the role of striatal Shati/Nat8l in the depression pathology and stress sensitivity. Methods: Male C57BL/6 J mice were exposed repeated social defeat stress (RSDS) using male ICR mice, and the stress susceptible or resilient group were classified by social interaction test. We generated dorsal striatal Shati/Nat8l overexpression (dSTR-Shati OE) mice using adeno associated virus vectors. Depression-like behaviors were assessed using these mice after RSDS or sub-threshold social stress (micro social defeat stress; MSDS). 5-HT content in the dorsal striatum were measured using in vivo microdialysis. The effects of 5-HT in the dorsal striatum to stress sensitivity were investigated by microinfusion of selective serotonin reuptake inhibitors, fluvoxamine (FLX), into the dorsal striatum. We also investigated the contribution of 5-HTergic neuron projected from raphe nucleus to dorsal striatum to stress sensitivity using DREADD system. Results: We found that Shati/Nat8l mRNA levels were increased in the dorsal striatum of stress susceptible, but not resilient, mice exposed RSDS. We also found that only stress susceptible mice showed the downregulation of 5-HT content in the dorsal striatum. The proportion of stress resilient group after RSDS decreased to 50% in dSTR-Shati OE mice compared to control mice, and these mice showed the vulnerability to even MSDS. The reduction of 5-HT content in the dorsal striatum was observed in dSTR-Shati OE mice. The vulnerability to stress in dSTR-Shati OE mice was recovered by microinfusion of FLX into the dorsal stratum or activation of 5-HTergic neuron projected from raphe nucleus to dorsal striatum by DREADD system. Conclusions: In the present study, we have demonstrated that striatal Shati/Nat8l-induced stress vulnerability is mediated by the reduction of striatal 5-HT content through 5-HTergic system in the raphe nucleus. Our study suggests that dorsal striatum-raphe nucleus circuit is involved with the stress sensitivity in the depression pathogenesis, and that striatal Shati/Nat8l could be a novel therapeutic target for depression. Keywords: Depression, Social Defeat Stress, Dorsal Striatum, Dorsal Raphe, Serotonin Disclosure: Nothing to disclose. P358. A Non-Hallucinogenic LSD Analog With Therapeutic Potential for Mood Disorders Argel Aguilar Valles, Vern Lewis, Andre Telfer, Emily Arsenault, Fatimeh-Frouh Taghavi-Abkuh, Fatema El Sayegh, Alfonso Abizaid Carleton University, Ottawa, Canada Background: Major depressive disorder (MDD) is the leading cause of disability worldwide. Current pharmacotherapy treatments, such as SSRIs and SNRIs, have long effective latencies, require chronic administration, and show an estimated 30-40% treatment resistance rate, necessitating the search for more effective, alternate treatments. Interest in psychedelic hallucinogens (e.g., lysergic acid diethylamide [LSD], psilocybin) has seen a resurgence due to their potential for treating neuropsychiatric diseases, including anxiety and depressive disorders. Unlike first line MDD treatments, hallucinogens target serotonin receptors directly and show rapid and persistent antidepressant effects after only one or two treatments, potentially due to activation of synaptic and structural plasticity mechanisms. However, psychedelics induce hallucinations that can last for hours, making efficient and cost-effective treatment difficult. Therefore, discovering non-hallucinogenic derivatives with antidepressant properties is of paramount importance. 2-Bromo-LSD (2-Br-LSD, BETR-001) is an LSD derivative with 5-HT2A partial agonist activity that lacks hallucinogenic effects and has been safely used for cluster headache treatment. Thus, 2-Br-LSD may represent a possible novel therapeutic for treating neuropsychiatric diseases whose pathologies involve dysfunction in cortical serotonergic pathways. Methods: We investigated the effects of 2-Br-LSD on dendritogenesis in primary rat cortical neuron cell cultures. Cortices from embryonic day 18 rat embryos were enzymatically and mechanically disaggregated; cells were then plated at 50 000 cells/well and treated with different concentrations of 2-Br-LSD (1, 10 and 100 nM, 1 and 10 uM), 10 uM ketamine, or vehicle for 3 hours on days in vitro 3 (DIV3). On DIV6, cells were either processed for immunofluorescence to detect MAP2 or for cell survival using the Neurite Outgrowth Staining Kit (Thermo Fisher). Sholl analysis was used to quantify changes in arbor complexity in imaged cells. To assess spinogenesis, neurons were kept in culture until DIV18, treated as above and stained for MAP2 and F-actin. To assess the contribution of the serotonin 5-HT2A receptor on 2-BR-LSD dendritogenesis, DIV3 neurons were pretreated with the 5-HT2A selective antagonist volinanserin (VOL) at 100 nM, 500 nM, or 1 uM for 1 hour, followed by vehicle or 2-Br-LSD (1 uM). Neurons from 6 different wells per treatment and from at least 2 independent experiments were used for analyses. To determine the effect of 2-Br-LSD on stress-coping behaviour, adult male and female mice (n = 12 group/sex) were treated with vehicle or 2-Br-LSD at 0.3, 1 or 3 mg/kg (IP). 24 hours after treatment, mice were tested in the open field (OF, 10 min) and 1 hour later in the forced-swim test (FST, 6 min). Immediately after, brains were harvested and processed for Golgi staining (FD Rapid GolgiStain Kit, FD NeuroTechnologies). A separate cohort of mice (n = 11/group) was treated with VOL (IP, 0.125 mg/kg) or vehicle 15 minutes before 2-Br-LSD (IP, 1 mg/kg), then tested in the OF and FST the following day. Finally, a group of female mice were subjected to chronic variable stress (CVS; 2 stressors per day in variable order) for 5 weeks (n = 12/group). Following the last stress day, mice were treated with vehicle or 2-Br-LSD (IP) once at 3 mg/kg or 3 times, every 48 h, at a 1 mg/kg dose. Mice were tested in the open field and splash test at two time points post-treatment (3-4 h and 4 weeks). Results: 2-Br-LSD induced a dose-dependent increase in dendritic arbour complexity, as reflected by the increases in the number of process crossings in the Sholl analysis and the increased length of the dendritic arbour. The maximal effect was achieved with the 1 uM dose comparable to the 10 uM ketamine treatment. 2-Br-LSD also increased spine density in DIV18 neurons, but only at the 10 uM dose. Neither of the treatments decreased cell viability in cultured neurons. In vivo, 2-Br-LSD decreased immobility in the FST at the 1 mg/kg dose, in both male and female mice, without affecting general locomotion in the OF. Furthermore, spine density in the prefrontal cortex was increased by this treatment. 2-Br-LSD treatment also reversed the effects of CVS in female mice, increasing exploration of the centre of the OF and increasing self-grooming in the splash test. Promotion of dendritogenesis in vitro and active stress-coping behaviour by 2-Br-LSD depended on 5-HT2A receptors, as VOL blocked both effects. Conclusions: Our findings demonstrate that 2-Br-LSD induces structural plasticity in cortical neurons in vitro and in vivo, promotes active stress-coping behaviours and reverses the behavioural effects of CVS. These results show that 2-Br-LSD may possess a therapeutic potential and represents a promising alternative to psychedelics in treating MDD. Keywords: LSD, Dendritic Arborization, Spine Morphogenesis, Stress Coping, 5-HT2A Receptor Disclosure: BetterLife Pharma: Contracted Research (Self) P359. Cariprazine, a Dopamine D3 Receptor-Preferring Partial Agonist, Reduces Footshock-Induced Ultrasonic Vocalizations in a Rat Model of Anxiety: Synergy With Serotonergic and GABAergic Mechanisms Nasser Haddjeri, Bence Farkas, Béla Kiss, Viktor Román, Balázs Lendavi, Nika Adham* Abbvie, Madison, New Jersey, United States Background: Symptoms of anxiety are frequent in patients with schizophrenia, bipolar I disorder, and major depressive disorder, highlighting the need for treatments that can address the complex symptomatology characteristic of these disorders. Cariprazine is a dopamine D3 receptor-preferring D3/D2 receptor partial agonist approved by the US Food and Drug Administration to treat adults with schizophrenia as well as manic, mixed, and depressive episodes associated with bipolar I disorder. In addition to dopaminergic actions, cariprazine also acts as a partial agonist at serotonergic 5-HT1A receptors, which are implicated in the development of anxiety symptoms. The objective of this study was to assess the anxiolytic-like effects of cariprazine in rats, tested alone and in combination with two clinically used anxiolytics: the selective serotonin reuptake inhibitor (SSRI) escitalopram and the benzodiazepine diazepam. Methods: Anxiety-like symptoms were tested using an electric footshock-induced ultrasonic vocalization (USV) model in male Wistar Unilever rats (150–200 g). The testing apparatus was a 20 cm×20 cm x 25 cm chamber equipped with a grid floor, through which footshocks (1 mA; 4 s) could be delivered. Day 1 consisted of two 10-min sessions, separated by 60 min, during which rats received 1–4 randomly distributed footshocks until USV emission was detected. The following day, rats were placed in the chamber and received a single shock 30 min after injection of vehicle (saline with 10% 2-hydroxypropopyl-β-cyclodextrine; i.p.). On day 3, the test was repeated 30 min after i.p. injection of cariprazine (1, 5, 50, 100, or 300 μg/kg), escitalopram (0.2, 1, or 3 mg/kg), or diazepam (1, 3, or 6 mg/kg). In a second experiment, sub-effective doses of the drugs (determined in the first experiment) were administered alone or in combination 30 min before testing on day 3. USVs were defined as vocalizations with frequencies higher than 20 kHz. For both experiments, duration of USVs on each testing day was determined using the 5-min period beginning immediately after the first footshock-induced USV. For analysis, USV durations were converted to a percentage of USV duration after vehicle treatment on day 2. Results: Cariprazine treatment dose-dependently decreased the duration of footshock-induced USVs. Compared with vehicle, significantly lower USV durations were observed at doses ≥50 μg/kg (P < 0.05) and USVs were eliminated by the 300 μg/kg dose (P < 0.001). Dose-dependent decreases were also observed with escitalopram and diazepam and were statistically significant versus vehicle at doses ≥1 mg/kg (P < 0.001) and ≥3 mg/kg (P < 0.05), respectively. At the highest doses tested, maximum decreases in USV duration relative to vehicle were 89% with escitalopram and 55% with diazepam. Higher doses of diazepam produced confounding sedative effects, making it difficult to determine specific anxiolytic effects. In the second experiment, combination treatment using sub-effective doses of cariprazine (1 μg/kg) and escitalopram (0.2 mg/kg) reduced footshock-induced USV duration by 42% compared with vehicle (P < 0.001), whereas combination treatment using sub-effective doses of cariprazine (5 μg/kg) and diazepam (1 mg/kg) resulted in a 26% reduction (P < 0.01 versus vehicle). Conclusions: In a rodent footshock-induced USV model, cariprazine demonstrated potent anxiolytic-like activity, similar to the SSRI escitalopram and benzodiazepine diazepam. Synergistic anxiolytic-like effects were observed for cariprazine applied in combination with escitalopram or diazepam at sub-effective doses of each compound. Although further research is needed to understand the mechanisms underlying these synergistic effects, therapies including cariprazine, either alone or in combination with lower-dose anxiolytic agents, may be effective in the treatment of recalcitrant anxiety disorders or anxiety associated with schizophrenia. Keywords: Cariprazine, Dopamine, 5-HT, Anxiety, Ultrasonic Vocalization (USV) Model Disclosure: AbbVie: Employee (Self) P360. A Potent 5-HT2A Receptor Agonist TCB-2 Exerts Rapid Antidepressant-Like and Anxiolytic-Like Effects in Mice Hiroyuki Koike, Ichiro Horinokita, Mikio Suzuki, Takashi Futamura Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan Background: Classic serotonergic psychedelics have gained renewed interest both in clinical and preclinical fields because of robust antidepressant and anxiolytic responses after a single dose. As a result, understanding the mechanisms of action has been an area of great interest in the basic science. Recent studies emphasize the importance of 5-HT2A receptor agonism in the therapeutic effects of psychedelics, yet the exact mechanism remains unclear. Relevant and useful animal models are necessary to obtain a better understanding of behavioral outcomes and biological correlates associated with the antidepressant and anxiolytic effects of psychedelics, especially through activation of 5-HT2A receptor. In this study, we investigated behavioral effects of a potent 5-HT2A receptor agonist TCB-2 in corticosterone-induced anhedonia and novelty suppressed feeding test (NSFT) to evaluate the potential utility of these models for preclinical research on psychedelics. Methods: Male C57BL/6 J mice were used for this study. We treated mice with corticosterone to induce anhedonia, observed as reduced sucrose preference. NSFT was carried out in naive mice to measure anxiety-induced hypophagia. To evaluate antidepressant-like and anxiolytic-like effects of TCB-2 in these assays, we injected mice intraperitoneally with it 24 hours before the test. To evaluate hallucinogenic potency, we measured head-twitch response immediately after administration. Experiments employed 6-8 mice per treatment. The data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test (p < 0.05 was considered statistically significant). Results: TCB-2 improved the chronic corticosterone-induced reduction in sucrose preference; the response was maximal at an intermediate dose (0.1 mg/kg) but slightly declined at higher doses. TCB-2 also shortened the latency time until feed in the NSFT. The effective doses of TCB-2 in these behavioral assays elicited the head-twitch response. Conclusions: We demonstrated that TCB-2 exerted the antidepressant-like and anxiolytic-like effects after a single injection in a behavioral model of anhedonia, sucrose preference test in corticosterone-treated mice, as well as a model of anxiety, NSFT. Given that traditional antidepressants are effective in these models after chronic treatment, our findings indicate that the effects of TCB-2 is rapid onset. Furthermore, because the effects of TCB-2 were observed 24 hours after treatment and psychedelics are reported to promote long-lasting neural plasticity through 5-HT2A receptor activation, it is likely that TCB-2 at the effective doses induce neural plasticity which potentially mediates it’s antidepressant-like and anxiolytic-like effects via the same mechanism as psychedelics. Future work that leads to a precise delineation of the mechanisms underlying the observed effects of TCB-2 is needed to identify and develop effective treatment for alleviating symptoms of depression and anxiety, and the behavioral models we employed may be useful to detect the robust effects of psychedelics and elucidate the mechanisms of their action. Keywords: Serotonin 5-HT2A Receptor, TCB-2, Psychedelics, Animal Models Disclosure: Nothing to disclose. P361. In Vitro and In Vivo Profile of CYB003: A Novel, Deuterated Psilocybin Analog for the Potential Treatment of Major Depressive Disorder Michael Palfreyman, Joan Krakowsky, Michael Morgan, Clinton Canal, Pradip Pathare, Ken Avery, Amy Reichelt, Joshua Hartsel, Alex Nivorozhkin, Amir Inamder, Geoffrey Varty Palfreyman BioPharm Advisors, Inc., St. Petersburg, Florida, United States Background: Phase 2 clinical studies have demonstrated that short-term administration of psilocybin has beneficial effects on Major Depressive Disorder (MDD) and Treatment Resistant Depression (TRD). While psilocybin is efficacious and inherently safe, there is significant variability between patients in their psychedelic and side-effect experiences. This variability is likely due to psilocybin acting as a pro-drug that requires de-phosphorylation to the psychoactive metabolite, psilocin. CYB003 is a novel, deuterated analog of psilocybin that may offer benefits over psilocybin. The aim of these studies was to compare the in vitro and in vivo activity of CYB003 to psilocin and conduct Investigational New Drug (IND)-enabling studies to allow the initiation of clinical studies. Methods: Pharmacological profiles of CYB003 and psilocin were compared using serotonin (5-HT) receptor binding and functional assays to evaluate potency, efficacy, and selectivity at serotonin receptors; both compounds were also screened for activity at a panel of over 100 proteins. Additionally, both compounds were evaluated in the mouse head twitch response (HTR) assay (a behavioral model of psychedelic-like effects). ADME and pharmacokinetic (PK) studies were also completed followed by dose-range finding studies in rodents and non-rodents species. Finally, IND-enabling studies conducted to Good Laboratory Practice (GLP) standards were completed; specifically, repeat dose toxicological studies, and cardiovascular, respiratory, and CNS safety pharmacology assessments. Results: The pharmacological and selectivity profile of CYB003 was similar to that seen with psilocin. CYB003 binds and activates the 5-HT2A receptor (5-HT2A Ki: 37 nm; psilocin 31 nM) and induces HTR in mice (No.of HTR (0-30 min): CYB003 8.9 _+ 1.3; psilocin 12.6 + _1). ADME studies indicate little metabolism in vitro and PK/PD studies showed a good correlation between plasma levels and in vivo activity. Safety pharmacology study findings were consistent with activation of 5-HT receptors, reflecting findings seen with other psychedelic compounds, including psilocybin. GLP-toxicological studies established safe, and potentially effective doses, and will support multiple-dose clinical studies. Collectively, these data permitted the initiation of Phase 1/2 A clinical studies www.clinicaltrials.gov ID: NCT05385783: A study of a Psilocybin Analog (CYB003) in Participants with Major Depressive Disorder. The Phase 1/2 A protocol in participants with MDD is now ongoing (Clinilabs, Eatontown, NJ). Conclusions: Our findings confirm that CYB003 has the appropriate pharmacology and safety profile to proceed to clinical trials in patients with MDD. These data further support the potential of CYB003 to have superior properties to psilocybin and to offer considerable clinical benefits. Keywords: Psychedelics, Psilocybin Analog, Pharmacology, IND-Enabling GLP Studies, Major Depressive Disorder Disclosure: Cybin, Inc.: Employee (Self), Palfreyman BioPharm Advisors, LLC: Owner (Self) P362. Pharmacokinetic Profile of CYB003: A Novel, Deuterated Psilocybin Analog for the Potential Treatment of Major Depressive Disorder Amir Inamdar, Michael Morgan, Joan Krakowsky, Amy Reichelt, Clinton Canal, Tina Mueller, Ken Avery, Pradip Pathare, Joshua Hartsel, Alex Nivorozhkin, Geoffrey Varty, Michael Palfreyman Cybin, London, United Kingdom Background: Phase 2 clinical studies have demonstrated that short-term administration of psilocybin has beneficial effects on a range of psychiatric conditions such as Major Depressive Disorder (MDD), anxiety, and substance use disorders. While psilocybin is efficacious and inherently safe, literature shows there is significant variability between patients in their psychedelic and side effect experiences. This variability is likely due to psilocybin acting as a pro-drug that requires de-phosphorylation to the psychoactive metabolite, psilocin. Once metabolized, psilocin is absorbed into the bloodstream and crosses the blood-brain barrier to interact with central serotonergic receptors, including 5-HT2A receptors. Improving the absorption, distribution, and elimination of psilocybin, while retaining its therapeutic effects, may offer significant benefits to the patient. CYB003 is a deuterated analog of psilocybin that may offer such benefits. The aim of these studies was to compare the pharmacokinetic (PK) profile of CYB003, a novel psilocybin analog, to psilocybin and psilocin. Methods: To compare the PK profile, CYB003, psilocin, and psilocybin were administered orally to male Sprague-Dawley rats. Plasma and brain samples were collected over a 4-hour post-dosing period and were analyzed by LC-MS/MS for levels of CYB003 and psilocin. The PK profiles of CYB003 following both oral and intravenous administration to the mouse, rat, and dog were also determined. PK parameters were determined using noncompartmental analysis. Results: CYB003 demonstrates potential advantages over psilocybin in terms of its plasma PK profile. Specifically, compared to psilocybin, the rat plasma exposure profile of CYB003 has a shorter time to reach peak effect, has reduced half-life (T1/2), and importantly, results in reduced variability. The time to reach peak plasma exposure (Tmax) was 30 min for CYB003, compared to 60 min for plasma psilocin following psilocybin administration. The half-life (T1/2) for CYB003 was 45 mins, compared to greater than 240 mins for psilocin from psilocybin, and was corroborated by a reduction in CYB003 MRT of 36%. Furthermore, the inter-subject variability of CYB003 at the maximum achieved plasma concentration (Cmax) was reduced by 53% compared to psilocin from psilocybin. The rank order for oral bioavailability (%F) across species was mouse=dog»rat. The CYB003 brain to plasma ratio in the rat after IV administration was 11.5 compared to 8.1 for psilocin from psilocybin, suggesting improved penetration into the CNS, and therefore, the potential to reduce the systemic dose of CYB003 needed to achieve a target therapeutic level in the brain. This trend was also observed in mice. Conclusions: These findings suggest that the unique PK properties of CYB003 provide a meaningful advantage over psilocybin for the treatment of an array of psychiatric disorders, including MDD. Keywords: Psychedelics, Pharmacokinetics, Major Depressive Disorder, Psilocybin Analog, Psilocin Disclosure: Cybin: Employee (Self), Astrazeneca: Employee (Self) P363. CVL-354, a Novel, Brain Penetrant and Selective Kappa Opioid Receptor Antagonist Georgette Suidan, Galen Missig, Sokhom Pin, Srinivas Chakilam, Sridhar Duvvuri, Philip Iredale Cerevel Therapeutics, Cambridge, Massachusetts, United States Background: Kappa opioid receptors (KOR) have strong activity throughout many key regions of the brain whose activity defines psychological phenomena, including motivation incentivized by reward or addictive substances, as well as anxiety. As a regulator of both cellular excitability and synaptic transmission of key circuits involved in reward and mood, KORs present a unique therapeutic target for clinical investigation for the treatment of major depressive disorder and substance abuse disorder. Methods: In vitro pharmacology studies were conducted to evaluate binding affinity and functional antagonism (cAMP) of CVL-354 at human KOR and human mu opioid receptor (MOR). Pharmacokinetic studies were run in both mouse and nonhuman primate to determine brain penetrance. In mouse, target engagement studies were conducted to investigate the relationship between drug exposure and occupancy of CVL-354 at both mKOR and mMOR to determine in vivo selectivity. Opioid-induced thermal sensitivity measured by the tail flick assay was utilized to determine in vivo pharmacological selectivity for mKOR over mMOR. Lastly, CVL-354 reversal of KOR-agonist induced deficits in motivation were measured in the progressive ratio responding task. Results: In vitro binding data demonstrated that CVL-354 has 31-fold binding affinity for hKOR over hMOR indicating selectivity for KORs. Furthermore, CVL-354 was determined to be an antagonist at both KOR (IC50 = 0.042 nM) and MOR (IC50 = 9.1 nM) and, importantly, did not demonstrate agonist activity at either receptor up to 1uM (0.01 nM-1 µM tested). Pharmacokinetic studies in mouse, rat and nonhuman primate demonstrated that CVL-354 is brain penetrant and suggested greater brain penetration in higher species. In vivo target engagement studies in mouse revealed 27-fold selectivity for KOR (IC50 = 2.2 nM) over MOR (IC50 = 59.7 nM), similar to human in vitro binding studies. The thermal sensitivity (tail flick) assay was utilized to demonstrate functional antagonism at both KOR and MOR of analgesia (p < 0.05) and, although qualitative, suggested ~10- fold pharmacodynamic selectivity. Lastly, the effects of CVL-354 on progressive ratio responding were evaluated. Administration of the KOR agonist, spiradoline, in mice induces an anhedonic-like phenotype in which the animals are less motivated to seek rewards in the progressive ratio paradigm (p < 0.05). CVL-354 dose dependently reversed the effects of spiradoline on motivation (ED50 = 0.09 mg/kg, ~2 nM). One-way ANOVA (tail flick and progressive ratio responding) were used and Dunnett’s posthoc tests comparing treatment to control groups were done when appropriate. Conclusions: In vitro and in vivo pharmacology assays demonstrated that CVL-354 is a novel, brain-penetrant, potent and selective kappa opioid receptor (KOR) antagonist. Additionally, CVL-354 reverses KOR agonist induced deficits in motivation. Keywords: Kappa Opioid Receptor, Kappa Opioid Receptor Antagonist, Motivation, Anhedonia Disclosure: Cerevel Therapeutics: Employee, Stock / Equity (Self) P364. Fibroblast Growth Factor 21 (FGF21) is Increased in Patients With Bipolar Depression Krista Wartchow, Giselli Scaini, Tina Li, Giovana Zunta-Soares, Rodrigo Machado-Vieira, Jair Soares, João Quevedo UTHealth Science Center, Houston, Texas, United States Background: One leading hypothesis is that Bipolar Disorder pathology is in part due to failure of mitochondrial function to support adequate neurotransmission and synaptic plasticity, thus affecting mood regulation, memory, and executive function. Several studies support this hypothesis, showing BD patients present atypical mitochondrial metabolism, oxidative stress, and mitochondrial DNA (mtDNA) damage. Moreover, studies in primary mitochondrial diseases have shown a high prevalence of self-reported affective syndromes. Plasma levels of Fibroblast-growth-factor 21 (FGF-21) is an established biomarker for mitochondrial disorders. Methods: In this pilot study, we included 31 healthy controls and 34 patients with BD (11 BD euthymic and 13 BD depressed). All subjects underwent a comprehensive clinical interview and diagnosis of BD and TRD according to the DSM-IV-TR. Mood symptoms and functional status were assessed with the Montgomery Asberg Depression Scale (MADRS), Young Mania Rating Scale (YMRS), Global Assessment of Functioning (GAF) and Functioning Assessment Short Test (FAST). Quantitative analysis of FGF-21 plasma levels was performed using commercial kit. Results: One-Way ANCOVA after controlling for age, gender, BMI, ethnicity, and smoking status showed that FGF-21 plasma yielded no statistically significant between BD patients and HCs. However, after stratifying patients between depressed and euthymic, we found that patients with depressed BD present higher levels of FGF-21 compared to euthymic BD patients. Another notable finding was that FGF-21 plasma levels predicted changes in the depression-rating scale and functional status. Conclusions: In summary, our results suggest that increased plasma FGF-21 may act as a mood state marker in BD. Also, our findings corroborate previous studies and support the notion that mitochondrial dysfunction plays a role in the pathophysiology of BD and may play a role in clinical and functional outcomes. Keywords: Mitochondria, Bipolar Disorder, FGF-21, Bipolar Depression Disclosure: Nothing to disclose. P365. Epigenetic GrimAge Acceleration and History of Suicide Attempt in Bipolar Disorder Camila De Carvalho-Lima, Emese H. C. Kovács, Alexandra Del Favero-Campbell, Alexandre Paim Diaz, Salahudeen Mirza, Consuelo Walss-Bass, Benney M. R. Argue, Joao Quevedo, Jair C. Sores, Marie E. Gaine, Gabriel R. Fries UTHealth Houston, Houston, Texas, United States Background: Bipolar disorder (BD) is associated with reduced life expectancy, with excess mortality in nearly all categories of natural causes. Suicide is a major cause of mortality in BD, and a previous history of suicide attempt has been associated with decreased lifespan and worse clinical outcomes. Although the risk of suicide in BD is high, excess mortality is mainly explained by somatic comorbidities, with recent studies suggesting that premature cellular senescence contributes to the shortened life expectancy seen in psychiatric disorders. Therefore, this premature mortality may be explained by observations that BD is associated with changes in age-related biomarkers of inflammation, oxidative stress, epigenetics, and metabolism. In this study, we investigated the acceleration of GrimAge, a novel epigenetic clock trained on time-to-death data and uniquely associated with mortality and lifespan, as well as its subcomponents, in patients with BD with and without a lifetime history of suicide attempt. Methods: Study participants from a discovery cohort (Houston) included BD patients with no history of suicide attempt (BD/non-SA, n = 66), BD patients with a lifetime history of suicide attempt (BD/SA, n = 77), and healthy controls (HC, n = 51) matched for age, sex, and race. An index of GrimAge acceleration (AgeAccelGrim) was computed based on peripheral blood genome-wide DNA methylation (DNAm) levels measured by the Infinium MethylationEPIC Beadchip (Illumina) and chronological age for all participants. ANCOVA models were used to compare groups for AgeAccelGrim as well as DNAm-based smoking pack-years and seven age-related plasma proteins (adrenomedullin, beta-2-microglobulin, cystatin C, growth differentiation factor 15 (GDF-15), leptin, plasminogen activation inhibitor 1 (PAI-1), and tissue inhibitor metalloproteinases 1). Results from the patient-specific comparisons were independently validated in a replication cohort (Iowa) including BD/non-SA (n = 47) and BD/SA (n = 47). Results: In the discovery cohort, HC, BD/non-SA, and BD/SA significantly differed for AgeAccelGrim after controlling for age, sex, population genetic stratification, years of education, body mass index, smoking status, and blood cell counts (F(2,175) = 7.864, p < 0.001), with the highest AgeAccelGrim found in BD/SA, with an excess of mortality of 3 years compared to HC (p = 0.001). BD/SA also showed a significantly higher AgeAccelGrim than BD/non-SA in the unadjusted model (P = 0.002) and after adjusting for covariates in the discovery cohort (p = 0.027). Furthermore, this between-group difference was also replicated in an independent cohort in both unadjusted (P = 0.02) and adjusted models (P < 0.001). Finally, in the discovery cohort, unadjusted models showed that BD/SA had significantly higher DNAm-based PAI-1 levels (P = 0.019) and smocking pack-years (P = 0.029) compared to HC. After covariate adjustment, BD/SA showed significantly higher PAI-1 levels (P = 0.016), smocking pack-years (P = 0.022) and GDF-15 (P = 0.035) compared to HC. Conclusions: Epigenetic GrimAge acceleration may contribute to premature morbidity and mortality in BD patients with a lifetime history of suicide attempt. These findings pair with existing evidence that not only BD, but also suicide attempt, may be associated with an acceleration of biological aging, and provide putative biological mechanisms for premature mortality in these conditions (for example, through the actions of PAI-1 and GDF-15). Future studies are warranted to explore the role of epigenetic aging in the pathophysiology of BD and suicidal behavior, as well as to dissect their shared and unique biological underpinnings. Keywords: Bipolar Disorder, Suicide Attempt, GrimAge Acceleration Disclosure: Nothing to disclose. P366. Reduced Glutamatergic Cortical Facilitation Associated With Glutamate-Related Gene Expression Abnormalities in Treatment-Resistant Depression Masataka Wada, Shinichiro Nakajima, Shiori Honda, Mayuko Takano, Keita Taniguchi, Yu Mimura, Sakiko Tsugawa, Masaru Mimura, Yoshihiro Noda Keio University School of Medicine, Sinuku-ku, Japan Background: One third of patients with depression do not respond to optimal antidepressant treatment, defined as treatment-resistant depression (TRD). Recent studies noted the benefit of ketamine on TRD and abnormal glutamatergic neurometabolite levels in TRD suggest that abnormalities in glutamatergic neurotransmission are implicated in the pathogenesis of TRD. Furthermore, a recent lesion network mapping study reported that the symptom network of depression may involve the dorsolateral prefrontal cortex (DLPFC) on which transcranial magnetic stimulation (TMS) treatment is also effective on TRD. The intracortical facilitation (ICF) paradigm with a combined TMS and electroencephalography (TMS-EEG) method allows the evaluation of primarily N-methyl-D-aspartate receptor-mediate neural function in target brain regions. The objectives of this study were 1) to compare glutamatergic neural activity as indexed by the ICF paradigm at the DLPFC between patients with TRD and healthy controls (HCs) using TMS-EEG and 2) to explore the links between the cell-specific glutamatergic gene expression and abnormalities of glutamatergic neural activity in TRD using virtual histology approach. Methods: This study was approved by the ethical committee at Keio University School of Medicine and the registration identification number of this study is UMIN000028863. Sixty patients with TRD and thirty HCs received 80 single-pulse TMS and paired-pulse TMS at an interstimulus interval of 10 ms at the left DLPFC to measure the evoked potential. The degree of ICF was assessed by the difference between paired-pulse and single-pulse evoked potentials. Differences in ICF between TRD and HCs groups were calculated by cluster-based permutation analysis. Additionally, we calculated the correlations of interregional profiles between altered glutamatergic neural activity and the glutamate-related gene expressions derived from the Allen Human Brain Atlas dataset. Results: ICF level at the electrode sites above the left DLPFC was not significantly different between the two groups; however, ICF level at the left DLPFC just beneath the TMS coil was decreased compared with that of HCs (p = 0.014). Furthermore, the distribution on the brain surface of the decreased ICF level correlated with the distribution on the brain surface of the specific glutamate-related gene expression (p = 0.016). Conclusions: A reduced level of ICF at the left DLPFC represents a pathophysiological endophenotype of TRD and may be associated with glutamate-related gene expression. Keywords: TMS-EEG, Glutamate, Treatment-Resistant Depression, Imaging-Genetics Disclosure: Nothing to disclose. P367. Developing Neuroimaging Biomarkers for Depression Secondary to Neurological Illnesses Mark Kvarta, Yizhou Ma, Si Gao, Heather Bruce, Joshua Chiappelli, Peter Kochunov, Elliot Hong University of Maryland School of Medicine, Maryland Psychiatric Research Center, Catonsville, Maryland, United States Background: Depression is highly prevalent in individuals with neurological disease, in particular epilepsy, migraine, multiple sclerosis, and stroke, with lifetime depression rates after neurological illness onset as high as ~50%. Co-morbidities between depression and neurological insults are well described, although data on the neurobiological underpinnings are lacking. DSM-defined Major Depressive Disorder (MDD) is associated with well replicated structural brain changes, e.g., reduced volumes in frontal cortices and hippocampus, while the neurobiological origin of Depressive Disorder Due to Another Medical Condition (incl. neurological) is poorly understood and rarely rigorously studied. If biophysical aspects of brain function contribute to MDD, then a neurological illness might change brain structures in a way that resembles patterns associated with MDD as a systems-level neurobiological explanation why some neurological patients develop depressive symptoms. Using neuroimaging and longitudinal behavioral data from the UK Biobank (UKBB) and applying a Regional Vulnerability Index (RVI) to quantify individual brain-wide phenotypic similarity to MDD (RVI-MDD), we tested the hypothesis that a neurological insult can cause brain changes to resemble the deficit patterns observed in MDD to increase risk for depressive symptoms. Methods: UKBB is a large prospective study to identify determinants of health in middle to old age. We used data from two UKBB visits: the initial assessment visit (v0) and the second visit with brain imaging (v2). A continuous measurement of depression symptomatology obtained at v0 and v2 was obtained using the Recent Depressive Symptoms-4 (RDS-4) scale, a measure validated for neuroimaging study within UKBB with questions corresponding to several MDD criteria (Dutt et al., 2022). RDS-4 has high agreement with other scales like PHQ-9 when obtained concurrently, with an advantage that RDS-4 questions were collected at repeat UKBB visit dates including when imaging data was collected. Total depressive symptom scores at either visit were the sum of the answers given (including up to 1 imputed missing score). 46,910 participants (51.3% female) in UKBB completed at least 3 of the RSD-4 depressive symptom questions at both visits and were included. We identified individuals with new report of neurological illness between v0 and v2, a prospective approach in which v0 data can be considered baseline prior to onset of neurological illness (NI). The list of neurological diagnoses included infectious, multiple sclerosis, epilepsy, migraine, intracerebral hemorrhage, and stroke. Imaging data was obtained from UKBB neuroimaging data release version 1.6. RVI calculations require large imaging samples aggregated from independent imaging studies of an illness to establish its ‘gold standard’, here using the ENIGMA consortium data for MDD. UKBB phenotypes included 24 regional white matter tract FA values, 33 regional estimates of cortical gray matter (GM) thickness, volumes of the lateral ventricles, and 7 subcortical gray matter volumes per hemisphere corresponding to these derived by ENIGMA workflows. RVI-MDD scores were calculated using ‘RVIpkg’ in [R]. After regressing out covariates, imaging phenotypes were transformed to z-scores, and the Pearson correlation coefficient calculated between a participant’s z-scores and corresponding effect sizes for MDD patient-control group differences from the ENIGMA consortium for MDD (Schmaal et al., 2017). Results: In first validating RVI-MDD in UKBB data outside of neurological illness, we found individuals with MDD had higher cortical RVI-MDD compared to those without MDD (p = 4×10-7). Amongst the non-neurological control group (NC), those with an increase in depressive symptoms between visits had higher RVI-MDD than those with no worsening in mood (p = 3×10-6). Thus, both cross-sectional and longitudinal data supported validity of applying the RVI-MDD derived cortical deficit pattern to UKBB depression symptom data. There were no significant differences in age and sex ratio between NI (n = 890) and NC group (n = 46,020). NI had higher RDS-4 depression scores at v0 prior to neuro illness onset compared to NC (p = 4×10-7), while both NI and NC showed reduced depression from v0 to v2 (p = 3×10-15). A higher proportion of individuals in the NI group reported worsening depression scores (30.8%) than the NC group (24.9%) at v2 (p = 6×10-5). Among NI, RVI was higher for those with worsened depressive score compared to those with no worsening (p = 0.021). In a full 2×2 ANCOVA model, there was a significant groupdepression change interaction (F(1,35550)=4.02, p = 0.045). NI with worsened depression had the highest RVI-MDD, supporting that RVI can separate NI by depression score status. In comparison, polygenic risk score for MDD was not different between NI and NC groups (p = 0.70). Conclusions: This study demonstrates longitudinal, quantitative changes in mood scores before and after serious neurological events, and that these changes drive cortical deficit changes towards a brain pattern similar to MDD. Those individuals with a neurological insult during the study period who reported worsening depression scores had a higher RVI than those who did not. These data add new neurobiological insight into the etiology of depression after neurological illness. RVI for MDD may provide a biomarker to index those who are vulnerable to develop depression associated with neurological illnesses. Keywords: Human Neuroimaging, Brain Based Markers for Depression, Neurological Disorders, Depression, Big Data Disclosure: Nothing to disclose. P368. Persistent Brain Connectivity Changes in Healthy Volunteers Following Nitrous Oxide Inhalation Charles Conway, Peter Nagele, Thomas Zeffiro, Britt Gott, Thomas Nguyen, Charles Zorumski, Ben Julian Palanca Washington University School of Medicine, Saint Louis, Missouri, United States Background: Nitrous oxide (N2O), an N-methyl D-aspartate (NMDA) receptor antagonist with similarities to ketamine, has shown promise as a therapeutic in treatment-resistant major depressive disorder (TRMD) and PTSD. Similar to ketamine, the antidepressant effects persist well beyond a single one-hour inhalation. Previous studies have demonstrated that during N2O inhalation changes in electroencephalographic signals and altered brain networks occur. Of note, electroencephalographic studies of ketamine in depressed patients demonstrate persistent plasticity changes in occipital cortex. This brain imaging trial examines sub-acute and persisting network brain connectivity changes 2 hours and 24 hours following exposure to one hour of inhaled N2O using resting state functional magnetic resonance imaging (rs-fMRI). Methods: Sixteen healthy volunteers of both sexes were recruited in a double-blinded crossover study. Individuals underwent randomized inhalation sessions of 50% nitrous oxide/oxygen or oxygen/air mixture for one hour. Three 10-minute rs-fMRI scans were obtained prior to inhalation, and at 2-hours and 24-hours after inhalation sessions. Based on literature support of involvement in mood disorders, four a priori canonical network seeds were chosen: default mode, dorsal attention, affect, and reward networks. rs-fMRI inter-regional correlations were measured using echo-planar imaging. Inter-regional correlations were computed and converted to Fisher z scores from time series of preprocessed, motion artifact-scrubbed, and nuisance covariate-regressed time series data. To avoid bias introduced by serial testing, false discovery rate (FDR) corrections were employed. First-level whole brain modeling estimated change in connectivity strength (pre- vs post-N2O) for both a local connectivity strength using local correlation (LCOR) metric and global connectivity strength using a global correlation metric (GCOR). Seed-based functional connectivity was subsequently used to characterize the spatial pattern of connectivity changes associated with areas that showed changes in LCOR and GCOR. Second-level modeling (pre- vs post-N2O) built upon the first-level model findings used a repeated measure, mixed-effects model, with fixed effects of replication, session, condition and subject as a random effect, selecting the primary visual cortex as the seeded region. Results: In comparisons of pre- to post-nitrous oxide inhalation, the a priori selected canonical networks did not reveal significant changes in resting-state functional connectivity. However, exploratory whole brain analyses demonstrated that, compared to placebo, N2O inhalation was associated with statistically significant changes in global brain connectivity that persisted in occipital cortex at 2- and 24-hours post inhalation (p-FDR = 0.019). Further, second-level modeling across 200 grey matter regions demonstrated stronger post-N2O correlations with the bilateral insula, lateral parietal, and middle cingulate regions, as well as bilateral cerebellar regions (all p < 0.05, FDR-corrected with region-based statistics). Further, analysis of resting-state networks demonstrated robust strengthening of connectivity between regions of the visual network and those of the dorsal attention network, both at 2- and 24-hours after inhalation (Cluster TFCE = 55.94 p = 0.041). Conclusions: N2O inhalation in healthy volunteers revealed persistent increases in global connectivity between regions of primary visual cortex and dorsal attention network. These findings suggest that N2O inhalation induces neurophysiological cortical changes for at least 24 hours and are consistent with reports of synaptic plasticity in visual networks induced by ketamine. The sustained nature of these findings is compelling, as it parallels the observed N2O antidepressant clinical findings as well as the previous neuronal plasticity ketamine studies and suggests that these NMDA-antagonists may bring about their antidepressant effects, in part, by persistently altering network connections in the visual and attention realms. Further brain imaging studies of N2O in depression are warranted. Keywords: Nitrous Oxide, Antidepressants, Functional Connectivity, Networks, NMDA Disclosure: Nothing to disclose. P369. Longitudinal Effects of Subcallosal Cingulate Deep Brain Stimulation on the Resting Electroencephalogram in Treatment-Resistant Depression Tanya Nauvel, Sankar Alagapan, Mosadoluwa Obatusin, Stephen Heisig, Jacob Dahill-Fuchel, Patricio Riva-Posse, Christopher Rozell, Allison Waters, Helen Mayberg Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Major depressive disorder affects approximately 14.8 million adults in the US (Kessler et al, 2005). While most cases of depression are treatable, up to 10% of the patients do not respond to conventional interventions using various combinations of medication, psychotherapy and somatic treatments including ECT. Deep brain stimulation (DBS) of the subcallosal cingulate (SCC) white matter is an emerging new treatment strategy for such treatment resistant depression (TRD) with published studies demonstrating sustained long-term antidepressant effects in 73% of implanted patients (Riva-Posse 2018). While long-term effects are generally maintained once achieved, clinical observation and ratings as well as local LFP electrophysiological recordings in the SCC (Alagapan et al, 2021) show that recovery appears to have multiple stages: depressed (defined as <50% Hamilton change); first well (acute behavioral effects in the OR); rough (improved but unstable); and well (>50% Hamilton Depression Ratings Scale). These changes occur with a variable timecourse across individuals. Here we test if the resting EEG demonstrates comparable changes to the LFP and behavioral changes previously observed. Methods: EEG was recorded in two cohorts of TRD patients receiving chronic SCC DBS using two DBS systems: Medtronic Activa PC + S r (n = 8) and Medtronic Summit RC + S (n = 5). All patients were tested at 8 time points: before DBS implantation surgery; 4 weeks post-surgery without ongoing DBS; once a month for 6 months during active stimulation. Recordings consist of 5-minute eyes open and eyes closed resting EEG data (EGI, 256 channels), collected with bilateral stimulation both ON and OFF. Time frequency domain analyses of the resting EEG data over time were first calculated over 3 second windows using the Thomson multitaper method (5 tapers) and average power over 5 frequency bands (θ: 4-8 Hz, α: 8-12 Hz, low β: 12- 20 Hz, high β: 20-30 Hz and γ: 30-50 Hz) was calculated and compared across timepoints. To track the time course of recovery, we clustered the spectral information from a subset of the 256 channels and tracked the percentage of the “well” cluster (extracted from the final 6 months visit) compared to all the other timepoints. We then compared this time course to the transitions defined by a neural network classifier used to distinguish SCC LFP dynamics that reflect differences in clinically defined ‘depressed’ and’well’ states. Results: Changes in EEG power over time show that there are differential early (1 month) and late (2-6 month) band-specific EEG changes in the first PC + S cohort of patients (Two Group Test, P ≤ 0.05). These are similar to the timelines reported in PET and LFP studies within the same cohort of patients. The EEG spectral findings are replicated in the second cohort. Topoplots show different patterns of power changes across each frequency band: alpha and low beta frequency changes localize to parietal/occipital channels, whereas beta power increases are more centrally located around Cz. Resting EEG spectral clusters transitions from ‘sick’ to ‘well’ occur at similar timepoints as the LFP classifier transitions. Conclusions: These findings provide evidence that the resting EEG can identify distinct early and late response change patterns over the course of successful SCC DBS treatment for TRD. The response trajectory over 6 months involves 3 to 4 specific brain states transitions. The similarity of the identified sick-well transitions using EEG to those previously identified in the SCC LFP recordings suggest a nonlinear evolution of SCC-cortical network changes over time with ongoing DBS. These findings invite further consideration of the role of serial resting EEG to identify treatment milestones that might guide treatment optimization. References: 1. Kessler et al. Arch Gen Psychiatry, 2005. 62:593–602. 2. Riva-Posse et al. Mol Psychiatry, 2018. 23:843-849 3. Alagapan et al. Brain Initiative Poster, 2021 Keywords: Brain Based Markers for Depression, Deep Brain Stimulation, Quantitative Electroencephalography (qEEG) Disclosure: Nothing to disclose. P370. Sex and Drinking: Sex Differences of the Effects of Chronic Stress on Drinking Behavior of C57Bl6 Mice in the Sucrose Preference Test Measured With Lickometry Andreas Wulff, Scott Thompson University of Colorado School of Medicine, Aurora, Colorado, United States Background: Depression is a devastating and prevalent mental illness. Rodent models are widely used to understand the neurobiology of depression and in the testing of potential novel antidepressants. As chronic stress is a major risk factor for the development of depression in humans, it is often used to precipitate depression-like behaviors in rodents. A wide range of behavioral outputs are used to measure cognitive, rewarding and other behavioral impairments following chronic stress. Among these is the sucrose preference test used to measure an anhedonic-like phenotype following chronic stress. However, sex as a biological variable has largely been overlooked in these rodent experiments and it is still not clear how the drinking behavior of male and female mice during the sucrose preference test relates to hedonic state. Methods: We used a custom-built homecage lickometry setup to record the drinking behavior of male and female C57Bl6 mice undergoing a sucrose preference test (n = 8-17 per group and sex). We analyzed the changes in drinking as the night progressed and further identified ‘bursts’ of drinking, which has been used as a measure of palatability in prior studies of licking microstructural analysis. We then perturbed the sucrose preference test by either replacing 1% sucrose with 0.1% sucralose, or by subjecting the mice to acute stress or mild food restriction. Finally, we tested how drinking behavior was affected following chronic multimodal stress. When testing the effects of chronic stress, we further complemented the sucrose preference test with other behavioral tests of hedonic state, curiosity, and pain sensitivity. Results: At baseline, male and female mice exhibited a phasic drinking behavior with increased drinking at the beginning and end of the dark phase of a 12:12 light:dark cycle. Both male and female mice exhibited a strong preference for the 1% sucrose solution as observed by a preference in volume consumed as well as by increased drinking at the sucrose bottle and increased number and length of drinking bouts. Mice also exhibited a strong preference for the non-caloric 0.1% sucralose which could also be observed in the number of licks, as well as number and length of lick bouts. Neither acute stress nor mild food restriction appeared to affect the drinking behavior significantly in either male or female mice but chronic stress precipitated a decrease in sucrose preference. When comparing the drinking behavior of male and female mice, we observed that female mice drank in more, but shorter, bouts compared to the males. The female mice were also less susceptible to the chronic stress with males exhibiting a strong decrease in drinking at the sucrose bottle. However, while male mice had a strong decrease in the number of drinking bouts at the sucrose bottle following chronic stress the length of the bout was increased at both the water and sucrose bottle and the relative length of the bout was unchanged between sucrose and water following chronic stress. This effect of stress was also seen in female mice. In this cohort however, we also did not see impairments of other tested hedonic behaviors or of curiosity, but we did observe a heightened pain sensitivity in both male and female mice following chronic stress. Chronic stress also affected the phasic drinking with a stronger peak in drinking at the beginning of the dark phase but no increase in drinking towards the end of the dark phase. Because the length of drinking bouts is usually taken as a measure of acute rewarding response to a palatable solution, our data indicates that a loss in sucrose preference following chronic stress may not be due to a lack of reward responsivity. Instead we saw a decline in the number of lick bouts which could indicate impairment of metabolic or cognitive processes instead. What causes a loss to the sucrose preference may depend on species, strain and/or stressors. Conclusions: Male and female C57Bl6 mice both exhibit a preference for sucrose, but the microstructural drinking behavior is slightly different. We further found that female mice were less susceptible to chronic stress than male mice. Finally, we found indications that under the conditions used in this experiment, the impaired sucrose preference following chronic stress may be due to impairments of cognitive or metabolic impairments rather than impairments in reward responsitivity. Keywords: Reward Deficit, Anhedonia, Stress Disclosure: Terran Biosciences: Patent (Self), Ambit: Patent (Self) P371. Sex-Specific Anti-Angiogenic Mechanisms Unite Brain and Vascular Dysfunction in the RGS2 Knockout Model of Psycho-Obstetric Risk Serena Gumusoglu, Michaela Kiel, Brandon Schickling, Kaylee Weaver, Marisol Lauffer, Hannah Sullivan, Kaylie Coulter, Yuping Zhang, Eric Devor, Donna Santillan, Mark Santillan University of Iowa Carver College of Medicine, Iowa City, Iowa, United States Background: Gestational hypertensive disorders such as preeclampsia share high rates of comorbidity with mood disorders and anxiety, which in-turn appear to increase risk up to 3-fold for preeclampsia. This bi-directional risk suggests shared mechanisms, which may reveal insights into potential treatment targets for these often-dangerous disorders. Prior work by our group revealed that loss of RGS2, a risk gene for preeclampsia and psychiatric disorders including anxiety, depression, and suicide, results in preeclampsia-like obstetric phenotypes such as gestational hypertension and placental dysfunction in a murine model. Here we examine conserved, RGS2-mediated mechanisms that might disrupt both brain and vascular function to increase risk for preeclampsia and psychiatric disease in females in particular. Methods: Adult RGS2 knock out (KO) and wildtype (WT) littermate controls (n = 6 per genotype per sex) were behaviorally tested using the Y-maze, open field, sucrose preference test, tail suspension, social approach, and elevated plus maze (EPM) assays. Brains from these animals were hemisected, with half reserved fresh for qPCR (dissected cortex, midbrain, and hypothalamus/ paraventricular nucleus) via qPCR and half formalin-fixed and labeled with IB4 lectin (cerebrovascular marker), NeuN, and DAPI. ImageJ and stereology were used to calculate vascular and cellular density. Platelet-free plasma serotonin (5-HT) was measured by ELISA. A separate cohort of animals (n = 3-7 per genotype per sex) were tested for peripheral vascular dysfunction. Blood pressure was assessed by tail cuff plethysmography (CODA) and aortas were dissected and studied for redox and angiogenic factors by qPCR. Males and females were analyzed separately. PRISM was used for data analysis and depiction. Results: Male RGS2KO mice exhibited hyperlocomotion (P = 0.05), increased latency to explore (Y-maze) (P = 0.048), and impaired social memory (P = 0.02). Female RGS2KOs exhibited decreased center exploration (P = 0.05) and increased struggle on the tail suspension assay (P = 0.005). Both male and female RGS2KO mice exhibited anxiety-like (EPM) and hedonic (increased sucrose preference) behaviors (P = 0.04, 0.02 respectively). RGS2KO mice also exhibited significant reductions in cortical vascular density (males P = 0.05, females P = 0.144). Cortical thickness, white matter, commissural thickness, and cortical cell density were unchanged. Molecular profiling of the cortex, midbrain, and hypothalamus/paraventricular nucleus via qPCR revealed a significant increase in cortical HTR2A (P = 0.006) and MAOA (P = 0.03) in females, and in hypothalamic SLC6A4 in males (P = 0.02). Peripheral, platelet-free plasma 5-HT was unchanged by sex or genotype. In preliminary assessments, blood pressure was also unchanged by sex or genotype. Aorta assessments revealed significant upregulation of NOX4 (P = 0.002) and HIF-1A (P = 0.04) in females, and PDGFRb (males P = 0.07, females P = 0.05). MMP-9 was also significantly downregulated in RGS2KO males (P = 0.05). Conclusions: Collectively, these results demonstrate that in the RGS2KO, animals exhibit sex-specific alterations in mood and other behaviors, serotonergic gene expression in the brain, in peripheral vascular redox and angiogenesis, and in cerebral vasculature density. Some of these results demonstrate female-specific vulnerabilities. The disruptions we report are not driven by hypertension, rather shared vascular redox and angiogenic dysregulation mechanisms may underlie both peripheral and cerebrovascular and brain function changes. These mechanisms may also link the shared pathogenesis of anxiety/depression to preeclampsia. This points to valuable future therapeutic targets and avenues for further mechanistic exploration. Keywords: Pregnancy, Cardiovascular Function, Serotonin, Depression and Anxiety, Angiogenesis Disclosure: Nothing to disclose. P372. Inflammation Moderates Antidepressant Response to Ketamine in a Rodent Model of Treatment-Resistant Depression: Bioenergetic and Inflammatory Markers of Response Susannah Tye, J. Blair Price, Adam Walker, Jennifer Kruse, Kathryn Cullen, Mark Frye, Lilly Schwieler, Sophie Erhardt The University of Queensland, St Lucia, Australia Background: Low dose ketamine directly stimulates neurotrophic signaling to promote neuroplasticity and antidepressant response. Despite a clear biological mechanism for target engagement, moderating factors are not well understood. The present study examined the effects of inflammation on response to ketamine in a preclinical rodent model of treatment resistant depression. Methods: Male Wistar rats (n = 86) received adrenocorticotropic hormone (ACTH; 100 µg/day, 22 days), lipopolysaccharide (LPS; 750-1250 µg/kg, days 17-22), and/or control vehicle saline (0.9% w/v, 22 days). Sucrose preference testing was conducted every second day across the course of this treatment. Animals were administered ketamine (10 mg/kg, final two days) before the open field and forced swim tests (FST) to screen for antidepressant effect. Prefrontal cortex (PFC) tissue was collected from animals’ postmortem to assay kynurenine, kynurenic acid (KYNA), and 3-hydroxykynurenine (3HK) using high-performance liquid chromatography. Enzyme-linked immunosorbent assays (ELISA) were used to determine protein levels in the PFC (i.e., mammalian target of rapamycin (mTOR), 5’adenosine monophosphate-activated protein kinase (AMPK), and glycogen synthase kinase-3 α/β (GSK3α/β). Serum levels of C-reactive protein (CRP) and corticosterone were determined via ELISA. Results: Significant interaction effects (p = 0.0493) and effects of LPS and ketamine treatment (p = 0.0017) on behaviors in the FST were observed for total immobility time. Notably, a robust antidepressant-like response was observed in animals receiving ACTH + LPS + ketamine, reducing immobility levels relative to ACTH-controls (p = 0.0017) and animals receiving ACTH + LPS (p = 0.0439). Similarly, immobility levels expressed by ACTH + ketamine treated animals were significantly lower than ACTH-controls (p = 0.0027), but not ACTH + LPS-treated animals. No significant effects of ketamine treatment on immobility time were observed among saline-treated animals, and no group differences were observed for other behavioral measures. ACTH + LPS treated animals had significantly higher levels of prefrontal KYNA than animals receiving ACTH + LPS + ketamine (p = 0.0037) or ACTH-controls (p < 0.0001). Conversely, ACTH + LPS + ketamine treated animals had significantly higher kynurenine/KYNA ratios compared to ACTH + ketamine treated animals (p = 0.0008) and ACTH + LPS-treated animals (p = 0.0001). ACTH-controls had higher kynurenine/KYNA compared to ACTH + ketamine treated animals (p = 0.0117) and ACTH + LPS-treated animals (p = 0.0014). ACTH + LPS-treated animals also had significantly greater KYNA/3-HK compared to ACTH-controls (p = 0.0393) and ACTH + ketamine treated animals (p = 0.0102). No significant differences were observed among saline-treated animals for tryptophan metabolites, however Saline+LPS + ketamine treated animals had significantly higher levels of prelimbic AMPK and GSK3α/GSK3β relative to saline-controls (p < 0.05). A significant positive correlation was observed between FST immobility time and serum CRP concentration across all treatment groups. Conclusions: Ketamine directly attenuated LPS-induced elevations in levels of tryptophan metabolites in the PFC of animals treated with ACTH, potentially contributing to the antidepressant-like behavioral response within the context of this model. Neither LPS nor ketamine had any significant effect on prefrontal tryptophan metabolite levels in control saline-treated animals. CRP levels across all groups were associated with immobility time in the FST, further supporting the role of inflammation in moderating stress coping. Targeted research in clinical populations is needed to determine the utility of inflammatory markers in precision medicine treatment stratification. Ketamine’s mechanism of action may optimally promote antidepressant response in individuals with elevated inflammatory markers within the context of TRD. Keywords: Treatment Resistant Depression, Ketamine, Insulin, mTOR, Immune System Disclosure: Nothing to disclose. P373. Dissociable Gene and Circuit Networks in VTA Dopamine Subpopulations Larry Zweifel, Jordan Elum, Barbara Juarez, Grigory Loginov, Scott Ng-Evans, Sam Golden University of Washington, Seattle, Washington, United States Background: Dopamine neurons of the ventral tegmental area (VTA) are genetically and functionally distinct. How the circuit connectivity and gene expression patterns within these populations endow their unique functionality is not resolved. We have endeavored to resolve how genes and circuits intersect at the level of the mesolimbic dopamine neuron subpopulations to establish a more granular understanding of the specialized roles of this system in motivated behavior, reinforcement learning, and decision making. Methods: Fiber photometry was used to measure calcium dynamics in genetically distinct dopamine populations during cue-induced reinstatement, probabilistic reinforcement, and reward valuation. Sice electrophysiology was performed to measured synaptic connectivity and intrinsic properties of these cells. Analysis of single nuclear RNA sequencing was employed to establish gene network differences. Cell-specific rabies tracing, light sheet microscopy, and brain wide connectivity analysis was used for 3D reconstruction of circuit connections. Results: We discovered that dopamine subpopulations that project to either the nucleus accumbens core or nucleus accumbens shell display differential calcium dynamics during cue-induced reinstatement, probabilistic reinforcement, and reward valuation. Whole-brain connectivity analysis revealed differential inputs to these cells that was confirmed by in vivo optical stimulation of these inputs and imaging of subpopulations. Electrophysiology revealed that these subpopulations have differential inhibitory connectivity and intrinsic excitability. Consistent with these findings disinhibitory networks evoke dissociable response profiles in these cells. Finally, our gene expression analysis reveals that differential ion channel expression within these cells contributes to their excitability and stimulus response profiles. Conclusions: By combining circuit, gene, systems, and behavioral analyses we have established that both distinct and graded difference in circuit connectivity, gene expression, and intrinsic excitability of VTA dopamine subpopulations converge to impart the unique functionality of these cell types within the mesolimbic system of the brain. Keywords: Dopamine, Behavior, Neural Circuits, Molecular Genetics, In Vivo Imaging Disclosure: Nothing to disclose. P374. The Bed Nucleus of the Stria Terminalis Mediates the Expression of Two-Way Active Avoidance Justin Moscarello, Diana Guerra, Wei Wang, Karienn Souza Texas A and M University, College Station, Texas, United States Background: Although avoidant behavior is a unifying symptom of many anxiety disorders, its underlying neural circuitry remains a mystery. Here, we report a series of experiments using a signaled active avoidance (SAA) procedure to model avoidant behavior in male rats. In SAA, the subject rapidly acquires an association between a conditioned stimulus (CS) and an aversive unconditioned stimulus (US). As training proceeds, the subject learns to perform a response (two-way shuttling) during the CS in order to prevent US delivery. Over the course of SAA training, the US presentation decreases drastically and thus transforms from a certain threat during initial acquisition into a distal threat as the avoidance response reaches asymptotic levels of expression. Prior research demonstrates that the bed nucleus of the stria terminalis (BNST) mediates defensive responses to possible/distal conditioned threats, suggesting a possible role for this region in the expression of active avoidance behavior. However, its role in SAA has yet to be examined. Methods: We used a virally mediated DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach to explore the role of BNST in SAA. Rats received stereotactic infusions of a solution containing one of the following AAVs, depending on the experiment: AAV5-hSyn-hM4D(Gi)-mCherry, AAV5-hSyn-hM3Dq-mCherry, or AAV5-hSyn-EGFP. Following recovery, subjects received SAA training in which a tone CS (15 sec, 70 dB) preceded a footshock US (0.7 mA, 0.5 sec). Training occurred in a rectangular chamber separated into two compartments by a divider with an open aperture that allowed free movement between compartments. If, during the CS, the subject shuttled across the chamber to the opposite side, the CS terminated, and the US was omitted. Following the completion of training, all subjects received test sessions preceded by administration of 3 mg/kg of the DREADD agonist clozapine-N-oxide (CNO) or vehicle (10% DMSO). Results: We first tested the hypothesis that BNST is necessary for the expression of the two-way active avoidance response. Male rats received intra-BNST infusions of AAV containing the gene construct for either the inhibitory hM4Di DREADD or GFP. After recovery, animals received four days of SAA training in order to reach stable levels of avoidance. This was followed by two additional days of SAA training preceded by counterbalanced IP injections of either the DREADD ligand CNO or vehicle. We found that CNO decreased avoidance responses and increased avoidance latencies in hM4Di subjects but not in GFP controls, demonstrating that the BNST is necessary for expression of the two-way avoidance response. Because some subjects in this experiment showed hM4Di expression in the medial septum (MS), we followed up with an anatomical control experiment specifically targeting this region. We found no evidence of a role for MS, indicating that the effects observed in our initial experiment were BNST-specific. We then compared DREADD activation (hM3Dq) and inhibition (hM4Di) of BNST in a test of avoidance under extinction conditions (10 CSs, no USs) in which all groups receive a common series of stimuli. After AAV infusions and recovery, subjects received six days of avoidance training. 24 hours later, all subjects were tested following the administration of either CNO or vehicle. This experiment confirmed that CNO decreased the expression of the avoidance response in hM4Di-expressing subjects. Indeed, CNO caused the hM4Di group to avoid at levels that were highly similar to the poor avoider group, which was comprised of subjects that never successfully expressed the response during training, illustrating the degree of the behavioral decrement induced by BNST inactivation. In contrast, administration of CNO to hM3Dq-expressing subjects caused a period of potentiated shuttling that extended beyond the presentation of the CS, whereas other groups quickly dropped back to baseline levels of shuttling following CS offset. Analyses of other ongoing behavior at test revealed that these effects on two-way shuttling in hM4Di and hM3Dq groups cannot be attributed to alterations in freezing or overall locomotor activity. Conclusions: We conclude that BNST is not only necessary for normal levels of avoidance but is also sufficient to potentiate the output of the response, establishing a region identified as a crucial substrate for distal threat processing as a key mediator of avoidant behavior. This is in contrast with prominent conceptual models of the behavioral processes underlying SAA, such as two-factor theory, which identify putative fear states most related to imminent threat processing as the primary trigger of the avoidance response. Our results argue for an updated multi-factor model in which post-encounter responses to certain threat are supplanted by pre-encounter responses to possible threat over the course of the SAA learning curve. Keywords: Fear, Anxiety, Avoidance, Bed Nucleus of the Stria Terminalis, Predatory Imminence Disclosure: Nothing to disclose. P375. (R,S)-Ketamine and (26,6 S)-HNK Attenuate Learned Fear by Differentially Modulating Brain-Wide Neural Activity Alessia Mastrodonato, Noelle Kee, Marcos Lanio, Michelle Jin, Andrea Muñoz Zamora, Christine Ann Denny Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Stress exposure is one of the greatest risk factors for fear and anxiety disorders, such as major depressive disorder and post-traumatic stress disorder (PTSD). We previously showed that a single injection of (R,S)-ketamine prior to stress reduces behavioral despair and attenuates learned fear by modulating hippocampus ventral CA3 activity in male mice. Recently, we found that a metabolite of (R,S)-ketamine ((2 S,6 S)-hydroxynorketamine (HNK)) also attenuates learned fear. However, the whole-brain regions mediating (R,S)-ketamine and (2 S,6 S)-HNK effects on fear behavior are still largely unknown. Methods: Here, we used a 3-shock contextual fear conditioning (CFC) paradigm as a stressor 1 week following a single injection of saline or (R,S)-ketamine (30 mg/kg) or (2 S,6 S)-HNK (0.075 mg/kg) to adult (8-week-old, n = 10 per group) 129S6/SvEv male mice. Five days later, mice were re-exposed to the aversive context and sacrificed an hour later to quantify neural activity (i.e., c-fos expression) across the whole brain using an analysis pipeline developed in our laboratory. We then transformed the c-fos cell counts separated by brain region into correlation matrices and created whole-brain networks to provide information on functional connectivity. Results: (R,S)-ketamine and (2 S,6 S)-HNK administration attenuate learned fear compared to saline mice (p < 0.01 for both drugs). (R,S)-ketamine increases fear-related neural activity and connectivity in several brain regions (e.g., ventral CA3, restrosplenial cortex and temporal associative area (p < 0.01)), while (2 S,6 S)-HNK increases the connectivity specifically within and between the prefrontal cortex and amygdalar regions (p < 0.05). Conclusions: Our results indicate that (R,S)-ketamine and (2 S,6 S)-HNK attenuate learned fear by differentially altering network correlated activity. We found new nodes in the network that are altered by fear behavior so that we can target with pharmacological manipulations to alleviate fear. This work contributes to the understanding of prophylactic drugs as therapeutic aids for fear-related disorders. Unique Data: All data are new and unpublished Keywords: (R,S)-ketamine, (2 S,6 S)-HNK, Fear Conditioning Disclosure: Nothing to disclose. P376. A Stress-Sensitive Frontostriatal Circuit Supporting Effortful Reward-Seeking Behavior Robert Fetcho, Puja Parekh, Margaux Kenwood, Laura Chalencon, David Estrin, Jolin Chou, Megan Johnson, Conor Liston Weill Cornell Medical College, New York, New York, United States Background: Effort valuation—a process for selecting actions based on the anticipated value of rewarding outcomes and expectations about the amount of work required to obtain them—plays a fundamental role in decision-making. Effort valuation is disrupted in chronic stress states and is supported in part by the anterior cingulate cortex (ACC), but the circuit-level mechanisms by which the ACC represents effort-related signals and regulates effort-based decision-making and reward-seeking are not well defined. Methods: We developed an automated, physiology-compatible platform for high-throughput assessments of effort valuation in freely moving mice. In an elevated T-maze apparatus, animals (male C57Bl/6 mice, age 8-12 weeks) were presented with the choice between water rewards of varying magnitudes that are associated with varying effort expenditure requirements. We utilized fiber photometry and optogenetics to record from and manipulate nucleus accumbens projecting ACC neurons (ACC-NAc) as mice performed this effort-based decision-making task. Following training, a subset of mice underwent chronic corticosterone exposure - a model of the neuroendocrine response to stress - and following exposure were retested on the behavioral paradigm alongside ACC-NAc recordings. Results: We found that ACC-NAc activity anticipates and responds to reward acquisition; however, the circuit does not appear to encode reward magnitude. Instead, the magnitude of ACC-NAc reward-related activity scaled with the level of effort expenditure required to obtain the reward (N = 16 mice, 4384 behavioral trials, 3 experiments; linear mixed effects model: significant interaction between reward type and effort level – F(2,4378) = 135.38 p < 0.0001). We next found that optogenetic silencing of this effort-sensitive reward signal led to significant reductions in future effortful decisions within a behavioral session (Two-way repeated measures ANOVA: N = 18 mice (11 control, 7 experimental), 2 experiments. Significant interaction F(1,16) = 13.06, p = 0.0023. Post-hoc bonferonni testing control: baseline vs. stim p = 0.7, experimental: baseline vs. stim p = 0.0001). Finally, chronic corticosterone treatment led to significant impairments in effortful reward-seeking behavior that strongly correlated with impaired ACC-NAc circuit function (N = 5 mice, 10 sessions from 2 experiments. Linear mixed effects model T(10) = 4.56, p = 0.002, R2 = 0.72). Conclusions: Our results show that ACC neurons support effort valuation behavior through projections to the NAc. Specifically, ACC-NAc circuit activity integrates both reward- and effort-related information and this activity is critical for reinforcing future effortful decisions. Chronic corticosterone leads to disruptions in this effort-sensitive reinforcement signal that correlate with impaired effortful reward-seeking behavior, suggesting one potential mechanism underlying stress-induced motivational deficits. Keywords: Anterior Cingulate Cortex (ACC), Effort, Reward, Stress Disclosure: Nothing to disclose. P377. Nucleus Accumbens Glutamatergic Afferents Integrate Outcomes in Reward-Learning Eshaan Iyer, Jessie Muir, Serena Wu, Karen Wassef, Rosemary Bagot McGill University, Montreal, Canada Background: Alterations in reward learning are associated with depression and other psychiatric disorders. The nucleus accumbens (NAc) is a key region implicated in motivation and reward and disrupted in depression. Susceptibility to depression-relevant behavior is mediated by afferent glutamatergic projections from the ventral hippocampus (vHip) and the medial prefrontal cortex (mPFC) to NAc. The role that these glutamatergic inputs to the NAc in supporting reward learning remains relatively unexplored. Methods: Here, using in vivo fiber photometry, we simultaneously record the population-level activity of mPFC and the vHip projections to the NAc in adult male (n = 12) and female mice (n = 12) in a two-armed bandit task. Results: Both neural projections dynamically encode information about the outcomes of a given trial. Rewarded vs non-rewarded outcomes are associated with greater suppression in the mPFC-NAc both shortly following a choice (p < 0.001) and throughout the inter-trial-interval (ITI) (p < 0.01). In the vHip-NAc, reward-associated suppression emerged at a longer delay following a choice (p < 0.01) and also continued through the ITI (p < 0.0001). In both pathways, this reward-associated suppression continued into following trials (mPFC: p < 0.0001; vHip: p < 0.001) revealing neural representations of outcome modulated by reward history. We observed notable pathway-way specific differences with mPFC-NAc projections tracking outcome history, while vHIP-NAc projections preferentially encode unrewarded outcomes, tracking history of loss but not reward. Conclusions: Together, these findings demonstrate that mPFC-NAc and vHip-NAc projections integrate outcomes over time in a task-dependent manner to support reward learning. In light of earlier evidence identifying altered neural activity in these pathways in stress-induced states, these neural circuits may be relevant to understanding alterations in reward learning in depression and other stress-related disorders. Keywords: Probabilistic Reward Learning, Glutamatergic, Nucleus Accumbens Glutamatergic Afferents, mPFC, Ventral Hippocampus Disclosure: Nothing to disclose. P378. Identifying the Mechanisms of Noradrenergic Modulation of BLA-mediated Avoidance Behaviors Sean Piantadosi, Veronica Porubsky, Madison Martin, Avi Matarasso, Tammy Nguyen, Michael Bruchas University of Washington, Seattle, Washington, United States Background: Acute stress and threat produce physiological anxiety thought to facilitate planning and allow an organism to adapt its behavior for future exploration of the environment. This serves as an adaptive mechanism that allows anxiety-like behavior and avoidance to be tuned and selected. However, in many mental health disorders this homeostatic behavioral response becomes maladaptive and dysfunctional, leading to excessive anxiety in scenarios where it is unwarranted or undesirable. Anxiety disorders, which are typified by this maladaptive response, are the most common mental illness in the U.S., affecting roughly 20% of the adult population. Clinically, we know that neuromodulators such as norepinephrine (NE) play pivotal roles in long-term outcomes following stress exposure. Despite this, the mechanism by which these monoamine neuromodulatory signals regulate circuit activity associated with anxiety-like behavior remains poorly understood. One key modulatory system well positioned to mediate these behaviors is the locus coeruleus noradrenergic system (LC-NE), as it is one of the first engaged following stressful events and stimuli and activation of the LC and its projections to the basolateral amygdala (BLA) is anxiogenic. However, we know very little about how this critical neuromodulatory LC-BLA circuit generates anxiety-like behavior at the network, circuit, cell type, transmitter, and receptor level. Methods: We conducted two-photon calcium imaging of individual LC-NE neurons using an endoscopic prism lens while mice (n = 5, 3 male, 2 female) were exposed to the potent predator stressor 2MT. Next, we modeled the effect of stress on LC activity and measured NE release in the BLA using a NE sensor (GRABNE2m) while activating LC terminals in the BLA at a tonic (5hz) frequency using combined optogenetics and fiber photometry (n = 6, 6 male). We then performed freely moving microendoscopic calcium imaging of BLA neurons in classic anxiety-like behavior assays while manipulating LC terminals optogenetically at the stress-induced tonic frequency (Dbh-cre mice; n = 8, 5 male, 3 female). Pharmacological manipulations (using selective α1 and β2 antagonists) were used to discern which adrenergic receptors were mediating the anxiogenic effect of LC-BLA stimulation. Support vector classifiers were trained to predict whether mice were in an anxiogenic context based solely on BLA population activity. Graph theory analyses were developed to test whether tonic LC terminal activation would cause correlated increases in activation of BLA neurons as might be expected of a gain control signal. Finally, CRISPR-SaCas9 knockdown of gene which encode adrenergic receptor (AR) subtypes (α1- and β2-ARs) within the BLA was combined with microendoscopic calcium imaging to evaluate the necessity of these receptors for the expression of anxiety-like behavior following stress and how each of these receptor subtypes on specific cell types contributes to BLA population encoding of anxiety-state. Results: LC-NE neuron activity was found to be highly synchronous when mice consumed an appetitive stimulus. When mice were exposed to the predator odor stressor 2MT, exploratory licking behavior was dramatically reduced (t(3)=2.82,p < 0.05). This reduction in exploratory licking coincided with an increase in tonic firing rate in a large subset of LC-NE neurons (t(37)=18.1,p < 0.0001), suggesting that in many LC-NE neurons stress induced tonic activation, though responses became less synchronous as a population. We next modeled the effect of this increased tonic activation on NE release within the BLA, finding that 5hz activation of LC-BLA terminal activation produced sustained increases in NE release as measured by GRABNE2m signal (p < 0.001). This effect was blocked by the α2-AR antagonist yohimbine. Using combined optogenetics and freely moving microendoscopy, we recorded the activity of individual BLA neurons while manipulating LC-BLA terminals at a tonic frequency and assessed anxiety-like behaviors. We found that tonic LC-BLA activation was anxiogenic (RM-ANOVA, p < 0.05; post-hoc t(7)=3.41,p < 0.05), promoted a shift in activity of BLA neurons that were activated when mice entered the anxiogenic context (center of open field, and open arms of elevated zero maze), and improved the ability of BLA population activity to classify mouse location within the arenas (t(6)=2.95,p < 0.05). These effects were blocked by the β2-AR antagonist propranolol (p > 0.05). Tonic LC-BLA activation produced sustained changes in the correlation structure of BLA neurons that lasted after stimulation ended, suggesting that mimicking stress by specific activation of LC inputs to BLA produces sustained changes in BLA network activity. CRISPR-SaCas9 knockdown of Adrb2 (gene encoding β2-AR) in Vglut1 neurons in the BLA was effective and resulted in a roughly 40% knockdown. Conclusions: Neuromodulation, and specifically the neuromodulator norepinephrine, has long been suggested to serve as a gain control signal capable of shifting the activity of large populations of downstream neurons. Here using a combination of behavioral, optical, and molecular approaches we show that sustained release of norepinephrine following stress or optogenetic activation produces lasting alterations in BLA network activity, consistent with this theory, and identify the potential receptor (β2-AR) that might be necessary for this type of network shift. Keywords: Locus Coeruleus (LC), Norepinephrine, In Vivo Calcium Imaging, Basolateral Amygdala, Stress and Anxiety Behavior Disclosure: Nothing to disclose. P379. Early Life Adversity Causes Fear Generalization by Impairing Serotonergic Modulation of the Ventral Dentate Gyrus Lauren Malave, Rushell Dixon, John Bickle, Christoph Anacker Columbia University and New York State Psychiatric Institute, New York, New York, United States Background: Having a history of early life adversity (ELA), such as physical or emotional trauma experienced during sensitive periods in early development, increases risk for psychiatric disorders later in life and reduces responsiveness to antidepressant treatment. ELA exerts long-lasting changes on the developmental trajectory of neural circuits and neurotransmitter systems, such as the serotonergic system, in a way that may be different from the effects of adult stress or genetic predisposition. Previously, we found that hyperactivity of the ventral dentate gyrus (vDG) region of the hippocampus increases stress responses. Here, we investigate the interaction between ELA, serotonin (5-HT), and DG function on fear overgeneralization, which is a form of cognitive distortion that is characteristic for variety of mental illnesses. Understanding the neurobiological mechanisms underlying the specific contributions of ELA to fear overgeneralization may thus help us develop better treatments or preventions for psychiatric disorders. Methods: To induce ELA in mice, we used the limited bedding and nesting (LBN) model from postnatal day (P) 3-10. LBN causes fragmented and unpredictable maternal care, as well as rough handling and stepping on pups. To examine if the effects of ELA can be rescued by increasing 5-HT signaling, we used a transgenic mouse model to conditionally knockdown 5-HT1A auto receptors on raphe 5-HT neurons (Pet1-tTS; Htr1atetO/tetO mice), resulting in increased 5-HT signaling starting at birth. Control and LBN-exposed offspring with and without 5-HT1A knockdown were tested during adolescence (P35) and adulthood (P56) in a fear discrimination task. Mice received a foot shock in context A on day 1 and were then re-exposed to context A on day 2. Two hours after re-exposure to the shock-associated context A, mice were placed in a new, safe context B. Freezing behavior was recorded using FreezeFrame software (Actimetrics) to compare fear expression between context A and context B on day 2. We also used immunohistochemistry for the immediate early gene, Fos, to measure the activity of raphe 5-HT neurons and of vDG granule neurons after exposure to context B. Tissue levels of 5-HT in the vDG of control and ELA-exposed mice were measured using HPLC. Results: Female, but not male, mice exposed to LBN overgeneralize between a foot-shock associated context A and a safe context B in adulthood at P56, as indicated by a lower discrimination ratio between the two contexts (Wild-Type (WT) control: 0.63 ± 0.04 n = 26; WT ELA: 0.34 ± 0.1 n = 14; 2way ANOVA ELA vs. genotype interaction F(1,55)=7.7, p = 0.01; Tukey’s post hoc, WT control vs. ELA p = 0.01). Increasing 5-HT levels rescued the behavioral deficits observed in adulthood (WT ELA: 0.34 ± 0.1 n = 14; 5-HT1A knockdown ELA: 0.66 ± 0.06 n = 11; 2way ANOVA ELA vs. genotype interaction F(1,55)=7.7 p = 0.01; Tukey’s post hoc, p = 0.02). These differences were only observed at P56 and not at P35 for all groups, indicating that LBN leads to cognitive deficits in adulthood. At P56 in females, we found increased Fos expression in the vDG of ELA-exposed mice following exposure to the safe context B compared to controls (WT control: 67.4 ± 4.1 n = 3; WT ELA: 42.5 ± 5.6 n = 6; 2way ANOVA ELA effect F(1,11) = 8.5, p = 0.01; Tukey’s post hoc, control vs. ELA p = 0.02). This increase in Fos expression was only found in the vDG, an area involved in anxiety-related behaviors, and not in the dorsal DG, an area involved in spatial navigation. This increase in vDG Fos expression was rescued by 5-HT1A knockdown, similar to the effect of 5-HT1A on rescued fear overgeneralization. Percentage of Fos+ 5-HT neurons in the median raphe nucleus (MnR), the nucleus projecting to the hippocampus, was reduced in mice that experienced ELA (Control: 16.4 ± 1.02 n = 5, ELA: 8.9 ± 1.3; n = 5; 2way RM ANOVA ELA vs. region interaction F(1,8)=28.2, p = 0.001; Sidak’s post hoc, control vs. ELA, p = 0.0004). No effects of ELA were observed in the dorsal raphe nucleus, or on the total number of 5-HT neurons, suggesting that ELA causes dysfunction in MnR 5-HT neurons independent of total neuron number. Additionally, we found that 5-HT levels in the vDG were decreased in ELA-exposed animals at P56 (Control: 232.1 ± 35.6 n = 6; ELA: 112.2 ± 13.9 n = 6; unpaired t test, p = 0.01) but not at P35, suggesting an impairment of 5-HT modulation of the vDG that manifests in adulthood. Conclusions: Our results show that ELA causes vDG hyperactivity and fear overgeneralization in adult female mice, and that increasing 5-HT levels from birth can rescue these neurobiological and behavioral deficits. Understanding how 5-HT regulation of the vDG mediates ELA-induced behavioral impairments may provide new potential treatment options for psychiatric disorders that have their origin early in life. Keywords: Early Life Adversity, Serotonin, Dentate Gyrus, Early Life Stress, Neural Circuits Disclosure: Nothing to disclose. P380. Frequency-Dependent Entrainment of Brain Networks Using Transcranial Magnetic Stimulation Juliana Corlier, Andrew Wilson, Cole Citrenbaum, Andrew Leuchter University of California - Los Angeles, Los Angeles, California, United States Background: Transcranial Magnetic Stimulation (TMS) is an effective treatment for several psychiatric and neurological conditions such as depression, chronic pain, obsessive compulsive disorder, smoking cessation and others. While the therapeutic efficacy is hypothesized to arise from the effects on widespread networks beyond the stimulation site, the role of the stimulation frequency is not clear. In the past, various frequencies have been successfully used for therapeutic purposes, including 1, 5, 10, 18, 20 Hz as well as theta-burst stimulation. It remains to be elucidated how the elicited patterns of activation vary by frequency. Additionally, it is unclear if brain circuits have resonant frequencies (RF) to which the networks respond in a preferential manner and if these frequencies differ between individuals. We hypothesized that different frequencies would elicit spatially distinct patterns of electroencephalographic (EEG) activity that would reflect how different brain circuits respond to stimulation. We have also hypothesized that preferred RFs vary across individuals. We are using a newly developed TMS-EEG interrogation paradigm to evaluate TMS-evoked spectral changes and the entrainment of oscillations in the source-localized EEG signal and the identification of RF across individuals. Methods: 80 subjects with Major Depressive Disorder (MDD) underwent a TMS interrogation at 71 frequencies ranging from 3-17 Hz (in steps of 0.2 Hz) administered to left dorsolateral prefrontal cortex (DLPFC). Each stimulation frequency consisted of 40 pulses with 26 s off period in between frequencies. Frequency order was randomized for all subjects. 64-channel EEG was recorded at baseline, during the entire interrogation procedure and after the session. We used a custom developed artifact removal pipeline to remove TMS-pulse artifact, decay artifacts and conventional EEG artifacts (electrical noise, eye, muscle, cardiac artifacts) following each TMS train. The period [-1000 0 ms] pre stimulation and [0-1000 ms] post every frequency was extracted and used for source localization using Brainstorm. We created a head model with the Boundary Element Method (BEM) using digitized electrode locations and the OpenMEEG toolbox. The source time series were reconstructed using the Minimum Norm Estimation (MNE) method to obtain the inverse solution, which addresses the ill-posed nature of EEG source estimation through regularization. For each frequency, we obtained an average cortical source activation map for each frequency band. Only circuit activation greater than z-scores > = 2.5 compared to baseline activity are displayed. Results: We present average source-localized activation maps (Figure 1) as well as individual subject examples (Figure 2) for all 71 stimulation frequencies in the broadband and the bandpass filtered frequencies of delta [1-4 Hz], theta [4-8 Hz], alpha [8-12 Hz] beta [12-20 Hz] and low gamma [20-30 Hz]. Different TMS stimulation frequencies elicit widespread cortical activations well beyond the stimulation site. The spatial distribution of the elicited patterns varies substantially between individuals based on the stimulation frequency. Conclusions: We demonstrate that different TMS stimulation frequencies elicit largely different spatial activation patterns in the source localized EEG signal and that these activation maps vary as a function of frequency and across individuals. These findings have clinical implications suggesting that a personalized treatment frequency should be used to enhance the entrainment of target brain circuits. Keywords: Brain Stimulation, TMS-EEG, Frequency Optimization Disclosure: Nothing to disclose. P381. Distinct Populations of Ventral Pallidal Cholinergic Neurons Encode Valence of Olfactory Stimuli Ronald Kim, Mala Ananth, Niraj Desai, Lorna Role, David Talmage National Institutes of Health, Bethesda, Maryland, United States Background: Basal forebrain cholinergic neurons (BFCNs) play a critical role in a wide array of behaviors. In addition to an established role of BFCNs in learning and memory, recent evidence demonstrates BFCNs encode innate behaviors. For example, we have shown that mice display approach to an appetitive odor (2-phenylethanol) and avoidance to an aversive odor (predator urine). These innate behaviors are associated with an increase in the number of activated cholinergic neurons in the ventral pallidum (VP). The goal of the present studies is to expand on these findings, and further examine the functional significance of VP cholinergic neurons. Methods: To better understand how VP cholinergic neurons respond to an appetitive vs. aversive odor, fiber photometry was used to characterize in-vivo calcium activity. Chat-cre mice were injected with AAV-syn-Flex-GCaMP6F and received a fiber optic implant targeting the VP. Following habituation, calcium activity of VP cholinergic neurons was assessed in response to either the appetitive or aversive odor one day, and the opposite odor the following day. To test whether same or distinct subsets of VP cholinergic neurons are activated in response to each odor, Chat-cre x cFos-tTA/GFP mice were injected with an activity- and cre-dependent viral vector in the VP (ADCD-hM4Di). This strategy utilizes a Tet-Off system, where in the absence of a doxycycline (DOX) diet, activated cholinergic neurons are permanently labeled with mCherry. cFos-GFP can be used to label activated neurons in a distinct context. Mice underwent behavioral testing across 3 days, Day 0 = habituation (DOX-on), Day 1 = odor exposure 1 or saline (DOX-off, activated VP cholinergic neurons labeled with mCherry), Day 2 = odor exposure 2 or saline (DOX-on, immunohistochemistry for GFP and Chat). To examine the role of VP cholinergic neurons in innate behavioral responses to odor exposure, Chat-cre mice were injected with syn-DIO-hM4Di. Fifteen minutes following an IP injection of a subthreshold dose of clozapine, preference to either the appetitive or aversive odor was assessed in a Y-maze. For selective chemogenetic inhibition of previously activated VP cholinergic neurons, Chat-cre x cFos-tTa/GFP mice were injected with ADCD-hM4di. Following habituation, mice were taken off a DOX diet and exposed to either the appetitive or aversive odor (activated VP cholinergic neurons labeled with mCherry). Twenty-four hours later, mice were injected with clozapine to inhibit the previously activated VP cholinergic neurons, and preference to the same odor was assessed in a Y-maze. Results: Preliminary fiber photometry results show that VP cholinergic neurons demonstrate reliable, time-locked increases in calcium activity in response to both the appetitive and aversive odor. We also examined if activated cholinergic neurons in the VP were the same or distinct neurons following exposure to appetitive vs. aversive odor. When exposed to the same odor on days 1 and 2, mice exhibited a significant increase in colocalization of mCherry (activated VP cholinergic neurons on day 1) and GFP (activated VP cholinergic neurons on day 2). This indicates re-activation of previously activated VP cholinergic neurons when exposed to the same odor. However, when mice are exposed to a distinct odor on day 2, no colocalization of mCherry and GFP were observed. These results are consistent with there being two distinct subpopulations of VP cholinergic neurons: one population activated in response to an appetitive odor and a second, distinct population activated upon exposure to an aversive odor. Our chemogenetic inhibition experiments with syn-DIO-hM4Di revealed that general inhibition of all subpopulations of VP cholinergic neurons abolished normal approach to the appetitive odor. Mice spent more time in the saline paired arm, signifying the appetitive odor was now aversive. In contrast to the VP cholinergic block of approach behavior, avoidance to the aversive odor was still observed. Similar results were observed when specifically targeting VP cholinergic neurons that were previously activated. Selective inhibition of VP cholinergic neurons that were previously activated in response to the appetitive odor abolished approach behavior and led to avoidance of the appetitive odor during the preference test. Targeted inhibition of VP cholinergic neurons that were previously activated after exposure to the aversive odor resulted in normal avoidance to the aversive odor. Conclusions: The results from the present studies reveal (1) increases in in-vivo calcium activity of VP cholinergic neurons in response to both an appetitive and aversive odor; (2) the VP contains two distinct and non-overlapping subpopulations of cholinergic neurons which are uniquely engaged by either appetitive or aversive stimuli; and (3) chemogenetic inhibition (either general inhibition or selective inhibition of previously activated neurons) of VP cholinergic neurons alters innate behavioral responses to appetitive but not aversive odor. In ongoing studies, we are exploring how these subpopulations of VP cholinergic neurons differ, with a focus on mapping their projections and measuring their baseline electrical properties. Keywords: Acetylcholine, Ventral Pallidum, Valence Disclosure: Nothing to disclose. P382. Differential Vulnerabilities to Acute and Chronic Variable Stress in Mice Carrying a Parkinson’s Disease-Linked LRRK2-G2019S Mutation Christopher Guevara, Kumayl Alloo, Romario Thomas, Alexander Tielemans, Kyomi Blake, Jamal Magoti, Swati Gupta, Alexandra Magee, Deanna Benson, George Huntley Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Parkinson’s disease (PD) is associated with psychiatric non-motor symptoms such as depression and anxiety that emerge early, appear to be independent of dopamine neuron loss, and are poorly understood. The G2019S mutation in the Lrrk2 gene is one of the most commonly associated PD risk gene mutations found in both sporadic and familiar late-onset PD. Risk for both PD and depression is increased by stress, and previous work in the lab has shown that young adult Lrrk2-G2019S knockin mice display behavioral, synaptic and non-synaptic plasticity adaptations to social stress that differ significantly from those of wildtype mice. Methods: In order to determine whether social stress-effects are generalized to other forms and magnitudes of behavioral stress, we subjected young adult wildtype (WT) and G2019S knock-in (GS) mice to a standard, daily variable stress (VS) paradigm consisting of one, 1-hr stressor per day over three days in the following order: 100 mild foot shocks, tail suspension and restraint. This stress paradigm was applied for 6 days (6d-VS) or 28 days (28d-VS). Following the last stressor, home-cage unstressed controls and stressed mice underwent a battery of assays (open field test, social interaction test, and novelty suppressed feeding) to probe for stress-induced behavioral changes. Results: In WT mice, we found no major changes in behavioral assays after 6d-VS, but significant stress-effects in post-stress behavioral assays emerged at 28d-VS, as expected from prior work. In contrast, GS mice already displayed significant stress susceptibility after 6d-VS which persisted after 28d-VS. These behavioral adaptations are driven specifically by stressful experiences, as no differences were observed between genotypes in unstressed control conditions. Conclusions: These data show that the G2019S mutation lowers the threshold for stress susceptibility broadly across stress paradigms, mounting a temporally evolving set of neural and behavioral adaptations that differ from WT. Such differential vulnerabilities may impact the onset of psychiatric symptoms in human PD patients. Future studies are probing how different brain regions are impacted at a cellular and synaptic level by the differing durations of stress. Understanding these interactions will provide insight into the neural adaptations of individuals harboring the G2019S mutation, revealing novel targets for ameliorating mood-related symptoms associated with PD. Keywords: Acute and Chronic Stress, Synapses, Glutamate, Parkinson’s Disease, Depression Disclosure: Nothing to disclose. P383. Contribution of Adult Born Granule Cells to the Mechanism of Action of Electroconvulsive Therapy Julia Castello Saval, Victor Luna, Hannah Chung, Rene Hen Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Major depressive disorder is the leading cause of disability worldwide and about one third of patients do not respond to pharmacotherapy. In cases of treatment-resistant depression, electroconvulsive therapy (ECT) is an effective alternative with a fast onset of action. It is known that ECT increases neurogenesis in the dentate gyrus (DG) of the hippocampus, a brain region highly vulnerable to stress and thought to play an important role in the development of depression. In this study, we investigate which is the contribution of adult born granule cells (abGCs) to the mechanism of action of ECT. Methods: Electroconvulsive stimulation was delivered on alternate days for 10 sessions. Mice were anesthetized with isoflurane and electrical stimulus were delivered through ear clipped electrodes (120 Hz, 0.5 mA, 1 sec). Behavior was assessed one week after the last session in the forced swim test and the novelty suppressed feeding test (males, n = 9-10 per group). In the forced swim test, mice were placed in a container with water (25°C) and allowed to freely swim. Immobility time was quantified using Videotrack. In the Novelty suppressed feeding test mice were starved for 19 hours and placed in a novel, bright arena with a food pellet in the center, the latency to eat the food pellet was measured. For hippocampal irradiation after anesthesia, mice were placed in a stereotaxic frame, and cranial irradiation applied using a lead shield for focal Xray. A cumulative dose of 5 Gy was given over the course of 3 sessions. Results between groups were compared using one-way ANOVA for statistical group comparisons or multiple t-tests when appropriate. For fluorescence images, animals (males, n = 5 per group) were perfused and brains were sectioned. Sections underwent washing steps, antigen retrieval with sodium citrate buffer and blocking step with 10% normal donkey serum. Slices were incubated overnight at 4°C in primary antibody (rabbit anti-doublecortin, 1:400, rabbit cFos, 1:400, Synaptic Systems). The next day, sections were washed and incubated in secondary antibody (anti-rabbit Alexa Fluor 488). Sections were prepared for confocal imaging. Slice electrophysiology was performed with a Nestin-CreERT2 line crossed with a Channel Rhodopsin line to drive expression of Channel rhodopsin in adult born neurons. Tamoxifen was injected 6 weeks before whole cell current clamp recordings to induce Cre recombinase. Whole-cell recordings (-70 mV) were obtained using a patch pipette (4.5-6.5 M). For optogenetic stimulation of abGCs, light pulses were delivered through a 40x objective directly into brain slices. Results: Electroconvulsive stimulation, the mouse model of ECT, rescues the depressive-like phenotype of mice administered chronically with corticosterone (CORT) in the novelty suppressed feeding test an anxiety related test. Vehicle/Sham 100.4 ± 48.61; Vehicle/ECS 113.5 ± 65.8 unpaired student’s t-test: p = 0.6325. CORT/Sham 272.3 ± 33.76; CORT/ECS 175.7 ± 41.58 unpaired student’s t-test: p = 0.0458). In the forced swim test, a stress coping test, ECS rescued the depressive-like phenotype in the Vehicle group and the CORT group. Vehicle/Sham 124.0 ± 11.02; Vehicle/ECS 80.86 ± 27.92 one-way ANOVA multiple comparisons test: p = 0.006683. CORT/Sham 126 ± 17.41; CORT/ECS 87.46 ± 11.04 p = 0.005860. X-Ray ablation of abGCs renders mice unresponsive to ECS novelty suppressed feeding and forced swim test. Sham/Sham 272.3 ± 33.76; Sham/ECS 164.1 ± 38.95 unpaired t-test: p = 0.0501; XRay/Sham 309.1 ± 29.15; XRay/ECS 318.2 ± 28.24 one-way ANOVA multiple comparisons test: p = 0.8283. ECS stimulates the production of abGCs as shown by increased levels of doublecortin (DCX), a marker for young neurons. In addition, using transgenic mice expressing YFP-Synaptophysin in the DG, we visualized presynaptic boutons. After ECS, there is an increase in the number of puncta in the granule cell layer, suggesting increased presynaptic terminal density In contrast, when abGCs are ablated, the ECS-induced increase in synaptic boutons is comparable to Sham treated animals Sham 0.922 ± 0.03984; ECS 1.240 ± 0.03745 XRay 0.3468 ± 0.03327 One way ANOVA multiple comparisons test p < 0.001. Intrigued by this finding, we used a Nestin-CreERT2 mouse line crossed with a Channelrhodopsin-2(ChR2)-EYFP floxed mouse line to express ChR2 in abGCs. This allowed whole cell clamp recordings in mGCs after optogenetic stimulation of abGCs. When comparing mice that received 10 sessions of ECS with Sham animals, we found that stimulation of abGCs induced an inhibitory current in mGCs that is revealed after pharmacological suppression of the excitatory current component using the AMPA antagonist, NQBX and the NMDA antagonist, APV. Furthermore, addition of the mGluR2/3 antagonist APICA suppressed the inhibitory current in mGCs All results shown as Mean±S.E.M Conclusions: To integrate these results, we propose that ECS induces neurogenesis and axonal sprouting from abGCs which results in an increase in the number of presynaptic boutons from abGCs in the granule cell layer which in turn results in increased activation of mGluR2 receptors on mGCs. The resulting decrease in DG activity may be responsible for the antidepressant-like effects of ECS. Keywords: Electroconvulsive Therapy, Adult Hippocampal Neurogenesis, Major Depressive Disorder, Dentate Gyrus Disclosure: Nothing to disclose. P384. VTA-NAc Neural Activity Underlying Chronic Stress-Induced Reward-Seeking Deficits Lucille Johnston, Amanda Amilcar, Rachel Ding, Alexander Harris Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Anhedonia, defined as reduction in the pursuit of pleasure, is one of the core symptoms of depression. Stress, a risk factor for depression, decreases reward seeking in both humans and mice. We recently demonstrated that ventral tegmental area (VTA)-nucleus accumbens (NAc) activity underlies the reduction in anticipation seen after a single episode of restraint stress (Lowes et al., Nature Communications 2021). However, the precise nature of the behavioral changes (e.g., ‘wanting’ vs ‘liking’) that underlie deficits in reward seeking after chronic stress remain unknown. Moreover, the impact of chronic stress on reward circuit activity remains controversial, with studies reporting both increases and decreases in VTA dopamine firing rates (Tye et al. Nature 2013; Chaudhury et al. Nature 2013). In this study, we investigate VTA and NAc firing rates during reward seeking after mice have undergone chronic stress. Methods: We implanted chronic electrodes simultaneously in VTA and NAc of male and female mice to record both local field potential (LFP) and single unit activity (C57BL/6 J 3 males, 4 females). We trained the mice to associate a tone with reward availability (CS + ) and another tone with no condensed milk reward (CS-) to a criterion of 70 percent correct responses to the CS + for two consecutive days. We then recorded neural activity as mice performed the task. The mice then underwent 10 days of chronic social defeat stress (CSDS) and 24 hours after the final day of defeat, performed a social interaction task and the reward task. We analyzed the data using custom MATLAB script and used Pearson’s correlation to correlate the impact of stress on social interaction and reward seeking. Results: We found that chronic stress produced reductions in anticipatory and post-consumption lick rates that were sustained for over 24 hours and correlated with SI ratio (N = 7, r = 0.79 for anticipation, r = 0.87 for consumption, p < 0.05). We also are sorting neurons into their putative identities (e.g., VTA dopaminergic, VTA GABAergic, NAc MSN, etc) and analyzing the lick- and cue-evoked neural activity and synchrony between regions to determine the neural activity associated with these reductions in reward seeking. Conclusions: Together, these data suggest that chronic stress induces longer-lasting reward processing deficits and allows for the dissection of the contribution of individual cell populations to anhedonia. Keywords: Reward, Neural Circuits, Chronic Stress Disclosure: Genetika: Advisory Board (Self) P385. Somatostatin Peptide Signaling Dampens Cortical Circuits and Promotes Exploratory Behavior Nicole Crowley* Penn State University, University Park, Pennsylvania, United States Background: Somatostatin (SST) neurons in the prelimbic (PL) cortex mediate a variety of behavioral states, ranging from alcohol consumption to fear learning and avoidance-related behaviors. However, little is known about the role of somatostatin peptide signaling itself to cortical functioning and behavior. Here, we sought to characterize the unique physiological and behavioral roles of the SST peptide in the PL cortex. Methods: We employed a combination of ex vivo electrophysiology, in vivo calcium monitoring, and in vivo peptide pharmacology to explore the role of SST neuron and peptide signaling in the mouse PL cortex. Whole-cell slice electrophysiology was conducted in C57BL/6 J male and female mice in pyramidal and GABAergic neurons of the PL cortex to characterize the pharmacological mechanism of SST signaling. Fiber photometry recordings of GCaMP6f fluorescent calcium signals from SST neurons were conducted to characterize the activity profile of SST neurons during exploration of an elevated plus maze (EPM) and open field (OF). We further used local delivery of a broad SST receptor (SSTR) agonist into bilateral PL cortex to test causal effects of SST signaling on these same exploratory behaviors. Results: SSTR activation broadly hyperpolarized layer 2/3 pyramidal neurons in the PL cortex in both male and female mice ex vivo, through both monosynaptic and polysynaptic GABA neuron-mediated mechanisms of action. This included reductions in the resting membrane potential in females (t13 = 2.205, p = 0.0460) and males (t16 = 2.889, p = 0.0107) of pyramidal neurons – an effect that was greater in the presence of TTX in both females (t6 = 5.095, p = 0.0022) and males (t4 = 3.448, p = 0.0261). Multiple other measurements of excitability were also significantly altered in both cases. Hyperpolarization was blocked by pre-application of the SSTR antagonist cyclo-somatostatin (cyclo-SST) and was non-reversible. SST neurons in PL were activated during EPM and OF exploration, indicating task-related recruitment of these neurons. Specifically, SST neurons were more active while mice were in the open arms as compared to the closed arms of the elevated plus maze (t10 = 6.101 p < 0.001), and were more active while mice were in the center as compared to the edges of the open field (t10 = 2.797, p = 0.0189). No sex differences were seen in the fiber photometry experiments. Lastly, in line with this exploration-related activity profile, SSTR agonist administration directly into the PL enhanced open arm exploration in the elevated plus maze of male mice, with no effect in female mice (2-way ANOVA; Fsex(1,26) = 0.6452, p = 0.4291; Fdrug(1,26) = 3.462, p = 0.0741, Fsex x drug (1,26) = 7.868, p = 0.0094). We also saw a significant increase in the number of dead dips over the open arms of the elevated plus maze in male mice (2-way ANOVA; Fsex(1,26) = 5.917, p = 0.0222; Fdrug(1,26) = 3.264, p = 0.0824, Fsex x drug (1,26) = 5.614, p = 0.0255). Conclusions: Here we reveal a novel role for the SST peptide system within the PL cortex, by demonstrating a peptide-induced hypoexcitability of PL circuits and modulation of PL-dependent exploratory behaviors. Keywords: Somatostatin, Prelimbic Cortex, Slice Electrophysiology, Fiber Photometry Disclosure: Nothing to disclose. P386. Changes in Emotion Regulation-Relevant Neurocircuitry Following Standard RTMS for Treatment Resistant Depression Kristen Ellard, Walker Pederson, Samadrita R Chowdhury, Tracy Barbour, Joan A Camprodon Harvard Medical School, Boston, Massachusetts, United States Background: Transcranial magnetic stimulation (TMS) of the dorsolateral prefrontal cortex (DLPFC) is an FDA-approved standard intervention for treatment resistant depression. However, the mechanisms by which TMS of the DLPFC relates to treatment response remains poorly understood. Examining the mechanisms of TMS is crucial towards improving treatment outcomes, which currently show high response but low remission rates (~30%). Deficits in emotion regulation (ER) have been repeatedly demonstrated in depression, linked to symptom severity and treatment response. Healthy ER includes detection of salience, the initiation of regulation, and cognitive control in accordance with internal and external contextual demands, processes that require adaptive coordination between neurocircuits comprising the salience, frontoparietal control, and default mode functional networks. Here, we examined the effects of a course of repetitive (r) TMS to the left DLPFC on resting-state functional connectivity across the broader neurocircuitry supporting ER, spanning canonical functional networks. Methods: Based upon existing literature (e.g. Kohn et al., 2014; McTeague et al, 2020), an analysis of functional connectivity between individually-derived DLPFC target ROIs and a priori selected ER-related regions of interest (ROIs) was conducted on data from 24 depressed patients (50% female, mean age 39.91 ± 3.04, 92% Caucasian) receiving an interventional course (36 sessions, 120% MT, 10hz rTMS, 3,000 pulses) of left DLPFC rTMS. Selected ROIs comprised regions within the limbic (LIMB) salience/ventral attention (SalVA), dorsal attention (DA), frontoparietal control (FPC) and default mode networks (DMN). Significant pre-post TMS changes in DLPFC target - ER ROI functional connectivity were followed up with linear regressions to elucidate the relationship between ER functional neurocircuit changes and changes in ER-relevant clinical measures in responders (Resp; n = 10) versus non-responders (Non-Resp; n = 14). To understand TMS-related changes within the broader ER-related neurocircuitry, ROIs showing significant pre-post TMS functional connectivity with the DLPFC target and demonstrating a significant relationship to changes in ER-related clinical response were further characterized using graph metrics of centrality, based upon whole-brain functional connectivity. Results: Significant pre-post TMS changes in functional connectivity were found between the DLPFC target and regions in the LIMB (l. amygdala, Resp: t = -1.82, p = .05, Non-Resp ns; r. hippocampus, Resp: -2.07, p = .03, Non-Reps ns), SalVA (r. anterior insula, Resp: t = -2.11, p = .03, Non-Resp ns; r. VLPFC, Resp: t = -1.12, p = .10, Non-Resp: t = 2.08, p = .02), and DMN networks precuneus/posterior cingulate, Resp: t = 2.12, p = .03, Non-Resp ns). Changes in target-ER ROI functional connectivity were significantly associated with improvements in ER measures (delta DLPFC-amygdala/delta negative affect, z = -1.96, p = .05; delta DLPFC-hippocampus/delta affective control, z = -2.50, p = .02; delta DLPFC-anterior insula/delta negative affect, z = -2.45, p = .01; delta DLPFC-VLPFC/delta affective control, z = 2.89, p = .004; delta DLPFC-precuneus, PCC/delta affective control, z = 2.15, p = .03). Follow-up graph analysis identified significant pre-post TMS increases in betweenness-centrality (a measure of the amount of influence a region has on the flow of information between regions and networks), in the right VLPFC/anterior insula (p = .03). Significant changes in functional connectivity between this node and the broader ER neurocircuitry were found following TMS (all p’s < .05), with the strongest effect between VLPFC/anterior insula and DMN functional connectivity (precuneus/PCC, t = -2.63, p = .007). A significant relationship was found between these functional connectivity changes and measures of negative affect (t = 2.00, p = .05) and rumination (t = 2.01, p = .05). Conclusions: Treatment with TMS applied to individualized DLPFC targets resulted in broad changes in functional connectivity between the DLPFC target site and regions of the limbic, salience and default mode networks in treatment responders. These changes were significantly related to changes in ER relevant clinical measures. The right VLPFC/anterior insula showed an increased influence on the broader emotion regulation neurocircuitry as measured by betweenness centrality, and TMS-related changes in functional connectivity between this region and regions of the DMN were significantly related to changes in negative affect and rumination, two core features of emotion dysregulation. These results suggest more precise targeting of emotion regulation related neurocircuitry with TMS may be one avenue towards increasing overall response rates in treatment resistant depression. Keywords: Repetitive Transcranial Magnetic Stimulation (rTMS), Emotional Regulation, Depression Disclosure: Nothing to disclose. P387. Sex Moderates the Relationship Between Functional Connectivity and Remission in Late-Life Depression Andrew Gerlach, James Wilson, Helmet Karim, Howard Aizenstein, Carmen Andreescu University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Major depressive disorder accounts for more disability-adjusted life years than any other mental illness, yet the predominant treatment approach is essentially trial-and-error. This resulting delays in recovery exacerbate the personal, social, and even economic costs. This problem is amplified in late-life depression (LLD), where the disorder presents with greater heterogeneity, longer time to response, lower remission rates, and higher risk of relapse. Thus, biomarkers of early treatment response in LLD to improve response time and outcome are a major strategic goal of neuroimaging research. However, the role of biological sex has not been adequately studied in assessment of MRI-based biomarkers. Methods: Demographic information, baseline depression severity, and resting state fMRI scans at baseline and after commencing antidepressant treatment (day 1) were collected in two LLD studies (NEMO [R01 MH076079-15]: n = 28, Circuits2 [R01 MH076079-09]: n = 51). Remission was defined as a final MADRS score of 10 or less for at least 2 weeks and subject to blinded clinician assessment. The Shen50 atlas was used to calculate region-to-region functional connectivity (FC) for 82 non-cerebellar regions (excluded due to poor coverage). Differential connectivity (DC) was calculated by subtracting the baseline FC from the day 1 FC. The role of biological sex was assessed in both explanatory and predictive frameworks. For the explanatory framework, sex-based differences in remitters and non-remitters were assessed using two-sample t-tests on the DC for each pair of regions. For the predictive framework, a random forest classifier was used to predict the remission status of each participant. Models were fit and tested separately using all participants, only females, and only males. Monte Carlo cross-validation was used to evaluate the predictive performance of clinical and DC covariates to predict remission status in seven different model specifications for each study. Variable importance was evaluated using the Gini importance measure assessed through the trained random forest models. The average accuracy and area under the receiver operating characteristic curve metrics were used to assess the predictive performance of each model. Results: The location and direction of differences in DC among remitters and non-remitters, as well as the brain regions important to the prediction of remission substantially differed by sex. Prediction of remission was significantly improved by fitting separate models for males and females. Further, males showed stronger group DC differences between remitters and non-remitters than females, with large differences in the bilateral caudate, bilateral temporal pole, and left postcentral gyrus. These regions were also important for prediction in male-only models. Females, on the other hand, showed weaker group differences in DC between remitters and non-remitters, but a much more diverse set of regions important for prediction. Connectivity of the left caudate nucleus was the leading predictor of remission for both males and females. Conclusions: Early FC indicators of antidepressant treatment response in LLD may differ fundamentally between males and females. Simply including sex as a covariate in the model is insufficient to capture these difference, as there is a clear moderating effect of sex. Separate models for males and females or sex by region interaction terms may be necessary to provide sufficient sensitivity and specificity. Keywords: Late Life Depression, Resting State Functional Connectivity, Antidepressant Response, Predictive Models, Sex Differences Disclosure: Nothing to disclose. P388. Utilizing the Systemic Immune-Inflammation Index and Inflammatory Cytokines in Predicting Treatment Responsiveness Amongst Patients With Treatment Resistant Bipolar Depression Kyle Decker, Nausheen Baig, Stephen Murata, Angelos Halaris, Jawed Fareed, Debra Hoppenstaed Loyola University School of Medicine, Saint Charles, Illinois, United States Background: Inflammation is associated with depressive illness and treatment resistance. These advances have prompted a search for predictive biomarkers of treatment response. The Systemic Immune-Inflammation Index (SII) has shown promise as a prognostic indicator for solid malignancies and cardiovascular disease while its use in psychiatric conditions remains under-explored. In a recent randomized, double-blind, placebo-controlled trial we found that combined treatment with celecoxib (CXB) and escitalopram (ESC) was more effective in reversing treatment resistance and augmenting antidepressant response in treatment-resistant bipolar depression (TRBDD) compared to ESC plus placebo. Our follow-up analysis characterizes treatment response in relation to SII, as well as inflammatory and kynurenine pathway (KP) biomarkers. Methods: The sample (N = 69) included 65.2% female, 65.2% white, mean age 42 years (SD = 12.7). The study included healthy controls (n = 32) and TRBDD subjects (n = 47). The TRBDD group consisted of an ESC + CBX arm (n = 26) and ESC + PBO arm (n = 21). SII was calculated from the complete blood count with differential (SII = platelets x neutrophils/lymphocytes) at baseline and completion (8 weeks). Plasma inflammatory and KP markers levels were also obtained at baseline and week 8. Depressive symptom severity (main outcome) was measured both continuously (HAMD17 total score) and dichotomously (treatment remission, defined as HAMD17 total score < 7 by week 8). Statistical analysis was conducted using R-3.6.3. Results: Group comparison revealed no significant differences in SII by treatment arm at baseline or remission status by week 8. SII at baseline was trended with an elevated inflammatory cytokine profile including higher IL-2 at baseline (p = 0.073), IL-1B at baseline (p = 0.051), hsCRP at week 8 (p = 0.08), and lower anti-inflammatory IL-4 (p = 0.086). Baseline SII significantly correlated with lower baseline vascular endothelial growth factor (VEGF) (p = 0.029). On multivariate linear modeling (R2/ R2 adjusted = 0.49/0.41) HAMD17 at week 8 (outcome) was significantly associated with ESC + CBX treatment (p < 0.008), an interaction with age and SII-baseline (Beta-estimate = 0.001, (95% CI [ < 0.001, 0.001]), p < 0.001, Cohen’s D = 0.5). In a separate model using individual cell counts (R2 /R2 adjusted = 0.413/0.334), HAMD17 by week 8 was associated with baseline neutrophil count amongst older patients (b-estimate = 0.17 (95% CI [0.06, 0.27]), p < 0.003, Cohen’s D = 0.48), but not baseline platelet or lymphocyte counts (p = 0.312, p = 0.201, respectively) Conclusions: On univariate analysis, baseline SII trended with elevated baseline proinflammatory markers (IL-2, IL-1B, hsCRP) and lower baseline anti-inflammatory markers (IL4). SII significantly correlated with lower vascular endothelial growth factor (VEGF) which is associated with neuroprotection and was previously shown to be elevated in our TRBDD cohort compared to healthy controls. In older patients, lower pretreatment SII predicted lower depressive severity by week 8 irrespective of treatment arm, and this association appeared driven largely by the neutrophil component of SII. Taken together, SII appears to be a candidate poor prognostic indicator in TRBDD. Future studies with a larger sample sizes should further investigate the potential clinical utility of SII as it is readily available and accessible through the routinely drawn blood counts. Keywords: Bipolar Depression, Treatment Resistance, Inflammation Disclosure: Nothing to disclose. P389. Assessing the Generalizability and Stability of Biologically-Based Subtypes of Depression Katharine Dunlop, Logan Grosenick, Jonathan Downar, Fidel Vila-Rodriguez, Faith Gunning, Zafiris Daskalakis, Daniel M. Blumberger, Conor Liston University of Toronto, Toronto, Canada Background: Major depressive disorder (MDD) is associated with considerable symptom variability; a comprehensive understanding of this variability may lead to individualized intervention approaches and therefore improved treatment response rates. Recent efforts by our group and others have sought to understand symptom heterogeneity in MDD using functional neuroimaging. Although these recent studies mark significant progress in understanding heterogeneity in MDD, replication and validation of these results is critical given the limitations imposed by sample size and depth of clinical characterization. To address these limitations, this study has three aims. First, we sought to understand the neurobiological basis of MDD symptom heterogeneity by extending our earlier work defining robust and reproducible brain-behavior dimensions to new data. An L2-norm regularized multivariate model was generated using a large MDD dataset recruited from a single site and incorporated additional items assessing anhedonia and anxiety symptoms. Second, we tested for the existence of MDD subtypes and evaluated their stability and reproducibility. Third, we characterized these MDD subtypes regarding atypical resting-state functional connectivity (RSFC), clinical symptoms, and antidepressant response to non-invasive brain stimulation. Methods: L2-regularized canonical correlation analysis (RCCA) was evaluated in a large, single-site MDD dataset (n = 328, 215 female (65.6%); mean age=40.35 ± 12.05 SD) using RSFC and clinical symptomatology. First, to optimize three RCCA hyperparameters, we performed a nested grid search (with training, validation, and test splits); the optimal hyperparameter combination was defined as the highest median canonical correlation in held-out validation data for the first dimension. Next, we examined the stability and hold-out performance of dimensions (on held-out test data not used for training or validation) and tested for significant dimensions using random permutation testing. Afterwards, we generated a final optimized RCCA model and evaluated the performance and stability of hierarchical clustering. Upon identifying the optimal clustering solution, we characterized latent variables representing co-occurring RSFC and symptomatology, and symptom/RSFC differences by subtype. Lastly, we identified subtype differences in repetitive transcranial magnetic stimulation response and remission rates. Results: The performance and stability of the first three RCCA dimensions were significant (p < 0.05, random permutation test). These three dimensions represented: depressed mood, and thalamic and default mode RSFC; anhedonia, and cingulo-opercular and higher-order visual and network RSFC; and insomnia, and sensorimotor and posterior insula RSFC, among other connectivity features. Hierarchical clustering identified four significant depression subtypes (p < 0.05, random permutation test), each with distinct clinical symptom profiles, abnormal RSFC patterns, and responsivity to repetitive transcranial magnetic stimulation (rTMS) over the dorsomedial or dorsolateral prefrontal cortex. Subtypes with lower anhedonic symptoms were most responsive to rTMS. Subtypes did not differ by age or sex. Conclusions: In an extension of our previous work, we sought to characterize regularized CCA and clustering performance in a large, single-site MDD dataset. RCCA yielded three significant, stable and generalizable brain-behavior dimensions that resembled well-documented MDD symptom-brain associations, and four categorical subtypes. Both categorical and dimensional approaches to parsing heterogeneity may be beneficial in different contexts. We note several study design choices that may affect RCCA models, including participant inclusion/exclusion criteria, medication use, and choice of symptom severity measures. Taken together, these results represent an important step forward in assessing data-driven subtyping methods and provide evidence that RCCA is an effective tool to identify stable and generalizable associations between RSFC and behavior. Keywords: Resting State Functional Connectivity, Depression Subtypes, Transcranial Magnetic Stimulation, Canonical Correlation Analysis (CCA) Disclosure: Nothing to disclose. P390. Probing Dopaminergic Deficits in Adolescent Depression David Pagliaccio, Emily Zhang, Alma Bitran, Kenneth Wengler, Guillermo Horga, Randy Auerbach New York State Psychiatric Institute, Columbia University, New York, New York, United States Background: Depression is a chronic and impairing mental health condition that often peaks during adolescence. Prior work implicates alterations within the RDoC Positive Valence Systems in adolescent depression, including anhedonia and blunted striatal reward response. These alterations generally rely on dopaminergic projections from the substantia nigra and ventral tegmental area in the midbrain, to striatal and prefrontal circuits, respectively. Yet, the hypothesized role of dopamine is largely based on animal, pharmacological, post-mortem human, and adult studies utilizing methods too invasive for pediatric research (e.g., lumbar puncture, positron emission tomography). Recently, a safe and non-invasive alternative means of characterizing midbrain dopamine has been developed using magnetic resonance imaging (MRI) to assess neuromelanin, a key byproduct of dopamine metabolism. Methods: As part of ongoing data collection, we are collecting high-resolution neuromelanin-MRI data from adolescents (13-18 years old; Target N = 60; no exclusions based on sex or gender), primarily with a history of major depressive disorder. Adolescent complete clinical assessment, self-reports, and smartphone-based data collection over the subsequent months. Preliminary analyses entail linear regression models among N = 29 adolescents. All analyses controlled for age, sex, and head motion. Results: Adolescents with current depression exhibit reduced neuromelanin signal in the substantia nigra pars compacta (b = -1.88, t = -2.51, p = .02). Lower SNpc neuromelanin-MRI signal associates with more severe depression (CDRS; B = -0.45, t = -2.67, p = .01), social anhedonia (ACIPS; B = -0.46, t = -2.89, p = .009), and suicide ideation (SSI; B = -0.59, t = -4.23, p < .001. Conclusions: These novel data support the role of midbrain dopaminergic deficits in adolescent depression. Ongoing analyses will examine associations with real-world affect and anhedonia via smartphone ecological momentary assessment as well as associations with other neuroimaging modalities, i.e., linking midbrain neuromelanin with reward response during functional MRI. We aim to probe the utility of neuromelanin-MRI, in combination with other measures, as predictors of course and continuity of depression symptoms in this time of rapid adolescent development. Future studies will build on this to probe potential dopaminergic risk markers in earlier child development to predict depression onset before this adolescent peak. Keywords: Adolescent Depression, Anhedonia, Neuromelanin-Sensitive MRI, MRI Disclosure: Nothing to disclose. P391. Development and Characterization of a Novel Fluorinated Etonitazene Analog as a Potential Radiotracer for Mu Opioid Receptors Juan Gomez, Agnieszka Sulima, Arianna Rizzo, Zelai Garcon-Poca, Emilya Ventriglia, Jordi Bonaventura, Kenner Rice, Mike Michaelides National Institute on Drug Abuse, Baltimore, Maryland, United States Background: Opiates have long been used and studied for their analgesic properties and synthetic mu opioid receptor (MOR) agonist medications are the most effective analgesics available. However, such medications have known adverse effects including constipation, respiratory depression, and abuse liability. As such, there is a desperate need to develop novel MOR agonists with lower adverse effect profiles. A critical tool for such an endeavor involves the development of a MOR-selective radiotracer for in vivo target engagement studies using positron emission tomography (PET). The only MOR selective PET radiotracer developed to date is [11 C]carfentanil. [11 C]carfentanil has been used in many studies to measure MOR binding in humans and laboratory animals. Despite its use, [11 C]carfentanil has two key limitations. It has very high potency, which necessitates achieving very high specific activity in its radiosynthesis, and its use is restricted to PET studies in centers with on-site cyclotrons. Accordingly, an 18F-labeled MOR selective agonist PET radiotracer which has longer half-life and lower potency than [11 C]carfentanil would be desirable, but such a radiotracer has not been previously developed. Etonitazene is a selective and potent MOR agonist that has not been studied extensively. The aim of these studies was to characterize a novel fluorinated etonitazene analog (aka fluornitrazene, FNZ) and assess its potential to be radiolabeled and used as a MOR selective in vitro 3H-labeled radioligand and 18F-labeled PET radiotracer. Methods: To assess it selectivity, FNZ was screened against a panel of >100 receptors and enzymes at 100 nM and 10 µM concentrations. To assess its propensity for brain entry, FNZ was also screened for its drug transporter inhibition activity against a panel of several drug transporters using these same concentrations. FNZ and [3H]FNZ were studied via competitive binding assays using rat brain membrane suspensions (minus cerebellum) (40 μg of protein/ml) incubated in 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2, 10 nM of [3H]DAMGO (46 Ci/mmol) or 1 nM [3H]FNZ (43 Ci/mmol) and increasing concentrations of the tested compounds (DAMGO, FNZ) incubated of 2 hours at room temperature. Non-specific binding was determined in the presence of 100 μM naloxone. In all cases, free and membrane-bound radioligand were separated by rapid filtration in a 96-well plate harvester and washed with 2 mL of ice-cold Tris-HCl buffer. Microscint-20 scintillation liquid (65 μL/well) was added to the filter plates, incubated overnight, and radioactivity counts were determined in a MicroBeta2 plate counter. One-site competition curves were fitted, and Ki values were calculated using the Cheng-Prusoff equation. FNZ was also evaluated in its propensity to stimulate cAMP and β arrestin-signaling using HEK293 cells transfected with hMOR cDNA and the respective genetically encoded sensors. Results: At 10 µM, FNZ inhibited binding to serotonin (5-HT1A, 5-HT2A, 5-HT2B), adrenergic (α1A, α1D), cannabinoid (CB2), opioid (delta, kappa, MOR) receptors, as well as to calcium (L-type, diltiazem site), and potassium (hERG) channels. At 100 nM, FNZ inhibited binding only to MOR. In the efflux transporter panel, at 10 µM, FNZ inhibited transporter activity of OCT2, BSEP, MATE1, MATE2-K, OAT3, OATP1B1, OATP1B3, and p-glycoprotein (P-gp). At 100 nM, FNZ inhibited the activity of only OCT2 and MATE2-K, which are restricted to the periphery. Competitive binding assays against [3H]DAMGO showed that FNZ had a Ki = ~1.0 nM. Similarly, [3H]FNZ showed a Kd = ~1.3 nM. FNZ showed an EC50 of ~0.1 nM and Emax ~100% for cAMP and EC50 of ~10 nM and Emax ~100% for β arrestin. Conclusions: FNZ is a selective agonist for MORs and [3H]FNZ exhibits favorable properties as an in vitro radioligand. FNZ exhibits minimal interaction with efflux transporters expressed in the periphery and therefore may have potential as a PET radiotracer. Keywords: Mu-Opioid Receptors, Functional Characterization, Fluornitrazene Disclosure: Nothing to disclose. P393. GPR6 as a Modulator of Striatal-Based Motor Plasticity Under Conditions of Dopamine Inhibition Brenna Williams, Anuj Patel, Zoe Frank, Maellie Midroit, Hailey Huddleston, Patrick LaChance, Michael J. Frank, Kevin G. Bath Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Parkinson’s disease stems from depleted striatal dopamine (DA), leading to motor impairments such as bradykinesia, akinesia, and tremor. Theoretical models have suggested that the source of these impairments is not in motor execution per se, but is rather motivational, whereby DA depletion enhances the brain’s representation of the cost of action. Moreover, DA depletion not only induces a performance effect (amplifying the motivational cost), but is associated with aberrant learning, such that motor performance degrades with experience as the cost is learned in a particular context. Experimental studies manipulating DA blockade or depletion separately during learning and performance phases have provided strong evidence for the aberrant learning hypothesis, resulting from a lack of D2 receptor binding in the indirect corticostriatal pathway. GPR6, a G- protein coupled receptor, is expressed highly specifically to D2-containing striatopallidal neurons of both mice and humans; hence manipulation of GPR6 function provides a novel opportunity to intervene with the aberrant learning process. However, the mechanisms by which GPR6 affects performance or learning remain unknown. Given the putative role of GPR6 in plasticity-dependent processes, together with its selective expression on D2 cells, we predicted that loss of GPR6 would diminish aberrant learning under pharmacological DA receptor blockade and would result in paradoxically more rapid recovery following drug removal than is observed in wildtype mice. Methods: Male and female wildtype C57Bl/6 N and GPR6 KO mice (n = 8-10/group/sex) were assessed for motor learning and performance degradation on an accelerating rotarod to assess the effects of D1 and D2 receptor antagonists (including a D1/D2 cocktail) on the acquisition of a novel motor learning task and subsequent performance when drug was removed. Ongoing experiments are underway to assess animals for biochemical markers of plasticity. Based on previous studies investigating reinforcement learning, and our knowledge of signaling pathways affected by GPR6, we will specifically test for the activation of plasticity markers DARPP-32, CREB, and MAPK. Results: We found that DA receptor blockade impaired motor performance and that D2 and D1/D2 cocktail, but not D1 blockade alone, significantly impaired recovery in motor performance following drug removal. All mice showed impaired performance under dopamine antagonists (Days 1-5, “direct performance effects”). Following drug removal (D2 or D1/D2 cocktail), wildtype mice showed slowed recovery (WT + Drug vs WT/KO + Sal, one-way repeated measures ANOVA, post-hoc Tukey: Days 8-12, p < 0.05). Consistent with our hypotheses, GPR6 KO mice demonstrated enhanced performance after drug removal and showed a more rapid and complete progression to “normal” performance when compared with wildtype mice (GPR6 KO + Drug vs WT/KO + Sal, one-way repeated measures ANOVA: Days 8-9, p < 0.05, D10-12, ns). This effect was most dramatic in conditions in which both D1/D2 receptors were blocked. Notably, GPR6 did not impact the direct performance effects of DA receptor blockade, and thus appears to selectively impact the aberrant learning component (GPR6 KO + Drug vs WT + Drug, one way repeated measures ANOVA: Days 1-5, ns). Conclusions: Together, these data demonstrate that loss of GPR6 receptors diminishes the behavioral learning effects that occur under D2 blockade, suggesting that variation in GPR6 function modulates the extent to which aberrant learning occurs via plasticity of D2 striatopallidal neurons. Further experiments will elucidate whether, in wildtype mice, motor learning under D2 blockade will lead to enhanced activation of proteins involved in plasticity in D2-positive cells. We anticipate that loss of GPR6 will attenuate motor learning induced expression of phosphorylated DARPP-32, CREB, and MAPK, indicating decreased plasticity of D2 positive cells. These findings provide evidence that GPR6 represents an under-studied but promising target and pathway for intervention in various frontostriatal disorders that affect motor, motivational, and cognitive function, including but not limited to Parkinson’s disease. Keywords: Neuropsychiatric Disorders [Schizophrenia, Parkinson’s Disease, Major Depressive Disorder], Dopamine, GPCRs, Motor Learning Disclosure: Nothing to disclose. P394. Nanoscale Imaging of pT217-Tau in Aged Rhesus Macaque: Trans-Synaptic Propagation and Seeding of Tau Pathology in Entorhinal Cortex Dibyadeep Datta, SueAnn Mentone, Yury Morozov, Rosalinda Roberts, Christopher van Dyck, Amy Arnsten Yale University School of Medicine, New Haven, Connecticut, United States Background: Tau pathology in Alzheimer’s Disease (AD) targets higher cortical circuits, with evidence that phosphorylated tau propagates from the entorhinal cortex (ERC) to “seed” pathology throughout the neuronal network. Recent discoveries indicate that tau phosphorylated at threonine 217 (pT217-tau) can be captured in cerebrospinal fluid (CSF) and plasma as an early biomarker of ensuing disease. pT217-tau levels correlate with disease stage, progression and longitudinal rates of change. Importantly, CSF pT217-tau permits early identification of at-risk presymptomatic individuals and shows high specificity for conversion to mild cognitive impairment and dementia. More recently, pT217-tau in blood plasma has been shown to distinguish AD from other neurodegenerative disorders with high diagnostic accuracy (>98%) and was more accurate than blood-based tests for pT181-tau, neurofilament light or Aβ-42/40 ratio. Plasma pT217-tau collected during life correlated with neuropathological neurofibrillary tangle (NFT) density measured postmortem. CSF and plasma pT217-tau is increased ~7-fold in AD, and the levels increase 20 years before onset of cognitive impairment. Recent high-resolution quantitative proteomics map of post-translational modifications (PTMs) on multiple isoforms of tau revealed pT217-tau as a crucial epitope distinguishing AD from other neurodegenerative disorders and indicative of disease progression. However, the role of pT217-tau in brain tau pathology is unknown, especially as soluble tau species are dephosphorylated postmortem in humans. Rhesus macaques naturally develop the same qualitative pattern and sequence of tau and amyloid pathology, with NFT’s comprised of paired helical filaments, identical to human AD. Perfusion fixation of monkey tissue preserves phosphorylation state and allows imaging of molecular location and interactions with nanometer resolution not possible in humans due to postmortem degradation. The current study examined the ultrastructural localization of pT217-tau in layer II ERC of the aged rhesus macaques, focusing on potential evidence of propagation between neurons, and exposure to the extracellular space. Methods: We used immunohistochemistry to examine the anatomical localization pattern of pT217-tau in aged rhesus macaques and compared to human postmortem AD subjects. We performed high spatial-resolution immunoelectron microscopy (immunoEM) in aged rhesus macaques (18-31 years) to localize pT217-tau in the stellate cell islands in ERC layer II, which show the earliest signatures of tau pathology in AD. Results: Our data at the light-level reveals dense pT217-tau immunolabeling in stellate cells in ERC layer II, pyramidal cells in hippocampus CA3, CA1, and pyramidal cells in dlPFC layer III, all of which show tau pathology in human AD. The labeling shows aggregated, filamentous fibrillated structures within apical dendrites and basilar dendrites, often with a twisted morphology common in NFTs. pT217-tau immunolabeling was predominantly observed in postsynaptic compartments in macaque ERC layer II. pT217-tau accumulated on the calcium-storing smooth endoplasmic reticulum spine apparatus near axospinous asymmetric glutamatergic synapses in dendritic spines. We observed extensive, trans-synaptic pT217-tau trafficking between interconnected neurons within omega-shaped bodies and endosomes in ERC layer II, specifically near excitatory, but not inhibitory synapses. Within dendritic shafts, pT217-tau aggregated on microtubules often in concordance with autophagic vacuoles indicative of neurite dystrophy. Conclusions: pT217-tau accumulates in ERC layer II subcompartments known to be the earliest to show pathology in humans. The data provide the first evidence of pT217-tau trafficking between neurons to “seed” tau pathology in higher brain circuits, potentially interfacing with the extracellular space to become readily accessible and captured in CSF and blood as a robust AD biomarker. Illuminating patterns of neurodegeneration with pT217-tau could potentially guide earlier intervention of therapeutics that might mitigate tau hyperphosphorylation in AD. Keywords: Alzheimer’s Disease, Tau, Entorhinal Cortex, Protein Trafficking, Neurofibrillary Tangle Disclosure: Nothing to disclose. P395. Replication of the N170 Response to Faces for Use as a Potential Stratification Biomarker in Clinical Trials for Autism Spectrum Disorder Matthew Klein, Wen-Hsuan Chan, Molly Lucas, Srinivasan Vairavan, Abigail Bangerter, Nikolay V. Manyakov, Gayle Wittenberg, Gahan Pandina Janssen Research and Development, San Diego, California, United States Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by impairments in social communication, and restricted and repetitive behavior. It often occurs in combination with other psychiatric conditions. There are no approved medications for the core symptoms of ASD and, partly, this is due to the highly variable symptom presentation. Stratification of the heterogeneity could allow for more targeted interventions. Currently, there are no validated biomarkers for ASD. The N170, an early-stage neural response to faces, has been proposed as a potential stratification biomarker. Longer latency, or slower response, to faces has been found to be related to social communication skills, making it a relevant candidate. For utility in clinical trials, biomarkers must also be reliable and robust. In order to move towards the validation of the N170 as a biomarker, results must be replicable within the context of use. In this abstract, we present an event-related potential (ERP) study for N170 obtained in a large sample of autistic and typically developing (TD) children and adults within a clinical trial context. In addition, we use age-adjusted modeling to create subgroups based on N170 response and investigate whether there are phenotypic differences between groups. Methods: Biosensor data, including electroencephalogram (EEG), was obtained from ASD participants (n = 144) as part of a battery of passive viewing tasks at 3 time points (0, 4, 8 weeks) during an observational study (NCT02668991). Data from a group of TD participants (n = 41) was obtained at a single time point. For the face task design, static upright faces stimuli with either direct or averted gaze were presented. EEG recording was conducted using ActiCHamp 32 with 19 electrodes placed in accordance with the standard 10-20 system. ERPs were computed for epochs extending from 200 ms pre-stimulus to 1000 ms post-stimulus onset. ERPs were averaged by stimuli type after subtraction of the 200 ms pre-stimulus baseline. We focused on N170 at the parieto-occipital sites (P7, P8, O1, O2). We calculated group differences (all ages, 6-12, 12 + ) in averages for N170 peak latency and amplitude including sex and age as covariates. We assessed stability of these measures for the ASD group across 3 timepoints using intraclass correlation coefficient (ICC) from a linear mixed model. Correlations between ASD N170 features, and phenotypic data (IQ, ADOS, Autism Behavior Inventory) were calculated. Finally, we used a linear mixed effect model to estimate age-adjusted N170 latency for both TD and ASD groups, and calculated residual differences between expected and measured N170 latency (Webb, 2022). Using a 90th percentile cut off based on TD residuals, we categorized ASD participants into either “slowed” or “standard” groups. This approach allowed us to stratify ASD subgroups and to evaluate these N170 latency defined subgroups in relation to the phenotypic behaviors. Results: The final number of participants with data for analysis was TD 30/ASD (97, 95, 91) for each respective timepoint, TD 15/ASD (43, 47, 38) and TD 15/ASD (54, 48, 53) respective to the age groups under and over 12 years old. There were no between group differences in response to condition (averted or direct gaze). For the ‘all ages’ group there was a group difference in N170 to all faces, where the ASD group had longer latency and reduced amplitude. The latency group difference remained when age and sex were included in the analysis, and when the groups were analyzed by age. The ICC value was 0.81 (0.61 and 0.85 for age under and over 12 years old), suggesting moderate stability over 4 and 8 weeks for the measured N170 latency in the ASD. There were no correlations between N170 latency and phenotypic data, other than a small negative correlation with restricted interests, which was observed at all 3 timepoints. We observed significant differences in latency defined ASD (slow/standard) subgroups analyses. For the under 12 s, increased severity of ASD core behaviors was seen in the slowed N170 group and for over 12 s, increased irritability was associated with the standard N170 group. There was a difference in IQ between the subgroups, with a slowed response related to a lower IQ. Conclusions: These findings are an important step in demonstrating the reliability and robustness of the N170 latency as a potential stratification biomarker for ASD. The relationships observed between subgroups and ASD core symptom severity in the younger age group confirm the relevance of this biomarker to the ASD phenotype. The relationship of this biomarker to age and IQ needs continued exploration. Keywords: Autism, EEG, N170, Biomarker Disclosure: Janssen Research and Development, LLC: Employee (Self), Johnson and Johnson: Stock / Equity (Self) P396. Identification of Stable Clinical Subtypes in Autism Spectrum Disorder Using the Autism Behavior Inventory Srinivasan Vairavan, Savannah Gosnell, Abigail Bangerter, Matthew Klein, Molly Lucas, Nikolay V. Manyakov, Gayle Wittenberg, Gahan Pandina Johnson and Johnson, Titusville, New Jersey, United States Background: Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by impairments in social communication, and restricted and repetitive behavior/interests. Other psychiatric comorbidities are common in ASD. Variability in etiology and phenotypic outcomes of ASD contribute to clinical heterogeneity. Identification of subtypes that maximize homogeneity using clinical characteristics could potentially improve detection of changes in response to treatment. In this abstract, we identified clinical subtypes in ASD using 5-core domains (i.e., social communication, restrictive behaviors, mood and anxiety, self-regulation, and challenging behavior) as part of the Autism Behavior Inventory (ABI) and replicated findings across two different cohorts. Methods: Baseline ABI data were obtained from three large studies, including an online survey, an observational study (NCT02668991), and an efficacy study (NCT03664232). Age, gender, and symptom severity (5-core domains in ABI) of the ASD subjects across three studies were subjected to propensity score matching with the efficacy study as reference, leading to a sample size of 180, 45, 45, in the online survey, observational study, and efficacy study, respectively. The distribution of intelligence quotient (IQ) across studies was comparable with mean IQ ≥ 90. The 5-core domains of the ABI were subjected to Uniform Manifold Approximation and Projection (UMAP) based dimensionality reduction. The number of clusters was chosen in a data-driven way using eigen gap metric. The UMAP parameters, namely, number of neighbors and minimum distance, were optimized based on distance between training and validation cluster, computed as mean distance of each point in training cluster (online survey) to its centroid minus the mean distance of each point in validation cluster (observational study) to the training cluster centroid. The UMAP parameters corresponding to the minimum number of clusters corresponding to the top three positive distances between training and validation cluster were chosen as the optimal parameters. Results: Three clusters were identified corresponding to high, medium, and low symptom report across different domains in ABI and were stable across validation (observational study) and testing datasets (efficacy study). Conclusions: These findings highlight the inherent clinical subtypes in ASD using ABI. The stable three-cluster profile across ASD will be further evaluated in the efficacy study to understand the enrichment in treatment response. In addition, the three-cluster profile will also be evaluated for enrichment in digital signatures observed via heart rate variability, evoked response (N170 corresponding to neural processing of faces), eye tracking and facial expressions. Keywords: Autism, ASD, ABI Disclosure: Johnson and Johnson: Stock / Equity (Self), Janssen Research and Development, LLC: Employee (Self) P397. Dysregulation of the Kynurenine Pathway is Related to Persistent Cognitive Impairments After Tick Borne Encephalitis (TBE) Lilly Schwieler, Jacob Ahlberg Weidenfors, Vytautas Griška, Xueqi Li, Fredrik Piehl, Aukse Mickiene, Sophie Erhardt Karolinska Institutet, Stockholm, Sweden Background: Tick-borne encephalitis (TBE) is caused by neurotropic flavivirus infection and is one of the most serious neurological tick-transmitted diseases. TBE patients present long-term post-encephalitic neurologic and neuropsychiatric symptoms including cognitive decline. Importantly, clinical biomarkers and targeted treatments for post-encephalitic symptoms are currently unavailable. It is well known that virus infections induce tryptophan degradation via a cascade of enzymatic steps known as the “kynurenine pathway” (KP). Modulation of kynurenine metabolites during infection represents a finetuned system for regulating immune responses. This KP is also responsible for the biosynthesis of neuroactive compounds such as quinolinic acid (QUIN) and kynurenic acid (KYNA), both capable of impacting cognition. Indeed, altered KP activity has repeatedly been demonstrated in several diseases from which patients suffer cognitive decline. CSF KYNA associates with both poor cognition and psychosis in psychiatric disorders, while concentrations of CSF KYNA and QUIN are found elevated in HIV-1 infected patients and in patients with COVID-19, disorders with a high incidence of long-term cognitive dysfunctions. The present study aims to measure KP metabolites in cerebrospinal fluid (CSF) and serum of TBE patients and to investigate their relation to long-term neurocognitive performance. Methods: TBE patients were recruited upon admission to Lithuania University of Health Science hospital, Kaunas during which serum and CSF samples were obtained, and the severity of encephalitic illness was classified according to clinical criteria as mild, moderate, or severe. During two follow-up occasions, 6 and 18 months after hospital discharge, patients underwent neurocognitive performance testing (MATRICS Consensus Cognitive Battery, MCCB) and additional serum samples were obtained. Serum and CSF concentrations of tryptophan, kynurenine, KYNA, QUIN, 3-hydroxykynurenine, picolinic acid, and nicotinamide were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in TBE patients and a non-inflammatory neurological disease (NINDC) control group. Following data collection, statistical analysis was carried out to investigate relationships between KP metabolites and patient neurocognitive performance and symptomatology. Linear regression models using log-transformed metabolite concentration data were employed to assess differences in KP metabolite levels between the control group and TBE patients of different clinical severity. Interactions between measured KP metabolite levels and neurocognitive performance were assessed via Spearman rank-correlation tests. Results: TBE patients (n = 87, median age 53, median BMI 25.9, 39% females) did not significantly differ from NINDC controls (n = 12, median age 52.5, median BMI 25.3, 58% females) with regard to age, gender, and BMI. Statistical analysis revealed extensive induction of the KP in TBE patients compared to the control group. In CSF, concentrations of all kynurenine metabolites were significantly increased with notable drastic elevations of the neurotoxic metabolite QUIN (TBE: median 544.24 nM (222.85, 1199.63), NINDC: median 10.31 nM (7.82, 11.29), p < 0.001). The kynurenine/tryptophan ratio, (rKT) a proxy for kynurenine pathway activity, was greatly elevated in the CSF of TBE patients. Similar alterations in KP activity were observed in the serum of TBE patients, with elevations of all measured kynurenine metabolites and notable exceptions of KYNA and QUIN. Interestingly, serum rKT remained significantly elevated among TBE patients up to 18 months, indicating persistent, long-term activation of the KP in TBE patients. In addition, increasing disease severities corresponded with significant elevation of CSF kynurenine and rKT. Conclusions: Our results demonstrate inductions of the KP in both serum and CSF of TBE patients, with persisting serum rKT that has not normalized 18 months after infection. Statistical analyses showed the importance of the magnitude of KP induction for the performance within several neurocognitive domains among TBE patients measured at 18-month follow-up. Confirming that kynurenine metabolites are elevated during TBE infection as well as understanding their role in symptomatology and cognitive decline may open up for new treatment interventions with the aim of dampening the activity in the kynurenine pathway. Keywords: Cognitive Decline, Virus Infection, Kynurenine Pathway Disclosure: Nothing to disclose. P398. A Reliable Measure of Excitatory / Inhibitory (E/I) Balance in Alzheimer’s Disease Juan Molina, Joyce Sprock, Dylan Iwanaga, Jo Talledo, Yash Joshi, Bradley Voytek, Lisa Delano-Wood, Christopher Gonzalez, Steven Huege, Gabriel Leger, Greg Light, Neal Swerdlow University of California, San Diego, La Jolla, California, United States Background: The balance of cortical excitatory and inhibitory synaptic activity (“E/I balance”) has been proposed to be a critical mechanism for fine-tuning neural network activity within a narrow temporal window associated with the processing of sensory stimuli. Several studies have proposed that aberrant E/I balance in Alzheimer’s Disease (AD) may: 1) reflect pathology in cortical glutamatergic, cholinergic and GABAergic transmission; 2) contribute to cognitive decline in this disorder; and 3) be a target for AD therapeutics. The study of E/I balance typically requires invasive methods, such as single-unit or voltage-clamp recordings in rodents and/or non-human primates, which have precluded their application to human studies. Recent findings suggest that the aperiodic, 1/f-like component of the neural power spectra may index tonic E/I balance and can be studied non-invasively using electroencephalographic (EEG) recordings. Using this approach, we reported that the uncompetitive NMDA receptor antagonist, memantine (MEM), normalizes E/I balance in patients with schizophrenia. MEM is used clinically to treat moderate-to-severe Alzheimer’s Disease (AD); as part of a longitudinal study of biomarker predictors of MEM sensitivity in AD, we assessed the effects of acute MEM (0 vs. 20 mg, po) on E/I balance in 18 patients with AD. Methods: Subjects to date are 18 carefully screened individuals with AD (mean (range): age = 72.8 (61-82 y); MoCA = 16.4 (6-23); education = 16.6 (12-20 y); M:F = 9:9). Baseline neurocognitive measures included the MoCA and ADAS-cog; GDS and NPI-Q scales assessed depression and general psychiatric symptoms. Subjects completed a double-blind order-balanced study of MEM (placebo (PBO) vs. 20 mg; 2 test days separated by 1 week) on subjective, autonomic, cognitive and electroencephalographic (EEG) measures. At 275 min post-pill, neurocognition was assessed via the RBANS. At 345 min post-pill, EEG measures were used to assess mismatch negativity (MMN), P3a amplitude and the Auditory Steady State Response (40 Hz coherence and power; ASSR). To analyze the aperiodic and periodic spectral EEG features, EEG signals were decomposed into their frequency-domain components via power spectral density (PSD) estimation using Welch’s method. PSDs from the 4–50 Hz range were used to characterize the aperiodic “background” or 1/f-like signal and oscillatory components using a robust linear regression algorithm per our published methods. Results: Aperiodic slope (E/I) measurements were reliable, i.e., significantly correlated across weeks (R = 0.58, p < 0.015), and were not significantly impacted by acute MEM ingestion (F < 1) nor by ongoing AChE-I use (F = 1.72, df 1,16, ns). PBO-week E/I values were not significantly associated with cognition, whether assessed by baseline MoCA (R = 0.27, ns) or ADAS-cog total scores (Core: R = -0.34, ns; Optional: R = -0.41, ns), or PBO-week RBANS score (R = 0.12, ns). PBO-week E/I values were also not significantly associated with baseline GDS (R = -0.375, ns) or NPI-Q scores (R = 0.05, ns). Acute MEM effects on E/I values did not correlate significantly with those on cognition (RBANS Index Total Score; R = 0.24, ns) or on either MMN, ASSR or P3a amplitude (all ns). After completing testing, all subjects began a 24-week open-label trial of MEM (10 mg BID), with neurocognitive assessment at 8, 16 and 24 weeks; any predictive effects of E/I measures on sensitivity to therapeutic response to MEM will be reported. Conclusions: An EEG-based measure of “excitatory/inhibitory balance” can be acquired in patients with AD and shows significant test-retest reliability in these patients. In this modest sample, E/I balance is not associated with baseline neurocognitive deficits, is not sensitive to acute challenge with MEM (20 mg) and appears to index processes that are distinct from other evoked EEG measures (MMN, ASSR and P3a). Ongoing studies will address whether baseline E/I values, or their sensitivity to acute MEM challenge, predict therapeutic response to a 24-week trial of MEM. Keywords: Alzheimer’s Disease, Memantine, Excitatory / Inhibitory Balance Disclosure: Nothing to disclose. P399. Dysregulation of the Chromatin Environment Leads to Differential Alternative Splicing as a Mechanism of Disease in a Human Model of Autism Spectrum Disorders Calvin Leung, Shoshana Rosenzweig, Brian Yoon, Nicholas Marinelli, Ethan Hollingsworth, Abbie Maguire, Mara Cowen, Michael Schmidt, Jaime Imitola, Ece Gamsiz Uzun, Sofia Lizarraga* University of South Carolina, Columbia, South Carolina, United States Background: Autism spectrum disorder (ASD) affects 1 in 44 children. Chromatin regulatory proteins are overrepresented among genes that contain high risk variants in ASD. Disruption of the chromatin environment leads to widespread dysregulation of gene expression, which is traditionally thought of as a mechanism of disease pathogenesis associated with ASD. Alternatively, alterations in chromatin dynamics could also lead to dysregulation of alternative splicing, which is understudied as a mechanism of ASD pathogenesis. The anticonvulsant valproic acid (VPA) is a well-known environmental risk factor for ASD that acts as a class I histone deacetylase (HDAC) inhibitor. However, the precise molecular mechanisms underlying defects in human neuronal development associated with exposure to VPA are understudied. Methods: To dissect how VPA exposure and subsequent chromatin hyper-acetylation influence molecular signatures involved in ASD pathogenesis, we conducted RNA sequencing (RNA-seq) in human cortical neurons that were treated with VPA. Neurons of male origin from three independent neuronal inductions were used at day 65. Differentially expressed genes (DEGs) were detected using DESeq2 software package (version 1.32.0) (adjusted P < 0.05 and a fold change ≥ \|1.5 \| ). Differential transcript usage (DTU) events were identified using DRIMseq software package (version 1.20.0). Statistically significant DTU events were identified following stageR (version 1.14.0) post-processing analysis as events that has an adjusted P < 0.05. Differential alternative splicing events were identified using rMATS (version 4.1.1). Statistically significant differential alternative splicing events were identified as events with FDR < 0.05 and IncLevelDifference ≥ \|0.1 \| . ClusterProfiler (version 4.0.5) was used for functional enrichment analysis (GSEA, GO, and DisGeNET) of DEGs and DTU events. Significant enrichment results were obtained using a q-value cutoff of < 0.05. Results: We observed that differentially expressed genes (DEGs) were enriched for mRNA splicing, mRNA processing, histone modification, and metabolism related gene sets. We observed widespread and distinct changes in gene expression in the VPA-treated samples compared to control samples with 3545 upregulated and 2663 downregulated DEGs. Furthermore, we observed widespread increase in the number and the type of alternative splicing events. Skipped exon (SE) and retained intron (RI) events were the most frequent splicing events detected in VPA-treated neurons at 40% and 22% of total events, respectively. Analysis of differential transcript usage (DTU) showed that exposure to VPA induces extensive alterations in transcript isoform usage across neurodevelopmentally important genes. Finally, we find that DEGs and genes that display DTU overlap with known ASD-risk genes. Conclusions: In summary, our work highlights the importance of the chromatin environment in the regulation of alternative splicing in the pathogenesis of ASD associated with environmental risk factors and potentially genetic risk factors (chromatin regulators). Keywords: Autism, Epigenetics, Alternative Splicing Disclosure: Nothing to disclose. P400. Inconsistent Patterns of Alzheimer’s Disease- and Anxiety-Targeting Medication Use are Associated With Faster Cognitive Decline and Higher Tau Pathology Burden in Cognitively Impaired Elderly Daniel Felsky, Hyun-Sik Yang Centre for Addiction and Mental Health, Toronto, Canada Background: Effective and tolerable pharmaceutical management of illness in elderly individuals with cognitive impairment is a major clinical challenge. Over time, psychological and somatic changes manifest as a broad set of symptoms requiring intervention, sometimes with agents that may impact mental or general wellbeing. Therefore, decisions to start or stop treatment are complex, and little is known about the consequences of these decisions on longer term outcomes for patients. We sought to understand how different patterns of medication prescription were related to cognitive changes in elderly who developed mild cognitive impairment (MCI) or dementia due to Alzheimer’s disease (AD). Methods: 1,035 elderly individuals (age 80.7, sd=7; 71% female) from the Religious Orders Study and Memory and Aging Project (ROS/MAP) were assessed annually (range 3-28 yrs follow-up, mean=9 yrs) with 19 cognitive tests. Analyses only included participants who received a clinical diagnosis of MCI (n = 416) or dementia (n = 619) at their last study visit. Prescription status for 47 drug types among 20 broad classes (Medi-Span database coding; e.g., anti-AD medications, such as cholinesterase inhibitors, and anti-anxiety medications, such as SSRIs, benzodiazepines, etc.) were ascertained at each time point by direct inspection of containers. Individuals were classified into five discrete trajectory categories for each drug type: 1) never prescribed during study (“never”); 2) prescribed at all study visits (“always”); 3) not prescribed at study entry, transitioning to prescribed during study (“transition on”); 4) prescribed at study entry, transitioning to not prescribed (“transition off”); 5) prescription status fluctuated at some point during study between [on-off-on], or [off-on-off] (“unstable”). Trajectories of diagnostic stability were calculated in a similar fashion, identifying individuals who: 1) transitioned only from CN to MCI or MCI to AD; 2) transitioned from a more impaired diagnosis to a lesser impaired diagnosis at any point during study. Medication trajectories were associated with longitudinal rates of cognitive decline - per-individual random slopes of a linear mixed model for composite scores of global cognition - using ANCOVA, adjusting for sex, education, APOE ε4 status, age at baseline, smoking and alcohol intake at baseline, number of self-reported medical conditions at baseline, cognitive performance at baseline, depressive symptoms at baseline, clinical diagnosis at last visit, and number of follow-up visits. For medication trajectories with effects on cognitive decline, associations were tested for 11 neuropathologies in a subset of 757 individuals with autopsy data, including postmortem interval and age at death as additional co-variates. Results: After Bonferroni correction for multiple testing, significant associations with cognitive decline were found for AD (F4 = 12, p = 1.5×10-9) and antianxiety medication (F4 = 11, p = 7.6×10-9) trajectories. For AD medications, the always prescribed group (n = 30) had the slowest cognitive decline and those who transitioned off medication (n = 19) experienced the most rapid decline (post-hoc comparison corrected p < 0.001). Significant differences were also found between both the always and transition on (n = 177) groups vs. the unstable (n = 132) group; the unstable group had the second most rapid cognitive decline following the transition-off group. For anti-anxiety medications, those never prescribed antianxiety medication (n = 847) showed the slowest cognitive decline, a significant difference from the unstable (n = 132) group (corrected p < 0.001), which had the most rapid decline. In both analyses, the always-off and transitioned-off groups were small – removing these groups from analysis finds consensus that unstable trajectories of medication are linked to faster rates of cognitive decline than stable. Neuropathologically, associations were observed for AD medication trajectories and both neuritic plaques and neurofibrillary tangles, whereby the transitioned-off group had the highest pathological burden – possibly reflecting the common discontinuation of these medications at more severe stages of impairment. The unstable trajectory group showed no difference in pathology compared to those who transitioned onto medication. For antianxiety medication trajectories, the unstable group was the only one significantly different from the never medicated group, showing elevated counts of neurofibrillary tangles (corrected p = 0.013). Finally, the AD medication trajectory was weakly associated with stability of AD diagnosis over time (Fisher’s p = 0.054), whereas antianxiety trajectories were not (Fisher’s p = 0.89). Conclusions: Unstable longitudinal patterns of AD and antianxiety prescription are associated with faster rates of cognitive decline, independent of a multitude of clinical and biological risk factors. Antianxiety medication trajectory specifically was not related to the stability of an individual’s clinical diagnosis but may be linked to increased neurofibrillary tangle accumulation. Our results suggest that fluctuations in AD medication or antianxiety medication use might have a detrimental effect on cognitive prognosis, although reverse causation cannot be fully ruled out. Further study is needed to fully understand potentially causal relationships between these fluctuations and the acceleration of decline in elderly suffering from cognitive impairment. Keywords: Cognitive Decline, Prescriptions, Trajectories, Alzheimer’s Dementia, Neuropathology Disclosure: Nothing to disclose. P401. Opioid Use Disorder is Associated With Alterations in Circadian Pathways: Proteomics Analysis of Human Postmortem Brains Stephanie Puig, Ryan Salisbury, Anastasia Yocum, Jill Glausier, David Lewis, Zachary Freyberg, Marianne Seney, Matthew MacDonald, Ryan Logan Boston University School of Medicine, Boston, Massachusetts, United States Background: Prevalence rates of opioid use disorder (OUD) have increased dramatically, and many patients being treated for OUD relapse within first year. Vulnerability to opioid relapse is associated with severe disruption to sleep and circadian rhythms. Thus, improving circadian rhythms may be an effective intervention for reducing relapse. While relationships between OUD and circadian rhythm alterations have been studied in rodents, an understanding of the molecular alterations that occur in human brains of people diagnosed with OUD remains limited. Methods: We used mass-spectrometry based proteomics to investigate protein alterations in human postmortem brains of OUD and unaffected subjects. Postmortem brains, provided by the Department of Psychiatry at the University of Pittsburgh School of Medicine, were collected from people diagnosed with OUD and unaffected control subjects. Subjects were matched on age, sex, postmortem interval (PMI), brain pH, and RNA integrity (RIN). To enable analysis of rhythms of protein expression, we used time of death information to build cohorts distributed across 24-hour timescale. We collected the nucleus accumbens (NAc), and on the dorsolateral prefrontal cortex (dlPFC), two structures strongly interconnected and heavily implicated in OUD. Quantitative proteomics with TMT was used to measure protein in NAc and dlPFC tissue homogenates and synaptosomes fractions. Limma was used to analyze differentially expressed proteins with adjusted and unadjusted p-values and log-fold-changes (logFC). Results: Identification of rhythmic proteins showed that, in homogenates, the total number of rhythmic proteins was reduced in OUD subjects in both the NAc and the dlPFC (NAc: 115 rhythmic proteins in unaffected controls vs. 56 OUD subjects; dlPFC: 88 rhythmic proteins in unaffected controls vs. 53 in OUD subjects). Conversely, in synaptosomes of OUD subjects, total rhythmic proteins in NAc and dlPFC were decreased or increased, respectively. Interestingly, rhythmic proteins were largely different between groups in both, homogenates and synaptosomes. Notably, in synaptosomes, pathways enriched in OUD rhythmic proteins included membrane potential, vesicle-mediated transport, and GPCR signaling, primarily involved synaptic function. Circadian pathways were also identified as top enriched pathways in synaptosomes. Analysis of differential rhythmicity that compares periods phases, amplitude and variance (R2), highlighted proteins that strictly change rhythmicity between unaffected and OUD subjects. In the NAc 25 and 23 proteins changed rhythmicity in homogenates and synaptosomes respectively and the majority lost rhythmicity. In the dlPFC, only 10 and 18 proteins changed rhythmicity in homogenates and synaptosomes, respectively. Interestingly, changes were equally distributed towards gain or loss of rhythm. In NAc synaptosomes, top pathways enriched in OUD proteins that changed rhythm included membrane trafficking, response to light stimulus and signaling by Rho GTPases. In dlPFC synaptosomes, they included platelet-derived growth factor beta signaling, tyrosine phosphorylation and GTPase signal transduction Conclusions: Our study is the first to analyze changes in protein rhythms caused by OUD in human postmortem brains at a large scale. Our findings demonstrate significantly altered circadian rhythms in synaptic function and signaling in the NAc and dlPFC associated with OUD. These results provide insight on processes mediating alterations in circadian rhythms and sleep/wake cycles caused by OUD. Ongoing studies are investigating the possible functional links between these pathways in mouse models of OUD. Keywords: Postmortem Human Brain Study, Proteomics, Circadian Rhythms, Opioid Use Disorder, Synaptosomes Disclosure: Nothing to disclose. P402. Multimodal Investigation of Structural and Functional Connectivity Changes Associated With 7q11.23 Copy Number Variations Michael Gregory, J. Shane Kippenhan, Tiffany Nash, Daniel Eisenberg, Philip Kohn, Zachary Trevorrow, Madeline Hamborg, Oliva Kline, Danya Adams, Carolyn Mervis, Karen Berman National Institute of Mental Health, Bethesda, Maryland, United States Background: Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are caused by hemideletion (leaving one gene copy) or duplication (yielding three gene copies), respectively, of ~25 genes on chromosomal locus 7q11.23. Previous research has demonstrated alterations in both functional connectivity and white-matter microstructure in WS (i.e., Gregory 2019, Marenco 2007). However, despite the opportunity to test for gene-dosage effects in research by comparing individuals who have hemideletions to individuals who have duplications of the same set of genes, little investigation of this nature has been carried out. Neuroimaging of myelination is a particularly good candidate for such investigation because rodent studies have demonstrated that knockout of the GTF2I gene, which is affected by these 7q11.23 copy number variations (CNVs), is associated with decreased brain myelination (Barak 2019). Here, we took a data-driven approach to identifying both structural and functional connectivity alterations associated with 7q11.23 CNVs by combining data from three neuroimaging modalities: After searching across the brain for regions with altered resting-state functional connectivity that was related to 7q11.23 CNV dosage, we identified white-matter tracts connecting these regions in each participant, and then tested for changes in myelination within these tracts, again, as a function of gene dosage. Methods: Resting-state fMRI, diffusion tensor imaging (DTI), and mcDespot sequences for quantitative myelin imaging were collected for 70 participants (WS: N = 20, age=13.8 ± 4.4, 15 females; typically developing: N = 34, age=14.3 ± 3.8, 21 females; Dup7: N = 16, age=14.8 ± 2.7, 8 females). Quality control measures identified 11 participants whose mcDespot data were not suitable for processing (four individuals with WS, five typically developing individuals, and two individuals with Dup7). First, for resting-state fMRI data, multivariate distance-based matrix regression (MDMR) controlling for age, sex, and motion was used to identify brain regions where whole-brain functional connectivity patterns significantly related to CNV dosage (p < 1×10-9). Second, after preprocessing of DTI data to calculate voxel-wise tensors, significant regions emerging from the first step, the MDMR resting fMRI analysis, were inflated such that they extended to include underlying white matter; these inflated regions were then used as seed regions in probabilistic tractography analyses using AFNI’s FATCAT software to identify white matter tracts connecting each pair of regions in each participant. Only white matter connections that could be identified in 90% of participants were carried forward in the analysis. Finally, voxelwise myelin water fraction (MWF) maps were computed for each participant from the mcDespot data using the publicly available QUIT pipeline; MWF was averaged across each tract identified from probabilistic tractography in each participant, and compared across CNV groups while controlling for age and sex using linear regression in R. Results: Resting-state functional connectivity patterns that significantly related to CNV dosage were found in 22 gray matter regions. These regions were predominantly in areas known to subserve visuospatial and social functions, consistent with the neurobehavioral phenotypes observed in individuals affected by these CNVs. Probabilistic tractography identified 50 white matter tracts connecting these regions that were common across >90% of the participants. MWF in three of these tracts significantly related to CNV status (gene-dosage). These included tracts connecting posterior cingulate cortex with anterior cingulate cortex (p = 0.0018), right angular gyrus with right fusiform gyrus (p = 0.0028), and right parietal lobe with right prefrontal cortex (p = 0.0038). Myelination of all three tracts showed a pattern of increasing myelination with increasing copy number (Dup7 > typically developing > WS). No tracts showed the opposite pattern of significantly decreasing myelination with increasing copy number. Conclusions: These data take a multimodal neuroimaging approach to identifying structural and functional connectivity changes associated with 7q11.23 CNVs. First, the 22 regions found to have altered resting-state functional connectivity largely subserve visuospatial and social functions, which are key components of the neurobehavioral phenotypes of WS and Dup7. Second, consistent with previous reports of decreased myelin basic protein in postmortem brain samples from individuals with WS (Barak 2019), we found that myelination of three tracts connecting these regions increased with increasing 7q11.23 copy number. Among the white matter tracts investigated, these three are particularly notable for their role in connecting regions known to be major hubs of structural and functional brain networks, including posterior cingulate cortex, anterior cingulate cortex, angular gyrus, parietal lobule and prefrontal cortex (Oldham 2019), supporting the notion that the genetic mechanisms underlying 7q11.23 CNVs lead to altered myelination and that these neural features may contribute to the behavioral phenotypes in WS and Dup7. Further work may better characterize these structural and functional connectivity changes and may relate the findings to neuropsychological or behavioral measures. Keywords: Williams Syndrome, 7q11.23 Duplication, Multimodal Neuroimaging, Myelin Imaging, Resting State Functional Connectivity Disclosure: Nothing to disclose. P403. Genetic Influences on Psychotic and/or Affective Neuropsychiatric Symptom Phenotypes of Alzheimer’s Disease Inga Antonsdottir, Mary Ann DeMichele-Sweet, Lambertus Klei, Byron Creese, Clive Ballard, Bernie Devlin, Constantine Lyketsos, Robert Sweet Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Neuropsychiatric symptoms (NPS) including delusions or hallucinations (psychosis) and depression, anxiety, and/or irritability (affective disturbance) are common among people living with Alzheimer’s disease (AD). Symptoms such as these contribute to more rapid and severe cognitive decline, while causing significant disability, morbidity, and mortality. Methods: This genome-wide association meta-analysis examined data from persons with AD assessed on the Neuropsychiatric Inventory or Questionnaire (NPI or NPIQ) who participated in one of the following six source studies: the Fundació ACE Barcelona Alzheimer Treatment and Research Center (ACE/GR@ACE), a Consortium of National Institute on Aging Alzheimer Disease Centers (ADC), Eli Lilly and Company (LILLY), the Norwegian, Exeter and King’s College Consortium for Genetics of Neuropsychiatric Symptoms in Dementia (NEXGENS), the National Institute on Aging’s Late Onset Alzheimer’s Disease Family Study (NIA-LOAD), and the University of Pittsburgh Alzheimer Disease Research Center (PITT ADRC). The presence of psychotic symptoms (AD + P; hallucinations or delusions) or affective symptoms (AD + A; depression, anxiety, and/or irritability) was indicated if participants had at least one of these symptoms at any visit as assessed by the NPI or NPIQ. A GWAS analysis was performed separately on each of the two phenotypes. Results from the AD + P and AD + A phenotypes were combined, and genetic correlation and heritability analyses were conducted using GenomicSEM. Results: Data from 8,714 individuals with AD (60.2% female) were analyzed. Of these individuals, 32% had neither psychotic (AD-P) nor affective symptoms (AD-A), 28% had affective but not psychotic symptoms (AD + A; AD-P), 28% had psychotic and affective symptoms (AD + P; AD + A), and 12% had psychotic but not affective symptoms (AD + P; AD-A). Significant association occurred between affective and psychotic symptoms, with affective symptoms present in 70.1% of participants with psychotic symptoms versus 46.9% in participants without psychotic symptoms (χ2 = df=1, p < 0.001). There was significant heritability to both the AD + P and the AD + A phenotypes as well as to the joint phenotype. Based on a prevalence of AD + P and AD + A in AD subjects of 0.50 and 0.40, respectively, the estimated h2 on the liability scale was 0.23 + 0.06 and 0.06 + 0.07 for AD + P and AD + A, respectively. The estimated correlation between the two phenotypes on the liability scale was 0.55 + 0.44. The estimated h2 on the liability scale for the joint phenotype was 0.155 + 0.065. Genetic correlation of the joint phenotype with AD + P was 0.95 + 0.31 and with AD + A was 0.78 + 0.64. When assessing specific genetic associations, we first contrasted AD-P to AD + P where two loci achieved genome-wide significance. One was in ENPP6 (rs9994623, O.R. (95%CI) 1.16 (1.10, 1.22), p = 1.26 × 10 − 8) and the other at 19q13 spanning three genes, APOE, TOMM40, and NECTIN2 (of note, rs429358 is one of two SNPs defining the APOEε4 genotype [O.R. 0.84 (0.79-0.90), p = 2.34 = 10-8]). These findings are similar to those previously reported in a GWAS analysis of AD + P, with changes in p-value significance likely attributed to the change in sample size. When contrasting AD-A to AD + A, no SNP reached genome-wide significance; however, a locus at 9q31 spanning RAD23B approached significance (rs1805331, O.R. (95% CI) 0.80 (0.74, 0.87), p = 1.331×10-7) and may become significant if the sample size were larger, as assessed by probability-probability plot. When bivariate association tests were performed, no SNP reached genome-wide significance, however three approached significance. The first was at 9q31 spanning RAD23B (rs1805331, O.R. 0.80 (0.74, 0.87), p = 1.33×10-7), a single SNP, rs112368830, at 1q42 (O.R. 1.39 (1.22, 1.57), p = 2.53×10-7), and a third locus at 15q22 (best SNP rs35669194, O.R. 0.78 (0.70, 0.86), p = 6.11×10-7), spanning GTF2A2 and the 3’ portion of BNIP2. Conclusions: These results show common genetic variation accounts for a significant portion of heritability in both phenotypes. These findings confirm prior work in associating AD + P with common genetic variation and extend the association to AD + A. Of note is the novel finding that the joint AD + P/AD + A phenotype is associated with common genetic variation. AD + P samples had a larger maximum absolute Z, and smaller minimum p-value, than observed values for AD + A. This suggests affective symptoms in AD are less distinctly driven by genetics as opposed to psychotic symptoms and could instead be more environmentally driven. We note that the bivariate analysis was slightly better powered to detect genetic associations of NPS in AD than in either phenotype alone, conditional to the sample size being equivalent. This illustrates the importance of utilizing a joint affective-psychotic phenotype when conducting research on genetic associations of NPS in AD. Given the near universal prevalence and adverse impact of NPS for patients with AD and considering the relative paucity of effective treatment options further study of emerging genetic influences on distinct NPS phenotypes has the potential of contributing to better treatments. Keywords: Alzheimer’s Disease, GWAS, Neuropsychiatric Symptoms (NPS) Disclosure: Nothing to disclose. P404. Development of a CSF1R Pet Ligand [18 F]JNJ-4249 Hartmuth Kolb, A. Katrin Szardenings, Wei Zhang, Chunfang Xia, Tamara Berdyyeva, Su-Tang Lo, Mani Salarian, Christian Constantinescu Janssen Pharmaceutical, San Diego, California, United States Background: There is increasing evidence that neuroinflammation promotes and modulates neuropsychiatric disorders. Microglia play a critical role as key cellular components of the inflammatory process. Being able to image activated microglia in-vivo would provide a valuable tool to study neuroinflammation and responses to new targeted treatments in the brain. Colony-Stimulating Factor 1 Receptor (CSF1R) is a receptor tyrosine kinase, predominantly expressed on macrophages and microglia, that regulates microglia activation and density. It therefore provides a target that could be used not only to measure the extent of inflammation in the brain, but also to develop new effective therapies. Here, we describe preclinical studies of the PET ligand [18 F]JNJ-4249, a selective and potent CSF1R inhibitor. Methods: The novel CSF1R PET ligand, [18 F]JNJ-4249 was evaluated in a series of standard in vitro assays and in-vivo imaging experiments in rodents and non-human primates. To induce neuroinflammation, two preclinical rodent models were introduced. A local neuroinflammatory model was induced by intracranial injection of Lipopolysaccharide (LPS) into the striatum of Sprague Dawley rats. A systemic neuroinflammatory model was induced by intraperitoneal injection of LPS into C57BL6 mice. Both models were investigated by imaging with [18 F]JNJ-4249, followed by post-mortem analysis of the brains by IHC (Iba-1) and Western Blotting (CSF1R). The tracer distribution and radio-metabolite analysis were further investigated in non-human primates. Results: JNJ-4249 is a highly potent (IC50 1.2 nM) and selective (400 kinase panel) PET ligand. In-vivo imaging of healthy rats showed good initial brain uptake and fast washout. In a locally injected LPS rat model, an increased (30%) uptake on day 2 and 4 post-LPS injection (20 µg, right striatum) is reproducible and consistent. On the other hand, 48 h post-IP injection of LPS in mice showed 50% increase in whole brain compared to naïve mice. Western blotting of brain tissue shows low levels of CSF1R expression in normal tissue, but increased expression in LPS treated animals (47 kDa, cytoplasmic domain). A baseline scan in healthy non-human primates shows a similar brain uptake and washout pattern as in rodents. The distribution was moderately homogeneous and demonstrated reversible kinetics. The VT (240 min) at baseline in whole brain was 12.4 mL/cm3, with a range of 11.1 mL/cm3 (cerebellum) to 15.2 mL/cm3 (frontal). Metabolism of the tracer was fast, forming more polar metabolites (65% and 4% parent remaining at 6 and 60 minutes, respectively). Conclusions: The preclinical evaluation of [18 F]JNJ-4249 demonstrates that this tracer is a suitable candidate for imaging CSF1R in the brain and has potential to be used as a biomarker of microglia activation and neuroinflammation. Further experiments in neuroinflammation models are ongoing in preparation for first-in human studies. Keywords: Molecular Imaging, CSF1R, Neuroinflammation, Microglia Disclosure: Janssen: Employee (Self) P405. Calbindin- And Parvalbumin-Positive Neurons and Microglia Display Sexual Dimorphism in Long-Term Responses to Methamphetamine and HIV-Associated Brain Injury Marcus Kaul, Rohan Shah, Ricky Maung, Daniel Ojeda-Juárez, Ana Sanchez, TMARC Group School of Medicine, University of California, Riverside, California, United States Background: Methamphetamine (METH) use is a frequent comorbidity of infection with human immunodeficiency virus type-1 (HIV-1) and exacerbates HIV-associated neurocognitive disorders (HAND). However, the combined pathological mechanisms of METH and HIV-1 are incompletely understood. Transgenic mice expressing the HIV-1 envelope protein gp120 in the brain (gp120tg) share characteristic neuropathological features and gene expression patterns with neurocognitively impaired HIV/AIDS patients. Methods: We previously exposed age-matched female and male gp120tg mice and non-tg controls to an escalating METH binge regimen for 25 days (HIV-1 gp120tg SAL n = 16, HIV-1 gp120tg METH n = 14, WT SAL n = 15, WT METH n = 18) and analyzed the animals 7 months later, at about 12 months of age using behavioral assessments, neurohistopathology and gene expression (Hoefer et al., Exp. Neurol. 2015). Both, HIVgp120 and METH compromised neurites, synapses and behavioral performance. In this follow-up study, we analyzed calbindin (Calb)- and parvalbumin (PV)-positive neurons and glial cells in a subset of the animals (n = 3 - 4 each of males and females per experimental group) using quantitative immunofluorescence and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Statistical analysis employed ANOVA and Fisher’s PLSD post hoc tests for comparison of the experimental groups. Results: Calb and PV revealed sex-dependent differences in cerebral cortex and hippocampus with regard to the number of neurons, the quantity of the cell marker’s expression, and the response to gp120 and/or METH exposure (RNA and protein). Similarly, pronounced sexual dimorphism was observed in microglia, including in the response to METH. In contrast, astrocytosis indicated only in cerebral cortex a sex-dependent difference associated with gp120 expression but not in response to METH. Conclusions: In summary, METH exposure and viral gp120 compromise learning and memory function and induce neuropathology in both sexes. While injury to MAP-2 and synaptophysin-positive neurites and presynaptic terminals, respectively, does not reveal sexual dimorphism, Calb- and PV-positive interneurons and microglia display significant sex- and brain region-specific differences in the long-term response to METH exposure and HIVgp120 expression. Supported by NIH, MH087332, MH104131, MH105330, DA052209 and DA026306 (P5) to M.K. Keywords: HIV-Associated Neurocognitive Disorder, Methamphetamine, Drug Use Disorders, Microglial Activation, Astrogliosis Disclosure: Nothing to disclose. P406. Exploring Codon-Edited tRNA Approaches to Correct Premature Stop Codons in Mouse and Human Models of SCN2A-Related Autism and Intellectual Disability Olivia Klein, Jonathan Sikora, Marisol Lauffer, Karina Kruth, Christopher Ahern, Aislinn Williams* University of Iowa, Iowa City, Iowa, United States Background: The SCN2A gene encodes the alpha subunit of the neuronal voltage gated sodium channel NaV1.2, which is necessary for action potential generation and propagation. Allelic variants of SCN2A are associated with autism spectrum disorder (ASD), intellectual disability, and seizure disorders. Loss of function mutations, such as nonsense mutations, can cause ASD and intellectual disability. Nonsense mutations create a premature termination codon (PTC) in the reading frame of NaV1.2 and result in functional haploinsufficiency. Therapeutic repair of these nonsense mutations could restore NaV1.2 sodium channel expression. Currently, treatment of PTCs is limited to using small molecules with to promote ribosomal readthrough of the nonsense codon, which often results in generation of a missense mutation. These therapies would likely offer minimal clinical improvement for SCN2A and other ion channelopathies since many ion channels are intolerant of mutations, and missense mutations in NaV1.2 are strongly associated with epileptic encephalopathy. We have generated a novel mouse model of an SCN2A PTC, R1626X, as well as a human patient-derived neuronal model of a different PTC, C959X, to test therapeutic approaches. We have also developed a novel codon edited transfer RNAs (tRNA) approach to correct PTCs. Codon edited tRNAs bind to the target PTC in mRNA but are charged with the correct amino acid, thereby generating full-length wild-type protein. Codon edited tRNAs have limited interaction with native stop codons. Here, we show that heterozygous SCN2A R1626X mouse display SCN2A autism-associated behaviors, and that our codon edited tRNA system can rescue PTCs in mouse and human neurons. Methods: Mice heterozygous for the SCN2A R1626X mutation were generated through the University of Iowa Genome Editing Facility. To record ultrasonic vocalizations (USVs), pups were separated from dams at postnatal day 6 (P6) and recorded for 5 minutes (n = 10-15 per genotype). For behavioral work in adult mice, mice were tested between 10-20 weeks of age (n = 9-15 per genotype) on open field, novel object exploration, Erasmus Ladder, three-chamber social interaction test, and free social interaction using standard protocols. Both male and female mice were used for all experiments and results were analyzed by two-way ANOVA or linear mixed models, where appropriate. For mouse neuronal cultures, cerebella were dissected from P6 pups to isolate cerebellar granule neurons (CGNs). Plasmids were transfected into neurons using PolyJet or Mirus reagents or were nucleofected (Lonza). Cells were analyzed by immunofluorescence 96 hours after transfection. For human neuronal cultures, induced pluripotent stem cells (iPSCs) were generated from a patient with an SCN2A C959X mutation and a neurotypical control. iPSCs were differentiated into neural progenitors using dual-SMAD inhibition, and progenitors were differentiated into neurons in BrainPhys medium with supplements. Plasmids in this study contain fluorescent reporters (mOrange2 or eGFP) engineered to encode a PTC and codon-edited tRNA to correct the PTC. Plasmids were transfected into human neurons using Lipofectamine STEM transfection reagent. For quantitative PCR, SCN2A expression was quantified using PrimeTime primers (Integrated DNA Technologies), normalized to GAPDH. For Western blot, cerebella were dissected from adult SCN2A R1626X mice and WT littermates, then lysed and run on a gradient gel under standard conditions. NaV1.2 protein was detected using a custom antibody (gift of Dr. Geoff Pitt, Cornell University). Results: Heterozygous SCN2A R1626X pups had lower mean frequency and longer USVs compared to WT littermates (p < 0.01), with no differences in total USV number. Adult heterozygous SCN2A R1626X mice were more likely to explore the center of the open field than WT littermates (p < 0.05), with no differences in total distance traveled. Erasmus Ladder testing showed that SCN2A R1626X mice display improved gait adaptation (p < 0.05), a form of motor learning, with no evidence of ataxia. We observed no differences in novel object exploration or social behaviors using the three-chamber social interaction test. SCN2A R1626X mice have decreased NaV1.2 protein in the cerebellum, consistent with the PTC causing functional haploinsufficiency. Although human SCN2A C959X neurons contain a PTC, SCN2A mRNA levels were not lower in C959X neurons compared to control neurons throughout their maturation in vitro, suggesting that human SCN2A transcripts containing PTCs do not undergo substantial nonsense mediated decay. Codon-edited tRNA were able to rescue PTCs in fluorescent reporters in both mouse and human iPSC-derived neurons, showing that codon-edited tRNA can be expressed in neurons and facilitate PTC rescue. Conclusions: Heterozygous SCN2A R1626X mice display behaviors consistent with aspects of autism and intellectual disability, some of which can be detected at very young ages, along with reduced NaV1.2 protein. Human SCN2A C959X neurons express SCN2A and, to our knowledge, this is the first study to show that codon edited tRNA can rescue PTCs in human neurons. These models will be useful for testing codon-edited tRNA therapeutic approaches. Future directions include transitioning from plasmids to viral vectors, which have greater translational potential, and attempting to correct endogenous PTCs in these human and mouse models. Keywords: Ion Channels, Autism Spectrum Disorder and Related Syndromes, Intellectual Disability, Behavioral Tasks, Induced Pluripotent Stem Cells (iPSCs) Disclosure: Nothing to disclose. P407. Optimization and Validation of Profiling Efforts Within the NINDS Preclinical Screening Platform for Pain (PSPP) to Accelerate the Development of Novel Non-Opioid, Non-Addictive Pain Therapeutics Smriti Iyengar, Mark Varney, Mark Urban, Elizabeth Dugan, David Budac, Qing Chang, Taleen Hanania, Sarah Woller NINDS, NIH, Rockville, Maryland, United States Background: The NINDS Preclinical Screening Platform for Pain (PSPP) was developed under the NIH HEAL Initiative to facilitate the identification and development of new non-opioid, non-addictive pain therapeutics. PSPP provides researchers from academic, industry, and government institutions worldwide, an efficient, rigorous, resource to accelerate development of novel small molecule, biologic, natural product, and device treatments for pain. Such novel assets accepted into the program are profiled to evaluate their effects in vivo in rats in extensively validated pain related models and endpoints, paying close attention to pharmacokinetic parameters, understanding potential for neurological deficits in vivo and drug abuse liability in vitro as well as in vivo. Methods: Male and female SD rats (Envigo, Indianapolis, IN) 180-250 g, were acclimated for at least a week prior to testing and appropriately housed. During the study, 12/12 light/dark cycles were maintained with lights on at 6 am. The room temperature was maintained between 20 and 23°C with a relative humidity maintained around 50%. Chow (Lab diet 5001, LabDiet, St. Louis, MO, USA) and water were provided ad libitum for the duration of the study. The tests were performed during the animal’s light phase between 8 am – 4 pm. Pharmacokinetic studies were conducted to guide dosing, select the route of administration, and to determine the time course, supporting subsequent behavioral studies. The modified Irwin (n = 4) and rotarod tests (n = 10) were conducted to evaluate potential neurologic, physiologic, and fine motor effects that may impact outcome measures in the pain models. Following side effect profile assessment, efficacy was evaluated in the plantar incisional pain (n = 10) and L5/L6 spinal nerve ligation (SNL; n = 10) models. The plantar incision model is an established model of acute post-operative pain induced by incision of the skin and the plantaris muscle (Brennan et al. 1996). The model is characterized by transient hind paw tactile allodynia and spontaneous guarding behaviors. SNL is a model of peripheral neuropathic pain resulting from chronic nerve compression in which tactile and cold allodynia are produced (Kim and Chung, 1992). All experiments were conducted in a blinded manner with both sexes included. Statistics: Data were analyzed by either one-way ANOVA or Repeated Measures ANOVA using GraphPad Prism (Version 9.2.0), followed by Dunnett’s or Bonferroni’s post-hoc comparisons when appropriate. An effect was considered significant at p < 0.05 level. Data are presented as the mean and standard error of the mean (s.e.m). Power analysis was used to determine the group sizes for the various assays. Results: Results from the evaluation of duloxetine, celecoxib and diazepam suggest that all three drugs had differing profiles of efficacy in pain related models. Administration of duloxetine (10, 30, 60, 100 mg/kg PO) did not affect performance on the rotarod in male or female rats at the doses or times assessed. In the modified Irwin test evaluating similar doses, the 10 mg/kg dose was well tolerated, but the 100 mg/kg treated animals consistently displayed decreased body position, decreased locomotor activity, and sedation. In both the plantar incision model and in the L5/L6 SNL model, duloxetine was evaluated at 3, 10, 30, 60 mg/kg PO. In the plantar incision model, the 60 mg/kg dose of duloxetine robustly reduced mechanical allodynia and guarding behaviors in male and female rats with the maximal effect occurring at 1 hour while in the SNL model duloxetine (60 mg/kg) robustly reduced mechanical allodynia and acetone cold sensitivity in both male and female animals compared to vehicle treated animals with maximal effect occurring between 1 and 2 hours post treatment. Ongoing studies with celecoxib (3, 10, 30, 100 mg/kg PO) show that the drug did not affect rotarod performance in male or female rats at the doses or times assessed. In the plantar incision model (10, 30, 60 mg/kg PO), celecoxib produced a significant attenuation of guarding behaviors in male and female animals at 1, 2, 4 and 6 hours but did not affect mechanical allodynia behaviors. At doses of 1, 3, 10 and 30 mg/kg PO, diazepam treatment produced sedative effects, including decreased body position, decreased locomotor activity, ataxia, visual placement after nose contact, and decreased grip strength in a dose dependent manner in both male and female animals in the modified Irwin test and significantly impaired rotarod performance in males at 1 hour and in females at 1 and 2 hours, post-treatment. In the plantar incision model, while diazepam (1, 3, 10 mg/kg PO) showed a statistically significant effect on PWT in male and female rats and on guarding behavior in female rats, it did not robustly alter these behaviors. Administration of diazepam showed no effect on either PWT or acetone cold sensitivity in male and female animals in the L5/L6 SNL model at similar doses across a 6-hour period post administration. The results will be presented in the context of pharmacokinetic parameters. Conclusions: In summary, the evaluation of clinically used drugs duloxetine, celecoxib and diazepam showed differing profiles in the PSPP paradigm, that were consistent with their utility clinically. The NINDS PSPP program strives to accelerate the development of novel non-opioid, non-addictive therapeutics for pain. Keywords: Pain Therapeutics Profiling, Clinical Pain Standards, Pain Models Disclosure: Retiree, Eli Lilly and Company: Other Financial or Material Support (Self) P408. Oliceridine Demonstrates a Reduced Effect on Neurocognitive Function in Humans, Compared to Morphine: A Phase 1 Randomized, Placebo-Controlled, Dose-Ranging Partial Block Cross-Over Study Mark Demitrack, Michael Kramer, Cathy Michalsky, Hemme Hijma, Laurence Moss, Geert Jan Groeneveld Trevena, Inc., West Chester, Pennsylvania, United States Background: Oliceridine is an agonist at the mu-opioid receptor, with preferential post-receptor engagement of G-protein signaling, while showing reduced beta-arrestin recruitment and receptor internalization (DeWire, 2013). Opioid medications are an essential component of pain management after surgery, though a variety of opioid-induced adverse events (AEs) complicate their use. Notable among these is the development or exacerbation of cognitive dysfunction, which may range from sedation to confusion or progress to delirium. Cognitive dysfunction can therefore have potential implications for post-operative recovery and health outcomes, and in some instances may result in deficits that persist beyond the immediate post-operative period. The mechanism of these cognitive complications is unclear, though it has been hypothesized that opioids, such as morphine, can bind to the toll-like receptor 4 (TLR4), and the subsequent neuroinflammatory response may contribute to these postoperative cognitive sequelae (Muscat, 2021). Rats treated with oliceridine demonstrate reduced levels of spinal cord TLR4 after experimental fracture compared to morphine-treated animals (Liang, 2018). The present study was designed to characterize the neurocognitive impact of IV oliceridine versus IV morphine using a validated cognitive test battery. We hypothesized that IV oliceridine would demonstrate a reduced effect on cognitive measures compared to IV morphine. Methods: Twenty-three healthy subjects (13 males, 10 females; median age 26 years), provided their informed consent and were randomized to receive 3 of the 5 possible treatments as single IV doses in a partial block cross-over design: placebo, oliceridine 1 mg or 3 mg, or morphine 5 mg or 10 mg. The dose range for each drug was selected based on prior data confirming a relative potency of oliceridine to morphine of approximately 1 to 5, and the maximum approved single dose of oliceridine (3 mg; Olinvyk® product label). Neurocognitive function using the NeuroCart® test battery was assessed at: baseline, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h and 6 h. The primary outcome of the study was saccadic eye movement peak velocity, a sensitive measure of sedation. Secondary outcome measures included saccadic eye movement reaction time and % inaccuracy, smooth pursuit eye movement, an adaptive tracking test of eye-hand coordination, postural stability measured by body sway, and the symbol-digit substitution test. Right and left pupillometry, and analgesia measured with the cold pain test assessed adequacy of target engagement. Outcomes were examined with a mixed-model ANOVA, with treatment, period, time, and treatment by time as fixed factors, subject, subject by treatment and subject by time as random factors and average baseline value as a covariate. For a significant main effect of treatment, between-group pairwise comparisons of the treatment conditions were performed. Results: Consistent with the known relative potency of oliceridine and IV morphine, across the dose ranges studied, both drugs demonstrated expected effects on opioid-induced pupillary constriction, and analgesia in response to cold pain testing. There was a statistically significant effect of treatment on the primary outcome measure of saccadic eye movement peak velocity (main effect of treatment, P < 0.0001), driven by a favorable, reduced impact of oliceridine versus IV morphine (LS mean treatment difference [95% CI]: -11.40 degrees/s [-21.19, -1.61], P = 0.0236). Similar outcomes favoring oliceridine were observed on the secondary outcome measures of saccadic eye movement reaction time (main effect of treatment, P = 0.0201; LS mean treatment difference [95% CI]: 0.0088 sec [0.0010, 0.0166], P = 0.0273), reduced body sway (main effect of treatment, P = 0.0314; LS mean treatment difference [95% CI]: 16.1% (mm) [-2.7%, 38.5%], P = 0.0951), and improved performance accuracy on the adaptive tracking test, a measure of eye-hand coordination (main effect of treatment, P = 0.0011; LS mean treatment difference [95% CI]: -1.519% [-3.505, 0.467], P = 0.1303). Additional neurocognitive outcome measures, visual tracking and the symbol-digit substitution test, did not show statistical differences between oliceridine and IV morphine. No serious adverse events were observed in the study. Common AEs included expected opioid-related adverse events of nausea, vomiting and somnolence. Events were assessed as mild in all cases with the exception of one AE of moderate nausea in a morphine-treated subject. Conclusions: IV oliceridine has a reduced impact on several clinically relevant measures of cognitive performance, compared to IV morphine, including certain measures of sedation, motor performance, and eye-hand coordination. Keywords: Cognition, Opioids, Clinical Psychopharmacology, Postoperative Cognitive Dysfunction Disclosure: Trevena, Inc.: Employee (Self) P409. Kappa Opioid Receptor Antagonism Suppresses Biochemical and Behavioral Phenotypes in Mice Expressing an ADHD and ASD-Associated Variant of the Dopamine Transporter Felix Mayer, Adele Stewart, Michelle Velez, Maximilian Rabil, James Foster, Roxanne Vaughan, Lynette Daws, Sammanda Ramamoorthy, Randy Blakely Florida Atlantic University, Jupiter, Florida, United States Background: The availability of extracellular dopamine (DA) is tightly regulated by the dopamine (DA) transporter (DAT). The rare DAT Val559 variant has been identified in individuals with attention-deficit hyperactivity disorder (ADHD), bipolar disorder and autism spectrum disorder (ASD). DAT Val559 exhibits anomalous DA efflux (ADE), a property that is shared by multiple disease-associated DAT variants. Mice expressing DAT Val559 display elevated extracellular DA in the striatum and present with hyperreactivity to imminent handling, waiting impulsivity, working memory deficits, and enhanced motivation for reward. ADE supports continuous activation of D2-type DA autoreceptors (D2AR), leading to elevated surface expression and hyperphosphorylation of DAT Val559 in a sex and circuit specific manner. The synthesis of dynorphin, the endogenous ligand of kappa opioid receptors (KORs) can be elevated postsynaptically by increased DA signaling at D1 receptors. In turn, activation of KORs on DA terminals leads to elevated DAT surface trafficking. Consequently, we hypothesized that antagonism of KORs could, by reducing dynorphin signaling, diminish surface levels of the efflux prone DAT Val559, and thereby suppress neurochemical and behavioral phenotypes in DAT Val559 KI mice. Methods: We performed surface-biotinylation and immunopurification assays using acute coronal slices containing the dorsal and/or ventral striatum of DAT Val559 and wild-type (WT) control mice to assess the effect of the KOR agonist U69,593 and the KOR antagonist nor-binaltorphimine (norBNI) on surface expression and phosphorylation of WT DAT and DAT Val559 ex vivo. To measure the effect of systemic norBNI (10 mg/kg, i.p.) on DA-release in vivo, we performed microdialysis and fiber photometry, the latter effort performed using virally-expressed, genetically-encoded sensors for DA. Following vehicle or norBNI (10 mg/kg, i.p.) administration, WT and DAT Val559 expressing mice were assessed in the Y-maze and open field test. All experiments utilizing mice were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Florida Atlantic University. Results: Using acute slices from DAT Val559 and WT mice, we found that DAT Val559 remains amenable to regulation via KOR. Regardless of genotype, exposure to U69,593 enhanced DAT surface expression that was accompanied by phosphorylation at DAT threonine 53 (Thr53). In contrast, exposure to norBNI normalized the basal elevated DAT Val559 surface expression and hyperphosphorylation at Thr53 but was without effect on WT DAT. Assessment of DA dynamics in DAT Val559 in vivo compared to WT revealed that systemic norBNI normalized vesicular DA release in the dorsal striatum of male mice. Finally, norBNI was found to restore the alternation-deficit (Y-maze) and anxiety-related behaviors (reduced time in center of the open field) observed in DAT Val559 mice. Conclusions: DAT Val559 mediated ADE occurs independently from neuronal activity and results in the tonic activation of D2ARs, supporting a feedback loop that recruits additional efflux-prone transporters to the plasma membrane. By targeting KOR, a well-established regulator of DA neuronal activity and DAT surface expression, we identified antagonism of KOR as a promising strategy to ameliorate neurochemical and behavioral phenotypes that arise from the expression of a disease-associated DAT variant. More generally, our observation that antagonism of KOR was without gross effects on WT animals for the measures obtained supports KOR antagonism as a potentially useful strategy to offset biochemical and behavioral deficits that are linked to hyperdopaminergic states. Keywords: Dopamine Transporter, ADHD, Kappa Opioid Receptor, ASD, Dopamine Disclosure: Nothing to disclose. P410. TRV045, a Novel, Selective S1P Receptor subtype-1 Modulator that Does Not Cause lymphopenia, Is Efficacious in Acute and Chronic Rodent Epilepsy Models Michael Kramer, Ruihua Chen, Mark Demitrack Trevena, Inc., Chesterbrook, Pennsylvania, United States Background: Epilepsy is caused by the aberrant synchronized firing of neurons resulting from the imbalance in excitatory and inhibitory neurotransmission. The mainstream antiepileptic drugs (AEDs) are direct modulators of ion channels, but they do not control seizures in 30% of patients. There is a need to discover and develop AEDs with novel mechanisms to address refractory epilepsy. S1P and its receptors, especially subtype 1 (S1PR1), play important roles in neuroinflammation, a process underlying seizures and epileptogenesis. S1P receptor expression is increased in experimental post status epilepticus (SE) TLE mouse models. Fingolimod, a nonselective S1PR modulator but with high potency for S1PR1, has shown anti-epileptic effects in a diverse range of preclinical epileptic models, possibly through anti-inflammatory mechanisms as well as preservation of neuronal and blood brain barrier integrity. However, nonselective S1PR modulators such as fingolimod cause lymphopenia. TRV045 is a highly selective S1PR1 modulator that does not cause lymphopenia in animal models. Through collaboration with the NINDS Epilepsy Therapy Screening Program (ETSP) we previously showed that subcutaneous dosing of TRV045 reduced seizures in the corneal-kindled (CK) mouse model and the rat maximal electroshock seizure (MES) model, but other models were confounded by a vehicle effect. Here, a new vehicle (10% cremophor, 20% Captisol in water) was used for oral (PO) dosing in the mouse CK, rat MES, Theiler’s murine encephalomyelitis virus (TMEV) and post-kainic acid spontaneous recurrent seizures (KA-SRS) rat model of temporal lobe epilepsy. Methods: In the MES model, 60 Hz of alternating 150 mA current was delivered to male Sprague Dawley (SD) rats for 0.2 seconds by corneal electrodes. Rats (n = 8/gp) were dosed PO with 30 or 60 mg/kg TRV045 and tested for seizure activity 0.25 to 2 hr postdose to identify the time of peak effect (TPE). An animal was considered “protected” from convulsant activity upon abolition of the hindlimb tonic extensor component of the seizure. A full dose response was then performed with testing at the TPE. In the CK seizure model, male C57BL/6 mice were fully kindled to reach the criterion of 5 consecutive stage 5 seizures. Mice (n = 8/gp) were dosed PO with 10 or 15 mg/kg TRV045 5-7 days after the last stimulation and tested 1 and 2 hr postdose to determine the TPE. A full dose response was then performed with testing at the TPE to identify an ED50 dose. In the TMEV model, male C57BL/6 J mice (n = 18-20/gp) were pretreated with TRV045 for 2 days (10 or 30 mg/kg, b.i.d., PO) and then infected with TMEV on Day 3. Daily dosing continued with assessment of seizure severity using a modified Racine scale through Day 7. In the KA-SRS model, status epilepticus was induced in male SD rats with repeated low-dose kainate treatment. Following a 7-day baseline period, rats (n = 6/gp) received intraperitoneal (IP) doses of TRV045 or vehicle for 5 days. Following a 2-day washout period, animals were crossed over to the opposing treatment arm for a second 5-day period. Seizure burden was calculated as the summation of all Racine scale seizures during treatment divided by the number of treatment days. Seizure freedom was based on an animal having zero seizures from the time of first dose through 12 hr post-final dose. Results: TRV045 prevented seizures in the rat MES model of generalized tonic-clonic seizures, with a TPE of 0.5 to 1 hr postdose. Tested 0.5 hr postdose, PO dosing of 20, 30, 40, 50 and 60 mg/kg TRV045 prevented generalized seizures in 1, 4, 3, 1 and 0 out of 8 rats, respectively. Tested 1 hr postdose, PO doses of 3, 10, 20 and 40 mg/kg TRV045 prevented generalized seizures in 0, 3, 2, and 0 out of 8 rats, respectively. An ED50 was not identified due to the inverted U-shaped dose response curve. TRV045 protected mice against chronic secondarily generalized focal seizures in the CK mouse model. In the initial time-course screen, 10 mg/kg TRV045 PO protected 5 out of 8 mice at both 1 and 2 hr postdose, while 15 mg/kg protected 6 out of 8 mice at 1 hr and 4 out of 8 mice at 2 hr postdose, with average seizure scores ranging from 1.25 to 2.5. In the full dose-response study with testing at the TPE 1 hr postdose, 1, 2.5, 10, 15 and 20 mg/kg TRV045 PO protected 0, 4, 5, 6 and 6 out of 8 animals, respectively. TRV045 produced a dose-dependent effect on seizure scores, ranging from 2.5 (2.5 mg/kg) to 1.25 (20 mg/kg), resulting in a calculated ED50 of 5.97 ± 0.47 mg/kg. TRV045 did not significantly reduce cumulative seizure burden in the TMEV model. Cumulative seizure burden was 88 ± 10% of vehicle in the 30 mg/kg group, with no effect on seizure freedom. Cumulative seizure burden was 90 ± 18% of vehicle in the 10 mg/kg group, with a nonsignificant increase in seizure freedom (7 out of 20 vs 4 out of 20 in the vehicle group). In the KA-SRS model, 10 mg/kg TRV045 IP significantly reduced mean seizure burden compared to baseline (3.1 ± 0.9 vs 13.4 ± 6.6; p < 0.05 Wilcoxon rank sum) but did not significantly improve seizure freedom (3 out of 12 protected vs 2 out of 12 vehicle-treated). A 15 mg/kg dose significantly reduced mean seizure burden compared to the vehicle-treated group (5.0 ± 4.0 vs 9.0 ± 2.7; p < 0.05) and significantly improved seizure freedom (6 out of 12 protected vs 1 out of 12 vehicle-treated; p < 0.05 Fisher’s exact test). Conclusions: The S1PR1 modulator TRV045 prevented generalized seizures in the CK mouse and rat MES models and reduced seizures in the KA-SRS chronic spontaneous seizure model, indicating that it is a viable candidate for human research in the treatment of epilepsy. Keywords: Epilepsy, S1P Receptor, Seizure, Preclinical Pharmacology, Pharmacotherapy Disclosure: Trevena, Inc.: Employee (Self) P411. Global Electrophysiological Excitatory to Inhibitory Imbalance in Hippocampus and Temporal Cortex in the Continuum of Alzheimer’s Disease Pietro Scaduto, William Russell, Agenor Limon* University of Texas Medical Branch of Galveston, Galveston, Texas, United States Background: Individuals at distinct stages of Alzheimer’s disease (AD) show abnormal electroencephalographic activity, which has been linked to network hyperexcitability and cognitive decline. However, whether pro excitatory changes at the synaptic level are observed in brain areas affected early in AD, and if they are emergent in mild cognitive impairment (MCI), is not clearly known. Equally important, it is not known whether global synaptic E/I imbalances correlate with the severity of cognitive impairment in AD. Methods: Using electrophysiology and proteomics of human synapses from the hippocampus and temporal cortex of control, mild cognitive impaired (MCI) and AD individuals we aimed to determine the global synaptic balance in hippocampus and temporal cortex at distinct stages of neuropathology. Electrophysiological synaptic E/I ratios in post-mortem samples from the temporal cortex of individuals with MCI (n = 6) or AD (n = 6) compared to non-demented controls (n = 6), and the hippocampus (MCI, n = 8; AD n = 11, CTRL = 8) were assessed by microtransplantation of synaptic membranes (MSM). Proteomics of synaptosomes from temporal cortex were analyzed in the context of their electrophysiological responses using Electrophysiologically-anchored Dataset Analysis (EDA). Results: We found that the higher the amplitude of GABA receptor currents the better the cognitive performance score. A similar association was observed for AMPARs currents. The electrophysiological E/I ratio was significantly higher in the TCx of AD subjects and was negatively associated with the MMSE in the TCx but not in the hippocampus. The synaptoproteome revealed the impact and directionality of protein alterations and neuropathology on the amplitude of synaptic receptors responses and cognitive MMSE scores. By using electrophysiologically anchored analysis of the synapto-proteome in the same individuals, we identified a group of proteins sustaining synaptic function and those related to synaptic toxicity. We also found an uncoupling between the function and expression of proteins for GABAergic signaling in the temporal cortex underling larger E/I and worse cognitive performance in this region Conclusions: These findings indicate that early shifts of the E/I balance contribute to the loss of cognitive capabilities in the continuum of AD symptomatology. Keywords: Excitation-Inhibition Balance, Alzheimer’s Disease, Postmortem Brain Tissue, Excitatory Synapses, Inhibitory Synaptic Transmission Disclosure: Nothing to disclose. P412. Familiarity and Identity Representations in Hippocampal Area CA2 Lara Boyle, Lorenzo Posani, Sarah Irfan, Steven Siegelbaum, Stefano Fusi Columbia University, New York, New York, United States Background: Memory is the basis of all complex social relationships. The hippocampus is critical for remembering interactions with, and information about, other individuals; in other words, it is critical for social recognition memory. Social memory consists of two related processes: the general sense of whether and to what extent one has previously encountered another individual (familiarity) and the specific recall of details or episodes that involve another individual (recollection). To date, the neural mechanisms underlying familiarity and recollection remain unclear, including whether familiarity and recollection depend on the same brain region. Prior studies in rodents have shown that the hippocampal CA2 subregion is of particular importance for social memory as assessed by social novelty detection. However, it is not known how familiarity and identity coding are represented within CA2 and relate to other variables known to be encoded in the hippocampus (space, time). Here, we investigated the neural mechanisms of familiarity and identity coding within hippocampal CA2, including consequences of different neural representation models for memory. Methods: We injected a Cre-dependent virus into the dorsal CA2 (dCA2) region of Amigo2-Cre male mice to express the genetically encoded calcium indicator GCaMP6f selectively in dCA2 pyramidal neurons. We implanted a grin lens above CA2 to measure calcium events in a large number of dCA2 pyramidal neurons in awake, behaving animals. We imaged behaving mice exposed to different combinations and placements of novel and familiar individuals to explore changes in neural activity during experiments with variable combinations of familiarity, identity, position, and time. These combinations included: novel and familiar individuals in two trials with position swapped (same identity across trials, n = 12), novel and familiar individuals in two trials with position swapped (different identity across trials, n = 5), two novel individuals (n = 11), and two familiar individuals (n = 11). We characterized CA2 neural population representations using a decoding approach (linear support vector machine). In each case, we used data across the two trials to determine the degree that social, spatial, and trial information could be decoded. We then employed a technique called cross condition generalization performance, which gives information about how variables interact in the neural code (the dimensionality of these representations), to determine the relationship (the degree of entanglement) between different variables (familiarity, identity, space, and time) in the neural code. Results: We found that the degree of familiarity of interacting partners and their locations can be decoded from dCA2 population activity (social decoding performance = 0.88; chance = 0.49 ± 0.02, p < 0.001; spatial decoding performance = 0.90; chance = 0.51 ± 0.02; p < 0.001). Trial decoding was lower than social or spatial decoding (trial decoding performance = 0.62; chance = 0.54 ± 0.02; p < 0.001). Trial decoding also informs the dimensionality of social and spatial coding, with low trial decoding suggesting low dimensionality, and high trial decoding supporting high dimensionality. We calculated the cross-condition generalization performance (CCGP), another marker of dimensionality. CCGP is inversely proportional to the dimensionality of represented variables. We found that familiarity and spatial CCGP were significantly above chance (CCGP = 0.77, chance level = 0.51 ± 0.03 (mean ± SD); p < 0.001; spatial CCGP = 0.76; chance level = 0.49 ± 0.03, p < 0.001). We then examined CCGP and trial decoding for equally novel and familiar individuals. We found that while novel individuals were represented with low dimensionality, with high CCGP and low trial decoding (Novel identity CCGP = 0.77; chance = 0.50 ± 0.04; p < 0.001; Trial decoding = 0.62; chance = 0.49 ± 0.02, p < 0.001), familiar individuals were represented in higher dimensions, with relatively low CCGP and high trial decoding (Familiar identity CCGP = 0.62; chance = 0.50 ± 0.04; p < 0.001; Trial decoding = 0.69; chance = 0.50 ± 0.032; p < 0.001). We generated a geometrical model that re-capitulated these experimental findings. In addition, we developed a theoretical argument that captures the advantages of higher dimensional representations for recollection memory. Conclusions: Our experiments and analyses demonstrate that dCA2 encodes both social familiarity and the social identity of individuals with similar degrees of novelty or familiarity, the latter being a key requisite of recollection. We find that familiarity is coded in a low dimensional space, meaning that as a variable this information is disentangled from other variables (identity, space, time). While novel identities are similarly encoded in a low dimensional framework, familiar identities in contrast are coded in higher dimensional representations, meaning that the variables of identity, space, and time are entangled. The distinct geometrical arrangements of familiar and novel animal representations provide a neural mechanism for how the brain may rapidly distinguish social familiarity from social novelty while storing detailed information of social experiences with familiar animals, thus enabling the same brain structure to encode the two key components of social recognition memory: social familiarity and social recollection. Keywords: Dorsal Hippocampus, Social Recognition Memory, Recollection and Familiarity Disclosure: Nothing to disclose. P413. Correlating the Behavioral Sequelae of Blast Traumatic Brain Injury With Brain Structure and Function in Rats Moriah McGuier, Crystal Noller, Diana Wallin, Lucas Dwiel, Emily Sullivan, Patrick McCunn, Katelyn Salotto, Armando Braz, John Kanter, Krista DiSano, Paul Holtzheimer, Wilder Doucette Geisel School of Medicine, Lebanon, New Hampshire, United States Background: Nearly 69 million people worldwide are affected by traumatic brain injury (TBI). Military personnel are at high risk of experiencing TBI, especially shockwave-induced blast injury (bTBI), as results from proximity to explosive devices. bTBI increases the risk of developing posttraumatic stress disorder, mood and anxiety disorders, and substance use disorders. Current psychotherapeutic and pharmacologic treatments for these psychiatric consequences provide some symptom relief but may not address the underlying neural dysfunction. The use of pre-clinical models will allow us to identify neural correlates of blast and relate these biomarkers to emergent post-blast behavioral phenotypes. Through this understanding, we hope to guide the implementation of novel interventions (e.g., brain stimulation) to promote recovery from the chronic neuropsychiatric consequences of bTBI. We hypothesize that bTBI induced disruption of communication in the frontal-striatal-limbic system (using measures with translational relevance [e.g., MRI and electrophysiology]) will identify biomarkers of blast injury that relate to behavioral phenotypes. Methods: 55 male Sprague-Dawley rats were exposed to a blast overpressure injury using a blast tube (ORA, Inc) or were naïve controls. We titrated blast parameters, including varying overpressure targets and interblast intervals, until significant changes in delay discounting task performance were observed (n = 15). We then used these parameters (three events ∼126kPa [18 psi] with a ~3 min interblast interval [IBI]) on a large cohort (n = 40) and assessed an array of behaviors starting one month post-blast: 1) open field test with automated behavioral scoring; 2) sucrose preference; 3) Morris Water Maze (MWM) and 4) context fear conditioning. Following behavioral assessment, a subset of rats (12 bTBI and 12 control) were implanted with electrode arrays targeting the bilateral infralimbic cortex, orbitofrontal cortex, nucleus accumbens (NAc) core and NAc shell for LFP recording. We quantified 216 LFP features (power and coherence) across 6 established frequency ranges (delta through high gamma) and used machine learning to determine if blast and control LFP data could be classified more accurately than permuted datasets. Permute-and-relearn feature importance testing was then used to identify the most important LFP features. Another subset (8 bTBI and 8 control) underwent MRI for resting state functional (rs-fc) and diffusion tensor imaging (DTI) analyses. We used seed (medial frontal and NAc) based analyses to determine if significant connectivity differences existed between groups (blast versus control) and region of interest analyses corresponding to our electrode locations for DTI metrics (e.g., median diffusivity [MD]). Results: We found that none of the rats in the 24 hour IBI group had significant DDT performance changes from pre-blast to post-blast while roughly half of the rats in the 3 min IBI group made significantly more impulsive choices (paired t-test p < 0.05) following blast. No anxiety or movement related differences were detected from the open field metrics but the automated behavioral scoring revealed that blasted rats groomed themselves significantly less (unpaired t-test p = 0.0035). Blast injured rats demonstrated higher sucrose preference (two-way ANOVA p = 0.0182) than control rats. We did not detect any group differences in the average latency to the platform across training days (p = 0.1243) for MWM. Following conditioning to aversive foot shock, no group differences during the 3-minute post shock interval (p = 0.5106) were detected but blast injured rats exhibited significantly more freezing (two-way ANOVA p = 0.0458) when returned to the conditioned context 24 hours later. Machine learning models revealed that LFP features could classify animals as blast versus control with average model performance (area under the receiver operator characteristic curve [AUROC] = 0.7656), which gives p = 0.0348 when compared to the permuted distribution. Feature importance testing indicated reduced low and high gamma power in frontal regions compared to controls. Rs-fc and DTI analyses revealed an array of structural and functional differences between blast injured animals and controls. For example, rs-fc analysis revealed the NAc seed had reduced connectivity to frontal regions and DTI analysis suggests (p = 0.02) decreased MD in frontal brain regions of blast injured animals. Conclusions: Our data aligns with published work showing that blast injury in rodents is associated with a myriad of neurobehavioral consequences, including increased impulsivity and fear. Importantly, we found distinct neural signatures in injured animals that relate to the observed behavioral deficits, supporting a potential role for neural biomarkers in guiding rehabilitation post-injury. Future work will determine how these biomarkers predict recovery and response to treatment, including focal brain stimulation. Keywords: Traumatic Brain Injury, Neuronal Oscillations, Functional and Structural MRI, Delay Discounting, Contextual Fear Disclosure: Nothing to disclose. P414. Activation of Astrocytes in the Paraventricular Nucleus of Thalamus Modulates Social Behaviors Seungwoo Kang* Augusta University Medical College of Georgia, Augusta, Georgia, United States Background: Social behaviors are critical for animals to sustain healthy living patterns. Consequently, deficit or dysfunctional social behaviors are correlated with multiple psychiatric and neurological disorders. The paraventricular nucleus of thalamus (PVT) has been considered a hub brain area controlling context-dependent salience and aversion processing including the social behaviors. Indeed, recent studies suggest that the PVT presents a variety of neural signals according to social condition and the activities are significantly correlated with social behaviors. Accumulating evidence demonstrate that astrocytes have not only the microstructural supporting role in neural circuit also physically form tripartite synapses with adjacent neurons and directly modulate the neuronal activities via release of gliotransmitters or uptake overflowed neurotransmitters. However, it remains unknown the coordinated activities of astrocytes and neurons in the PVT encode social behaviors. Methods: To investigate the astrocyte activity-driven changes in the adjacent neuronal activities and behavioral consequences, we chemogenetically activated the astrocytes in the PVT using GFAP promoter-driven expression of hM3Dq, the excitatory designer receptors exclusively activated by designer drugs (DREADDs). First, we used ex vivo and in vivo calcium imaging to examine chemogenetically induced cellular activity in the PVT astrocytes of GFAP-GCaMP6s calcium fluorescence indicator-expressing mice. Then, we recorded electrophysiological changes in the firing and synaptic activity of the PVT neurons. To evaluate the behavioral consequences, we exposed mice in three-chamber social approach tasks. The mice were exposed to the chamber with three different social conditions: without any social factors for habituation and evaluation of place preference, with a strange mouse, or with a strange mouse and a familiar mouse. Social preference was measured as the comparison between the time the experimental animal spent with a stranger mouse and a novel object (sociability) or a familiar mouse (social recognition). Results: Chemogenetic activation of the PVT astrocytes increased the intensity and frequency of calcium signaling. Interestingly, the astrocyte activation enhanced the firing and the evoked excitatory postsynaptic currents (eEPSCs) in the neurons of the PVT. The changes in eEPSCs induced by the astrocyte activation were significantly inhibited by astrocyte-specific deletion of a glutamate transporter, GLT1 (as known as EAAT2, slc1a2) via the expression of GFAP-promoter driven Cre recombinase in the PVT of GLT1 floxed mouse. The changes in eEPSCs were also occluded by the pretreatment of DHK, a GLT1-specific blocker, suggesting that GLT1 is, at least partly, required for astrocyte-induced changes in the neuronal activity in the PVT. In social behavioral evaluation, we found that the astrocyte activation significantly reduced the social recognition in the three-chamber social approach test. Importantly, mice lacking GLT1 selectively in the PVT astrocytes mimicked the astrocyte activation-induced impairment in social behaviors. Conclusions: Together, our observation indicates that the astrocyte activity in the PVT modulates the adjacent neuronal synaptic events via not only the homeostatic passive activities but the direct modulation of the glutamatergic signaling, at least partly, through the GLT1, leading to shape social behaviors. This implies the importance of astrocytes as another layer to modulate social behaviors and the interaction between astrocytes and neurons via glutamatergic signaling could be a potential therapeutic target for related disorders. Keywords: Social Behavior, Astrocyte, Glutamate Transporter, Paraventricular Nucleus of the Thalamus Disclosure: Nothing to disclose. P415. Cognitive Control Predicts Alleviation of OCD Symptoms by Ketamine Peter van Roessel, Sepehr Asgari, Booil Jo, Leanne Williams, Carolyn Rodriguez Stanford University School of Medicine, Stanford, California, United States Background: Obsessive-compulsive disorder (OCD) is a common and impairing illness. First-line pharmacologic treatment with serotonin reuptake inhibitors helps most patients, yet benefit may take weeks to accrue, and many patients don’t achieve adequate response. Ketamine and other glutamatergic interventions offer great promise as alternative and more rapidly acting treatments. The growing diversity of treatment options, however, increases the need for data to support goals of precision medicine, matching the right treatment to the right patient. OCD has been associated with alterations in both cognitive and affective processing, assessed using both neuroimaging and behavioral tests. These alterations include deficits in cognitive control and a valence bias reflected in increased threat responsivity and decreased reward responsivity. In secondary analysis of data from a randomized, active-placebo-controlled trial of intravenous ketamine for treatment of OCD, we explored whether validated behavioral measures of cognitive control and valence processing have utility as predictive biomarkers or as clinically meaningful targets of treatment. Methods: Data were analyzed from N = 45 unmedicated individuals with DSM-5 obsessive-compulsive disorder who participated in a randomized controlled trial exploring the mechanisms of ketamine as a rapid-acting treatment. Participants were randomized 2:1 to receive a single 40 min intravenous infusion of ketamine (0.5 mg/kg) or midazolam (0.045 mg/kg). Clinical OCD symptoms were assessed using the Yale-Brown Obsessive Compulsive Scale (Y-BOCS) at baseline, at post-infusion Day 1, and at post-infusion Day 7. Participants additionally completed a validated, computer-based neurocognitive test battery (‘WebNeuro’) one day prior to and one day after infusion. Testing included measures of cognitive control (performance on Stroop color-word interference and Go/No-Go tests) and of valence processing (performance on tests of emotion identification and of implicit priming by facial expressions of fear and happiness), quantified as z-scores relative to age-, sex-, and years of education-matched healthy norms. We explored whether baseline performance on cognitive control and valence processing tests moderate symptom alleviation in response to ketamine vs midazolam, and whether pre-post change in test performance is associated with symptom alleviation. MacArthur criteria were used to define potential moderator and mediator variables. Results: Treatment had a significant effect on OCD symptoms at Day 1 (β = -7.894, p = .004, representing a nearly 8 point mean difference in Y-BOCS change for ketamine vs midazolam) and at Day 7 (β = -6.048, p = .005). In moderation analyses, the interaction between a measure of Stroop interference by response speed and treatment had a significant effect on the change in Y-BOCS at Day 1 (β = -4.0618, Cohen’s f2 = 0.14, p = .04), but not at Day 7. That is, the effect of treatment at Day 1 was considerably larger for those with faster normalized performance on a color-naming relative to color word-reading task. Performance on the Go/No-Go and valence processing tests did not moderate the treatment effect on outcomes at Day 1 or Day 7. In mediation analyses, treatment did not have a significant effect on pre-post change in cognitive control or valence processing; these measures did not fulfill criteria as potential mediators of Y-BOCS change. Pre-post change in Stroop interference by response speed did, however, correlate with change in Y-BOCS at Day 1 (r = 0.36, p = .03). Conclusions: Ketamine robustly reduced symptoms of OCD at Day 1 and Day 7 post-infusion, compared to the active-placebo midazolam. Our data suggest that the short-term effect of ketamine vs midazolam on Y-BOCS may be influenced by baseline inhibitory cognitive control, such that greater control (less Stroop interference for color-naming) predicted greater benefit from ketamine vs midazolam. Our data may accord with published findings suggesting that Stroop task performance moderates the effectiveness of other OCD treatment modalities (e.g., cognitive behavioral therapy vs fluoxetine). Keywords: Obsessive-Compulsive Disorder, Ketamine, Neurocognitive Assessment, Moderators, RCT Disclosure: Nothing to disclose. P416. Efficacy of Ketamine in Unmedicated Adults With Obsessive-Compulsive Disorder: A Randomized Controlled Trial Carolyn Rodriguez, Chi-Ming Chen, Gary Glover, Booil Jo, Daniel Spielman, Leanne Williams, Peter van Roessel, Charles DeBattista, Pamela Flood, Alan Ringold, Max Wintermark, Kelley Anderson, Omer Linkovski, Anthony Lombardi, Andrea Millen, Anthony Pinto, Hannah Raila, Keara Valentine, Maria Filippou-Frye, Jessica Hawkins, Elizabeth McCarthy, Pavithra Mukunda, Andrea Varias, Jordan Wilson, Brianna Wright Stanford University, Stanford, California, United States Background: Developing novel, robust Obsessive Compulsive Disorder (OCD) treatments is an urgent public health need, given OCD typically starts in childhood, leads to lifelong morbidity, and costs the economy $2.1 billion (direct costs) and $6.2 billion (indirect costs such as lost productivity) annually. OCD is characterized by an inability to inhibit intrusive thoughts (obsessions) and repetitive behaviors (compulsions). Serotonin reuptake inhibitor (SRI) treatment of OCD exhibits a long lag time (2-3 months) before clinical benefit, and this benefit is typically only partial. Identifying effective, fast-acting treatments will help reduce OCD morbidity and its life effects. We previously reported the rapid OCD symptom reduction of ketamine, a glutamate N-methyl-d-aspartate (NMDA) receptor antagonist, versus saline infusions in a proof-of-concept crossover trial (n = 15) in unmedicated adults with OCD. Building on this initial finding, we evaluated the efficacy of ketamine in a larger group of unmedicated OCD patients with improved control conditions (active placebo control condition). Methods: This was a randomized controlled trial of a single infusion of ketamine compared to an active placebo condition (midazolam, an anesthetic). With institutional review board approval, unmedicated adult patients (age 18-65) with OCD were randomly assigned under double-blind conditions to receive a single intravenous infusion of ketamine (0.5 mg/kg) or midazolam (0.045 mg/kg) in a 2:1 ratio (total n = 45). Participants met DSM-5 criteria for OCD with at least moderate symptoms (Yale-Brown Obsessive Compulsive Rating Scale score of at least 16). Exclusion criteria included severe depression (17-item Hamilton Depression Rating Scale was less or equal to 18 to enter the study) and comorbid psychiatric or medical conditions that made participation unsafe. The primary outcome was change in OCD severity 1 week after drug administration, as assessed by the Y-BOCS. Duration of effect was explored with weekly Y-BOCS up to 4 weeks post-infusion. We focused on estimating intention to treat effects based on longitudinal mixed effects modeling. For both moderator and mediator investigation, we employed the MacArthur approach embedded in mixed effects modeling. Results: Regarding the primary outcome, the ketamine group had significantly greater improvement in Y-BOCS score than the midazolam group 1 week after treatment (Cohen’s d = 1.25, p < 0.001). The effects from a single intravenous infusion of ketamine persist up to 3 weeks post-infusion (Cohen’s d = 0.59, p = 0.007), gradually reducing each week and then becoming insignificant by Week 4. We examined age, sex, and race as potential moderators of treatment effects, although none were identified as significant moderators. We also examined change in dissociation as a potential mediator of treatment effect, although it did not turn out to be a significant mediator. Conclusions: To our knowledge, this is the largest clinical trial to date of ketamine in unmedicated OCD patients. Ketamine demonstrated rapid and durable OCD symptom improvement compared to the active control condition. By using an optimized active placebo design to control for nonspecific anesthetic effects, this study provides new supporting evidence for the specific OCD therapeutic effects of ketamine. Keywords: Ketamine, OCD, Midazolam Disclosure: American Psychiatric Association Publishing: Other Financial or Material Support (Self), Biohaven Pharmaceuticals, Osmind: Consultant (Self), Biohaven Pharmaceuticals: Contracted Research (Self) P417. Investigating the Role of Medial Orbitofrontal Cortex (mOFC) in Deterministic and Probabilistic Negative Reinforcement Brittany Chamberlain, Matthew Geramita, Susanne Ahmari University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Obsessive compulsive disorder (OCD) is a chronic, severe psychiatric illness that is characterized by obsessions – recurrent intrusive thoughts and images – and compulsions – repetitive behaviors performed to relieve anxiety associated with obsessive thoughts. Negative reinforcement-based theories of OCD suggest that the manifestation and maintenance of compulsions may be driven by this temporary relief from obsession-evoked anxiety. A recent clinical study by our group (Panny et al., under review, Biological Psychiatry) found heightened activity in the medial orbitofrontal cortex (mOFC) when OCD patients removed compulsion-related images in a novel negative reinforcement paradigm. To further dissect the cellular responses contributing to these bulk regional changes in mOFC activity, we measured neural activity via single-photon calcium imaging in wild-type mice during a negative reinforcement task in this exploratory study. Methods: C57Bl6/J mice (n = 6; 4 male, 2 female) were trained on a novel negative reinforcement task. During the task, a light cue predicted a foot shock, and mice could avoid shocks with 100% probability by pressing a lever within the 20 sec cue period (“avoid response”). If mice did not press the lever during the cue period, a series of shocks commenced. Mice could escape remaining foot shocks by lever pressing during this period (“escape response”). Mice performed 50 trials per day for 7 days. This was followed by 5 days of probabilistic reinforcement of responses, in which lever pressing led to shock avoidance on 50% of trials. Prior to training, mice were injected with virus encoding GCaMP6f (AAV5-CaMKII-GCaMP6f-WPRE-SV40, titer 2.2×10^12) into mOFC and implanted with gradient-index (GRIN) lenses to visualize mOFC activity using miniature microscopes (Inscopix). Single-cell calcium activity was extracted using CNMF-e algorithm, converted to ∆F/F, and time-locked to several timepoints within each trial (i.e. presentation of avoidance cue, lever extension, lever press, shock onset). Calcium transients aligned to behavioral events of interest that exceeded >1 SD from null distribution were considered significant. Results: Mice quickly learned to avoid foot shocks, showing significantly more avoidance responses (59.3%) than either escape responses (29.3%) or trial failures (11.3%) beginning on the first day of training (avoid vs. escape: p < .05; avoid vs. failure: p < .005). By day 7 of training, mice avoided 99.8% of potential shocks, performing an avoid response on 97.3% of trials. Calcium imaging on day 1 of training demonstrated that 13.3% of neurons (69/518) were activated at onset of lever pressing to initiate an avoidance response; this effect was maintained over time, with 15.3% of neurons (107/699) activated at onset of avoidance responses on day 7. Ongoing analyses of cells tracked over days will determine whether activity of individual mOFC neurons changes as 1) avoidance behaviors become well-learned and maintained over time and 2) after changing from deterministic to probabilistic negative reinforcement. Conclusions: In OCD, compulsive behaviors are reinforced by the temporary relief provided from anxiety brought on by intrusive thoughts and images. Preliminary findings from our lab show that patients with OCD display heightened activity in mOFC in response to removal of compulsion-related images, suggesting this may be a key locus of negative reinforcement in OCD. Here, we developed a novel task which leads to rapid acquisition of active avoidance responses in mice and coupled it with single-photon in vivo calcium imaging in mOFC. Preliminary analyses show that a subset of neurons in mOFC are selectively responsive during engagement in an active avoidance behavior. Further analyses will establish how mOFC responses are modulated throughout negative reinforcement training and following changes in task contingencies. Keywords: Active Avoidance, Negative Reinforcement, Medial Orbitofrontal Cortex, Obsessive-Compulsive Disorder (OCD), In Vivo Calcium Imaging Disclosure: Nothing to disclose. P418. Precision Functional Mapping in Obsessive-Compulsive Disorder Using Dense Sampling Scanning Matilde Vaghi, Sunjae Shim, Jaime Ali Rios, Patrick Bissett, Pavithra Mukunda, Carolyn Rodriguez, Russell Poldrack Stanford University, Stanford, California, United States Background: One of the roadblocks in translating imaging research findings into clinical practice biomarkers is represented by shortcomings of traditional approaches. These generally entail analysis of limited data from each subject. Accordingly, inferences are based on cross-subjects averages which might obscure patterns of brain organization specific to each individual. Additionally, by focusing on an individual time point there is limited understanding of whether observed effects are stable over time or state dependent. Here, we used a recently pioneered ‘dense sampling’ scanning acquisition where imaging data are collected on the same subject repeatedly over time, resulting in more data per individual. This experimental design enables conclusions to be drawn at the individual level and to capture brain fluctuations in the intermediate timescale of weeks to months, which might bear relevance to fluctuations in symptoms. We examined magnitude and anatomical distributions of network variability across subjects and sessions from seven high-quality, highly sampled patients with Obsessive Compulsive Disorder (OCD). Imaging data were collected while subjects performed cognitive tasks as well as during resting-state, but results presented here pertain to resting-state only. Methods: Data were collected from 7 patients with a diagnosis of OCD (4 males; age: 22-48). For each subject, data were acquired on 10 sessions, each on a separate day, approximately one week apart. To minimize within-subject variability due to external factors, scans were performed at fixed times of the day, with rare variations due to participant or scanner’s scheduling constraints. Each session entailed psychiatric evaluation as well as functional MRI. Overall, this resulted in approximately 6 h (3 h resting-state) of functional imaging per subject. MRI data were acquired on a research-dedicated GE 3 T MRI scanner using a 32-channel head coil. All functional imaging scans were performed using a multi-echo, multi-band, gradient-echo EPI sequence. Additionally, a T1 and a T2-weighted image were obtained for each subject. Imaging data were pre-processed via fMRIPrep 22.0.0 and denoised with Tedana. Functional connectivity (FC) was measured via time-series correlations among cortical regions parcellated using the Yeo’s 17 networks template. Accordingly, a FC connectivity matrix was obtained for each subject and session. Effects were calculated per subject and were compared with one another using paired two-tailed t tests, with p values FDR corrected for the number of comparisons. Results: Group and individual effects on network similarity were investigated by calculating correlations among FC connectivity matrixes. To quantify group effects, correlations between FC connectivity matrixes from different individuals and sessions were inspected. This analysis indicated a shared common basic structure, as functional networks from different individuals and sessions showed substantial similarity (mean Spearman’s correlation = 0.76). However, networks from the same individual and different sessions were even more like each other, with an additional effect of mean Spearman’s correlation = 0.86 over the group effect, demonstrating a large influence of individual identity on functional networks. We also investigated within-subject variability across sessions by computing, for each subject, the standard deviation of the correlation between each parcel-pair across all 10 sessions. Average variability across all correlations showed a pattern of relatively larger variability in visual, somato-motor and dorsal attentional regions, which is in line with analogous studies in healthy subjects. However, in contrast to studies in healthy subjects, variability in these networks was not significantly different from the one observed in executive control areas, which have been traditionally implicated in OCD. Conclusions: We find that, in patients with OCD, functional networks are dominated by common organizational principles as well as prominent individual features. These results mirror previous investigations in healthy subjects and extend it to patients with OCD highlighting the importance of individualized approaches for studying properties of brain organization. In contrast to previous reports in healthy subjects, variability was similar in processing and executive control regions, setting the stage for relating longitudinal dynamics of brain functions to behavioral and psychiatric symptoms variability. Keywords: Obsessive-Compulsive Disorder (OCD), Precision Psychiatry, Functional Connectivity Disclosure: Nothing to disclose. P419. Resting-State Functional Connectivity Signatures of Obsessive-Compulsive Symptom Profiles Tracey Shi, Seonjoo Lee, Nikolaus Kriegeskorte, David Pagliaccio, H. Blair Simpson, Rachel Marsh Columbia University, New York, New York, United States Background: Obsessive-compulsive disorder (OCD) is a disabling illness that exhibits bimodal timing of onset, with up to half of cases beginning in childhood. Pediatric subclinical obsessive compulsive (OC) symptoms often increase risk for the development of OCD, but the neural bases and longitudinal trajectory of OC symptoms remain poorly characterized. Capitalizing on the Adolescent Brain Cognitive Development (ABCD) study to perform a well-powered whole-brain analysis, herein we identify “profiles,” or groups of OC symptoms with similar neural underpinnings and characterize resting state functional connectivity (rsFC) patterns that correlate with these symptom profiles across the severity spectrum. Methods: ABCD data was divided into matched training and test subsamples. Participants completed rsFC scans, CBCL, and KSADS. Eight individual items from the CBCL (CBCL-OC Symptom scale) were used as clinical data for the primary analyses. RsFC data were preprocessed, and connectivity matrices were calculated by the DCAN Lab. Participants with at least 10 minutes of motion-free data were included in analyses (training: n = 2,846; ages 8-11; 1,495 female; test: held out from current work). Using only the training subsample, age, sex, race, and head motion were regressed out of rsFC and clinical data (“features”). 501 rsFC features were selected for elastic net canonical correlation analysis (eCCA) based on stable correlations with clinical features across 100 subsamples. ECCA hyperparameters were optimized using cross-validation, eCCA was performed, and significance was assessed via permutation testing. For post-hoc validation, the canonical variates were t-tested for group differences across participants with and without KSADS-diagnosed OCD. Results: ECCA revealed 8 significant pairs of canonical variates (“profiles”). Canonical variate 1 (r = .47, p = .01) represented a clinical profile with both obsessions and compulsions, in addition to general symptoms like worries and fears. RsFC dysconnectivity centered around the dorsolateral prefrontal cortex (dlPFC), with symptoms associated with more positive connectivity between task-positive and negative regions and weaker positive connectivity within the task-negative default mode network. Canonical variate 2 (r = .39, p = .01) corresponded to obsessions, perfectionism, and worries, but not compulsions. This profile was associated with rsFC features involving motor and sensory areas, including weaker negative connectivity with the insula and weaker positive connectivity within sensory and motor networks. The 3rd (r = .33, p = .01) profile focused on perfectionism and fear of doing something bad, specifically without obsessions. As with the first profile, the rsFCs centered around the dlPFC. However, the connections were in the opposite direction: stronger symptoms in this profile were associated with less positive connectivity between task-positive and negative regions and stronger positive or weaker negative connectivity within the default mode network. Profile 4 (r = .36, p = .01) was predominately compulsions without general strange behavior. This was most associated with connectivity involving the frontal eye field in the frontoparietal network, including more positive connectivity with task-negative regions and more negative or weaker positive connectivity with task-positive regions. In post-hoc analyses, the rsFC patterns for the first 3 profiles significantly differed between participants with and without KSADS-diagnosed OCD (p < .03 after Bonferroni correction). Specifically, OCD diagnoses were associated with a rsFC pattern indicating higher OC symptoms according to CBCL scores. Profiles 5-8 were statistically significant (r ~ .30, p < .02) but not discussed due to space limitations. Conclusions: We identified 8 significant “profiles,” or sets of OC symptoms and associated patterns of rsFC dysconnectivity. The first 3, derived from quantitative relationships with subclinical to clinical OC symptoms, had clinical validity in that they distinguished children with OCD diagnoses from those without. One possible interpretation of these findings is that profile 1 may represent a more traditional presentation of OC symptoms including both obsessions and compulsions, while profiles 2-4 represent atypical presentations and/or related disorders such as obsessions-predominant, general fears or anxiety, and compulsions-predominant, respectively. The distinct rsFC patterns associated with each clinical profile suggests different underlying network disturbances giving rise to different symptoms, possibly denoting heterogeneity that could predict longitudinal symptom course or responsiveness to interventions or treatment. The rsFC patterns for profiles 1 and 3 center around the dlPFC, a target for transcranial magnetic stimulation for OCD due to its rich connectivity with the cortico-striatal loops that are altered in OCD. Furthermore, rsFC patterns detected herein corroborate prior work showing that an altered balance between task-positive and negative networks may be central to OCD pathophysiology. Future work will include publicly preregistering the findings from this training subsample, assessing replication of the canonical variates in the matched test subsample (held out from present analyses), and evaluating whether these profiles predict longitudinal progression of OC symptoms or response to OCD treatment in a separate sample of participants. Keywords: Resting State fMRI, Obsessive Compulsive Symptoms, Obsessive Compulsive Disorder Disclosure: Nothing to disclose. P420. Imaging Transcriptomics in Obsessive-Compulsive Disorder Leonardo Saraiva, João Sato, Roseli Shavitt, Euripedes Miguel, Carolina Cappi Institute of Psychiatry of the Sao Paulo University Medical School, Sao Paulo, Brazil Background: Robust genetic and neuroimaging studies have implicated genetic and neural alterations, respectively, in OCD. However, few studies have performed investigations integrating genetics and neuroimaging in the disorder. In this respect, imaging transcriptomics is a pioneering field that explores gene expression signatures underlying imaging-derived phenotypes. This association is performed by using the neurotypical brain-wide gene expression data from the Allen Human Brain Atlas (AHBA), which was mapped in the stereotaxic space. The morphometric similarity network (MSN) is a recently proposed methodology that captures anatomical connectivity between cortical regions with similar cytoarchitecture and gene expression profiles. Imaging transcriptomics studies uncovering genetic and molecular factors underlying abnormalities in MSN characteristics have been performed in major depression, schizophrenia, and neurodevelopmental disorders, but not in OCD. Therefore, this study is the first imaging transcriptomics study using the MSN in OCD. Its central aim is to probe the gene expression signatures, and its biological correlates, underlying neural abnormalities associated with the disorder. Methods: T1-weighted MRIs of 116 OCD cases and 74 healthy controls were used in this study. These MRIs were processed using the Freesurfer standard automated processing pipeline. A cortical parcellation subdividing the Desikan-Killiany atlas into 308 regions of approximately equal surface area was computed in the native surfaces of the MRIs. Intra-individual MSNs were computed by calculating pairwise Pearson correlations coefficients between vectors of z-scored MRI features (surface area, gray matter volume, cortical thickness, intrinsic curvature and mean curvature) for each pair of cortical regions. Intra-individual regional morphometric similarity (MS) measures were computed for each region by averaging its correlation coefficients with all other regions. OCD case-control differences in regional MS were computed by using linear regression, with sex and age as covariates. Regional brain gene expression data from the AHBA were processed using the abagen toolbox. Partial least squares (PLS) regression was performed to assess the contribution of regional brain gene expression, as measured by the AHBA, to the observed OCD case-control differences in regional MS for the left hemisphere. It should be noted that only the left hemisphere was included in this analysis since the right hemisphere is undersampled in the AHBA dataset. The PLS first component (PLS1) thus represents the linear combination of weighted brain gene expression measures whose spatial distribution mostly contributes to the spatial distribution of observed OCD case-control differences in regional MS for the left hemisphere. Permutation testing was used to assess the statistical significance of the variance explained by the PLS1. Bootstrapping was used for the estimation of z-scores for the PLS1 gene weights. Genes with significant positive and negative contributions to the PLS1 were defined as having z-scores > 3 (PLS1 + ) and < -3 (PLS1-), respectively. The R package gProfileR was used to perform enrichment analysis for the PLS1 + and PLS1- genes. Finally, a OCD case-control differential gene expression (DGE) analysis was performed in RNA-sequenced peripheral blood samples of 19 OCD cases and 19 healthy controls. Pairwise Spearman correlation coefficients were computed between the PLS1 + and PLS1- genes z-scores and their respective logarithms of folds changes (logFC) obtained in DGE analyses. Results: Statistically significant (not corrected for multiple testing) OCD case-control differences in regional MS were found for cortical regions in both hemispheres. In the left hemisphere, these regions include frontal lobe regions (parsopercularis [part1], rostralmiddlefrontal [part4], and precuneus [part6] cortices) and parietal lobe regions (superiorparietal [part5], superiorparietal [part9], and supramarginal [part2] cortices). In the right hemisphere, these regions include frontal lobe regions (fusiform [part4], and inferiorparietal [part3] cortices) and parietal lobe regions (postcentral [part7], and superiorparietal [part1] cortices). The PLS1 explained 16.22% of the variance in the spatial distribution of the observed OCD case-control differences in regional MS, which was statistically significant (p = 0.035) in permutation testing. After the estimation of z-scores for the PLS1 gene weights by bootstrapping, 461 PLS1 + genes and 472 PLS1- genes were identified. The PLS1 + genes were mostly enriched for cerebral cortex in the Human Protein Atlas (HPA) database and for regulation of cellular process in the Gene Ontology Biological Process (GO:BP) database. The PLS1 + genes were mostly enriched for cerebral cortex in the HPA database and for generation of precursor metabolites and energy in the GO:BP database. Finally, the z-scores of the PLS1 + and PLS1- genes were negatively correlated (rho = -0.15 and -0.042, respectively) with their respective logFC obtained in the OCD case-control DGE analysis in peripheral blood. Only the PLS1 + genes correlation was statistically significant (p = 0.007). Conclusions: This study provide compelling evidence that regional brain gene expression profiles contribute to neural abnormalities associated with OCD. Moreover, this study suggests that these brain gene expression profiles are reproduced in peripheral blood. Keywords: Gene Expression, Imaging-Genetics, Obsessive-Compulsive and Related Disorders, Transcriptomics, Brain Transcription Disclosure: Nothing to disclose. P421. Recent Advances in the Genetic Architecture of OCD: Genetic Maternal Effect, Ultrarare, Rare, and Common Variation Contribute to Risk Behrang Mahjani, Seulgi Jung, Carolina Cappi, Marina Natividad Avila, Lily Cohen, NORDiC Consortium, EGOS Consortium, James Crowley, Kathryn Roeder, Bernie Devlin, Joseph Buxbaum, Dorothy Grice Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Obsessive-compulsive disorder (OCD) is a multifactorial disorder, meaning that the risk of OCD is influenced by multiple genetic and other factors. The heritability of OCD, historically estimated by the analysis of twin and family studies, is reported to be 35-50%. Heritability can also be estimated from individuals drawn from a population who have no obvious familial relationships, as long as they have been characterized for genetic variation across their genomes. Usually, this genetic characterization employs genotypes of single nucleotide polymorphisms (SNPs) for which alleles are common in the population. When the heritability of OCD is computed in this manner, estimates range from 25-43%, which mostly arise from covariance due to common genetic variants. Among the environmental factors, different studies have linked maternal conditions before and during pregnancy to the risk of OCD. Such factors could represent maternal effect. Maternal effect is defined as a causal effect of the mother’s phenotype on the phenotype of the offspring, and it can be partitioned into genetic maternal effect (maternal genetic nurture) and environmental maternal effect. Genetic maternal effect occurs when the genotype impacting the phenotype of the mother influences the phenotype of the offspring, usually independent of the child genotype. Environmental maternal effect occurs when the environment impacting the phenotype of the mother (independent of genotype) influences the phenotype of the offspring. Despite these findings, the contribution of inherited genetic variation across the allelic frequency spectrum and role of maternal effects in the risk of OCD remains uncertain and worthy of further study, as it impacts both our understanding of processes underlying OCD risk architecture and rational study design. In our studies, we partitioned the risk architecture of OCD by a common set of latent genetic and environmental factors (e.g., maternal effects). In addition, we identified rare potentially damaging structural variations (e.g., potentially damaging copy number variation; pdCNV) and significant OCD risk genes impacted by rare variation. Methods: To study the genetic risk architecture of OCD, we studied family data for 822,843 individuals in Sweden [7,184 (0.9%) with OCD], as well as SNP data from a sample of 2,090 Swedish-born individuals diagnosed with OCD and 4,567 controls. Using genotypes of these SNPs to estimate distant familial relationships among individuals, we estimated heritability of OCD, both overall and partitioned according to minor allele frequency (MAF) bins. We used a subsample of these data to evaluate the distribution of pdCNV in OCD, examining associations between pdCNV and the phenotypes of probands, including a consideration of early- vs. late-diagnoses. We used analytical approaches to estimate the number of genes that could increase risk for OCD through de novo mutations and used this estimate for power calculations for sequencing studies. In ongoing studies, these Swedish, and additional, samples are being sequenced for rare association studies. Results: Using family data, we observed that 35-40% of the liability for OCD is due to direct genetics (heritability), and 7-7.6% due to genetic maternal effect. We observed evidence for substantial assortative mating among individuals with OCD. Additionally, our results demonstrated associations between parental age and maternal smoking during pregnancy with risk of OCD. Using SNP data, we estimated narrow-sense SNP-heritability of 29% (SE = 4%). Contrary to earlier studies, however, SNPs with MAF between 0.01 and 0.05 accounted for 10% of heritability and estimated heritability per bin roughly follows expectations based on the infinitesimal model (where risk is influenced by a large number of loci distributed across the genome and across MAF bins). Nine percent of OCD probands carried a pdCNV, showing that 1 in 11 OCD cases may have a genetic diagnosis from CNV. The most frequent pdCNV found was at the 16p13.11 region. There was no significant difference in pdCNV frequency between early- vs. late-diagnosed OCD probands. Using published data, we estimate that deleterious mutations in about 200 distinct genes can increase risk for OCD, motivating high-throughput sequencing for gene discovery in OCD. Sequencing of 8000 samples is ongoing. Conclusions: Our results provide new insights into the risk architecture of OCD. We showed that maternal effect, combined with the effects of rare and common variants, shape a large portion of the risk architecture of OCD. We observed that genetic variation across a very broad allelic frequency spectrum influences the risk of OCD. Studies by other groups have shown that ultrarare de novo mutations are also part of OCD risk and we expect to observe this in our ongoing sequencing studies. Keywords: Obsessive Compulsive Disorder, Heritability, Genetics, Rare Genetic Variation, Copy Number Variants Disclosure: Nothing to disclose. P422. De Novo Mutations in Conserved Genes Contribute to OCD Risk Seulgi Jung, Carolina Cappi, Marina Natividad Avila, NORDIC Consortium, EGOS Consortium, Kathryn Roeder, James Crowley, Behrang Mahjani, Joseph Buxbaum, Dorothy Grice Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: OCD is a neurodevelopmental psychiatric disorder that affects ~3% of the population. For years, our understanding of the genetic architecture and risk factors for OCD has lagged compared to other major psychiatric disorders. However, as OCD genetic cohorts and sample sizes increase, we have the opportunity to expand our knowledge regarding OCD risk architecture. Early studies provide convincing evidence for the importance of genetic risk factors in OCD. In family studies, the odds of OCD in case versus control relatives were 4.6 - 5.0 in first-degree relatives, 1.5 – 2.3 in second-degree relatives and 1.4 in third-degree relatives. The heritability of OCD has been estimated at 27-56% in family-based studies and SNP-based heritability at 25-43%. While earlier studies suggested that inherited risk variation was skewed toward the most common variants, our group recently showed that variants across the allelic spectrum all contribute meaningfully to the heritability of OCD. Early and recent studies of copy number variation (CNV) supported a role for rare de novo mutations in OCD. Taken together, these findings suggest that additional inherited and rare variants contribute to risk for OCD. Indeed, whole-exome sequence (WES) studies using OCD cases support the role for rare single nucleotide variation (SNV) in risk for OCD, including de novo mutations. Cappi et al. observed that rates of de novo mutations, likely protein-truncating variation (PTV), are significantly elevated in OCD trios. Similarly, Halvorsen et al. observed a 1.3-fold increase in damaging de novo variants in OCD cases relative to controls, including a 2.6-fold increase in de novo PTV in constrained genes. To identify genes that increase risk for OCD when harboring rare deleterious mutations, we are sequencing 8000 samples, while integrating data from existing and emerging OCD WES studies. Currently, we are analyzing published and unpublished data from OCD cases, with parental or ancestry-matched controls. Methods: To date, we have new WES data including 160 cases from Mount Sinai and 480 Swedish OCD cases, together with over 2,800 ancestry-matched controls. We also analyzed previously published WES data. For calling SNV and indel variants from WES data, we used the current Genome Analysis Toolkit (GATK) protocol. We performed quality control steps, including checking the accuracy of the reported pedigree information (samples with a discrepancy are dropped) using Hail (an open-source framework for scalable genetic data analysis). Variants were annotated with the Variant Effect Predictor (VEP), prioritizing coding canonical transcripts. We then performed genetic analyses using the Transmitted and De novo Association (TADA) model for gene discovery. Results: All genes were ranked by P value, and constraint scores were noted, including constraint for PTV and missense variation in the TADA analysis. The top 10 genes were CHD8, INO80, SCUBE1, ZMYM2, ADIPOR2, EIF3G, TP53BP2, KMT2B, PITPNM2, and TRRAP. Three potentially novel OCD genes - EIF3G, KMT2B, and TRRAP, are already known neurodevelopmental genes. (1) EIF3G, with deleterious missense variation in Cappi et al. and in a Mount Sinai sample, is also a likely autism risk gene. (2) Cappi et al report a de novo PTV in KMT2B, and we observe 1 missense with MPC > 2 in a Swedish case. Interestingly, a de novo missense mutation in KMT2B was also observed in a Chinese case (Lin et al.). KMT2B codes for lysine methyltransferase 2B. Interstitial deletions or pathogenic mutations of KMT2B cause a dystonia syndrome, which onsets in the first or second decade of life and can include psychiatric comorbidities such as mild intellectual disability (ID), attention-deficit/hyperactivity disorder (ADHD), anxiety, and OCD. (3) TRRAP encodes a large multidomain protein of the phosphoinositide 3-kinase-related kinases family with de novo mutations reported in autism, ID, schizophrenia, and early-onset psychosis with OCD. Additionally, based on parameters such as de novo mutation rates, and relative risk ratios estimated from the data underlying these studies, we estimate that roughly 200 OCD genes can ultimately be discovered using these approaches. Conclusions: As seen in ID and autism, de novo mutations in OCD are strongly enriched in highly conserved genes. This represents less than 10% of all genes, when comparing individuals with OCD to individuals without a known psychiatric phenotype. Leveraging this, we are beginning to discover genes that increase risk for OCD, when these genes are impacted by rare deleterious variation. We are also seeing overlap with known ASD and ID risk genes, as well as interesting associations with known syndromal genes. Keywords: OCD, Genetics, Rare Variation Disclosure: Nothing to disclose. P423. Interaction of Excitatory Neuromodulation and Perceptual Modification of Self-Images in Body Dysmorphic Disorder Wan-wa Wong, Rangaprakash Deshpande, Reza Tadayon-Nejad, Joel Diaz, Andrew Leuchter, Gerhard Hellemann, Jamie Feusner* University of Toronto, Centre for Addiction and Mental Health, Toronto, Canada Background: Body dysmorphic disorder (BDD) is marked by preoccupation with misperceived appearance flaws, which they believe render them ugly and disfigured. Disturbances of visual information processing in BDD seem to be core neurobiological contributors to the psychopathological feature of perceptual appearance distortions. Previous neuroimaging studies have found abnormally reduced dorsal visual stream (DVS) activity in BDD when viewing filtered images that conveyed configural/holistic information, contributing to a model of imbalances in global vs. local visual processing. Some evidence has shown that magnocellular pathways in the DVS are tuned to rapid presentation of images, thereby allowing global/holistic, but not local/detailed, visual processing. Moreover, ventral visual stream (VVS) regions seem to reduce activation magnitude with higher stimulus frequency/shorter stimulus duration. Accordingly, we investigated if DVS/VVS systems in individuals with BDD could be altered during rapid face presentation. In addition, we tested if intermittent theta-burst stimulation (iTBS), a form of repetitive transcranial magnetic stimulation, could further enhance the effects of rapid face presentation, as quantified through dynamic effective connectivity (DEC) modeling. Methods: Fourteen unmedicated adults with BDD, were randomly assigned to receive active (n = 7) or sham (n = 7) iTBS. Stimulation targets were left and right lateral parietal cortices, (corresponding to CP3 and CP4 respectively, on the EEG 10-10 system). Stimulation was applied at 100% active motor threshold (AMT) for the active group and 10% AMT for sham. We then immediately acquired fMRI data from participants while they viewed photos of their own faces for short (125 ms, 250 ms, 500 ms) and long (3000 ms) durations. As an additional comparison, given the possibility of sham stimulation effects, the data were also compared to the data collected separately from 38 BDD participants and 29 healthy controls during an identical task of face viewing, but without iTBS beforehand. The BDD participants met DSM-5 criteria for BDD with face concerns. Fourteen regions of interest (ROIs) in DVS and VVS were selected: 2 ROIs in primary visual cortex (V1) [bilateral calcarine], 6 ROIs in VVS [bilateral inferior occipital gyrus (IOG), fusiform gyrus (FG), and inferior temporal gyrus (ITG)], and 6 ROIs in DVS [bilateral superior occipital gyrus (SOG), inferior parietal gyrus (IPG), and superior parietal gyrus (SPG)]. Hemodynamic deconvolution was then performed on the timeseries extracted from these ROIs to minimize intra-subject hemodynamic response function variability, and to improve estimation of effective connectivity. DEC, a dynamic measure of directional connectivity between pairs of ROIs, was computed at each time point using Kalman-filter based time-varying Granger causality (GC). Twelve intra-hemispheric connections were chosen and divided into 4 categories: 1) VVS Lower (Calcarine to IOG), 2) VVS Higher (IOG to FG; IOG to ITG), 3) DVS Lower (Calcarine to SOG), and 4) DVS Higher (SOG to IPG; SOG to SPG). Linear mixed model was used to analyze the data (fixed factors: group [BDDActive_iTBS, BDDSham_iTBS, BDDNo_iTBS or HCNo_iTBS], duration [125 ms, 250 ms, 500 ms or 3000 ms], category [VVS Lower, VVS Higher, DVS Lower or DVS Higher]; random factor: participant). Results: For positive GC values, there was significant three-way interaction between group, duration and category from tests of fixed effects (F[27, 135614]=1.89, p = 0.004). For DVSHigher, the active group exhibited stronger DEC than the BDD without iTBS across the four durations, and they achieved similar levels as the controls. Conclusions: In this proof-of-concept study, we explored the effects of interactions between excitatory neuromodulation and rapid face presentation on dorsal visual stream and ventral visual stream connectivity in BDD. Excitatory neuromodulation induced by iTBS enhanced dynamic connectivity for DVSHigher, achieving similar levels as the healthy controls. These results, showing target engagement and modulation, have implications for designing novel perceptual retraining treatments to remediate perceptual distortions of appearance in those with BDD. Keywords: Body Dysmorphic Disorder, Repetitive Transcranial Magnetic Stimulation (rTMS), Visual Perception, Effective Connectivity, Perceptual Distortion of Appearance Disclosure: Nothing to disclose. P424. The Psychosocial and Educational Burden of Obsessive-Compulsive Disorder in Youth McKenzie Schuyler, Bowie Duncan, Daniel Geller, Amitai Abramovitch Massachusetts General Hospital, Boston, Massachusetts, United States Background: Obsessive-compulsive disorder (OCD) is associated with significant multi-domain impairment. Indeed, OCD is associated with poorer long-term educational attainment, vocational problems, and cognitive dysfunction. Compared to adults, youth with OCD exhibit relatively better cognitive functioning. Nevertheless, they may struggle more in family and peer relationships and may require more assistance in school settings compared to healthy controls. Only a few studies have directly examined these issues. Furthermore, research investigating the impact of comorbidities on psychosocial functioning in pediatric OCD is limited. The goal of the present study was to directly assess psychosocial functioning in a large, well-characterized sample of children and adolescents with OCD. Methods: A sample of 100 children and adolescents with OCD (Mage = 11.42; 42.0% male) and 138 non-OCD controls (Mage = 11.45; 42.8% male) participated in the study. Participants were administered a battery of measures including the Kiddie Schedule for Affective Disorders and Schizophrenia (KSADS) to assess diagnostic status and comorbidities, the Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) to assess OC symptom severity, the Social Adjustment Inventory for Children and Adolescents (SAICA) to assess psychosocial adjustment, and the Family Environment Scale (FES) to assess family functioning. In addition, information regarding repeated grades, special placement classes and extra help/tutoring was obtained from teachers and parents. We conducted independent samples t-tests to examine group differences in psychosocial, family, and school functioning variables. Results: Compared to controls, youth with OCD had significantly higher levels of school behavior problems (p = .001; Cohen’s d = .43), problems with spare time activities (p = .000; d = .89, problems with peer activities (p = .000; d = .71), and problems with parents (p = .001; d = .49). Compared to controls, families of youth with OCD also had significantly elevated current and past family conflict scores (p = .035, d = .28 for both), and reduced current and past family cohesion scores (p = .000, d = .51; p = .018, d = .32). There were no differences in the family expression subscale. Significantly more youth with OCD attended special classes (0.7% vs. 4.3%, p < .0001) and received extra help (10.2% vs. 64%, p < .0001) compared to controls. There were no differences in rates of repeated grades. OCD severity was significantly correlated with attending special classes and with several SAICA subscales. However, it was not correlated with FES subscales. Finally, comorbidity with attention deficit hyperactivity disorder, conduct disorder, and oppositional defiant disorder resulted in a different pattern of difficulty, primarily in the domain of school behavior problems. Conclusions: Although pediatric OCD is not associated with substantial cognitive impairments, this population receives more extra help in school, exhibits a higher frequency of enrolling in special classes, and displays significantly more psychosocial and family problems than controls. In addition, there is a differential impact of several comorbid disorders on psychosocial problems in this population. These results highlight the need for careful screening for OCD in this age group, including assessment of comorbid conditions. More research is needed to examine how reassurance-seeking versus measurable academic deficits contribute to help-seeking in this population. Keywords: Obsessive-Compulsive Disorder (OCD), Pediatric, Family Study Disclosure: Nothing to disclose. P425. Tripping Mice and Stoned Fish: Head Twitch Response (HTR) and Behavioral Phenotypic Evidence of Effect Differences Between Synthetic Psilocybin and Psychedelic Mushroom Extract Leonard Lerer, Alexander Botvinnik, Katherine Spear, Orr Shahar, Patryk Lipski, Haley Calderon, Karin Blakolmer, Tzuri Lifschytz, Bernard Lerer Back of the Yards Algae Sciences, Chicago, Illinois, United States Background: Anecdotal reports suggest that the behavioral and pharmacological effects of psilocybin-containing, “full spectrum” psychedelic mushroom extract (FSME) differ from those of chemical psilocybin (PSIL) in their nature and intensity. A limited number of rodent studies have compared synthetic psilocybin (or psilocin) with crude psychedelic mushroom extract. Furthermore, psychedelic mushrooms contain intermediate products of the psilocybin biosynthetic pathway such as baeocystin, norbaeocystin and aeruginascin that may influence the nature of the effect of psilocybin (“entourage effect”) along with other components such as harmines with monoamine oxidase inhibiting properties. In the current study, we compared the effect of PSIL to that of FSME on the mouse head twitch response (HTR), which is correlated with psychedelic effects in humans, on a rodent screening test for antidepressant effect and in a behavioral phenotypic zebrafish model. Methods: Male C57Bl/6j mice were used in all head twitch experiments. PSIL (98.75% purity) was provided by Usona Institute. FSME, a methanol extract of Psilocybe cubensis with a psilocybin content of 1.5%, was provided by BYAS-PEB. Drug doses were calculated so that equal injection volumes of PSIL and FSME contained equal concentrations of psilocybin on a mg per kg basis. Control mice received vehicle (VEH) injections (0.9% NaCl solution). HTR was measured over 20 minutes in a magnetometer-based system using ear clip magnets. The tail suspension test (TST) was conducted using a Noldus Ethovision system by observers blind to treatment status, 48 hours after drug administration. Individual male zebrafish (Danio rerio) were used in an open arena, behavioral phenotyping experiment. The drug dose, 3 mg/L of PSIL and FSME was administered in a beaker containing 200 ml of water over 10 minutes. Control fish were placed in a beaker containing 200 ml of water for 10 minutes. The fish were then placed in an 50x50x4cm arena and video-tracked using idTracker software, with trajectories recorded for 20 minutes immediately after treatment and for 20 minutes at 80 minutes after treatment. Results: FSME induced a significantly greater number of head twitches over 20 minutes at a psilocybin dose of 4.4 mg/kg than PSIL at the same dose (F = 4.41, df 1,21, p = 0.04; FSME n = 11, PSIL n = 12). The difference was evident over the entire time course. On the tail suspension test (TST) conducted 48 hours after i.p. administration of PSIL (4.4 mg/kg), FSME (psilocybin content 4.4. mg/kg) or VEH, both PSIL (209.3 ± 93.6 sec, n = 11, p = 0.01) and FSME (198.14 ± 61.3 sec, n = 10, p = 0.0006) showed significantly less inactivity than VEH (296.0 ± 33.8 sec, n = 11). On the highly active measure of the TST, FSME (11.38 ± 10.13 sec, n = 10) induced significantly more activity than both VEH (0.04 ± 0.12, n = 11, p = 0.0007) and PSIL (0.53 ± 1.04 sec, n = 11, p = 0.001). In the zebrafish experiment, during the first 20 minute recording period, 2D spatiotemporal reconstructions of the zebrafish swim paths demonstrated clearly visible differences in swimming patterns including velocity, distance swum, average distance to perimeter and middle and changes in direction, between the control and the FSME/PSIL groups. There were also a number of clear differences in swimming patterns between the FSME and PSIL groups, especially related to the mean time spent in the corners of the arena (P < 0.05; FSME/PSIL n = 9). At 80 minutes, the swimming pattern of the PSIL group closely resembled that of the control group, whereas the FSME group continued to show a similar, slightly attenuated swimming pattern to that observed in the initial 20-minute recording period. Conclusions: A prior study by Zhuk et al (2015) suggested that mushroom extract has greater potency in inducing HTR than psilocin (the active metabolite of psilocybin). Our findings in mice are in accordance with this observation. We have further shown that on the TST, a screening test for antidepressant potential, FSME induces a stronger effect than PSIL when the same dose of psilocybin is administered with both preparations. Furthermore, this work provides evidence of a robust and measurable zebrafish response to PSIL and FSME. The more sustained effect of FSME may be indicative of an “entourage effect”. Further studies are indicated to elucidate a possible therapeutic advantages of full spectrum psychedelic mushroom extract as compared to chemical psilocybin and to identify the entourage molecules that contribute to this effect. This work also raises a tantalizing and important challenge related to the possible development of a zebrafish behavioral model equivalent to the mouse HTR test. Keywords: Psilocybin, Psychedelics, Entourage Effect, Head Twitch Response, Tail Suspension Test Disclosure: Back of the Yards Algae Sciences: Founder (Self) P426. Translational Implications of Marble Burying in ICR Mice for the Anti-Obsessional Effects of Psilocybin Sandeep Singh, Alexander Botvinnik, Orr Shahar, Gilly Wolf, Amit Lotan, Bernard Lerer, Tzuri Lifschytz Hadassah-Hebrew University Medical Center, Jerusalem, Israel Background: Initial clinical findings, supported by preclinical studies employing behavioral paradigms such as marble-burying, suggest that the psychedelic tryptamine, psilocybin, may be effective in treating obsessive-compulsive disorder (OCD) The aims of this study were to further evaluate the role of 5-HT2A receptors in the effect of psilocybin on marble-burying, to explore the role of 5-HT1A receptors in this effect and to examine potential use the 5-HT1A receptor partial agonist, buspirone, as a concurrent treatment for OCD with psilocybin Methods: Male ICR mice were administered psilocybin 4.4 mg/kg, escitalopram 5 mg/kg; the 5-HT1A agonist, 8-OH-DPAT 2 mg/kg; the 5-HT2A antagonist, M100907 (volanserin) 2 mg/kg; the 5-HT1A partial agonist, buspirone, 5 mg/kg; or the 5-HT1A antagonist, WAY100635 2 mg/kg; or combinations. Drugs were administered intraperitoneally, and the mice were tested on the marble burying test (MBT) for 30 minutes after treatment. Head witch response (HTR) induced by psilocybin alone or in combination with buspirone, was examined in a magnetometer-based assay. Results: 1) Both psilocybin (p < 0.01) and the positive control, escitalopram (p < 0.01), significantly reduced marble-burying. The effect of psilocybin was not attenuated by the 5-HT2A antagonist, M100907. The 5-HT1A agonist, 8-OH-DPAT, reduced marble-burying (p < 0.01) as did the 5-HT1A partial agonist, buspirone (p < 0.01). The effect of 8-OH-DPAT was additive to that of psilocybin (p < 0.01) but that of buspirone was not. The 5-HT1A antagonist, WAY100635, attenuated the effect of 8-OH-DPAT and buspirone on marble burying but not the effect of psilocybin. 2) Psilocybin injections over 3.5 hours had no effect on marble-burying and the effect of bolus injection was not persistent. 3) Co-administration of buspirone with psilocybin blocked the effect of psilocybin on HTR but not its effect on marble burying. Conclusions: Neither 5-HT2A nor 5-HT1A receptors are pivotally implicated in the effect of psilocybin on marble-burying. Co-administration with buspirone may block the psychedelic effects of psilocybin without impeding its anti-obsessional effects. Concurrent treatment of OCD with buspirone and psilocybin is a feasible strategy to achieve anti-obsessional effects while avoiding or minimizing the psychedelic trip. (Supported in part by Back of the Yards algae sciences and Parow Entheobiosciences) Keywords: Psychedelics, Obsessive-Compulsive Disorder (OCD), Psilocybin, Buspirone, 5-HT1A Receptors Disclosure: Back of the Yards Algae Sciences: Contracted Research (Self), Parow Entheobiosciences: Contracted Research (Self), Negev Capital: Consultant (Self) P427. Translating the Persistent Avoidance Circuitry: Implications for Obsessive Compulsive Disorder Lucas Remoaldo Trambaiolli, Freddyson J. Martínez-Rivera, Wei Tang, Mary Phillips, Gregory Quirk, Suzanne Haber McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: The caudal area 47/12 of the ventrolateral prefrontal cortex (vlPFC) was recently described as a node of the salience network in humans and nonhuman primates (NHP) (Trambaiolli et al. 2022 eLife). This region supports stimulus-outcome predictions associated with salient stimuli and prepares appropriate responses later selected by the anterior cingulate cortex (ACC). In the rodent persistent avoidance circuit, the agranular insular/lateral orbital (AI/LO) cortex has excitatory inputs to the rostral prelimbic (rPL) cortex (Martínez-Rivera et al. 2022 Biol. Psych.). These neurons project to the ventral striatum (VS) to control avoidance expression. Hypoactivity in AI/LO decreases excitatory drive from rPL to the VS, increasing VS susceptibility to basolateral amygdala (BLA) excitatory inputs. This drives avoidance expression. Herein, we hypothesize that (i) the primate caudal 47/12 includes the AI/LO region identified in rats and (ii) a similar avoidance circuit can be identified in NHP using anatomical tract-tracer data starting from caudal 47/12. Methods: We injected bidirectional tracers in the vlPFC area 47/12 (four injection sites) and the ACC (two injections in area 32 and three in area 24), anterograde tracers in the BLA (two injections), and retrograde tracers in the VS (two injections). We used bright-field microscopy to identify which sublocations in the vlPFC and ACC are highly interconnected. We quantified the number of cells labeled in 23 cytoarchitectonic regions in the frontal cortex and amygdala. The connectivity strength between each cortical area and the injection location was measured as the ratio between the number of cells in this area by the total number of labeled cells. We used dark-field microscopy to delineate axonal projection zones between all nodes of the avoidance circuit (vlPFC, ACC, VS, and BLA), starting from the locations identified in the previous step. Finally, we validated the connectivity strength of projections from vlPFC, ACC, and BLA to the VS using the same retrograde approach described before. Results: Area 24 was the ACC portion with the strongest projections to the vlPFC, showing a rostro-caudal gradient peaking at the caudal-most part of 47/12. The strength of connections from the vlPFC to the ACC peaked in the pregenual-genu portion of area 24 (rostral 24). Dark-field analysis validates the bidirectional projections between caudal 47/12 and rostral 24, with dense axon terminal fields in both locations. Rostral area 24 has dense projections to the VS (core extending to the shell) and BLA. Other ACC regions and the caudal 47/12 present weak or no projections to these regions. The BLA projects densely to the VS (mainly the shell), vlPFC, and ACC. Importantly, BLA and rostral 24 projections overlap in the border between the VS shell and core. Retrograde injections in the VS validate these patterns. Conclusions: These findings suggest that the anatomical connections relevant to the persistent avoidance circuitry in rodents are also present in NHP. The NHP caudal 47/12 likely includes this circuit’s AI/LO node, and rostral 24 is thought to be homologous to the rPL. Caudal 47/12 has weak-to-absent projections to the subcortical regions of interest, focusing its control over the circuit on interactions with the rostral ACC. Abnormal computations in the caudal 47/12 could lead to pathological behaviors, such as the persistent avoidance observed in patients with obsessive-compulsive disorder (OCD). Importantly, caudal 47/12 receives dense projections from the BLA, which may cause biased attention to negative stimuli and impaired response preparation. This is in line with recent studies reporting that OCD patients have hyperconnectivity between the caudal 47/12 and the amygdala compared to healthy controls (Thorsen et al. 2020 Neuroim. Clin.). This connection could also be associated with the hypoactivity in AI/LO observed in avoidant rodents (Martínez-Rivera et al. 2022 Biol. Psych.) or in caudal 47/12 from OCD patients (Chase et al. 2020 Neuroim. Clin.). The VS is a downstream region where avoidance behaviors are triggered or stopped. Rostral 24 and BLA converge to the same VS location (border between the shell and core). This region is a common target for deep-brain stimulation in patients with OCD, with rodent models suggesting its effectiveness is due to enhancing fear extinction (Rodriguez-Romaguera et al. 2020 PNAS). Given its role in the avoidance circuit and the homologies between NHPs and humans, caudal 47/12 can be an additional target within this circuit for treating these symptoms in OCD. Keywords: Ventrolateral Prefrontal Cortex, Persistent Avoidance, Obsessive Compulsive Disorder, Anterior Cingulate Cortex (ACC), Ventral Striatum Disclosure: Nothing to disclose. P428. Dorsal Striatum Modulates the Expression of Uncertain, but Not Certain, Value-Based Behaviors Matthew Geramita, Susanne Ahmari, Eric Yttri University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Patients with obsessive compulsive disorder (OCD) display deficits in making decisions based on both value and certainty. The dorsomedial striatum (DMS) likely plays a critical role in these disruptions as the DMS encodes both certainty and value and displays abnormal activity in patients with OCD. Yet it is unknown how DMS representations of value and uncertainty contribute to behavior. To answer this question, we recorded and manipulated DMS activity during a Pavlovian conditioning task in which value and uncertainty were modulated. Methods: Mice (n = 10; 5 female) expressing either channelrhodopsin-2 in either direct or indirect pathway medium spiny neurons (dMSNs or iMSNs) were implanted with bilateral optical fibers in the DMS (anteroposterior: +0.7 mm, mediolateral: +/-1.5 mm, dorsoventral: −2.5 mm) and trained to perform a head-fixed auditory Pavlovian conditioning task in which three cue types predicted reward on 0%, 50% or 100% of trials. To manipulate activity, dMSNs or iMSNs were briefly stimulated bilaterally (473 nm; 2 mW; 15 Hz for 1 s) during either the cue, delay, or outcome period. In a separate cohort of animals (n = 3; 1 female), MSN activity was recorded using Neuropixels electrodes. Results: Mice learned the value of each auditory stimulus as the rate of anticipatory licking during the cue and delay period correlated with reward probability (0%: 1.2 ± 0.1 Hz; 50%: 4.4 ± 0.2 Hz; 100% 5.1 ± 0.1 Hz; One-way ANOVA, p < 10-32). In a population of 343 putative MSNs, 51% were modulated equally by all three cues (ie salience), 40% were modulated by the value of the cue, and 8% were modulated by the certainty of the cue. Unsupervised clustering of activity during the cue and delay periods indicated four distinct response patterns – two that were positively modulated and two that were negatively modulated by cues. Value, salience, and certainty neurons were distributed equally across all four clusters. Interestingly, cue activity from neurons belonging to the two excitatory clusters was negatively correlated with licking for 50% cues (Cluster 1: r = -0.091 ± 0.01, p < 10-8; Cluster 2: r = -0.085 ± 0.01; p < 10-11), but not 0% or 100% cues. These data suggest that a subpopulation of DMS MSNs negatively modulated anticipatory licking during uncertain cues regardless of whether neurons encoded information about cue value, salience, or certainty. Cell-type specific modulation of MSNs corroborated in vivo data. Photostimulation of either dMSNs or iMSNs during the cue period of the 50% cue decreased anticipatory licking (No stim: 4.4 ± 0.3 Hz; vs Stim: 2.8 ± 0.3 Hz; t-test, p < 0.001). Surprisingly, there was no change in anticipatory licking when dMSNs or iMSNs were stimulated during the 100% or 0% cue. Additionally, photostimulation of either population during the delay or outcome period of any cue type did not alter licking. Conclusions: The DMS plays an important role in decision-making in a variety of contexts yet how value and certainty regulate value-based behavior is unknown. Here, using in vivo electrophysiology and optogenetics, we find that both DMS dMSN and iMSN activity inhibits anticipatory licking during uncertain, but not certain, cues. These findings suggest that the certainty of reward gates the influence of DMS activity on behavior. Keywords: Dorsal Striatum, Uncertainty, Value-Based Decision-Making Disclosure: Nothing to disclose. P429. Approach to Use Data of Diverse Ancestries to Uncover Generalizable Genotype Driven Fetal- and Adult-Specific Brain Transcriptomic Mechanisms in Psychiatric Disorders Aarti Jajoo, Christos Chatzinakos, Cindy Wen, Tade Souaiaia, Tim Bigdeli, Michael Gandal, Nikolaos Daskalakis McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Brain transcriptomic imputation utilizes genotype-expression reference panels to build predictive models of genetically regulated gene expression (GReX). GReX models can be applied to GWAS to conduct transcriptome-wide association studies (TWAS) to prioritize gene-trait associations (GTAs) with functional importance. However, reference panels and GWAS studies have been biased toward European Ancestry (EA) samples, posing limitations for the identification of generalizable GTAs. To overcome this challenge, we propose meta-analyzing TWAS results across the ancestries by applying ancestry-specific models to the respective ancestry GWAS. Methods: To this effect, we trained ancestry-specific GReX models, of Admixed African Ancestry (AA) and EA, separately for healthy adult DLPFC tissue (NAA = 165, NEA = 453 subjects) and fetal brain tissue (NAA = 164, NEA = 292). Results: Fetal models predict fewer genes and have lower R2 model performance for overlapping genes compared to same ancestry adult models (R2 = 0.17 for EA_adult vs R2 = .11 for EA_fetal, P < e-16). Within each tissue type, R2 was comparable between ancestries, but AA models predicted 45% fewer genes compared to the same tissue EA models likely due to less power. EA models compared to AA models performed worse in the AA test set, while AA models performed comparably in EA test set. We applied these models to GWAS of bipolar disorder (BD), major depressive disorder (MDD), post-traumatic stress disorder (PTSD), and schizophrenia (SCZ), using at least one GWAS for each ancestry within each disorder. The average correlation of GTA z-scores was Rho=0.49 between adult and fetal analyses. Fetal tissue yielded a higher percentage of GTAs for SCZ which was expected because of neurodevelopmental origins, but surprisingly this was true for PTSD which has a later age of onset and lower heritability. The common GTAs from fetal and adult analyses tended to cluster in particular genomic regions (e.g, 17q21.31 in PTSD). Furthermore, the general observation is that bi-ancestry TWAS meta-analysis using just EA GReX models yields the least percentage of Bonferroni significant GTAs while using ancestry-specific GReX models for respective ancestry GWAS yields the highest percentage (e.g., 2x increase in SCZ, 30x increase in PTSD). Conclusions: Our work shows that fetal and adult brain tissue-based GTAs reveal shared and distinct genetic underpinnings of psychiatric disorders that operate in multiple stages, and that bi-ancestral TWAS is more beneficial when ancestry-specific GReX models can be applied to respective ancestry-specific GWAS. Keywords: Genetic Ancestry, Gene Expression, Fetal Brain, Adult Brain Disclosure: Nothing to disclose. P430. Chemogenetic Inhibition of VTA Gaba Neurons Lead to Persistent but Sexually Diergic Changes in Reactivity to Ethanol Laverne Melón, Han Bin Kwon, Isabella Ahearn, Bilge Buyukdemirtas Wesleyan University, Middletown, Connecticut, United States Background: Sensitivity to alcohol’s stimulant effects predicts diagnosis with AUD and symptom severity (King et al., 2021). Preclinical models of repeated alcohol exposure that sensitizes this stimulant effect are useful tools for understanding maladaptive plasticity underlying the transition from use to disordered use. Moreover, sex differences in these preclinical models may be leveraged to better dissect which biological systems altered by alcohol sensitization are relevant for specific drug’s consequences. To this end, we used a chemogenetic approach to dissect a role for GABAergic inhibition in the VTA in the expression of sensitization and the sex-specific reduction in preference for social reward that follows alcohol-induced sensitization. Methods: Male and female C57BL6/J mice (5-8/group) were administered alcohol (2.0 g/kg) repeatedly for 14 days, with locomotor activity assessed on the first and final days of exposure. Animals were given an opportunity to interact with a sexually-immature juvenile for 5 minutes during early (1 day following last exposure) or protracted (14 days following last exposure) withdrawal. Brains and bloods were harvested 30 minutes following this social interaction opportunity. To probe the role that GABAergic inhibition in the ventral tegmental area plays in this sex difference, male and female VGAT-cre mice (7-9/group) received 250 nL of the Gi-DREADD virus (pAAV-hSyn-DIO-hM4D(Gi)-mCherry) or the sham mCherry virus (pAAV-hSyn-DIO-mCherry) into the VTA. Animals received a single administration of CNO (1.0 mg/kg, ip) and were tested 7 or 14 days later for their reactivity to alcohol (2.0 g/kg). Results: Both males and females demonstrated a sensitized response to ethanol at this dose, though the effect was more pronounced for females. Although both groups showed no change in preference for social interaction during early withdrawal from this sensitization protocol, females show a significant reduction in social preference two weeks after their final exposure to ethanol. We found that a singular inhibition of VTA GABA neurons induced heightened baseline activity seven days later in both males and females. However, two weeks following this acute chemogenetic inhibition of VTA GABA neurons only males display significantly greater locomotor response to ethanol (p = 0.014). Current analysis of the effect that this manipulation has on sensitivity to social interaction is currently underway. Conclusions: The results demonstrate that acute inhibition of VTA GABAergic neurons has a sexually diergic effect on later reactivity to ethanol and this may underlie sex-specific plasticity in behaviors following exposure to the drug. Keywords: Alcohol, Sensitization, GABA, VTA, Sex Difference Disclosure: Sage Therapeutics: Grant (Self) P431. A Human Laboratory Study on the Effects of a Novel Ghrelin Receptor Inverse Agonist Competitive Antagonist on Alcohol and Food-Related Behaviors in Individuals With Alcohol Use Disorder Monica Faulkner, Mehdi Farokhnia, Lisa Farinelli, Sara Deschaine, Brittney Browning, Fatemeh Akhlaghi, Lorenzo Leggio National Institute on Drug Abuse, Baltimore, Maryland, United States Background: Ghrelin is a 28 -amino acid peptide that acts as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a). In preclinical studies, ghrelin has also been found to increase ethanol intake by modulating the neural mechanism of reward processing. Clinical studies have also demonstrated the role of ghrelin in alcohol use behaviors by showing that ghrelin levels are suppressed by both oral and intravenous acute alcohol administration and that baseline ghrelin levels are positively correlated with craving for alcohol. In human laboratory studies, intravenous ghrelin administration significantly increased cue alcohol craving, and self-administration of alcohol and influenced increases in neural activation of the amygdala and medial orbitofrontal cortex during an alcohol related incentive delay task in non-treatment seeking individuals with AUD. Recently, we investigated of the safety and tolerability of a novel ghrelin receptor inverse agonist/competitive antagonist, PF-5190457, when it is co-administered with alcohol. While our focus was on safety, we conducted a preliminary investigation on the drug’s effect on alcohol cue elicited craving in a small subset of the study and found it reduced alcohol cue elicited craving in a bar like laboratory setting. Based on these preliminary findings, we expanded our investigations on PF-5190457 to specifically assess the drug’s effects on alcohol- and food-related behaviors in an inpatient sample of treatment-seeking individuals with AUD. Methods: The study was a within subjects, double blinded, placebo controlled clinical trial, comparing the effects of PF-5190457 100 mg b.i.d. up to steady state on alcohol/food-related behaviors. Participants were twenty-nine treatment-seeking, detoxified individuals with AUD (M = 21, F = 8). Participants completed 2 behavioral paradigms to 1.) assess whether the drug would reduce alcohol and food cue-elicited craving assessed in a “bar-like” laboratory and 2.) to assess whether the drug would reduce food choices in a “virtual buffet” conducted in a virtual reality environment. To assess alcohol and food cue-elicited craving, participants were exposed to their preferred alcohol-containing beverage and snack in a bar like laboratory. The procedure began with a baseline assessment of alcohol craving, measured by the Alcohol Urge Questionnaire (AUQ), and food craving, measured by the General Food Craving Questionnaire-State (GFCQ-S) outside of the bar like laboratory. After the baseline assessments, participants were escorted into the bar and exposed to alcohol, food, and neutral (water) cues. There was 1 exposure to the neutral cue (water), 1 exposure to the food cue, (their preferred snack), and 2 exposures to the alcohol cue, (their preferred alcohol beverage). After each exposure, participants completed the Alcohol Urge Questionnaire (AUQ) to assess craving for alcohol, General Food Craving Questionnaire-State (GFCQ-S) to assess craving for food, and one additional questionnaire after each alcohol exposure, the Alcohol Attention Scale (AAS), assess their attention to the alcohol-containing beverage. To assess food choice behavior, participants engaged in a virtual lunch buffet where they chose as many and as much of the virtual food and beverages during one trip to the buffet as they would normally choose during lunch and then completed questionnaires to assesses the current state of alcohol craving (AUQ), food craving (GFCQ-S), and affective mood state (POMS). Participant’s food choice behavior was assessed by calculating the total calories selected during the virtual lunch buffet. Repeated measures ANOVAs were used to analyze the results of the outcomes of the study and all analysis controlled for body mass index (BMI), acyl-ghrelin levels, and age on day 1 of the study as well as gender and experimental condition order. Results: The drug did not reduce alcohol cue-elicited craving as measured by the AUQ (F1,5 = .11, p = .738) and there was no drug × time interaction (F1,5 = .72, p = 0.607). There was a significant effect of the drug on attention to alcohol, with participants reporting paying less attention to the smell of alcohol (F1,31 = 4.88, p = 0.035) and having to exert less effort to stop thinking about drinking the alcohol (F1, 46 = 5.94 p = 0.02). There was also a significant effect of the drug on food choice behavior, with participants selecting fewer calories during the virtual lunch compared to the placebo condition (F1,29.6 = 4.49, p = .043. Conclusions: We found that the novel ghrelin receptor inverse agonist PF-5190457 had a significant effect on food choice behavior during a virtual lunch buffet and an effect on attention to alcohol cue during a cue reactivity paradigm in a bar-like laboratory. However, there was no effect of the drug on alcohol or food cue elicited craving in the bar-like laboratory. These preliminary findings suggest that ghrelin may play a role in the attention to reward related cues and stimuli but not craving in detoxified individuals with AUD. Future investigations should explore different stages of the AUD cycle to assess if ghrelin antagonism has similar effects across all stages. Keywords: Alcohol Use Disorder - Treatment, Clinical Trial, Ghrelin Disclosure: Nothing to disclose. P432. Molecular Characterization of the Mouse Lateral Septum Lionel Rodriguez, Matthew Nguyen Tran, Elizabeth Pattie, Heena Divecha, Leonardo Collado-Torres, Stephanie Cereceo Page, Keri Martinowich Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: The lateral septum (LS) is a midline, basal forebrain region that is surrounded by the lateral ventricles. The LS is a sensory and affective integrator that utilizes environmental stimuli to modulate behavior. In the rodent, LS circuitry plays roles in reward and fear learning, stress responses, context-dependent feeding, and various social behaviors, including aggression and response to social novelty. In line with these functions, the LS is connected, often reciprocally, to regions such as the ventral tegmental area, the dorsal raphe, the amygdala, various hippocampal sub-regions, and many cortical regions. While it is known that the LS contains diverse populations of GABAergic cells, which express combinations of neurotensin, somatostatin, and calbindin but are devoid of parvalbumin, the overall chemoarchitecture of the LS is understudied. To better understand and characterize cellular composition in the LS, we used single-nucleus RNA-sequencing (snRNA-seq) to generate a molecular atlas of LS cell types. Methods: Here, we used the 10X Genomics 3’ single cell platform to generate molecularly defined LS cell-types using snRNA-seq. For each sample, LS tissue from 2 naive mice of the same sex were pooled and processed together [N = 4 total samples (n = 8 mice,4 female, 4 male)]. Brains were extracted, flash-frozen, and sectioned into two 1 millimeter coronal brain slabs encompassing the span of the LS across the anterior- posterior axis. The LS was dissected from each slab, and nuclei were isolated using the “Frankenstein” nuclei isolation protocol developed by Martelotto et al. (2020). Samples were sorted on Bio-Rad S3e Cell Sorter, with 9000 nuclei sorted directly into reverse transcription reagents from the 10x Genomics Single Cell 3’ Reagents v3.1 kit. 10x Chromium was performed and libraries prepared according to the manufacturer;s protocol, and then sequenced on an Illumina NovaSeq 6000. FASTQ data generated from sample libraries were aligned to the mouse genome using cellranger (version 6.1) utilizing the –include-introns flag to account for the nuclear transcriptome settings. Using the Bioconductor suite of single-cell analysis packages by Amezquita, et al. (2021), nuclei calling was performed using emptyDrops on the raw feature-barcode matrices. Specifically, we used a sample-driven threshold to call nuclei as the default threshold failed and called ~10-fold greater nuclei than were sorted. Next, we performed mitochondrial rate adaptive thresholding using a 3x median absolute deviation from the median to threshold and filtered called nuclei from each sample. Feature selection was computed using deviance residuals and the total deviance was used to calculate the top 2000 highly deviant genes. In this feature space, PCA was conducted as by Townes et al (2019). Using the top 50 PCs, we performed graph-based clustering, using k = 20 nearest neighbors and the walktrap community detection algorithm yielded 35 preliminary clusters. We annotated clusters based on expression of the mouse orthologs of broad cell class markers from Tran, Maynard et al (2021). For cluster marker detection we employed the approach outlined in Tran, Maynard et al (2021) to characterize strict pairwise test-significant markers per cluster, in addition to cluster-enriched markers. The identified gene markers that were uniquely expressed by each cluster were surveyed using Allen Brain’s Genome-wide atlas of in-situ hybridization (ISH) data to assign clusters to regions by exploring marker spatial distributions. Results: We identified 33 cell-type clusters, 24 of which were neuronal. Within the neuronal populations 18 were from the septum and six were from other, adjacent regions (the striatum (four), thalamus (one), and islands of Calleja (one)). The septal populations were subclustered and further classified. Nine GABAergic clusters were annotated as LS including a distinct GABAergic population that lined the ventricle. Three glutamatergic clusters were annotated as medial septum including a small cholinergic population. Three glutamatergic clusters were annotated as a mix of neurons from the dorsocaudal septum where the tenia tecta, induseum griseum and septohippocampal nucleus meet. Two GABAergic clusters were annotated as septal broadly as their cluster markers were not confined to a previously identified septal sub-division. One glutamatergic cluster was annotated as the triangular nucleus of the septum. A curated set of genes, identified from previous studies of the chemoarchitecture of the LS, were used to characterize the LS-specific clusters. These genes included receptors for neuromodulators including dopamine, norepinephrine and serotonin as well as receptors for neuropeptides such as oxytocin, vasopressin and corticotropin releasing hormone. We then identified the cellular expression patterns of these markers in our annotated clusters. Finally, we generated an interactive web browser to allow exploration of these data for the neuroscience community. Conclusions: Together, these data create a molecular atlas of septal cell types. We describe cell-specific gene expression patterns, and identified novel LS neuronal cell-types. This resource provides cell-type-specific information that will be useful in better understanding the biology of the LS, and which cell types may underlie behaviors that are mediated by the LS. Keywords: Lateral Septum, Single-Nucleus RNA Sequencing, GABA Neuron, Septum Disclosure: Nothing to disclose. P433. Gender Differences in Adult ADHD Symptoms: A Network Analysis of Real-World Data Kira Griffiths, Gunjan Batra, Li Tong Low, Emily Palmer, Miguel Renteria, Rashmi Patel, Scott Kollins Holmusk, New York, New York, United States Background: Symptoms of ADHD can be classified as inattentive (IA) or hyperactive/impulsive (H/I). However, significant heterogeneity in the clinical presentation of ADHD has been reported across development and between genders. Network analysis offers a more detailed understanding of symptom structure compared to traditional factor analytic approaches. Electronic health record (EHR) data captures real-world symptom presentation in large and representative cohorts. The current study applied network analysis to real-world symptom data from adults with ADHD. This study aimed to explore and compare ADHD symptom networks in adult males and females. Methods: Symptoms recorded by mental healthcare professionals as part of clinical assessment were obtained from a de-identified EHR dataset from 25 US mental healthcare systems (WCG Institutional Review Board Ref: WCG-IRB 1-1470336-1).The cohort included adult patients (>18 years) with a diagnosis of ADHD (ICD9/10: 314.00, 314.01, F90.0, F90.1, F90.2, F90.8 and F90.9) and symptom data within the first six months of diagnosis. Symptoms were defined according to the 18 symptoms outlined in the DSM-5. Networks for males and females were constructed separately, with each symptom reflecting a unique node within a network. Symptoms were entered as binary variables indicated by their presence or absence within individual patients. Analyses were conducted using R version 4.1.3, Bootstrap package 1.5 and Isingfit package v0.3.1. Networks were estimated using the eLASSO procedure and then visualized. In addition to the network graphs the centrality measures of betweenness, closeness and strength were calculated. In networks of psychiatric symptoms, centrality measures allow the identification of symptoms of high importance within a network and may therefore highlight key symptoms to target for intervention. Results: Data were available for 2,398 patients (53% female) with a diagnosis of ADHD. Networks clustered into H/I and IA symptoms. In adult males, hyperactive/impulsive symptoms form a relatively tight network, whereas inattentive symptoms are less central. H/I and IA symptom clusters in females were relatively more dispersed than in males. In terms of strength, the top three unique symptoms for males and females both included two symptoms from the H/I cluster and one from the IA cluster. However, these differed by gender with the strongest symptoms associations in males being excessive/inappropriate movement, being “on the go” and failure of close attention. Whereas for females’ symptoms of difficulty remaining seated, frequently interrupting and being easily distracted had the highest strength in the network. In terms of closeness, the most central symptom was in the IA cluster for females (does not seem to listen) and the H/I cluster for males (difficulty waiting their turn). H/I symptoms were the most central in terms of betweenness for males and females, both of which reflected inappropriate movement. Conclusions: Clustering of H/I and IA symptoms is generally consistent with the current conceptualisation of ADHD. Differences in unique symptoms identified as most central in each network add further evidence that symptoms may vary in terms of clinical relevance for males and females, which may impact the identification, diagnosis, and treatment of ADHD between genders. These differing networks may also be important for identifying unique treatment targets for males and females. In addition, the general approach to characterizing networks could also be important for more precisely defining ADHD symptoms in other subgroups (eg., different racial/ethnic groups, different developmental levels). Keywords: ADHD, Gender Differences, Network-Analysis Disclosure: Holmusk: Employee (Self), Holmusk: Stock / Equity (Self) P434. An Experimental Program to Determine the Effects of Viloxazine on Cortical Serotonin Neurotransmission at Doses Relevant for ADHD Treatment Jami Earnest, Jennie Garcia-Olivares, Brittney Yegla, Vladimir Maletic, Chungping Yu Supernus Pharmaceuticals Inc, Crownsville, Maryland, United States Background: FDA-approved ADHD treatments are presumed to work by augmenting dopamine (DA) and norepinephrine (NE) signaling in the brain. The focus on these neurotransmitter systems stems largely from the pharmacology of effective treatments. However, the breadth of symptoms and comorbidities in ADHD, emerging genetic and imaging studies, and the demonstrated robust efficacy of viloxazine ER (despite its moderate ability to inhibit norepinephrine transporters (NET)) all suggest a more complex neuropathology may be involved. In a prior microdialysis study, we found viloxazine augments serotonin (5-HT) in the prefrontal cortex (PFC) in addition to increasing NE and DA. Similarly, an early in vitro functional activity study suggested viloxazine can act as a partial agonist at 5-HT2C receptors and antagonist at 5-HT7 and 5-HT2B receptors. However, it was unclear to what extent these effects occurred at clinically relevant concentrations. To further understand these serotonergic effects, we initiated a series of experiments to build upon this earlier work. Our objectives are three-fold: 1) Can we confirm and better elucidate the previously observed serotonergic effects of viloxazine and determine whether they occur at clinically relevant concentrations? 2) Are these effects observed in species with close physiology to humans? Methods: To answer objective 1, viloxazine was assessed in in vitro binding competition assays for 5-HT2C, 5-HT2A., and 5-HT7 receptors using the specific radioligands [3H]-mesulergine, [3H]-ketanserin and [3H]-LSD, respectively, and corresponding reference compounds. An in vivo PK/PD microdialysis study was also conducted in Sprague-Dawley rats by administering 1, 3, 10, or 30 mg/kg doses of viloxazine, to determine the relationship between viloxazine concentrations in the interstitial fluid (ISF) and changes in concentrations of NE, DA, 5-HT, and their metabolites in the PFC. To answer objective 2, a PET imaging study using a 5-HT2A/2 C radioligand agonist, [11 C]CIMBI-36, was conducted in anesthetized cynomolgus monkeys to evaluate viloxazine’s effect on endogenous cortical 5-HT release and direct binding to 5-HT2C receptors in the choroid plexus. Animal research was performed at Charles River Laboratories (South San Francisco, CA, USA), the Karolinska Institute Centre for Psychiatry Research (Stockholm, Sweden), and approved by local ethics and animal care committees. Animals were cared for according to international standards. Results: Objective 1: In vitro binding competition assays in cell lines expressing the human isoforms of 5-HT2C, 5-HT7 and 5-HT2A confirmed viloxazine’s affinity towards these receptors with Ki values of 1.5, 1.9 and 16.3 µM, respectively. In addition, the microdialysis study in rats was conducted using a 30 mg/kg dose, at which the Cmax of viloxazine in ISF was 3.5 ± 1.6 µM, approximating the unbound viloxazine plasma concentrations (2.1-3.3 µM) observed in pediatric ADHD patients receiving 400 mg/day of viloxazine ER. At this clinically relevant concentration, viloxazine increased NE levels up to 558% over baseline and significantly decreased its DHPG metabolite from baseline, confirming NET inhibition. Serotonin levels were significantly increased up to 213% over baseline; however, no significant changes were observed in its 5-HIAA metabolite, demonstrating that serotonergic effects were not due to 5-HT reuptake inhibition. Objective 2: Administration 3 mg/kg of viloxazine to non-human primates produced an unbound plasma concentration of 3.9 µM (Cmax), aligning with concentrations measured in pediatric ADHD patients. At this clinically relevant viloxazine concentration, changes in binding potential of [11 C]CIMBI-36 in the choroid plexus and cortical regions were 60% and 25-36% respectively, indicating viloxazine may directly bind to 5-HT2C receptors in choroid plexus, and elevate endogenous 5-HT levels in cortical regions. Conclusions: To date, our experiments to further elucidate the potential serotonergic effects of viloxazine have successfully shown that 1) the previously observed in vitro effects of viloxazine on serotonin receptors and the in vivo augmentation of serotonin in rat PFC are present at clinically relevant concentrations and 2) In vivo, serotonin modulatory effects are also seen in non-human primates, suggesting that they may translate clinically. Overall, it appears viloxazine can increase serotonin concentrations in cortical regions, including the PFC, at concentrations that have been shown to be therapeutic in pediatric ADHD trials. Data from in vitro receptor studies and the PET study are consistent with viloxazine binding to the 5HT2C receptor. Keywords: Viloxazine, ADHD, PET Imaging, Microdialysis, Serotonin Disclosure: Supernus Pharmaceuticals: Employee (Self), Johnson and Johnson, Alkermes, Merck: Stock / Equity (Self) P435. Peripheral Tumor Presence Produces Impairments in Hippocampal-Dependent Cognition and Persistent Elevated Cytokines in Plasma When Treated With Docetaxel Amber Asher, David Morilak The University of Texas Health Science Center, San Antonio, Texas, United States Background: Prostate cancer (PC) is the second most common cancer in American men such that 1 out of every 8 men will be diagnosed with PC at some point in his life. Despite this high prevalence, treatment of PC is effective, and the 5-year survival rate is over 99%. Unfortunately, however, many survivors of cancer experience persistent cognitive impairments associated with their life-saving treatment, and there are few treatment options available to these patients to prevent or reverse impairments. In particular, patients who receive chemotherapy exhibit impairments in working memory, cognitive flexibility, attention, and visuospatial memory that can last after chemotherapy ends in a phenomenon colloquially referred to as ‘chemobrain’. Docetaxel (DTX) is a microtubule-stabilizing agent approved for the treatment of late-stage PC and is associated with cognitive impairments in both human cancer patients and in studies of healthy rodents. Mechanisms behind chemobrain remain to be elucidated. Moreover, in preclinical investigations, the additional factor of cancer pathophysiology is rarely considered, and the contribution of a peripheral tumor to these impairments is also unknown. Here, we use a novel syngeneic rat PC model to characterize cognitive impairments produced by the interaction of a tumor and DTX. Moreover, we investigate changes in two circulating proinflammatory cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-6 (IL-6) as potential physiological changes produced by a tumor that could render the brain susceptible to DTX. Methods: Male Copenhagen rats, aged 3 months, were used in this study. To generate the PC model, a small fragment of a tumor grown from Dunning R-3327G cells, syngeneic with Copenhagen rats, was implanted subcutaneously along the dorsal midline between the scapula and pelvis. Sham animals received a sham surgery. When the tumor became palpable, animals were treated with DTX (4.5 mg/kg/injection; i.p.) or its vehicle (i.p.) three times over five days followed by a two-week recovery period. Hippocampal-dependent visuospatial memory and working memory were assessed using the novel object location task and spontaneous alternation on a y-maze, respectively. Plasma was collected after behavioral testing, and concentrations of TNFα and IL-6 were quantified using commercially available enzyme-linked immunosorbent assays. Tumors were measured using digital calipers every 7 days and tumor volume was calculated. Results: Tumor-bearing rats exhibited impairments in visuospatial memory regardless of treatment. Increases of both TNFα and IL-6 were observed in the plasma of tumor-bearing rats compared to sham rats, regardless of treatment. DTX treatment alone in sham-implanted rats also produced an elevation in plasma IL-6 still evident two weeks after cessation of treatment. Conclusions: The presence of a peripheral tumor produced impairments in hippocampal-mediated visuospatial memory and increases in TNFα and IL-6 in plasma compared to sham animals. DTX administration in sham-implanted rats produced modest impairments in hippocampal-dependent behavior that appeared to be worsened in animals with tumors. In peripheral measures of inflammatory cytokines, DTX treatment in sham animals produced an elevation in IL-6 in plasma still evident 2 weeks after the last injection, indicating lasting inflammation in sham animals. TNFα and IL-6 were elevated in both vehicle-treated tumor-bearing rats and those treated with DTX, despite the tumor volume being significantly reduced in the latter group. Taken together, the presence of a peripheral tumor produces impairments in hippocampal-dependent cognition and elevates TNFα and IL-6 in plasma even when treated effectively with DTX. Experiments are ongoing to examine more acute changes in cytokine production, as well as changes in blood-brain integrity produced by the combination of tumor and DTX. Keywords: Cancer, Cognitive Impariment, Hippocampus, Inflammation Disclosure: Nothing to disclose. P436. Astrocyte-Neuronal Metabolic Coupling in the Cingulate Cortex Promotes Chronic Pain Development Giannina Descalzi, Kaitlin Scherer, Paige Reid, James Tang University of Guelph, Guelph, Canada Background: Chronic pain is a major risk factor for anxiety and depression, with over sixty percent of people with chronic pain experiencing severe depression and anxiety; significant reductions in physical and social functioning; and underemployment or job loss due to their pain. Tragically, the risk of death by suicide is doubled in people with chronic pain. Present treatment options are woefully inadequate, with less than half of all patients reporting pain relief with current treatments. Human and rodent neuroimaging studies indicate that chronic pain corresponds with reorganization of an emotion-pain brain circuit, and evidence indicates that neuroplasticity of the anterior cingulate cortex (ACC) is a critical step in this reorganization. We previously showed chronic pain and fear learning enhance neuronal excitability and induce similar plasticity-related gene expression changes in the anterior cingulate cortex of mice. We recently showed that fear learning requires astrocyte-neuronal lactate shuttling (ANLS) in the dorsal hippocampus, and that ANLS is necessary for learning-induced associated molecular changes, including increases in plasticity-related gene expression. Here we present data that indicate that ANLS in the ACC may also be involved in neuroplasticity associated with murine models of chronic pain. Methods: Male adult mice were exposed to chronic inflammatory pain, modelled by injection of complete Freund’s adjuvant (CFA) into the hindpaw. Vehicle injections were used as control. Pain thresholds were measured at 3 hours (h) 24 h, 72 h, and 7 days post-injury, and ACC samples were extracted immediately after threshold testing. Lactate levels were quantified through a colorimetric lactate assay, and western blot analyses determined the expression of proteins involved in astrocyte-neuronal lactate shuttling. Temporary, continuous inflammatory pain was modelled through formalin injections into the hindpaw, with vehicle injections serving as control. We used antisense oligonucleotides (AS-ODN) targeting monocarboxylate MCT4, which is exclusively expressed on astrocytes, to decrease the export of lactate out of astrocytes. Results: Male (n = 10/group) mice showed a rapid increase in lactate levels in the ACC, detectable at three hours post CFA injection compared to vehicle injected controls. These levels returned to baseline by one day post injury, but steadily increased thereafter, resulting in significantly larger levels of lactate seven days post injury, compared to non-pain, vehicle-injected control mice. Mice injected with MCT4 AS-ODN in the ACC showed significant reductions in MCT4 protein expression in the ACC 24 hours (hrs) after injection, compared to injections of a non-coding, control scrambled ODN. We found that male mice that had intra-ACC MCT4 AS-ODN 24 h prior to formalin injection showed significantly less pain-related licking behavior in the formalin test, compared to non-coding, scramble control, which was rescued by intra-ACC injection of lactate 15 min prior to formalin injection (n = 9/group). Conclusions: Our data indicates that astrocyte-neuronal coupling is critically involved during the early stages of chronic pain development. Furthermore, our data indicate that disrupting astrocyte-neuronal lactate shuttling in the ACC blocks the persistence of pain in male mice. Keywords: Astrocyte, Chronic Pain, Astrocyte-Neuronal Lactate Shuttle Disclosure: Nothing to disclose. P437. α5-GABAA Receptor Positive Allosteric Modulation Reverses Cognitive Deficits and Neuronal Atrophy Across Animal Models Thomas Prevot, Ashley Bernardo, Michael Marcotte, Kayla Wong, Prithu Mondal, James Cook, Etienne Sibille University of Toronto, Centre for Addiction and Mental Health, Toronto, Canada Background: Multiple psychiatric, stress-related, and neurodegenerative disorders exhibit reduced GABA/somatostatin (SST) signaling. SST + interneurons from cortical layers and the hippocampus exert dendritic inhibition onto excitatory neurons, largely through α5-containing GABAA receptors (α5-GABAAR). Our group showed that α5-positive allosteric modulation contributes to alleviating working memory deficits and reverses neuronal atrophy in old mice, and in stressed mice. Here, we aimed to confirm our prior results in an independent aged cohort and further investigated the behavioral and neurotrophic effects of an α5-positive allosteric modulator (α5-PAM) in animal models of other conditions associated with reduced GABAergic function such as chronic stress and β-amyloid load. Methods: Three studies are presented, with ~N = 12 mice/group, 50% female: 1) Young C57BL6 subjected to unpredictable chronic mild stress (UCMS) to induce cognitive deficits. 2) 20-month-old C57BL6 with an existing cognitive decline. 3) 5xFAD transgenic mice with progressive amyloid-related cognitive decline. In all studies, the efficacy of chronic administration of the α5-PAM (30 mg/kg, p.o, for 4 weeks) at rescuing cognitive deficits across 3 domains was assessed. Working memory was assessed in an alternation task in the Y-maze, spatial learning and memory in the water maze, and cognitive flexibility in a set-shifting assay. Brains were then stained using the Golgi-Cox technique (n = 4brain/group; 8cell/brain), sectioned, and mounted for quantification of dendritic length and spine density in the prefrontal cortex and hippocampus (NeuroLucida). All techniques and studies employing laboratory animals were in accordance with Ontario Animals for Research Act (RSO 1990, Chapter A.22), Canadian Council on Animal Care (CCAC), and approved by CAMH Animal Care Committee. Results: Chronic treatment in stressed, old, or 5xFAD mice reversed cognitive deficits across domains in each model (ps<0.01) with a strong effect on working memory. Chronic treatment also consistently reversed UCMS-, age- or amyloid-induced dendritic shrinkage and spine loss at apical and basal dendrites (p < 0.001 in PFC and CA1). Conclusions: Together, the presented results support that selective targeting of α5-containing GABAA receptors overcomes chronic stress-, aging- or amyloid-related cognitive deficits upon chronic treatment, and reverses detriments in neuronal morphology. Altogether, the data suggest a positive impact on both symptomatic and disease-modifying dimensions, highlighting the high therapeutic potential of α5-PAMs. Keywords: Cognition, Dendritic Spine, Animal Models, α5 GABAA Positive Allosteric Modulator Disclosure: Nothing to disclose. P438. Valbenazine Effects on the Dopamine System in Humans, as Measured by [11 C]-PHNO Positron Emission Tomography (PET) Ryan Terry-Lorenzo, Daniel Albrecht, Satjit Brar, Graham Searle, Frans Van Den Berg, Ilan Rabiner, Dietrich Haubenberger Neurocrine Biosciences, Inc., San Diego, California, United States Background: Valbenazine, a potent, selective, orally active vesicular monoamine transporter 2 (VMAT2) inhibitor, is approved by the US Food and Drug Administration for the treatment of tardive dyskinesia (TD) at doses of 40 to 80 mg once daily. By inhibiting VMAT2, valbenazine disrupts the packaging of monoamines into synaptic vesicles, subsequently decreasing the release of monoamines including dopamine into the synaptic cleft. The reduction of dopamine is thought to be the foundational basis for the efficacy of valbenazine in treating TD and other hyperkinetic movement disorders. Reduction in synaptic dopamine is also a rationale for the potential utility of valbenazine in the treatment of psychosis. To our knowledge, however, there has not been a demonstration of dopamine reduction by valbenazine, or any other VMAT2 inhibitor, in humans. The aim of this study was to investigate the change in synaptic dopamine following valbenazine administration, using positron emission tomography (PET) imaging in healthy human volunteers. Methods: Imaging and tolerability data in this adaptive study were collected and analyzed in cohorts of 2-4 healthy volunteers. For each scan, participants received an injection of the D2/D3 dopamine receptor agonist radioligand [11 C]-PHNO, followed by 90 min of data acquisition using Siemens Biograph PET/CT. For post-valbenazine scans, participants received an oral dose of valbenazine 6-8 hours before the administration of [11 C]-PHNO, with PET imaging occurring around the time of maximal valbenazine plasma concentration. The binding potential relative to the non-displaceable binding (BPND) in the putamen, caudate, and ventral striatum, was used as the primary endpoint. The cerebellum was used as the reference region to estimate the regional BPND. Decreases in synaptic dopamine following valbenazine administration corresponded to an increase in [11 C]-PHNO BPND relative to baseline (ΔBPND). Plasma concentrations of valbenazine and (+)-α-dTBZ (dihydrotetrabenazine), the active metabolite of valbenazine, were measured at the start and end of each post-valbenazine PET scan. The mean plasma (+)-α-dTBZ concentration (Cave) was matched to the ΔBPND for each participant to provide an exposure- response curve. Results: To date, 9 participants (5 male, 4 female) have completed the trial. These participants received between 40-160 mg valbenazine, which resulted in plasma (+)-α-dTBZ Cave between approximately 10-60 ng/mL. Eight participants displayed valbenazine-induced, dose-dependent increases in [11 C]-PHNO ΔBPND (21-44%). Higher exposures to (+)-α-dTBZ from higher doses of valbenazine resulted in greater ΔBPND, revealing a monotonic exposure-response curve. Adverse events in this study were consistent with the known safety and tolerability profile of valbenazine, as reported in TD clinical trials. Conclusions: Valbenazine appears to decrease synaptic dopamine in a dose- and concentration-dependent manner, as indicated by an increase in [11 C]-PHNO ΔBPND. The approximately 20-40% [11 C] PHNO ΔBPND increase observed in this study is similar to the [11 C]-PHNO ΔBPND seen previously following treatment with a tyrosine hydroxylase inhibitor to deplete dopamine (Caravaggio et al, Neuropsychopharmacology 2014;39:2769). Thus, at pharmacological and therapeutic valbenazine doses, biologically meaningful dopamine decreases were observed in humans. These data will enable future exploration of the relationship between VMAT2 inhibition and the potential treatment of other central nervous system disorders. Keywords: Positron Emission Tomography (PET), VMAT2, Biomarker Disclosure: Neurocrine Biosciences, Inc.: Employee (Self) P439. Dose Dependent Effects of Acute Methamphetamine on EEG Alpha Power, Self-Reported Stimulation and Blood Pressure in Healthy Adults Connor Haggarty, Conor Murray, Royce Lee, Ilaria Tare, Harriet de Wit The University of Chicago, Chicago, Illinois, United States Background: Methamphetamine (MA) is a stimulant drug characterized by increased feelings of stimulation, cardiovascular effects, and changes in electrophysiological function. In particular, previous studies indicate that stimulants decrease alpha power in EEG studies, consistent with increased wakefulness. In the present study, the subjective, physiological, and cortical effects of acute MA (10 and 20 mg) were measured in healthy volunteers to examine relationships between its effects on alpha power and other measures. Methods: Healthy volunteers (N = 29), aged 18-35, participated in a within-subject, double-blind procedure in which they received a placebo, 10 mg, and 20 mg MA in 4-hour sessions at least four days apart. During the sessions, they completed subjective effects questionnaires and heart rate and blood pressure measurements were obtained. One hour after ingesting the capsule, resting state EEG measures were obtained to determine power at five frequency bands, with eyes open. Data were analyzed from electrodes representing the default mode network. Results: MA (10 and 20 mg) dose-dependently increased ratings of drug liking and vigor, and increased mean arterial pressure, compared to placebo. MA (10 and 20 mg) significantly decreased alpha power in a linear fashion, without significantly affecting other frequencies. The decrease in alpha power after MA (20 mg) significantly correlated with increases in feelings of vigor, but it was not correlated with increases in blood pressure. Conclusions: These data support the association between increased feelings of arousal and decreased alpha frequency power. Single doses of MA increased both subjective stimulation and blood pressure and decreased alpha power, but only the subjective effects were related to the EEG measure. This supports the EEG measure as an index of cortical processing relating to acute drug effects. Keywords: Methamphetamine, EEG, Subjective Effects Disclosure: Nothing to disclose. P440. Clinical Psychopharmacology: Racial and Ethnic Issues Andrew Olagunju, Jeffrey Wang McMaster University, Hamilton, Canada Background: There are advantages and positive contributions of ethnicity and race to health and wellbeing (e.g., through self/collective identity). However, belonging to a racial or ethnic group has also been linked with health-related disadvantages and negative perceptions —racism, stereotyping, and discrimination. A good knowledge of the ethnic/racial classification and groups in the particular setting of practice is beneficial for a culturally sensitive clinical practice and decisions in psychopharmacology. It is with this background that we pursue this study to survey and describe current literature on ethnic and racial issues in the practice of clinical psychopharmacology. Methods: This study is conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guideline. We searched major databases, including Pubmed/Medline, PsycINFO, Embase, CINAHL, and Web of Science for eligible studies published till date. The search string combined MeSH terms for clinical psychopharmacology, race and ethnic. This was supplemented by snowball searching of references in relevant studies and authors were contacted to request their work where necessary. All included studies will be rated with the National Institutes of Health Study Quality Assessment Tools based on study designs. Data extraction and quality assessment is conducted on the included studies. Results: A total of 57 reports are identified for title and abstract screening. This will be followed by data extraction and analysis. Conclusions: Through this review, we hope to highlight relevant issues to promote racial diversity and equity in the practice and training of psychopharmacology. The integration of ethnic matching and adequate racial/ethnic representation in study participants enrolled into clinical trials on psychotropics and other research activities will be addressed. Keywords: Racial/Ethnic Differences, Clinical Psychiatry, Psychopharmacology Disclosure: Nothing to disclose. P441. Old Rats With Fetal Alcohol Exposure Show Impaired Learning and Memory Functions and Elevated Levels of Various Biochemical Markers of Alzheimer’s Disease in the Brain Shaista Chaudhary, Dipak Sarkar Rutgers University, New Brunswick, New Jersey, United States Background: The influence of alcohol consumption during pregnancy could alter phenotype in the offspring and can have devastating and persistent consequences, such as central nervous system dysfunction. However, it is not known whether fetal alcohol exposure promotes physiological and biochemical characteristics of Alzheimer’s disease during aging. Methods: We employed an established first and second trimester human equivalent rat model of fetal alcohol exposure comprising of feeding liquid diet containing ethanol at a concentration of 6.7% v/v, or pair-feeding an isocaloric liquid diet or ad libitum feeding rat chow from gestational days 7 through 21 in isogenic Fischer 344 (F344) rats. Pups were weaned on postnatal day 21 and housed by sex, and were used for behavioral and biochemical studies at about 12 months of age. In each experimental group, only one male and one female offspring from a rat litter was used. Six rat offspring were assigned in each group. All animal care and procedures were approved by the Rutgers Institutional Animal Care and Facilities Committee and complied with National Institutes of Health policy. We measured learning and memory behavior using Morris water maze test. Since cognitive and memory impairments are often related to cholinergic dysfunction and acetylcholinesterase (AChE) activity, we evaluated the activity of AChE in cerebral cortex and hippocampus regions of the brain. Using Western blot assays we measured the levels of some biochemical markers of Alzheimer’s disease, including β-amyloid (Aβ) and Aβ1-42 proteins, total and phosphorylated tau proteins, β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and netrins receptor UNC5C proteins in cerebral cortex and hippocampus regions of the brain. Data were statistically evaluated using two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test in order to determine the time-dependent changes between groups. Multiple group comparisons at single time point were made using one-way ANOVA followed by Tukey’s post-hock test. A p value less than 0.05 was considered significant. Results: Determination of cognitive and memory functions using Morris water maze test showed enhanced escape latency in both male and female fetal alcohol exposed (alcohol-fed offspring) rats than control (both ad lib-fed and pair-fed offspring) rats at age 12 months. In addition, fetal alcohol exposed rats showed reduced time spent escaping the platform’s quadrant, indicating a significant deficiency in memory in these animals. The magnitude of fetal alcohol effects on learning and memory behaviors appears to be similar in both male and female offspring. These animals also had elevated levels of AChE activity, Aβ and Aβ1-42 proteins, hyper-phosphorylated tau, BACE1 and UNC5C proteins in the cerebral cortex and hippocampus regions of the brain during aging. The magnitude of enzyme and protein level responses to FAE was also similar in both male and female offspring. Conclusions: Overall, these findings provide evidence that fetal alcohol exposure increases the expression of some of the behavioral and biochemical phenotypes of Alzheimer’s disease during aging. Keywords: Fetal Alcohol Spectrum Disorder, Neurodevelopmental Disorders, Alzheimer’s Disclosure: Nothing to disclose. P442. Elucidating Brain Networks Subserving Working Memory Task Performance Using Interpretable Deep Learning Mario Serrano-Sosa, Philip Tubiolo, Thomas Hagan, John Williams, Chuan Huang, Jared Van Snellenberg* Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States Background: Working memory (WM) is a key cognitive domain and is disrupted in many psychiatric and neurological disorders. While a bilateral network of brain regions including dorsolateral prefrontal cortex (DLPFC), pre-supplementary motor area (pre-SMA), and intraparietal sulcus (IPS) reliably activate during WM tasks, less is understood about how activation in these regions relates to behavioral measures of WM ability. One major challenge is the high dimensionality of fMRI data, which generally limits analyses to single-voxel mass-univariate linear model approaches, or similar. However, recent work in computer vision has demonstrated that some neural networks (convolutional neural networks; CNNs) show good performance in classification and prediction from image data. CNNs learn hierarchical features of images across different levels of abstraction in a non-linear and data-driven manner. Critically, while deep learning algorithms are often “black boxes”, network back-propagation can be used in a CNN to determine which image locations are most salient for network performance. These “saliency maps” can be generated from a trained network for each input, and reflect the gradient of the output prediction, indicating which image locations most strongly drive network performance. Here we use data from an n-back WM task from the Human Connectome Project (HCP) and a CNN to identify cortical regions whose activation during WM are predictive of WM performance in a whole-brain, non-linear, data-driven manner. To our knowledge, this work reflects the first use of emerging developments in computer vision to identify brain regions whose activity is predictive of cognitive task performance using fMRI data. Methods: We used n-back task data from 419 subjects (male and female) of the HCP 1200 Subjects release, using only subjects with 3 T MR data, 100% WM task completion, no significant quality control issues (issues A, B, or C as per HCP documentation), and MSMAll-registered data. We used only one subject from each kinship set to prevent the CNN from learning features shared by genetically related individuals. Cortical surface data of the 2-back - 0-back contrast (all stimulus conditions) was extracted from CIFTI images and placed on a flattened surface. Left and right hemispheres were concatenated to form a single 2d image for input to the CNN. The CNN used a U-net architecture with 4 convolutional blocks followed by 3 fully connected layers. The network was trained to predict % correct scores on the 2-back condition for each participant. We employed 5-fold cross-validation to obtain blind predictions and saliency maps for each subject: 5 independent networks were trained, each using 335 subjects for training and 84 subjects for validation. Saliency maps were generated via back-propagation using the SmoothGrad algorithm to reduce spatial noise. To benchmark CNN performance we also trained a kernel ridge regression (KRR) model. Saliency maps (bounded between 0 and 1) were mean-centered for each subject to permit valid null-hypothesis tests. Maps were then used to identify regions with significantly above-average salience by testing the null hypothesis that salience < = 0 using Permutation of Linear Models (PALM) at P < 0.025, FWE corrected. Saliency maps were then z-scored and subjected to principal component analysis and correlated with task performance. The above analyses used dummy regressors for the 5 validation sets to remove systematic differences in salience across the 5 independent CNNs. Results: The CNN outperformed KRR on all metrics evaluated (R2, MAE, MAPE, and RMSE), with R2 of 0.389 (0.373 for KRR). We observed signficantly above-average salience for bilateral ventrolateral PFC, left premotor cortex, and several default-mode network (DMN) regions including bilateral anterior medial PFC (amPFC), precuneus and retrosplenial cingulate cortex, temporal-parietal junction, temporal poles, and left middle-temporal gyrus. The majority of left and all of right DLPFC and bilateral intraparietal sulcus did not exhibit above-average salience. Four principal components (1, 2, 3, and 9) were significantly associated with task performance, with PC1 showing the strongest relationship (r = -0.54). Loadings for PC1 revealed that strong task performance was associated with high salience in amPFC, medial temporal lobe, left middle temporal gyrus, and temporal pole, as well as low salience in bilateral DLPFC, IPS, and pre-SMA. Conclusions: These results demonstrate that interpretable deep learning using CNNs and saliency maps holds promise as a novel means to gain insight into brain regions whose activation is associated with task performance. Contrary to expectations, our results suggest that the magnitude of deactivation in DMN contains more information about WM task performance than does activation of DLPFC, IPS, or pre-SMA. PCA results further suggest that activation in these latter regions is predictive of performance, but only in low-performing individuals. Because greater activation in these regions was associated with high performance, this suggests that only low levels of activation are predictive of poor performance—once activation in this network reaches some level, the CNN instead attends to deactivation in amPFC and other DMN regions to identify the strongest performers. While in its infancy, this approach has clear potential to produce new insights into how the human brain subserves higher-order cognition in both health and disease. Keywords: Working Memory fMRI, Convolutional Neural Network (CNN), Human Connectome Project (HCP), Multivariate Approaches, Deep Learning Disclosure: Nothing to disclose. P443. A Novel Dissociation Between Two Parallel Thalamo-Striatal Pathways Encoding Distinct Motivational States Sofia Beas, Isbah Khan, Claire Gao, Emma McDonald, Alison Bashford, Shakira Rodriguez-Gonzalez, Mario Penzo The University of Alabama at Birmingham, Birmingham, Alabama, United States Background: Previous research has mainly centered on investigating the contribution of the dopaminergic mesolimbic system to these processes. Yet, the role that glutamatergic inputs to the NAc play in the control of motivation is far less clear. The paraventricular nucleus of the thalamus (PVT), a brain region that integrates bottom-up interoceptive signals with top-down cortical information, sends robust glutamatergic projections to the NAc. Recently, we identified two major distinct subpopulations of neurons in the PVT (Type1PVT and Type2PVT) that differ in their genetic identity, connectional features, and functionality. These subpopulations send divergent projections to the NAc, which raises the possibility that Type1PVT and Type2PVT neurons are likely to enable different but complementary aspects of motivated behavior. However, very little is known about the involvement of thalamic inputs to the NAc in the mediation of motivational processes. Methods: Here, we trained female and male food-restricted mice (n = 6 - 8) to perform a linear runway foraging task. In this task, mice are trained to run from a trigger zone to the reward zone in the maze to retrieve a food reward (strawberry Ensure). Using bulk calcium imaging and fiber photometry, we investigated the in vivo dynamics of the Type1PVT (using Drd2-Cre mice and Cre- dependent GCaMP6s) and Type2PVT (using Drd2-Cre mice and a CreOFFGCaMP6f) parallel thalamo-striatal pathways while mice performed the linear runway foraging task. Results: Our findings showed that the reward approach was associated with a prominent increase in GCaMP fluorescence in the Type1PVT–NAc pathway. This increase in fluorescence was quantified using the area under the curve, which revealed a significant increase in activity of this pathway when compared to baseline activity (t(18) = 4.6, p < 0.01) and when compared to GFP injected controls (t(18) = 4.6, p < 0.01). Moreover, we found that the activity of the Type1PVT–NAc pathway varies with aspects of motivation such as behavioral vigor and level of satiety. In contrast, Type2PVT–NAc neurons showed opposing in vivo dynamics and were not sensitive to motivational variables, suggesting that this pathway may participate in regulating other aspects of goal-oriented behavior. Conclusions: Altogether, the results gathered from this study show a novel dissociation between the Type1PVT–NAc and Type2PVT–NAc pathways and identify a specific neuronal subpopulation of the PVT that signals motivational states. Keywords: Motivation, Paraventricular Nucleus of the Thalamus, Nucleus Accumbens Disclosure: Nothing to disclose. P444. Neuropsychopharmacology (NPP) Special Projects Update: Efforts to Enhance Rigor in Clinical Trials Research and Promote Early Career Investigators Sofiya Hupalo, Chloe Jordan, Terri Bowen, Keri Martinowich, William Carlezon, Tony George National Institute of Mental Health, National Institutes of Health, North Bethesda, Maryland, United States Background: NPP engages in special projects which provide insights into journal function and support outreach efforts to promote researchers in the fields represented within NPP and ACNP. In this report, we describe recent activities the NPP Special Projects Team has spearheaded that aim toward improving rigor and transparency in clinical trials research and creating opportunities for early career investigators to publish in NPP. Methods: To examine the rigor and transparency of clinical research published in NPP, we examined the frequency with which authors provide CONSORT documents (Consolidated Standards of Reporting Trials) during the manuscript submission process. CONSORT documents report experimental details such as how the clinical trial was designed, analyzed, interpreted, and describe the flow of participants through the trial. We tracked NPP manuscript submissions to record the number of clinical trial reports being submitted and the proportion of submissions that provide complete CONSORT materials. To promote early career scientists in the field of neuropsychopharmacology, in October 2021 NPP launched the Early Career Commentary for trainees and early-stage investigators to share their scientific ideas and perspectives. Pre-submission inquiries are required and are reviewed by the NPP Special Projects Team and Editorial Board. Priority is given to inquiries that focus on novel and timely topics (e.g., scientific, social, policy, equity) of broad interest and importance to the NPP and ACNP community. Selected inquiries are invited to proceed with submission and undergo standard NPP peer review. The Special Projects Team has invited authors of each Early Career Commentary to participate in NPP’s “Meet the Author” Interview series. These interviews provide the authors an opportunity to promote their commentary and discuss their research and career goals. The recorded interviews are posted on NPP’s YouTube channel and shared on Twitter. Results: Clinical Trials Submissions: In 2020, NPP received 140 manuscript submissions that were identified as a clinical trial by the submitting author. Only 70 (50%) of these submissions included complete CONSORT documents. When journal staff followed up with corresponding authors of the 70 submissions that did not include complete CONSORT documents, 14 authors requested an exemption and 5 withdrew their submission. Ongoing tracking of clinical trial manuscripts submitted in 2022 reveals a similar rate of non-compliance (~55%) in providing complete CONSORT documentation on the first submission. Early Career Commentary: Since the launch, NPP received 36 pre-submission inquiries for the Early Career Commentary. Of these submissions, 7 were ineligible due to one or more of the authors not having early-stage investigator status. NPP additionally considered all pre-submission inquiries as topics for invited reviews unless the inquiry was withdrawn by the authors. To date, NPP has invited 4 groups to author an Early Career Commentary and an additional 3 groups to write an invited Perspective or Review article based on the proposed topic. Two Early Career Commentaries and one Perspectives article have been published. Collectively, these manuscripts have been accessed 7,162 times with Altmetric online attention scores ranging from the 85th-97th percentile. Conclusions: To improve adherence to transparency and accountability in clinical trial reports published in NPP, the journal is implementing changes to the manuscript submission process. Text describing the definition of a clinical trial is now included directly on the submission webpage with links to information on these definitions. Submitting authors are now required to confirm whether their manuscripts meet the definition of a clinical trial and verify that the submission includes CONSORT documents and the clinical trial registry number. These changes will improve the speed and thoroughness of peer review, improve transparency in reporting clinical trial methodology, and increase the impact of work published in NPP. The Early Career Commentary was created to provide a venue for early career investigators to share ideas and perspectives independently of senior mentors or collaborators. NPP has received many pre-submission inquiries and has selected additional topics for invited Reviews and Perspectives articles. The articles that have resulted from this initiative have been widely read and shared on social media, demonstrating that this is a successful approach for promoting and supporting early career scientists in the NPP and ACNP community. Keywords: Clinical Trial, Clinical Trial Methodology, Career Development Disclosure: Nothing to disclose. P445. Pharmacokinetics of Cannabidiol: A Systematic Meta-Regression Analysis to Guide Clinical Trials Ehsan Moazen-Zadeh, Alexandra Chisholm, Keren Bachi, Yasmin Hurd Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Cannabidiol (CBD) has exponentially gained attention in research and clinical applications as a potential treatment of several neuropsychiatric and general medical conditions. Today, many CBD-based formulations are in development aiming for FDA approval and numerous non-approved CBD preparations are available over the counter. However, many questions raised by clinicians, researchers and consumers of CBD products often relates to dosing and administration. One of the major challenges that has prevented quantitative aggregation of evidence in previous reviews of pharmacokinetic (PK) studies of CBD, has been different units and scales of reporting outcomes. We aimed to provide an updated systematic assessment of available evidence on the pharmacokinetics of CBD; provide comparable values of PK parameters from different studies on the same scale; demonstrate patterns in outcomes based on the CBD dose and route of administration; and explore the simultaneous impact of different factors on PK outcomes using meta-regression models. Methods: This systematic review and meta-regression analysis was pre-registered (PROSPERO: CRD42021269857). We systematically searched Medline, Embase, PsychInfo, and Web of Science Core Collection on September 19, 2021. Trials of CBD (pure CBD or in combination with THC) in healthy adults were included if they reported at least one of the PK parameters of interest in serum or plasma, including: time from medication administration to the maximum concentration of CBD in plasma/serum (Tmax), maximum concentration of CBD in plasma/serum (Cmax), the area under the curve of serum/plasma concentrations plotted against time from medication administration to a specific time-point, usually the end of the PK session (AUC0-t), the extrapolation of AUC0-t to the infinity time point (AUC0-inf), and the time needed for the concentration of the drug in the plasma to be reduced by 50% (T1/2). Studies of patient populations or CBD co-administration with other medications were excluded. The National Heart, Lung, and Blood Institute’s Quality Assessment Tool for Before-After Studies with no Control Group was used. Random-effects multivariable meta-regression analysis was conducted (alpha=0.05) with hierarchical models of different factors as independent variables and PK parameters as the dependent outcomes. Results: A total of 97 trial arms from 35 studies were included; 26 trial arms had a “Good” quality, 56 “Fair,” and 15 “Poor.” Six arms used inhalation CBD, 29 oromucosal, 61 oral, and 1 intravenous. CBD formulations could be categorized to nanotech (n = 13), oil-based (n = 19), alcohol-based (n = 10), water-based (n = 4), Sativex (n = 17), and Epidiolex (n = 21). For single-dose studies, CBD doses ranged between 2-20 mg in inhalation, 5-50 mg in oromucosal, and 0.42-6000 mg in oral administration. Sixty trial arms had only male participants or a higher number of males than females. The duration of the PK session was between 4h-164h. In meta-regression models, a higher CBD dose was consistently associated with a higher Cmax, AUC0-t, and AUC0-inf across all the models (p-value<0.001). Compared to oral administration, oromucosal administration was associated with lower Cmax, AUC0-t, and AUC0-inf (p-value between 0.001 and 0.007). Fed status was associated with higher Cmax and AUC0-t when compared to the fasting status (p-value<0.001). A higher ratio of female participants was associated with lower Tmax in oral administration and higher Cmax and T1/2 in oral and oromucosal administration (p-value between 0.001 and 0.023). Longer study duration was associated with higher AUC0-inf and T1/2 in all models (p-value<0.001). The highest percentage of variability in the data that could be explained by a model (R2) was 84% for Tmax, 53% for Cmax, 55% for AUC0-t, 54% for AUC0-inf, and 92% for T1/2. Conclusions: In exploring how different factors potentially influenced the PK outcomes of CBD, consuming food while taking CBD, female sex, and oromucosal administration were associated with higher bioavailability. Recommendations for future research would mainly concern conducting original systematic studies to elucidate the impact of biological sex, duration of PK session, and different formulations in single studies with multiple arms. It would also be beneficial for more studies to examine CBD doses in ranges currently being used clinically, given the increasing number of CBD preparations on the market and their potential application in clinical practice. Finally, reporting PK parameters in both arithmetic and geometric scales would help comparisons across studies, knowledge aggregation, and replicability. Keywords: Cannabidiol, Population Pharmacokinetics, Serum Levels, Therapeutic Drug Monitoring Disclosure: Nothing to disclose. P446. Genetic Basis of Paclitaxel-Induced Peripheral Neuropathy Model Traits in a C57BL/6 Reduced Complexity Cross Jacob Beierle, Bryan McKiver, Arthur Vanvalkenburg, Emily Yao, Jared Mann, Binh-Minh Nguyen, Kayla Richardson, William Johnson, Imad Damaj, Camron Bryant Boston University School of Medicine, Boston, Massachusetts, United States Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a heritable side effect in cancer therapeutics, yet the genetic basis is largely unknown. (PAC) exerts antitumor effects through stabilization of microtubules and interruption of cell division and is accompanied by CIPN in 60-70% of patients, resulting in numbness, cold hypersensitivity, and allodynia. Human genetic variants have been associated with CIPN within/near genes associated with cytoskeletal, neurodevelopmental, and pharmacokinetics. Discovery-based genetics in rodents offers a powerful, complementary approach to uncover new genetic and biological insight into CIPN, with the goal of improving chemotherapeutic profiles. Genetic crosses in closely related rodent substrains (reduced complexity crosses; RCCs) permit rapid identification of causal genes/variants underlying complex traits like CIPN, providing > 2-order magnitude reduction in the number of segregating variants within a locus and providing 2 nearly isogenic backgrounds to edit/validate necessity and sufficiency of causal variants. Our goal was to identify C57BL/6 substrain differences in CIPN model traits, conduct quantitative trait locus (QTL) mapping of behavior and gene expression and conduct transcriptome analysis of PAC Tx. We then identified candidate genes and downstream biological pathways associated with CIPN model traits. Methods: We assessed CIPN model behaviors over 1 month in C57BL/6 J (J) versus C57BL/6NCrl (NCrl) mouse substrains following a regimen of paclitaxel (PAC: 2 mg/kg, every 48 h, 4X) vs. vehicle (VEH: 1:1:18 – ethanol, kolliphor, water), including mechanical hindpaw hypersensitivity (von Frey), cold hindpaw hypersensitivity (acetone test), dorsal caudal nerve conductance, sucrose preference, and tactile hyposensitivity (plantar hindpaw surface adhesive test). Following identification of substrain differences, we made a reciprocal F2 cross and phenotyped 182-206 F2 mice (½ VEH-Tx, ½ PAC-Tx). Genotyping was conducted using the miniMUGA DNA array, with > 200 genetic markers distinguishing J vs. NCrl. QTL mapping was conducted in R/qtl in 182-206 F2 mice using Hayley-Knott regression (1000 perm) to determine significance thresholds. Bayes credible intervals comprised confidence intervals. Treatment (Tx) was included as an interactive covariate and Sex and Cohort as additive covariates. RNA-seq of J and NCrl was conducted on dorsal root ganglia (DRG), spinal cord (SC), and periaqueductal gray (PAG) using poly-A selected RNA. We used Illumina Nova-Seq, with 100 bp paired-end reads and yielding > 30 million paired-end reads per sample. Results: We identified robust inbred mouse substrain differences in CIPN model traits between NCrl and J, with J mice showing robust hypersensitivity to mechanical stimulation and cold hypersensitivity following PAC Tx. QTL mapping in an F2 RCC identified a major QTL on chr.1 mediating PAC-induced mechanical hypersensitivity on Day(D)7 (LOD = 7.7; p < 0.001; chr.1: peak 69 Mb:; Bayes: 47-76 Mb), and a trending QTL on chr.14 on D15 mediating cold hypersensitivity (LOD = 4.4; p = 0.095; chr.14: peak: 124 Mb; Bayes: 23-125 Mb). For the chr.1 QTL for mechanical hypersensitivity, there are 2 compelling candidate causal genes with coding variants, including Abca12 (splice site, chr1: 71 Mb; a gene encoding a known efflux transporter for PAC) and Bmpr2 (chr.1: 60 Mb; bone morphogenic protein receptor 2, missense). Differential gene expression analysis of parental substrains via RNA-seq within the chr.1 QTL interval identified a Substrain x Treatment interaction in Abca12 expression in SC (p = 0.02; J > NCrl). In contrast, neither RNA-seq nor protein analysis identified any differences in Bmpr2 transcript levels (DRGs, SC, PAG), BMPR2 protein levels (DRGs), or in the downstream signaling target SMAD 1/5/9 (DRG protein expression and phosphorylation). For the chr.14 QTL interval for cold hypersensitivity, we identified two candidate genes with missense mutations, including Abcc4 (chr.14: 119 Mb) and Nalcn (chr.14: 124 Mb). Abcc4 codes for an efflux transporter that transports chemotherapeutics to influence toxicity and Nalcn (Na+ leak channel, non-selective) codes for a voltage-gated Na+ channel implicated in chronic pain and was mapped in drosophila studies for chemotherapy toxicity. To identify biological pathways modulated by PAC in the CIPN-sensitive J substrain on D7 (chr.1 QTL), enrichment analysis identified GO enrichment terms (p < 0.01; padjusted<0.2) that included insulin-like growth factor receptor signaling, epithelial morphogenesis, centromeric sister chromatid cohesion, and elastic fiber (up in DRG), as well as tyrosine catabolic process (down in DRG). For SC, GO terms included skeletal muscle thin filament assembly, striated muscle hypertrophy, detection of/response to muscle stretch, skeletal myofibril assembly, muscle filament sliding, myotube cell development, and muscle tissue morphogenesis (up in SC). Conclusions: We identified distinct genetic loci for PAC-induced mechanical hypersensitivity and cold hypersensitivity. These loci contain candidate genes/variants involved in both pharmacokinetics (transport), pain/inflammation signaling pathways, and ion channels related to pain processing. Ongoing studies seek to identify additional functional evidence that would implicate a particular gene or variant prior to gene editing and causal validation studies. Keywords: Nociception, Hyperalgesia, Chemotherapy-Induced Peripheral Neuropathy, Pharmacokinetics, Allodynia Disclosure: Nothing to disclose. P447. Characterization and Optimization of Rat Models of Osteoarthritis Pain to Profile Novel Assets in Support of the NIH HEAL Initiative Preclinical Screening Platform for Pain (PSPP) Mark Urban, Elizabeth Dugan, Katelyn Buban, Jennifer Hagedorn, Sarah A. Woller, Smriti Iyengar, Taleen Hanania PsychoGenics, Inc., Paramus, New Jersey, United States Background: The National Institutes of Health Helping to End Addiction Long-termSM Initiative, or NIH HEAL InitiativeSM, Preclinical Screening Platform for Pain (PSPP) program aims to accelerate the discovery and development of novel non-opioid, non-addictive pain therapeutics. Under the HEAL initiative, the NIH is collaborating with PsychoGenics, Inc. to screen and profile novel assets in vitro and in vivo including small molecules, biologics, natural products, and devices. Assets are screened in vivo in rat models of pain to determine efficacy, and in additional tests to examine potential adverse effects and abuse liability. Efforts in the PSPP program are also focused on the characterization and optimization of disease-specific models of pain to evaluate select assets and provide further support for specific pain indications. Here, we describe one such effort to characterize and validate rat models of osteoarthritis pain, including the monoiodoacetate (MIA) and medial meniscal tear (MMT) models. Methods: Adult male and female Sprague Dawley rats (n = 10/group, each sex) were used in these studies. For the MIA model, MIA (0.3 – 4.5 mg) was injected intraarticularly into the left hindlimb knee joint. For the medial meniscal tear model (MMT) model, rats received a surgical procedure in which the medial collateral ligament was transected to reflect the meniscus towards the femur, and the meniscus was then cut at its narrowest point to simulate a complete tear. Behavioral pain endpoints included hind paw mechanical allodynia, knee joint mechanical allodynia, weight bearing deficits, and changes in gait. Behavioral pain phenotype in these studies was evaluated for 4-6 weeks following the induction of osteoarthritis. Pharmacology was examined by evaluating the effects of the reference analgesics morphine sulfate (3 mg/kg), ketoprofen (6 mg/kg), and duloxetine (60 mg/kg) after single and repeated administration at both early and later times following the induction of osteoarthritis likely representing initial and advanced disease states. Data were analyzed using two-way repeated measures ANOVA with Bonferroni’s or Dunnett’s post hoc test when appropriate. Effects p < 0.05 were considered to be statistically significant. Power analysis and effect size were determined using SAS/STAT, and appropriate sample size was based on a power value of 0.8 to ensure adequate power for F-tests for two-way interactions. Results: Intraarticular injection of MIA (0.3- 4.5 mg) into the hindlimb knee joint produced unilateral hind paw mechanical allodynia in male and female rats which was maximal at Week 2. Unilateral knee joint mechanical allodynia to pressure and pinch stimuli was observed in female, but not male, rats at Week 2. Weight bearing deficits associated with the affected hind limb were modest when measuring static weight bearing, but pronounced when measuring dynamic weight bearing with maximal effects observed at Week 1. In male rats, weight bearing deficits were stable for entire testing period through Week 6, whereas weight bearing deficits in female rats were observed during Weeks 1 and 2, and were no longer apparent by Week 4. Changes in gait were also observed in male and female rats following MIA injection at Weeks 1 and 2. In the MMT model, male rats that had received MMT surgery displayed unilateral hind paw mechanical allodynia that was maximal at Week 3, while changes in hind paw tactile sensitivity were not observed in female rats. Knee joint sensitivity to a pressure stimulus was unaffected in male and female MMT rats, and dynamic weight bearing was also unaffected in male and female rats that had received MMT surgery. Reference analgesic compounds were evaluated in the MIA model by examining effects on hind paw mechanical allodynia and weight bearing deficits at both early (Week 1) and later (Week 6) times following induction of osteoarthritis. In Week 1, single administration of morphine (3 mg/kg) significantly reduced mechanical allodynia and weight bearing deficits in male and female rats, whereas single administration of ketoprofen (6 mg/kg) or duloxetine (60 mg/kg) was less effective. In contrast, repeated administration of ketoprofen or duloxetine (4 days, b.i.d.) significantly reduced mechanical allodynia and weight bearing deficits in Week 1. In Week 6, morphine, ketoprofen, and duloxetine were ineffective in reducing weight bearing deficits. Conclusions: The results from these studies demonstrate that a variety of pain behaviors associated with knee joint osteoarthritis can be measured using the rat MIA model, while pain behaviors in the MMT model were less robust or not observable. Evaluation of novel assets following single and repeated administration in the MIA model using multiple pain endpoints may be a viable strategy to accelerate the development of non-opioid, non-addictive therapeutics for the treatment of osteoarthritis pain. The utility of evaluating novel assets at Week 1 vs Week 6 will continue to be examined. Keywords: Chronic Pain, Monoiodoacetate Model of Osteoarthritis, Preclinical Pharmacology Disclosure: Nothing to disclose. P448. A Novel Brain Permeable Epigenetic Inhibitor Ameliorates Neuropathic Pain in Mouse Changning Wang Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts, United States Background: Histone lysine residue acetylation/deacetylation represents an important epigenetic modification that affects gene expression without DNA sequence modification. Histone deacetylases (HDACs) are a class of amide hydrolases that catalyze a variety of substrates, including histones and other proteins. Eleven Zn2 + -dependent HDACs have been found in mammals, including class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10), and class IV (HDAC11), each of these isoforms has distinct functions in epigenetic regulation. Among them, HDAC11 is the most recently identified member of the class IV HDAC with accumulating evidence suggesting its connection with many diseases. A few HDAC11-selective inhibitors have been reported in recent years, yet none of them exhibited brain penetrability, nor have they been investigated in animal models of neurological disorders. Herein, we describe the development and characterization of a selective HDAC11 inhibitor PB94, which was identified by structural optimization of our lead compounds and evaluated its therapeutic efficacy on neuropathic pain. Methods: HDAC 1-11 Enzyme Inhibition Assays. HDAC inhibition assay of target compounds was carried out at Nanosyn (Santa Clara CA, United States). Test compounds were diluted in 100% DMSO using 3-fold dilution steps. TSA was tested in an identical manner. Chronic Constriction Injury (CCI). The mice (n = 8 in each group) were anesthetized in an anesthesia induction chamber. The left lower extremity was prepared with an alcohol swab. A 0.5 - 1 cm incision was made using a blade on the left lower extremity. The left side of the sciatic nerve was exposed in the mid-thigh. Four ligatures using 6.0 mm chronic gut sutures were loosely placed around the exposed sciatic nerve with a 1.0 - 1.5 mm interval between each ligature. Skin incision was closed with two 6-0 vicryl sutures. Mechanical withdrawal threshold. Mice were habituated by placing on a platform with a clear chamber individually 30 minutes daily for 3 consecutive days. Mechanical paw withdrawal thresholds (PWTs)) was carried out using calibrated manual Von Frey filaments. The positive response was recorded when the mice withdrew or shacked its paw during the stimulation. A negative response was followed by testing with the next larger filament. All CCI mice were tested before surgery (baseline) and 3, 5, 7, 10, and 14 days after surgery. Hindpaw withdrawal latency. Mice were placed on a preheated glass platform (28 - 29°C) and clear Plexiglas cubicles to acclimate to the testing room 30 minutes daily for 3 consecutive days before the testing. Paw withdrawal latency was defined as the time (seconds) from the initiation of heat exposure to the hind paw withdrawal. A cut-off time was set at 20 seconds to avoid tissue damage. Results: A series of lead compound analogues were synthesized to investigate the structure-activity relationship (SAR). Among them, PB94 had significant HDAC11 potency and the highest HDAC11 selectivity (IC50 = 108 nM and over 39-fold over other HDAC isoforms. Next, ADME assessments were carried out to evaluate the drug-like profiles of PB94. Our data indicated PB94 has good metabolic stability in human liver microsomal and mouse plasma, with half-lives (t1/2) of 54.6 min and 133.8 min, respectively. In addition, no significant inhibitory effects were observed on cytochrome P450 enzymes (CYPs) 1A2, 2C19, and 2D6 at 10 μM of PB94. PB94 has a reasonably good brain/plasma ratio of 2.3 at 30 min and 2.2 at 4 h post-injection by IP administrating PB94 at 1 mg/kg in C57BL/6 mice. We further tested its off-target binding in a panel of 46 targets (PDSP) and observed no significant off-target binding at 10 μM. To examine the potential roles of HDAC11 in a pathological status, we chose a mouse neuropathic pain model, chronic constriction injury to the sciatic nerve (CCI). At 14 days after surgery, HDAC11 protein was increased in the cortex compared with sham mice, indicating that HDAC11 is critically implicated in pain and that targeting HDAC11 might provide therapeutic effects. To test these, we administered PB94 in mice that underwent CCI to assess the development of nociceptive behavior. PB94 (10 mg/kg) was able to significantly alleviate mechanical and thermal pain-like behaviors during the entire experimental period of 14 days. Importantly, there were no significant side effects noticed during PB94 treatment. Mouse body weight remained similar between saline or PB94 treated animals. Conclusions: The promising drug-like properties of PB94 support its therapeutic potential for neurologic disorders. Since HDACs have been implicated in neurologic pain, we further determined whether PB94 treatment could ameliorate neurologic pain using the CCI mice model. The unilateral sciatic nerve chronic constriction injury surgery is associated with postoperative spontaneous pain, mechanical allodynia, and thermal hyperalgesia. In our study, a battery of tests was used to assess the analgesic effect of PB94 on pain-like behaviors, which led to findings supporting its effectiveness at 10 mg/kg. It has been indicated that HDAC11 is widely expressed in the brain in rodents. More interestingly, we found HDAC11upregulation in the somatosensory cortex of CCI mice. It is, therefore, reasonable to postulate that increased HDAC11 expression in the somatosensory cortex may be implicated in persistent nociception after CCI surgery, and inhibition of HDAC11 by PB94 in CCI could attenuate neuropathic pain. Keywords: Epigenetics, Pain Therapeutics, CNS Drugs Disclosure: Nothing to disclose. P449. Time-Dependent Differences on Pain Assessments After Orexin Receptor-1 Antagonism in a Mouse Model of Sickle Cell Disease Kimberlei Richardson, Alexa Ryan, Nia Sweatt, Victor Apprey, Robert Taylor, Kalpna Gupta Howard University, Washington, District of Columbia, United States Background: The orexins are neuropeptides that are synthesized in the perifornical area (PFA), lateral hypothalamus (LH) and dorsomedial hypothalamus (DMH) and mediate arousal, sleep, energy balance, food and drug reward. Data suggest that Orexin–A neuropeptide has an analgesic effect on inflammatory pain upon binding the orexin receptor-1. It may also affect mechanisms underlying the maintenance of hyperalgesia associated with neuropathic pain. Data from our prior study suggests that subpopulations of orexin neurons are preferentially recruited during behavioral assessments for hyperalgesia. We seek to determine whether there are differences in pain assessments after orexin receptor-1 antagonism (SB-334867) and whether that pain is potentiated in sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS-BERK) and age-/gender-matched controls (HbAA-BERK). Methods: Female transgenic HbSS-BERK and HbAA-BERK mice (n = 6/group, 20-30 g) were habituated to each test protocol for thermal/heat and mechanical hyperalgesia. Thermal/heat hyperalgesia was monitored using the Plantar Test (Hargreave’s Apparatus) for thermal stimulation. Latency was measured as the time (in seconds) for the mice to withdrawal their hindpaws from the heat stimulus. Mechanical hyperalgesia was monitored by the Von Frey filament test. Paw withdraw frequency was measured by the number of times that the mice lifted their hindpaws after tactile stimulation from the filament. The behavioral data were recorded prior to SB-334867 (20 mg/kg, IP), 1 hour, and 24 hours post-injection. Results: Significant time-dependent differences were observed in heat latency after SB-334867. Mean heat latency for HbAA mice was not significantly different between the various time points(p-value=0.103). Mean heat latency for HbSS-BERK mice was significantly different between time points (p-value=0.004) and post-hoc tests showed significant reductions in heat latency in HbSS versus HbAA-BERK mice before SB-334867 (p < 0.05) and 1 hour post-SB-334867 (p < 0.005). There was a significant reduction in heat latency exclusively in HbSS-BERK mice when comparing observations pre-SB versus 1 hour post-SB-334867 (p < 0.05). Statistical analyses are underway to determine time-dependent differences for mechanical hyperalgesia. Conclusions: These findings suggest differential orexin receptor responsiveness in HbSS-BERK mice vs. HbAA-BERK mice. These data may provide a mechanism for the increased hyperalgesia in HbSS-BERK mice, which contributes to neuropathic pain observed in sickle cell disease. Keywords: Hyperalgesia, Orexin Receptor Antagonist, Sickle Cell Disease Disclosure: Nothing to disclose. P450. Better Subject Recruitment: Can Aligned Messaging Improve Sample Accession? Gary Sachs, Jenicka Engler, D. Thorpe, Miriam Evans, D. Fagundo, S. Starling, S. Nicholas, H. Zandi, M. Moy, A. Vittoria Signant Health, Lincoln, Massachusetts, United States Background: Sample accession is key to clinical trial success. Social media advertisement is now a common means of initial recruitment outreach, with considerable success in increasing the number of pre-screening study inquiries, yet slow enrollment remains a chronic problem. Because efforts to combat slow recruitment by increasing compensation for time and travel risks increasing the enrollment of inappropriate participants and so called “professional subjects,” the goal of enrolling an appropriate study sample can be compromised by recruitment efforts to attract “clinical trial seeking” volunteers rather than participants representative of “treatment seeking” patients. Notably, Major Depressive Disorder (MDD) clinical trial recruitment is regarded as particularly challenging because depressive symptoms can be obstacles to attending screening visits. To begin addressing this complex issue, we ask a simple question: Does the “promise to refer” for post-study treatment impact show rates and participant quality for MDD trials? Most current quality control methodologies aim to disqualify inappropriate enrollment in clinical trials for MDD after the screening assessment. This pilot study examines the impact of prescreening recruitment messaging more aligned with the interests of “treatment seeking” subjects which clearly states an offer of referral for post-study clinical care as a benefit of clinical trial participation. This pilot experiment explores differences at various points in the recruitment-enrollment process (recruitment funnel) between participants who were promised referrals (experimental group) versus a control group who received usual study recruitment messaging. Methods: Starting in mid-May 2022, prospective research participants were recruited and randomized using the Facebook A/B testing algorithm. The algorithm randomly assigned users to a control group shown our usual Facebook advertisement content (information about MDD and clinical trials) or the experimental group shown advertisement text about MDD clinical trials and the promise that all respondents would receive referral to treatment providers and information about clinical resources regardless of trial eligibility. Both groups were offered phone screening interviews with Adams Clinical site staff and subsequently an in person pre-screening appointment. For those in the experimental group, recruiters and clinicians reiterated the promise of resources and referrals via IRB-approved scripts. Those in the experimental group who completed a phone screening but were not study candidates or declined were sent information for their local NAMI. Those who attended their in-person prescreening visit and were ineligible or declined were given a treatment referral letter with their prescreening assessment results and treatment recommendations, NAMI information, plus at least 2 referrals for in-network local clinicians listed as accepting new referrals on psychologytoday.com. The two groups were compared on rate of completing phone screens, completing the prescreening process and study eligibility using Chi Square. Results: A total of 1,358 prospective research participants clicked on Adams Clinical’s Facebook ads and submitted their contact information online as part of this A/B testing experiment (n = 751 in the experimental group, and n = 607 in the control group). Participants in the experimental group were significantly more likely to complete a phone screening (22%) than the control group (16%), X2 (1, N = 1358) = 6.46, p = .011. Participants in the experimental group were significantly more likely to have a prescreening visit scheduled (83%) than the control group (68%), X2 (1, N = 265) = 8.03, p = .005. The effect on prescreening visit attendance reached marginal significance, with participants in the experimental group being more likely to attend their prescreening visit (49%) than the control group (35%), X2 (1, N = 190) = 3.19, p = .074. The effect on study eligibility also reached marginal significance, with participants in the experimental group being more likely to be eligible (48%) than the control group (27%), X2 (1, N = 85) = 2.78, p = .096. At this time, scheduled prescreens and screening visits from both groups are still upcoming. Data from all participants will be available prior to the meeting, allowing full data on prescreening attendance and consenting to study participation to be presented in this poster. Conclusions: To our knowledge this pilot study represents the first empirical data on efforts to improve recruitment by offering a value proposition appealing to treatment seeking patients. We found a promise to refer and provide resources for prospective MDD study participants had positive impacts at all points examined in the recruitment funnel. Our results suggest that articulating and delivering on this value proposition has the potential to speed up trial recruitment by significantly improving potential participants’ likelihood of completing a phone screen and in-person prescreening visit. More work is needed to determine if the increase in willingness to engage with the clinical research screening process is accompanied by improved study participation rates, improved sample quality and enhanced potential for signal detection. Keywords: Clinical Trial Methodology, Recruitment, Social Media Use, Mood Disorders Disclosure: Signant Health: Employee (Self) P451. Risk of Major Malformations in Infants After First-Trimester Exposure to Stimulants: Results From the Massachusetts General Hospital National Pregnancy Registry for Psychiatric Medications Mercedes Szpunar, Marlene Freeman, Lauren Kobylski, Ella Rossa, Peter Gaccione, David Chitayat, Sonia Hernandez-Diaz, Adele Viguera, Lee Cohen Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: The prevalence of attention-deficit/hyperactivity disorder (ADHD) in adult women is 3-4%. ADHD is highly comorbid with other psychiatric disorders such as mood, anxiety, and substance use disorders. For reproductive-aged women, the treatment of ADHD with stimulant medications may be considered during pregnancy or breastfeeding, although historically, data are lacking to inform these decisions. The aim of this investigation was to determine the risk of major malformations in infants after first-trimester stimulant exposure. Methods: The Massachusetts General Hospital National Pregnancy Registry for Psychiatric Medications was established in 2008 to increase reproductive safety knowledge of psychiatric medications using rigorous, prospective data collection. The Registry systematically ascertains information from its participants including demographic information, medical and psychiatric history, use of prescription medications, and other information relevant to fetal outcomes. Participants provide verbal informed consent and are interviewed twice during gestation and again at approximately 3 months postpartum. The primary outcome of interest is the presence of a major malformation identified within 6 months after birth. Redacted cases of major malformations are reviewed by a dysmorphologist blinded to medication exposure. Women taking a stimulant during the first trimester of pregnancy (n = 233) were compared with controls (n = 1755 women) taking other psychiatric medication(s) during pregnancy to treat mental health disorders. The study is registered with ClinicalTrials.gov (NCT01246765). Results: N = 1988 women who were eligible for this analysis, including n = 173 exposed to mixed amphetamines, n = 40 exposed to lisdexamfetamine, n = 45 exposed to methylphenidate, and n = 3 exposed to dexmethylphenidate; the comparison group included n = 1755 women. Because of twin gestations, a total of 235 infants were exposed to one or two stimulant medications. The odds ratio of a major malformation among infants after first-trimester exposure to any stimulant was 0.57 (95% CI 0.17-1.85) compared to controls. The odds ratio of a major malformation after exposure to mixed amphetamines or lisdexamfetamine was 0.70 (95% CI 0.70-2.28). There were no major malformations observed in the infants exposed to lisdexamfetamine, methylphenidate, or dexmethylphenidate. The prevalence of major malformations after first-trimester exposure to mixed amphetamines was 1.2%. Conclusions: Although preliminary, this analysis from an ongoing pregnancy registry provides reassurance that these prescription stimulants do not appear to have major teratogenic effects. Keywords: ADHD, Stimulants, Pregnancy Disclosure: Intepros: Employee (Spouse) P452. Psychiatric Disorders and Income: A Population-Based, Longitudinal Study Mark Weiser, Ariel Asper, Adam Noy, Michael Davidson, Itzhak Levav, John M Davis Sheba Medical Center, Ramat Gan, Israel Background: Data suggest that having a psychiatric diagnosis is negatively associated with life-long income. We examined the effect of having a psychiatric diagnosis between ages 25-34, on income through ages 63-72. Methods: Baseline assessments were obtained from a population-based, epidemiological study of a random sample of 5000, 25-34 year-olds, conducted in Israel during the 1980s, the subjects are now aged 63-72. Data on mean annual income between 1983 to 2018 were obtained from the Israeli Social Security Administration. For each participant the median of his/her lifetime mean annual income was calculated; for each diagnostic group the mean of the medians of the participant was calculated. The difference (in standard deviations) was calculated between the mean annual income between healthy participant and each diagnostic group. Results: Healthy participant had the highest mean annual income. Mean income worked varied by diagnosis: Anxiety: -.2 SD, Depression: -.43 SD, Labile Personality Disorder -.49 SD, Bipolar Disorder -.52 SD, Schizotypal Personality Disorder -.58 SD, Anti-social personality disorder -.84 SD, Schizophrenia -.96 SD. Conclusions: Over their lifetime, young adults diagnosed with psychiatric disorders earn significantly less money, compared with young adults who do not suffer from psychiatric disorders. These longitudinal data indicate that having a psychiatric diagnosis increases risk for low income, but cannot dismiss the possibility that in parallel, low income increases risk for having a psychiatric disorder. Keywords: Psychiatric Disorders, Income, Population-Based Disclosure: Jansen: Advisory Board (Self), Teva: Consultant (Self), Dexcel: Consultant (Self), Lundeck: Advisory Board (Self), Minerva: Contracted Research (Self), MSD: Consultant (Self), Pfizer: Honoraria (Self), Orion: Consultant (Self), Clearmind: Consultant (Self) P453. The Effects of MDMA and Methamphetamine on Feelings of Closeness and Connection During Semi-Structured Conversations With Strangers Hanna Molla, Tang Li, Sonja Lyubomirsky, Harriet de Wit University of Chicago, Chicago, Illinois, United States Background: MDMA is classified as an “empathogenic” drug, as it increases feelings of empathy and closeness to others. These effects are thought to be partly mediated by increased oxytocin release via enhanced serotonergic transmission. Despite its prosocial effects, few studies in humans have examined MDMA in the context of interpersonal interactions. The objectives of this study were to determine the effects of MDMA on feelings of closeness and connection during a semi-structured dyadic conversation, and to investigate the relations between feelings of closeness and oxytocin levels. To investigate pharmacological specificity, a separate group of subjects was tested with the prototypic stimulant methamphetamine. Methods: Two studies were conducted in separate sets of healthy volunteers. In within-subject, double-blind, placebo-controlled designs, subjects aged 18-35 received a single dose of MDMA (100 mg) or placebo (Study 1; n = 18), or a single dose of methamphetamine (20 mg) or placebo (Study 2; n = 19), during two in-lab sessions separated by 72 hours. During peak drug effect, they engaged in a 45 min semi-structured conversation with a novel same-sex partner. Subjective mood, physiological, and oxytocin levels were obtained at regular intervals, and subjects rated feelings of closeness and connection with their partners at the end of each session and one week later. Results: Both MDMA and methamphetamine increased ratings of feeling connected to the partner, and increased ratings of enjoyment and meaningfulness of the conversation, relative to placebo (Study 1 MDMA; p = 0.009 and Study 2 methamphetamine p = 0.01). Both MDMA and methamphetamine increased subjective ratings of “sociable,” “loving,” and “friendly.” Only MDMA increased ratings of “trusting,” “grateful,” and “appreciated,” and only methamphetamine increased ratings of “understood” MDMA (p = 0.001), and to a lesser extent methamphetamine (p = 0.02) increased salivary oxytocin levels. Notably, oxytocin levels were positively correlated with feelings of closeness after MDMA but not after methamphetamine (Study 1; r = 0.52, p = 0.03). Conclusions: Both MDMA and methamphetamine increased feelings of closeness and connection to strangers during controlled interpersonal interactions, and both drugs increased subjective ratings of sociability. However, these feelings were related to oxytocin only after MDMA, suggesting that the mechanisms mediating prosocial effects may differ across drugs. This study provides a model for future studies assessing effects of drugs on interpersonal interactions. Keywords: MDMA, Social Interaction, Oxytocin, Closeness, Stimulant Disclosure: Nothing to disclose. P454. Inflexible Thinking Style Predicts COVID-19 Vaccine Hesitancy Naomi Fineberg, Pellegrini Luca, Aaron Clarke, Jemma Reid, Keith Laws University of Hertfordshire, Welwyn Garden City, United Kingdom Background: Mass vaccination against COVID-19 heralded the return to a state of normality following the recent pandemic. The success of this initiative needs to be set against the backdrop of a substantial minority who decline vaccination. Understanding the factors influencing vaccine attitudes and intentions represents an important plank in the armory against this and any future pandemics (1). Objective cognitive tasks hold certain advantages over clinical measurements as they are potentially less subject to rater-related error and may provide a window on the underpinning neuropsychological mechanisms. Few studies have investigated cognitive function in relation to vaccine hesitancy. One such study identified a relationship with executive function, broadly defined, using a Stroop task and the Montreal Imaging Stress Task (2). Another (3) identified a relationship with self-control on a Stroop-task. A third (4) found evidence suggesting a relationship with flexible adaptation to new concepts. In a fourth (5), parents’ psychological flexibility and positive coping-style were linked with reduced COVID-19 vaccine-hesitancy among a pediatric sample. This is the first study to our knowledge to investigate the relationship between cognitive inflexibility and COVID-19 hesitancy in a large adult sample using a specific digital neurocognitive task (Wisconsin Card Sort Task; WCST). Methods: This study is part of a larger research programme investigating post-pandemic adjustment. The study was approved by the University-of-Hertfordshire-Health-and-Human-Science-Ethics-Committee-With-Delegated-Authority-(ECDA)-(LMS/PGR/UH/04554) and a protocol was preregistered on Open-Science-Framework-(OSF)-10.17605/OSF.IO/XD5WZ. We surveyed adults UK-adult-population (>18 years) between June 2021 – July 2022 (using Gorilla-software) during a period when lockdown restrictions had eased. In order to recruit a sample that included traditionally underserved groups such as those with mental disorders, diverse groups were targeted. Sociodemographic information (age, gender, education and ethnicity) was collected. Cognitive inflexibility was measured using a digital version of the WCST. For this analysis, we focused on “WCST perseverative errors” as the most specific item for capturing attentional set-shifting problems. Vaccine hesitancy was measured with the Oxford Covid Vaccine Hesitancy Scale (OCVHS). We ran correlational and regression analyses to detect potential associations between cognitive inflexibility and vaccine hesitancy. Results: A sample of 207 individuals completed the survey and the WCST. The mean age of the sample was 37.3 years, 68% were females, 61% defined themselves as white ethnicity with 28% Asian, 5.8% South American and 6.3% of African background. The mean Total OCVHS score was 12.8 (SD:6.9), which is in line with the mean score found in a larger population-based sample (mean 13.6 (SD: 7.3))(6). A statistically significant correlation was found between WCST perseverative errors and Total OCVHS (Pearson’s r, p = 0.003). We ran a multivariate regression analysis in which we included OCVHS as the outcome, perseverative errors as the predictor and sociodemographic factors thought to have an impact on vaccine hesitancy (age, gender, education, ethnicity) as covariates. Perseverative errors significantly predicted increased vaccine hesitancy even after controlling for these covariates (p = 0.01). Conclusions: Cognitive inflexibility, measured with an objective computerized neurocognitive task in a large adult population sample, was a significant predictor of vaccine hesitancy, which applied irrespective of age, gender, level of education and ethnicity. Cognitive inflexibility is usually an enduring trait (7) and might represent a “red flag” for vaccine hesitancy, for use to identify those most at risk of refusing vaccination in the current and future pandemics. Public health education strategies should take into account the impact of an inflexible thinking style on the decision-making of those most at risk of vaccine hesitancy and adapt interventions accordingly. References: 1. Wang Y, Zhang X. Frontiers in Psychology 2021;12 2. Acar-Burkay S, Cristian D-C. Soc Sci Med 2022;301:114911. 10.1016/j.socscimed.2022.114911 3. Cao and Li 2022, Pers Indiv Diff 188:11147 4. Mazzuca C, et al. 2021. 10.31234/osf.io/4ndb8. 5. Wang Y, Zhang X. Frontiers in Psychology 2021;12. 6.Freeman D, Loe BS, Chadwick A, Vaccari C, Waite F, Rosebrock L, et al. Psychological Medicine 2020:1–15. 10.1017/S0033291720005188 7. Chamberlain SR, Fineberg NA, Menzies LA, Blackwell AD, Bullmore ET, Robbins TW, et al. Am J Psychiatry 2007;164:335–8. 10.1176/appi.ajp.164.2.335. Keywords: Cognitive Inflexibility, Vaccine Hesitancy, Public Health, COVID-19 Disclosure: Nothing to disclose. P455. The National Pregnancy Registry for Psychiatric Medications: Effects of Fetal Exposure to Second-Generation Antipsychotics on Risk for Major Malformations Lee Cohen, Marlene Freeman, Lauren Kobylski, Ella Rossa, Charlotte Clifford, Sonia Hernández-Díaz, David Chitayat, Adele Viguera Mass General Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Second-generation antipsychotics (SGAs), or atypical antipsychotics, are widely used by women of reproductive age to treat a number of psychiatric disorders. The National Pregnancy Registry for Psychiatric Medications (NPRPM) is a systematic prospective pharmacovigilance program used to collect reproductive safety data on second-generation antipsychotics, as well as other classes of psychotropic medications, in order to inform the care of reproductive aged women with psychiatric disorders. Methods: Data are prospectively collected from women, aged 18-45 years, with histories of psychiatric disorders. Two phone interviews are conducted during pregnancy and a third interview is completed at 3 months postpartum. Enrollment and longitudinal follow-up of participants is ongoing. In this analysis, the exposed group is comprised of women who took an SGA during the first trimester of pregnancy. The comparison group is composed of women who did not take this class of medication during pregnancy but who were prenatally exposed to other psychotropics. Information regarding the presence of major malformations is abstracted from medical records and identified cases of potential major malformations are adjudicated by a dysmorphologist blinded to drug exposures and psychiatric diagnoses. Results: As of July 25th, 2022, 2676 women were enrolled in the NPRPM, including 1031 in the exposure group and 1551 in the comparison group. Medical records were obtained for 78% of participants. A total of 1997 participants (787 exposed to an SGA in the first trimester, 1210 unexposed to an SGA during pregnancy) were eligible for analysis, having completed the postpartum interview. Of 810 infants in the exposure group, 22 confirmed major malformations were identified. 19 malformations were identified in the control group, which consisted of 1234 infants. No consistent pattern of malformation was seen in either group. The absolute risk of major malformations was 2.72% in the exposure group and 1.54% in the comparison group. The odds ratio was OR = 1.79 (95% CI: 0.96 – 3.32). Conclusions: The NPRPM offers a systematic way to collect prospective reproductive safety information which informs the care of women who may use second-generation antipsychotics to sustain psychiatric well-being. Current data suggest that it is unlikely that SGAs as a class have a major teratogenic effect. Future analyses will aim to better estimate risk of major malformations with even larger sample sizes and more generalizable cohort characteristics. We also continue to assess the reproductive safety of individual medications in this class. Additionally, we are studying other classes of medications, such as newer antidepressants and medications to treat ADHD and insomnia. Keywords: Pregnancy, Bipolar Disorder, Atypical Antipsychotics, Depression, Reproductive Psychiatry Disclosures: Alkermes Inc., Johnson and Johnson/Janssen Pharmaceuticals Inc., Otsuka America Pharmaceutical, Inc., Sage Therapeutics, Sunovion Pharmaceuticals Inc., Supernus Pharmaceuticals, Teva Pharmaceutical Industries Ltd., Forest/Actavis/Allergan:, AstraZeneca Pharmaceuticals, AuroMedics Pharma LLC, Aurobindo Pharma, Ortho-McNeil-Janssen Pharmaceuticals Inc., Pfizer Inc., MGH CTNI, Brain and Behavior Research Foundation, National Institute on Aging, National Institutes of Health, SAGE Therapeutics: Other Financial or Material Support (Self), JDS Therapeutics LLC: Consultant (Self) P456. Effects of Sex on the Relationships Between Visual Food Cue Ratings and Eating Behaviors, Weight, and Mood Kristina Legget, Marc-Andre Cornier, Lauren Sarabia, Eve Delao, Susan Mikulich, Christina Erpelding, Tessa Mitchell, Jason Tregellas University of Colorado School of Medicine, Aurora, Colorado, United States Background: Obesity rates are rapidly rising, giving rise to myriad health and quality-of-life concerns. Responsivity to food cues is a key factor underlying eating behaviors that may contribute to obesity. A critical factor in understanding these relationships, however, is how response to food cues may differ between men and women, including sex-based differences in factors that may relate to food cue responsivity, such as weight, eating behaviors, and mood. As such, the goal of the current study was to investigate sex-based differences in both appeal and “desire to eat” high-and low-calorie foods, including how sex may influence relationships between food cue ratings and BMI, eating behaviors, and mood measures. Methods: 334 adults (164 men, 169 women) completed this study, for which they completed an online survey in a neutral state of hunger. Survey measures included weight and height (from which BMI was calculated); the Three Factor Eating Questionnaire (TFEQ), which assesses trait-based eating behaviors relating to restraint, disinhibition, and hunger; the Eating Attitudes Test 26 (EAT-26), which assesses eating disorder-related behaviors; the Center for Epidemiologic Studies-Depression Scale (CESD-R), which assesses symptoms associated with depression over the past week; the Perceived Stress Scale (PSS), which assesses feelings of stress over the past week; and visual analog scale (VAS) measures assessing current hunger (“how hungry are you?” from “not at all hungry” to “extremely hungry”) and satiety (“how full do you feel right now?” from “not at all full” to “extremely full”), on a 0-100 scale. After these questionnaires were completed, participants began the Food Pictures Task, for which they viewed 96 different food images (48 high-calorie and 48 low-calorie) and were asked to rate how appealing each image was and how much they desired to eat each of the foods, using a visual analog scale (0-100). Independent samples t-tests were used to assess group differences (men vs. women) in questionnaire scores (TFEQ: restraint, disinhibition, and hunger scores; EAT-26 total eating disorder-related behavior score; CESD-R depression score; PSS perceived stress score; VAS hunger and satiety scores) and ratings of appeal and “desire to eat” on the Food Pictures Task, separately for high-calorie (HC) and low-calorie (LC) foods. Regression models investigated how questionnaire scores were associated with HC and LC ratings of appeal and desire to eat from the Food Pictures Task. Sex-based interactions were included in analyses; models with non-significant sex-based interactions were re-evaluated without the interaction component. Results: Two women were excluded from further analyses as outliers for BMI (BMI > 60 kg/m2), with 27 (12 men, 15 women) excluded due to being outliers on time to complete the survey (> 75 min). As such, 306 participants were included in the final sample (151 men, 155 women). Compared to men, women in the sample reported significantly greater dietary restraint (p = 0.004), disinhibition (p < 0.001), and trait-based hunger (p = .044), as measured by the TFEQ. Significantly higher scores on the EAT-26 were also observed in women compared to men (p < 0.001), as were significantly higher levels of perceived stress (p < 0.001) and depression (p < 0.001). We did not observe sex-based differences in “desire to eat” HC or LC foods or HC food appeal (p > 0.05 for all), but women did report greater appeal of LC foods compared to men (p = 0.002). A significant interaction with sex was observed in the relationship between perceived stress and HC food appeal (p = 0.006), such that higher stress was associated with increased HC appeal in men, but reduced HC appeal in women. For LC food appeal, we observed a significant sex-based interaction with disinhibition (p = 0.024), such that greater disinhibition was associated with lower LC appeal in women, but higher LC appeal in men. In both groups, increased satiety was associated with a decreased desire to eat LC foods, but a sex-based interaction was observed, such that stronger associations were observed in women compared to men (p = 0.049). Across sex, increased BMI was associated with increased HC food appeal (p = 0.042), with increased depression scores associated with increased desire to eat HC foods (p = 0.008). No association between BMI and LC foods was observed, but increased depression scores were associated with increased desire to eat LC foods (p = 0.008) and increased perceived stress was associated with increased LC food appeal (p = 0.009). Conclusions: We observed greater dietary restraint, disinhibition, and trait-based hunger in women compared to men, in addition to greater appeal of LC foods. Higher scores on eating disorder-related behaviors, depression, and perceived stress were also observed in women compared to men. Interestingly, we observed a sex-based interaction between perceived stress and HC appeal, with increased stress associated with reduced HC appeal in women, but increased HC appeal in men. This may indicate differential impacts of stress on eating behaviors in women compared to men, which may suggest stress as an important factor to consider in individualized weight management approaches. Across groups, BMI was associated with greater HC appeal, stress was associated with increased LC appeal, and depression with both HC and LC desire, further supporting the importance of considering the role of mood in eating behaviors and weight maintenance approaches. Keywords: Obesity, Sex-specific Effects, Food Cues, Eating Behavior, Mood Disclosure: Nothing to disclose. P457. Adolescent Stress Impairs Postpartum Social Behavior via Glucocorticoid Receptor-Mediated Anterior Insula-Prelimbic Pathway Kongpyung Kim, Shin-ichi Kano, Minae Niwa* University of Alabama at Birmingham, Birmingham, Alabama, United States Background: Postpartum social behavior is important not only for the mother’s health, but also for the child’s development. Unfortunately, postpartum social behavior is sensitive to stress that can occur at different points in the mother’s lifespan. Adolescent stress can be associated with changes in later postpartum behavior, including social behavior. Nonetheless, the neural circuit mechanisms by which adolescent stress leads to postpartum changes in social behavior are unclear. We recently found that mice exposed to social isolation in late adolescence (SILA), which alone causes no endocrine or behavioral changes, show long-lasting behavioral deficits only when accompanied by pregnancy and delivery. The behavioral deficits in dams exposed to SILA (stressed dams) were caused by an aberrantly sustained elevation of corticosterone levels during the postpartum period. More importantly, we have shown that short-term post-delivery treatment with a glucocorticoid receptor (GR) antagonist is sufficient to ameliorate the behavioral deficits observed in stressed dams (Niwa et al BioRxiv 2021). Using this mouse model, we also found that SILA, in conjunction with pregnancy and delivery, leads to hypofunction of anterior insula-prelimbic cortex (AI-PrL) glutamatergic pathway, which in turn alters activity patterns in PrL, resulting in behavioral changes in social novelty recognition (Kim and Niwa, ACNP 2021). Based on these findings, the present study further examined whether enhanced corticosterone signaling in AI-PrL pathway leads to social behavioral changes in stressed dams in the postpartum period. Methods: AAVretro-CaMKIIa-EGFP and a mix of CRE-DOG viruses (AAV1-EF1a-N-Cretrcint G and AAV-EF1a-C-Creint G) were bilaterally injected to PrL and AI of mice floxed with the GR gene (GRfl/fl mice), B6J, or Ai14 mice at eight weeks old. AAV1-Flex-tdTomato was also injected into AI for a validation study with B6J mice. For the Cre recombinase dependent on GFP (CRE-DOG) validation studies, mice were perfused at one, two, or three weeks after virus injections. EGFP + , tdTomato+EGFP + , EGFP + GR + , mCherry + , or mCherry+GR + cells were counted. For the behavioral cohort, animals were exposed to SILA (from 5 to 8 weeks of age) after surgery. Each mouse was then mated with a healthy C57BL/6J male and gave birth to pups. Three-chamber social interaction tests (SIT) were performed one week postpartum. Two hours after the last behavioral test, mice were perfused for c-Fos staining. Results: By combining the GFP-dependent Cre recombinase system and AAV-mediated retrograde tracing in GRfl/fl mice, pathway-specific GR knock-out (GR-KO) mice were successfully generated. The CRE-DOG method, in which GFP-binding proteins were used for the molecular assembly of Cre recombinase on a GFP scaffold, allowed us to express Cre recombinase in a GFP-dependent manner. We validated that Cre recombinase was successfully expressed in AI-PrL pathway when AAV-retro-CaMKIIα-EGFP and a mix of CRE-DOG viruses (AAV-EF1a-N-Cretrcint G and AAV-EF1a-C-Creint G) were injected into PrL and AI, respectively. As time passes after the injections, a gradual increase in the expression level of EGFP was observed in the AI, accompanied by a gradual decrease in the expression level of GR in AI neurons expressing EGFP. Such expression changes were not observed when AAV-retro-CaMKIIα-mCherry was injected instead of AAV2-retro-CaMKIIα-EGFP as a control virus. Deletion of GR in AI-PrL pathway ameliorated the behavioral changes in the social novelty-trial, but not the sociability-trial, of SIT in stressed dams. AI-PrL-specific GR-KO also normalized the reduced c-Fos immunoreactivity after social novelty-trials in stressed dams. These findings suggested that activation of GR signaling in AI-PrL pathway, but not in PrL itself, might play a causal role in PrL dysfunction and subsequent behavioral changes in social novelty recognition in the postpartum period. Conclusions: We successfully generated pathway-specific GR-KO mice using the CRE-DOC method. Using these GR-KO mice, we elucidated the causal role of GR-mediated AI-PrL pathway in postpartum behavioral changes during social novelty recognition. Further research on how GR signaling specifically regulates AI-PrL function and recognition of social cues would facilitate our mechanistic understanding of the pathological trajectory of adolescent stress leading to abnormal social behavior in the postpartum period. Keywords: Adolescent Stress, Social Behaviors, Glucocorticoid Receptor, Anterior Insula, Prelimbic Cortex Disclosure: Nothing to disclose. P458. The Role of Hypocretin/Orexin Neurons in Social Behavior Derya Sargin, Matthew Dawson, Dylan J. Terstege, Naila Jamani, Dmitrii Pavlov, Mio Tsutsui, Jonathan R. Epp, Gina M. Leinninger Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Canada Background: Intraspecific social interactions are integral for survival and maintenance of society among all mammalian species. Yet, our understanding of the neural systems and mechanisms involved in the establishment of social connectedness and the consequences of the detrimental effects of social isolation are limited. Since their initial discovery as regulators of sleep/wakefulness and appetite in the brain, the hypocretin/orexin neurons have also been shown to play an essential role in modulating energy homeostasis, motivated and emotional behavior. These neurons are located exclusively in the hypothalamus that regulates complex and goal-directed behaviours. The hypothalamus has previously been shown to play an important role in the modulation of social behavior by encoding internal states. However, the information for the role of hypocretin neurons in social behavior and deficits is limited. Methods: We infused AAV encoding GCaMP6s into the LH of female and male Hcrt-cre mice and performed fiber photometry to record the activity of hypocretin neuron population during social interaction. We next inhibited the activity of hypocretin neurons using optogenetics and determined the necessity of these neurons for social behavior in female and male mice. Using a mouse model of chronic social isolation, we additionally determined how hypocretin neuron activity is affected in mice with social deficits. Results: We identified hypocretin/orexin neurons to exhibit a robust increase in activity in response to social interaction in female and male mice. We demonstrate that the activity of hypocretin neuron population is differentially encoded during interaction between familiar and stranger conspecifics. Moreover, the optogenetic inhibition of hypocretin neuron activity during social behaviour leads to a reduction in the amount of time mice are engaged in social interaction in a sex specific manner. We also show that isolated mice have deficits in social behavior and disruption in hcrt neuron activity. Conclusions: Together, these data situate the hypocretin/orexin system as the part of a larger network that plays an integral role in the modulation of social behaviour. Here, we will additionally discuss the implications of these findings in an animal model of chronic social isolation that develops long-term social impairments. Keywords: Orexin/Hypocretin, Social Isolation, Social Deficits, Fiber Photometry, Optogenetics Disclosure: Nothing to disclose. P459. The Epigenetic Reader Plant Homeodomain Finger Protein 21B (PHF21B) Regulates Social Memory, Associated Behaviors, Glutamatergic Neurotransmission, and Synaptic Plasticity–Related Genes in the Hippocampus Julio Licinio, Eunice Chin, Qin Ma, Hongyu Ruan, Camile Chin, Aditya Somasundaram, Ma-Li Wong State University of New York, Syracuse, New York, United States Background: We have reported that the PHF21B gene is located in the genomic region of human chromosome 22q13.31. The 22q13.3 deletion syndrome, also known as Phelan-McDermid syndrome, is characterized by developmental delays, moderate to profound intellectual disability, decreased muscle tone (hypotonia), absent or delayed speech as well as autism or autistic-like behavior that affects communication and social interaction. We have hypothesized that while the full 22q13.3 deletion causes a severe syndrome, a specific decrease of PHF21B function might cause a more subtle and specific phenotype, possibly related to social interaction. In a model of stress, we reported significantly decreased hippocampal Phf21b gene expression in rats resilient to chronic restraint stress compared to non-chronically stressed rats. The PHF21B (aka PHF4) gene, a member of PHD finger proteins encodes a histone reader and is expressed in several brain regions, including the frontal cortex and the hippocampus; however, its functions in the brain have remained unclear. We generated a Phf21b knockdown mouse model to mechanistically understand how this gene regulates behavior, including social interaction, and hippocampal functions. Methods: All animal experiments were performed according to approved protocols by the State University of New York Upstate Medical University, South Australian Health and Medical Research Institute, Flinders University, and University of Adelaide. Male and female mice were studied. The following approaches were used – 1) CRISPR/Cas9 technology (SA Genome Editing Facility University of Adelaide, South Australia (SA), Australia) to generate a Phf21b mutant mice in which the exon 4 of the mouse Phf21b gene was mostly deleted and a frame shift mutation generated a premature stop codon. 2) A battery of cognitive and emotional behavioral tests was used to determine the effects of Phf21b deficiency (Phf21bΔ4/Δ4) compared to littermate wildtype (WT, Phf21b + /+) mice. 3) Standard whole-cell patch clamp was used to study long-term potentiation in hippocampal CA1 pyramidal neurons. 4) Transcriptome profile was performed using hippocampal tissues, followed by differential gene expression analysis and qRT-PCR confirmation studies. 5) Immunoblotting, immunohistochemistry, and Golgi staining. 6) Histone peptide array. Results: Phf21b WT and Phf21bΔ4/Δ4 mice were in generally good health. Phf21bΔ4/Δ4 expressed 60% less PHF21B than WT mice. Male and female mice had similar changes in behaviors, and their combined data were used for data analyses. Phf21b deficiency resulted in very specific social memory deficits (increased social novelty in the 3-chamber social test (P < 0.01); no decrease interaction time during the habituation trials in the 5-trial social memory test and the score difference was significantly lower (P < 0.05). Importantly, these animals did not display other memory, cognitive, or emotional differences. PHF21B deficit led to thinner cortices (P < 0.001) with lower astrocyte number (P < 0.0001), and reduced neurogenesis (DCX + cells, P < 0.001). Phf21bΔ4/Δ4 hippocampi had decreased synaptic protein expression (fewer PSD95 + clusters/unit area, P < 0.001; smaller PSD95 + puncta size, P < 0.01), diminished glutamatergic neurotransmission (reduced I/O relationship, P < 0.001; impaired LTP), and GluN2B (P < 0.05) and Grin2b (P < 0.01) levels. RNAseq showed that PHF21B modulates the expression of neurotransmission genes. Histone peptide studies showed that PHF21B regulates transcription through H3K9ac, H3K9Me2, and CREB (cAMP response element-binding protein), and that it interacts with H3K36me3. Conclusions: Our results demonstrate that PHF21B regulates social memory and that reduced PHF21B function brings about impaired hippocampal long-term potentiation. There are fewer AMPAR subunit GLUR1-expressing clusters in hippocampal tissues of Phf21bΔ4/Δ4 mice, resulting in decreased glutamatergic neurotransmission. We show that PHF21B is a critical upstream regulator of synaptic plasticity-related genes, functioning as an epigenetic reader. Furthermore, we characterized a potentially novel interaction of PHF21B with histone H3 trimethylated lysine 36 (H3K36me3), a histone modification associated with transcriptional activation and also with the transcriptional factor CREB. After identifying a novel function for PHF21B as a regulator of social memory, and dissecting the underlying neurobiology at the structural and functional levels, we now suggest that PHF21B may be a novel candidate translational therapeutic target for neurobehavioral disorders. Keywords: Social and Behavioral Deficits, Epigenetics, Neuroepigenetic Editing, Glutamatergic Transmission, AMPA Glutamate Receptors Disclosure: Nothing to disclose. P460. The Gene Expression Landscape of the Human Locus Coeruleus Revealed by Single-Nucleus and Spatially-Resolved Transcriptomics Lukas Weber, Heena R. Divecha, Matthew N. Tran, Sang Ho Kwon, Abby Spangler, Kelsey D. Montgomery, Madhavi Tippani, Rahul Bharadwaj, Joel E. Kleinman, Stephanie C. Page, Thomas M. Hyde, Leonardo Collado-Torres, Kristen R. Maynard, Keri Martinowich, Stephanie C. Hicks Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States Background: The locus coeruleus (LC) is a small bilateral nucleus located in the dorsal pons of the brainstem, which serves as the brain’s primary site for production of the neuromodulator norepinephrine (NE). NE-producing neurons in the LC project widely throughout the central nervous system, playing critical roles in arousal and mood, as well as various components of cognition including attention, learning, and memory. In line with its prominent role in a wide variety of core behavioral and physiological functions, the LC-NE system is highly implicated in multiple neurological and neuropsychiatric disorders. Importantly, LC-NE neurons are highly sensitive to degeneration in both Alzheimer’s and Parkinson’s disease. However, despite its prominent involvement in a number of critical brain functions, the small size and positioning of the nucleus deep within the brainstem has rendered it relatively intractable to a full cellular, molecular, and physiological characterization. Methods: Here, we used the 10x Genomics Visium Spatial Gene Expression platform to capture spatially-resolved transcriptomics (SRT) along with histology images to characterize the spatial gene expression landscape of the LC and surrounding region (N = 5 neurotypical adult male donors, 23,387 spatial locations, 8 capture areas), and the Chromium Single Cell Gene Expression single-nucleus RNA sequencing (snRNA-seq) platform to capture the transcriptomic profile of individual nuclei from LC-NE neurons and other neuronal and non-neuronal cell populations (N = 3 neurotypical adult male donors, 19,927 nuclei). Spatial regions in the SRT data containing LC neurons were annotated based on the histology images and validated by confirming expression of NE neuron marker genes (TH, SLC6A2, DBH), and used for further downstream analyses including differential expression analyses to identify LC-associated genes and for comparison with LC-associated genes previously identified in rodents. LC-NE neurons and other cell populations in the snRNA-seq data were identified by unsupervised clustering and validated by confirming expression of known marker genes, and used for analyses including integration with the SRT measurements and testing for genetic risk associations. Results: We identified sets of highly associated (false discovery rate, FDR < 10^-3, absolute fold-change, FC > 3) and statistically significant (FDR < 0.05, absolute FC > 2) genes characterizing the transcriptomic profiles of the LC regions in the human SRT samples, which included both known NE neuron marker genes (TH, SLC6A2, DBH) and other genes. A subset of LC-associated genes previously identified in rodents using alternative technological platforms were found to be highly expressed within the human samples. We identified a population of LC-NE neurons at single-nucleus resolution, and in addition, identified a population of 5-hydroxytryptamine (5-HT, serotonin) neurons that have not previously been characterized at the molecular level in human brain samples. We mapped the spatial distribution of the single-nucleus populations by integrating the SRT and snRNA-seq data, and investigated association of genomic risk for Alzheimer’s and Parkinson’s disease and attention-deficit hyperactivity disorder across the single-nucleus populations. Conclusions: We have generated a spatially-resolved and single-nucleus gene expression atlas of the LC and surrounding region in the neurotypical adult human brain, and provide a freely accessible data resource containing spatially-resolved and single-nucleus data in web-accessible and R/Bioconductor formats. This resource containing the full transcriptomic profile of LC-NE neurons and other cell populations within the LC region will contribute to understanding of neurodegenerative and other disorders, which is particularly relevant given that maintaining the neural density of LC-NE neurons prevents cognitive decline during aging. Keywords: Locus Coeruleus, Norepinephrine, Spatial Transcriptomics, Single-Nucleus RNA Sequencing, Postmortem Human Brain Tissue Disclosure: Nothing to disclose. P461. Noradrenergic Modulation of the Medial Prefrontal Cortex Alexandria Athanason, Marcis Scroger, Noah Paperny, Andrea Liss, Florence Varodayan Binghamton University, Binghamton, New York, United States Background: Norepinephrine (or noradrenaline; NE) is a major brain stress system, that tightly controls cognitive and affective behavior. During periods of arousal, stress and alcohol exposure and withdrawal, NE is released from the locus coeruleus (LC) into the medial prefrontal cortex (mPFC) where it binds to α1, α2 and β adrenergic receptors to modulate cognitive function. In particular, the prelimbic (PL) subregion of the mPFC plays a critical role in sustained attention, decision making, working memory and episodic memory, as well as coping with perceived stressful events. Unfortunately, most of these studies have focused on male subjects. This is particularly surprising as the LC is sexually dimorphic; females have a larger LC than males and their LC does not sensitize to repeated stress or alcohol exposure. Therefore, here we investigated potential sex differences in basal mPFC noradrenergic signaling in male and female mice. Methods: All experiments were performed male and female C57BL/6J mice (n = 10-18 per sex for each experiment). We used high-performance liquid chromatography to measure basal mPFC NE levels. Gene expression of α1 and β adrenergic receptors were also characterized using real-time polymerase chain reaction. To assess noradrenergic function, we conducted ex vivo patch-clamp electrophysiology recordings in PL layers 2/3 and 5 neurons. Finally, behavioral pharmacology experiments are currently probing the influence of noradrenergic signaling on cognitive and anxiety-like behavior (Barnes maze, elevated plus maze, etc.). Final values were analyzed for independent significance using one-sample t-tests and compared using unpaired t-tests or one-way ANOVA with post hoc analyses as appropriate with Prism software (GraphPad, San Diego, CA). Results: There were no significant sex differences in basal noradrenergic signaling in the mPFC. However, there were key sex differences in its modulation of PL glutamatergic synapses, with NE increasing glutamate release in layers 2/3 and 5 in males, but only layer 5 in females (all p < 0.05). Behavioral studies are ongoing. Conclusions: Thus, here we have identified key sex differences in noradrenergic influence over mPFC function that may need be taken into consideration during ongoing efforts to develop noradrenergic therapeutics to treat neuropsychiatric diseases, such as alcohol use disorder and posttraumatic stress disorder. Future studies should examine whether a role for NE emerges under conditions of increased cognitive load (e.g., stress or alcohol withdrawal). Keywords: Norepinephrine, Sex Differences, mPFC Disclosure: Nothing to disclose. P462. Selective Effects of Acute and Chronic Stress on Cortical Functional Connectivity Alexander McGirr, Donovan Ashby Hotchkiss Brain Institute, University of Calgary, Calgary, Canada Background: Depressive, traumatic and other stress-related disorders are associated with large scale brain network functional connectivity changes. Because it is unclear what functional connectivity changes occur during acute stress, it is unclear how these relate to the emergence of persistent large scale network organization. Methods: Using Thy 1-jRGECO1a transgenic mice, we repeatedly sampled mesoscale cortical calcium activity across dorsal neocortex during quiet wakefulness in several experimental conditions. First, animals were imaged in a homecage condition, after which animals underwent an acute foot-shock stress, a chronic variable stress protocol, an acute on chronic foot-shock stress, and treatment with the prototype rapid anting antidepressant ketamine. We focused on slow cortical activity (0.3-4 Hz) and theta-alpha cortical activity (4-15 Hz) to characterize functional connectivity. Results: Compared to homecage, acute foot-shock stress induced widespread increases in cortical functional connectivity in the 4-15 Hz temporal band both within functional modules (t(31)=-4.66, p < .001) and between functional modules (t(31)=-4.51, p < .001) before returning to control after 24 hours. Chronic stress produced a selective increase in between-module functional connectivity in the 0.3-4 Hz band (t(14)=-2.81, p < .05), which was reversed after treatment with the rapid antidepressant ketamine. Functional connectivity changes induced by acute stress in the 4-15 Hz band were strongly related to functional connectivity changes after chronic stress and to the pattern of reversal of functional connectivity by subanesthetic ketamine. Conclusions: Stress induces changes in functional connectivity, and these have specific effects that transcend spatiotemporal aspects of connectivity to link acute, chronic, and treatment effects. The mechanisms by which acute stress connectivity changes become embedded in a lower temporal band over time remain to be determined. Keywords: Functional Connectivity, Acute Stress, Chronic Stress Disclosure: Provisional Patent: Patent (Self) P463. Mesoaccumbal Core Circuit Dynamics During Aversive Processing Gabriela Lopez, Louis Van Camp, Talia Lerner Feinberg School of Medicine, Northwestern University, Chicago, Chicago, Illinois, United States Background: Behavioral adaptations to aversive stimuli are crucial for survival. The mesoaccumbal core circuit, which includes dopamine neurons in the ventral tegmental area (VTA) projecting to the nucleus accumbens core (cNAc) and their projection targets within cNAc, has been heavily studied for its role in motivation, cue associations, and goal-directed behaviors. Maladaptive changes in this circuit are associated with psychiatric disorders such as chronic pain, addiction, and depression. While the mesoaccumbal core circuit has most often been associated with reward processing, its role in aversive processing and different aversion scenarios remains unclear. Previous studies have largely investigated dopamine release in response to unavoidable aversive stimuli, observing inconsistent results. While understanding direct responses to aversive stimuli is important, it may be more ethologically relevant to study how an animal behaves when they have the chance to avoid an aversive stimulus, which can be accomplished with instrumental tasks. Additionally, many previous studies examined dopamine release and cNAc neuron activity separately, making it difficult to determine how cNAc dopamine influences local cNAc circuit dynamics. I used Pavlovian and instrumental behavioral paradigms in conjunction with multicolor fiber photometry to identify how variations in mesoaccumbal core circuit dynamics may predict differences in aversive processing. Methods: To investigate aversive processing in the mesoaccumbal core circuit, I used two shock-paired behavioral tasks – active avoidance, an instrumental behavior allowing mice to avoid shocks, and unavoidable shocks. I used fiber photometry to record dopamine release in cNAc by expressing a fluorescent dopamine sensor (pAAV9-CAV-dLight1.3b). To record the activity of dopamine D1 receptor (D1R)-expressing or dopamine D2 receptor (D2R)-expressing striatal projection neurons (SPNs), I also injected a virus carrying a Cre-dependent, fluorescent calcium sensor (pAAV1-CAG-FLEX-NES-jRCaMP1b) into the cNAc of D1-Cre and A2A-Cre mice, respectively. Fiber optic implants were then placed above the injection site and neural activity was recorded as mice freely behaved. During the active avoidance task, which used a 2-compartment chamber, mice were given light and sound cues that preceded administration of a footshock in whatever chamber they resided, and the mouse had 5 s to run to the opposite compartment to avoid the footshock. After 5 s, a 0.4 mA shock began and continued for 25 s or until the mouse moved to the opposite chamber. There were two behavioral scenarios in this task: 1) Avoided Shocks – in which the mouse moved to the opposite chamber before the shock began (<5 s), and 2) Escaped Shock – in which the mouse moved to the opposite chamber after the shock had begun (>5 s). Mice were tested on this task for 7 days, 30 trials per day. Finally, mice were subjected to an unavoidable shock task where a 5 s light and sound cue predicted 5 s of 0.4 mA shock that they could not avoid or escape. Mice were tested on this task for 1 session only consisting of 10 trials. Results: After 7 days of active avoidance testing, mice learned to avoid ≥80% of shocks. cNAc dopamine release increased during avoidance and escape events, as well as the time of shock cessation. The magnitude and duration of these increases changed as the animals learned the avoidance task. Dopamine release, in contrast, decreased in response to shocks and shock-predicting cues. These dynamics differed in magnitude depending on whether the mouse avoided or escaped the shock. In D1- and D2-SPNs, I observed ramping activity followed by prolonged decreases in response to avoidance and escape events. D1- and D2-SPNs additionally showed increased activity in response to shocks and shock-predicting cues. D1- and D2-SPNs often showed similar activity patterns rather than opposing activity that would be predicted by their differing responses to dopamine. During the unavoidable shock session, dopamine release dynamics and SPN activity differed in magnitude and duration in response to shocks and shock predicting cues compared to active avoidance trials and within unavoidable trials in a given session. Conclusions: My data illustrate how cNAc dopamine release dynamics as well as D1- and D2-SPN activity differ in response to varying aspects of Pavlovian and instrumental aversive learning. These data also highlight the importance of utilizing more complex and ethologically relevant behaviors to further understand how the mesoaccumbal core circuit is involved in neural and behavioral adaptations towards aversive stimuli. I am currently investigating how this circuit activity is impaired following spared nerve injury as a model of chronic pain, to understand how pain may modulate aversive processing, potentially contributing to opioid addiction and depression. Understanding fundamental processes important for survival, such as aversive learning, will allow us to better understand what may go awry in conditions where aversive processing is dysregulated such as in chronic pain, addiction, and depression. Keywords: Dopamine, Fiber Photometry, Mesolimbic Circuitry, Active Avoidance, Nucleus Accumbens Core Disclosure: Nothing to disclose. P464. Understanding Common Mechanisms in Suicide Across Different Psychiatric Disorders: The Role of CRH in the Central Amygdala Jonathan Vogelgsang, Kiely French, Anna Lally, Kerry Ressler, Sabina Berretta Harvard Medical School McLean Hospital, Belmont, Massachusetts, United States Background: Suicide is one of the most serious concerns, but also described as extremely burdensome for patients in psychiatry across all different psychiatric disorders. Emotional dysregulation, such as depression, anxiety, and impulsivity are one of the most important clinical features related to suicidal behavior. However, no biomarker that are clearly associated with suicide attempts have been identified so far and no common brain alterations have been described. As stress and its related factors and hormones are one of the most important risk factors in mental health, we have been focusing on corticotropin releasing hormone (CRH), its corresponding receptor (CRH1R), and the pituitary adenylate cyclase-activating peptide receptor (PAC1R) to identify common molecular patterns in suicide across different psychiatric disorders. Since all these markers have been described to be associated with emotional dysregulation, we postulate that changes in CRH, CRH1R, or PAC1R expression in the amygdala are involved in suicidal behavior. Methods: Protein lysates form human post-mortem brain tissue from the central amygdala nucleus of 150 brain donors, including controls, patients with major depression (MDD), and post-traumatic stress disorder (PTSD), were analyzed. Out of these 150 donors, 19 donors died by suicide, while 99 died by natural death or accident, and 32 were classified as “undetermined”. 52% of the donors were women. Four different races, Caucasian, Asian American, Hispanic, and African American donors were included. Western blotting analysis was carried out using antibodies raised against CRH, CRH1R, and PAC1R. Demographical information, such as age, sex, post-mortem interval (PMI) as well as clinical information on medication, medical history, or substance abuse were included in the analysis, using multiple linear regression models as well as elastic net analyses. Results: Using a regression model including diagnosis, suicide, and sex, we saw that donors diagnosed with MDD had lower CRH levels (p = 0.014) in the central amygdala. Donors died by suicide had significantly higher CRH levels in comparison to donors died by a natural death or accident (p = 0.024). Sex had no effect on CRH levels in the central amygdala. No associations between primary diagnosis, suicide, or sex and PAC1R or CRH1R levels were observed. We also performed elastic net regression analyses to include and control for all available covariates (n = 58) and using imputations (m = 1000) to take missing values into consideration. We calculated a pseudo-p-value as a surrogate for significant associations between a covariate and protein measurements. Death by suicide (p < 0.000) and childhood trauma (p = 0.019) were associated with increased CRH levels, while a primary diagnosis of MDD (p < 0.000), asthma (p = 0.019) and history of military service (p = 0.069) lead to decreased CRH levels in the central amygdala. Smoking, BMI, hypothyroidism (all p < 0.000), and a primary diagnosis of MDD (p = 0.008) were highly associated with decreased PAC1R levels in the central amygdala, while PMI (p < 0.000) and antipsychotic medication intake (p = 0.063) lead to increased PAC1R levels. Conclusions: These finding show that CRH might be a key component in suicidal behavior. This study suggests to further examination the role of CRH and its related pathways to be used as biomarkers but also trying to modify these circuits to treat patients with suicidal ideations or intents. Keywords: Suicide Risk Factors, CRH, MDD, PTSD Disclosure: Nothing to disclose. P465. Suicide Risk in a Transdiagnostic Outpatient Population: Interacting Long-Term and Immediate Action Regulation Nicholas Murphy, Marijn Lijffijt, Sidra Iqbal, Dania Armaneh, Nithya Ramakrishnan, Brittany O’ Brien, Amanda Tamman, Lynnette Averill, Sanjay Mathew, Alan Swann Baylor College of Medicine, Houston, Texas, United States Background: Suicide is the leading cause of trauma-related mortality in the US, exceeding vehicular accidents and homicides. While often associated with depression there is mounting evidence that suicide attempts (SA) result from transdiagnostic mechanisms. Delineating potential mechanisms critical challenge because many suicides are first attempts, and suicidal ideation and impulsivity can fluctuate unpredictably before an attempt. Addressing these challenges requires 1) defining a transdiagnostic behavioral model of suicidal behavior; 2) identifying indicators of potential suicidal behavior that do not require a previous attempt. In this investigation we used patients at high risk for a recurrent attempt based on their proximity to a previous medically severe SA (MSSA). Compared to people without history of SA, those with MSSA have elevated risk for all-cause mortality up to ten years post SA. This appears related to increased arousal and behavioral sensitization (BS) predisposing to impulsive behavior and reduced tendency for self-preservation. Accordingly, we investigated self-report behavioral measures of arousal, impulsivity, and potential sensitization, in people who had stabilized from MSSA compared with diagnostic controls. We focused on relationships between long-term (sensitization) and short-term (impulsivity, symptoms, arousal) regulation of action. Methods: We recruited 28 trans-diagnostic outpatients at high risk for SA, defined as MSSA in the past year (high-risk, HR), and 23 age and symptom-matched outpatients without a history of MSSA (low-risk, LR). Psychiatric diagnoses were based on the MINI; severity of affective, anxious, and psychotic symptoms was measured using the Schedule for Affective Disorders and Schizophrenia – Change (SADS-C) questionnaire, designed to measure these symptoms concurrently. Fifty patients reported symptoms consistent with a major depressive episode, anxiety symptoms were present in 45%, bipolar 35%, PTSD 41%, psychotic disorder 13%, BPD 4%, OCD 3.9%. All patients completed clinical and behavioral evaluations; 21 HR and 11 LR patients completed combined clinical, behavioral, and neurophysiological tasks. Clinical and behavioral analysis focused on 1) clinical symptoms, 2) aggressive behavior, 3) Impulsivity, 4) sensitization behaviors. Risk group comparisons used independent samples t-tests and Cohen’s D effect sizes. Correlations with Beck’s SSI-W were determined across all subjects to assess relationships to risk factors and suicidal ideation. Only HR subjects had suicide attempt histories, but we addressed the frequency and specificity of SI by allowing it in either group. Results: Groups were matched on symptom severity (SADS-C) and subjective self-rate symptoms (Internal State Scale (ISS)). Behavioral analysis revealed increased lifetime history of aggressive behaviors (p = 0.004,d = 0.87) and provoked aggressive behavior (PSAP, p = 0.02,d = 1.29) but not current aggressive behavior (MOAS). Trait (Barratt Impulsivity Scale, UPPS) and state (Immediate Memory Task (IMT)) did not differ significantly between groups across sub-domains. However, Cohen’s d effect sizes indicated trends for IMT correct detections (p = 0.059,d = 0.5; state impulsivity). History of abuse was significantly greater for physical abuse in HR patients (p = 0.04,d = 0.6) with a trend for sexual abuse (p = 0.055,d = 0.53). CAPS-5 scores did not differ between groups across domains. Lifetime cumulative adversity was greater in HR (p = 0.04, d = 0.67). HR patients also demonstrated stronger history of alcohol use disorder (Addiction Severity Index) than LR patients (p = 0.01, d = 0.69). Beck’s worst suicidal ideation (SSI-W) was significantly correlated with a broad pattern of clinical and behavioral scores. SSI-W differed significantly between HR and LR but ranged widely with substantial overlap between groups. Symptoms (SADS-C total r = 0.56, Depression r = 0.54, Anxiety r = 0.48), ISS (Depression r = 0.45, Perceived Threat r = 0.43), Aggression (PSAP r = 0.48), BIS (Attentional r = 0.55, Total r = 0.47), UPPS (Lack of Perseverance r = 0.43), IMT (Commission Error Reaction Time r = 0.48), CTQ (Emotional Abuse r = 0.46), and CAPS-5 (Arousal r = 0.41) correlated with SSI-W. Conclusions: We investigated behavioral markers of arousal and BS as indices of suicide risk using MSSA patients at HR for recurrent SA. Our findings suggest that lifetime history of aggression, PSAP provoked aggressive responses, childhood trauma, and alcohol use are potential behavioral markers of high suicide risk. In general, measures related to sensitization were more likely to be related to HR, while impulsivity related more to SSI-W scores. This suggests that a sensitization-like process may predispose to HR; overt suicidal behavior may require combined long-term (HR; sensitization) and immediate (SI, impulsivity) action dysregulation. Keywords: Suicide Risk Factors, Suicide Attempt, Suicide Mechanisms Disclosure: Nothing to disclose. P466. Meaningfully Addressing Suicide Requires a Sustained and Integrated Effort Comparable to the War on Cancer Sheldon Preskorn* University of Kansas, Wichita, Kansas, United States Background: Over 20 years, a year over year increase in the number of Americans who die from suicide (currently that is one out of 62) has occurred despite multiple US Surgeon Generals highlighting this problem and multiple attempts to address using public health approaches. However, these have been piecemeal rather that a sustained, integrated approach. The problems with such approaches is a biopsychosocial phenomenon with different components including different drivers and protective factors in different individuals who die (or do not) by suicide. This topic is of direct relevance to the many, if not most, members of American College of Neuropsychopharmacology (ACNP) and the ACNP which can be a vital voice in advocating for a comprehensive approach to this problem. Methods: The methods included: personal knowledge, focused literature reviews and conversation with individuals at organization such as the NIMH section on suicide. The material on War on Cancer came from personal experience augmented by focused literature review on the history of its establishment and evolution into a cohesive organization with central tissue and treatment databases, Cancer Centers at academic medical centers in most states, and community based cancer sites with bi-directional and collaborative interactions including collection of tissue for biological examination, data collection from multiple clinical protocols run, analyzed and then refined by new protocol in an iterative process. Additional literature reviews were focused on risk factors for suicide and the medicolegal death investigation system in the United States. Results: Given space constraints, an exhaustive review of the results is not possible but can be more fully presented in the poster. The following is a top-level summary: The War on Cancer has provided a blueprint for the development of a similar initiative to meaningfully address suicide and has established the amazing ability of such a structure to make amazing advances in the understanding and effective treatment of multiple different cancers. A criticism could be that cancer permits collection and analyze of pathological tissue but that same is true for suicide using blood and even brain tissue, if necessary. Of importance, there are two distinct populations who complete suicide or make multiple suicide attempts. Most individuals who die by suicide do so on their 1st attempt and approximately 95% of those who die by suicide do so by their 3rd attempt. There are three psychiatric diagnoses that account for the majority of suicide completers. Three different psychiatric diagnoses that are heavily represented in the population who is at high risk for multiple suicide attempts. In the interest of space, this data will be present in the poster rathe than here. Suffice to say: the genes in these two populations may be quite different because of the different underlying diagnoses but the bottom-line is that piecemeal studies which use multiple attempters for suicide completers can cause confusion rather than clarity. That can be addressed in the database that will be established under this initiative and will be great importance to dissecting the genetic based risk of suicide. Briefly, the biopsychosocial understanding of suicide includes the following by way of example but not limited to: Biological Data: 1) The individual’s DNA and epigenetic changes; 2) Messenger RNA reflecting the state of the individual at the time of death by suicide; 4) Markers of inflammatory processes and stress response systemically and in the brain particularly the hypothalamic-pituitary axis; 6) Analysis of whole brain samples (when possible) and of brain sections in high value regions when examination of whole brain samples is not possible. These samples will be analyzed to determine whether the brain circuits in these regions have been damaged by concussive damage versus chemical damage on a smaller scale caused by sound waves or chemical exposure); 7) Measures of cognitive and emotional processing when available; 8) Surrogate measures of impulsivity (e.g., CSF levels of serotonin and/or monoamine oxidase-A (MAO-A) activity; 9) Age and gender; 11) Co-morbid medical illnesses and substance abuse and/or dependence. Psychological Data: 1) The individual’s religious and/or philosophical beliefs when they can be ascertained with sufficient certainty. For example, some religions forbid suicide, whereas other religions may praise martyrdom suicide); 2) The individual’s sense of locus of control (i.e., primarily internally or externally driven); 3) Adverse childhood experiences;4) Psychiatric diagnosis(es); 5) Number of previous suicide attempts. Social Data: How the individual functioned within the nuclear family and subsequent social networks including how the individual functioned within society as assessed by educational attainment, marriage, and ability to sustain functioning and advance within her/his chosen profession. All the above information and more as results indicate will be included in the database and statistical models constructed to determine the highest risk combination(s) realizing there may be multiple pathways to suicide completion requiring different predictive profiles and treatment interventions. Conclusions: This abstract lays out a meaningful pathway forward in understanding and ability to meaningful treat individuals at high risk of death due to suicide. The ACNP organization and its members have a vital role in making this idea reality. Keywords: Suicide, Suicide Risk Factors, Database Disclosure: Nothing to disclose. P467. Protective and Risk Factors Among American Indian Peoples Modulate the Relationship Between Mental Health Problems and Multi-Modal Neural Markers During an Inhibitory Control Task Evan White, Ricardo Wilhelm, Nicole Baughman, Breanna McNaughton, Rayus Kuplicki, Jennifer L. Stewart, Martin Paulus, Robin Aupperle Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: American Indian (AI) communities suffer from increased burden of mental health risk factors as a result of historical and ongoing sociopolitical and environmental challenges (e.g., historical loss, discrimination, disenfranchisement). Although this has resulted in disproportionate rates of mental health conditions in AI populations, there is also evidence of high levels of positive mental health in AIs. A growing body of literature shows traditional cultural factors (e.g., spirituality) can be protective against poor mental or physical health outcomes. This is consistent with research conducted in the broader population that shows religiosity is associated with decreased prevalence of deaths of despair. Moreover, our recent work has demonstrated that when accounting for disproportionate risk factors (i.e., sociodemographic factors, trauma exposure), a heterogenous AI group had equivalent rates of substance use disorder (SUD) and lower rates of suicidal thoughts and behaviors (STB) relative to matched non-Hispanic white peers. Furthermore, our work demonstrated that among the AI sample, brain activation during an inhibitory control task differentiated both individuals with and without STBs from those without STB and individuals with SUD and those without SUD. This investigation used a systems neuroscience framework to examine the possible cognitive and neural processes that mediate the protective effects of cultural factors among AI peoples. Specifically, the current study investigated the potential modulation of the relationship between STB and multi-modal neural markers of inhibitory control by culturally relevant protective (i.e., spirituality) and risk (i.e., historical loss thinking) factors in a sample of AI individuals. It was hypothesized that 1) AI spirituality would diminish the influence of STB on inhibitory control and 2) that historical loss thinking would exacerbate the same relationship. Methods: Participants were drawn from an initial pool of 60 individuals who self-identified as AI to any degree in the Tulsa-1000 (T1000) study and agreed to participate in a follow-up session to assess culturally-relevant protective and risk factors. The T1000 study was a naturalistic longitudinal study that included assessment of mental health and substance use conditions using the Mini International Neuropsychiatric Interview as well as multi-modal neural markers of inhibitory control using concurrent functional Magnetic Resonance Imaging (fMRI) and electroencephalography (EEG) recording during a stop signal task (SST). Participants (n = 37 total; no STB = 17) were included in analysis if they had completed the MINI interview and had quality fMRI and EEG data for the SST. Neural correlates of inhibitory control included the contrast of percent signal change during hard versus easy stop signal trials in the dorsolateral prefrontal cortex (dlPFC) and the P300 event-related potential. The Historical Loss Scale (HLS) was used to measure self-reported historical loss thinking and the Native American Spirituality Scale (NASS) was used to quantify self-reported spirituality. Results: Linear mixed effect (LME) models were used to examine the interaction of STB and NASS on dlPFC PSC and P300 amplitudes separately. LMEs were also used to examine the interaction of STB and HLS on both neural outcomes. For spirituality the hypothesis was supported for the dlPFC, wherein results indicated significant main effects of both NASS (F = 5.144, p = .026) and STB (F = 12.76, p < .001) on dlPFC; this was qualified by a significant STBNASS interaction on dlPFC (F = 13.54, p < .001). Results also supported the hypothesis for the P300 demonstrating a significant STBNASS interaction (F = 4.41, p = .03). For historical loss thinking results also supported the hypotheses. dlPFC results indicated a significant main effect of STB (F = 10.65, p = .002) qualified by a significant STBHLS interaction (F = 9.47 p = .003). Finally, P300 results demonstrated a marginal main effect of HLS (F = 3.23 p = .07) qualified by a STBHLS interaction (F = 5.56, p = .02). Conclusions: These preliminary results indicate that in a heterogenous sample of AI adults (1) spirituality may attenuate the relationship between STB and inhibitory control disruption and (2) this relationship may be exacerbated by historical loss thinking. These findings extend our previous work demonstrating that inhibitory control may be an important mechanism for understanding mental health risk and protective factors for AI populations. Furthermore, results bolster a growing body of research indicating that traditional cultural engagement may serve as a protective factor among AI communities. The current analysis is limited to a cross-sectional empirical design, a relatively small sample, and does not consider comorbidities (physical or mental health) or other mental health or substance use concerns. This analysis does suggest that inhibitory control may be an important neural mechanism of risk and protective factors specific to AI mental health. Future work is needed to delineate the prospective and potential causal directionality of these effects. Keywords: Suicide, Native Americans, Protective Factor, fMRI, Event Related Potentials Disclosure: Nothing to disclose. P468. Plasticity in the Gustatory Insular Cortex Underlying Perceptual Learning in Behaving Mice Joshua Kogan, Ayesha Vermani, Memming Park, Alfredo Fontanini State University of New York at Stony Brook School of Medicine, Stony Brook, New York, United States Background: To adapt and survive, animals must learn to discriminate overlapping stimuli predicting different outcomes. This phenomenon, known as perceptual learning (PL), has been well described in the visual, auditory, somatosensory, and olfactory systems. Specifically, improved performance on PL tasks is associated with plasticity of both sensory representations and decision related neural signals. However, few studies have addressed PL in the gustatory system. The primary sensory region for taste, known as the gustatory insular cortex (GC), encodes both sensory and decision-related information, but how these representations might change with PL remains unknown. Additionally, as part of the larger insular cortex, GC has been implicated in many processes including homeostatic regulation of food consumption and eating disorders. Thus, the present study addresses important gaps in the literature by improving our understanding of PL in the gustatory system and enhancing knowledge of neural coding in a region involved in many important gustatory-related functions and pathologies. Methods: The experiments are based on a novel gustatory PL paradigm, which relies on a two alternative forced choice task. Mice first learn a sucrose (100 mM) vs NaCl (100 mM) discrimination, in which sucrose presentation at a central spout is associated with reward at one lateral spout and NaCl with reward at the other. They are then trained to discriminate between increasingly similar pairs of mixtures: 75/25 vs 25/75, 65/35 vs 35/65 and 60/40 vs 40/60 (%sucrose/%NaCl). When animals make an error, they receive a timeout and no reward. Before and after learning, mice are tested on a battery of mixtures to establish psychometric curves. To assess GC plasticity, ensemble activity is monitored with two photon calcium imaging. Single neuron and population-level analysis are used to identify changes in both sensory and decision-related neural responses that occur with PL. Results: After learning, performance increased for all mixture pairs, indicating an improvement in discrimination (n = 6 mice, two-way ANOVA, effect of learning p < 0.001). Previous work from our lab using a similar behavioral task has shown that GC neurons encode sensory and decision-related information in separate temporal epochs. Specifically, sensory information is encoded in the epoch directly after taste sampling, and decision-related in-formation is encoded in the delay epoch directly before the choice. Analysis of all recorded neurons (Pre-learning: 2337 neurons; Post-learning: 2658 neurons) showed an increase in the proportion of delay responsive neurons (Pre - 19%, Post – 24% Χ2 test, p < 0.001), but no significant change in the sampling responsive population. In the delay but not sampling period, responsive neurons became more selective to the upcoming choice after learning, and the magnitude of the activity difference between trial types in the delay was larger. These findings suggest that gustatory PL may be mostly associated with improvements in decision-coding. To test is this is true at the population level, we employed logistic regression to decode the choice from the population of recorded neurons, and similarly found an improvement in decoding performance after learning. Conclusions: These results demonstrate that improved behavioral performance on a gustatory PL task is associated with enhanced decision-related coding in GC. We confirm this result using single neuron and population level analyses of neural activity. Overall, these experiments further elucidate the GC plasticity associated with PL and provide novel information about neural processing in gustatory insular cortical circuits. Keywords: Insular Cortex, Taste, Two-Photon Calcium Imaging, Mice, Perceptual Decision-Making Disclosure: Nothing to disclose. P469. NMRA-511, a Novel Vasopressin 1a (V1a) Receptor Antagonist Reduces Threat Behaviors in Marmosets and Alters EEG Power Spectra Similarly in Marmosets and Humans Tanya Wallace, Filomene Morrison, Philip Gerrard, Larry Ereshefsky, Michael Ballard, Joel Posener, Katherine Tracy, Jane Tiller Neumora Therapeutics, Brisbane, California, United States Background: The arginine-vasopressin (AVP) neuropeptide system is involved in regulating complex social behaviors and emotional states across species. Within the brain, the vasopressin 1a (V1a) receptor is the predominant receptor subtype and has been implicated in mediating anxiety and threat processing. Given such emotional processing impairments are present in many brain disorders, modulation of the AVP system through the V1a receptor provides a viable approach for the treatment of these disorders. NMRA-511 has been extensively characterized in in vitro and in vivo systems and is a potent small molecule antagonist at the V1a receptor with selectivity over closely related V1b, V2 and oxytocin receptors (OTR) as well as to broad panels of off-target receptors, ion channels, and transporters, and is brain penetrant and orally bioavailable across species. The current studies were designed to evaluate the potential of NMRA-511 to modulate anxiety-related behaviors in marmoset monkeys (Callithrix jacchus) to a human intruder using the Human Threat Test (HTT). In the HTT, marmosets recognize the presence of a human observer as a threat and will present characteristic behavioral postures accompanied biochemically with elevated plasma cortisol concentrations. Additionally, using a translational pharmaco-electroencephalography (phEEG) approach, the effects of NMRA-511 on relative power across spectral frequency bands were evaluated in both marmosets under physiological and threat conditions and extended into a human healthy control population under physiological (resting state) conditions as an exploratory measure in a phase 1 single/multiple ascending dose trial. Methods: Adult marmosets (n = 8) treated with NMRA-511 (0-30 mg/kg, PO) or the positive control chlordiazepoxide (CDP; 2 mg/kg, SC) were used for the HTT. A separate cohort of marmosets (n = 6) was implanted with EEG electrodes above the frontal and parietal cortices and electromyography electrodes were fixed to the neck muscles. A single dose of NMRA-511 (10 mg/kg, po) or vehicle was evaluated in the marmoset phEEG study. Animals were treated according to a random blind crossover design, with a washout period of at least three days between treatments. Data were analyzed using RM-ANOVA (p < 0.05). In the healthy control study, NMRA-511 (5, 10, 15 mg dose) or placebo was administered once orally (n = 6:2; NMRA-511:placebo) and EEG from a 19-channel, 10/20 system was recorded for 6.5 hours. Tmax-centered mixed models for repeated measures served as the primary analysis (p < 0.1). Results: In the marmoset, orally administered NMRA-511 (10 and 30 mg/kg) significantly reduced anxiety-related behaviors as measured by a decrease in the number of threat-elicited postures observed in the HTT without affecting locomotor activity or causing sedation. Results from the phEEG study in the marmoset revealed significant changes specifically in the theta (4-8 Hz) and alpha (8-12 Hz) bands under physiological conditions that were further exacerbated under the threat condition. Non-significant trends were also observed in the beta (12-30 Hz) band. The phEEG studies were extended to a human healthy control population and in line with hypotheses, Tmax-centered analyses indicated significant increases from baseline in theta (eyes open [EO] and eyes closed [EC] conditions) and alpha (EO) in the midline frontal region of the 15 mg cohort (n = 6) on Day 1 compared to placebo (n = 11). Non-significant trends of moderate to large effect sizes suggesting an increase in beta were also noted at the 15 mg dose. At the 10 mg dose (n = 24), a significant increase in beta was observed (EO and EC conditions) with a moderate-sized non-significant increase in theta (EO) proximal to Tmax. The ascending single and multiple doses of NMRA-511 were safe and well tolerated by the healthy participants in this study. Conclusions: Oral administration of the V1a antagonist NMRA-511 produced anxiolytic-like activity without sedation in the marmoset threat test. The same dose of NMRA-511 (10 mg/kg) that reduced anxiety in the marmoset demonstrated increased relative power in the alpha and theta spectral bands with trends in the beta band in this species. Furthermore, using phEEG as a translatable biomarker, NMRA-511 was evaluated in a healthy control population and demonstrated similar changes in alpha and theta power and trends in the beta band as were observed in the marmoset. These studies support the utility of phEEG as a translational tool bridging preclinical and human populations; and the continued investigation of NMRA-511 in the treatment of brain disorders. Keywords: Vasopressin 1a Receptor Antagonist, EEG Biomarkers, Anxiety, Translational Neuroscience Disclosure: Neumora Therapeutics: Employee (Self), Neumora Therapeutics: Stock / Equity (Self) P470. Compassionate Imagery Practices Priming for Psilocybin Ceremonies: A Large Scale Field Study Carla Pallavicini, Lorena Llobenes, Federico Cavanna, Natali Gumiy, Maria Costa, Nicolas Bruno, Stephanie Muller, Laura Alethia De La Fuente, Paul Gilbert, Enzo Tagliazuchhi Computational Cognitive Neuroscience Lab, Universidad de Buenos Aires, Caba, Argentina Background: Classical psychedelics like psilocybin, N-N dimethyltryptamine (DMT), lysergic acid diethylamide (LSD) and mescaline are known to induce profound, transient alterations in consciousness. Psychedelic research has shed light on the neurophysiology of these altered states of consciousness, as well as on their potential for the treatment of several mental health disorders, and for the promotion of a sustained state of well-being. On the other hand, the increased focus on meditation in cognitive neuroscience resulted in a cross-cultural classification of standard meditation styles, which was validated by functional and structural neuroanatomical data. Compassionate imagery is a form of guided meditation focusing on building a compassionate image of oneself and others to work with and develop. This practice has been linked to psychological benefits and general increases in wellbeing markers; one of our main objectives was to understand whether these benefits can be facilitated by the sense of contentedness catalyzed by psilocybin mushrooms. Methods: After thorough screening, specially focused on participant safety, 105 healthy volunteers were measured in this experiment. We designed an ambitious setup in order to cover as many dimensions of the experience and its effects as possible. Participants were divided into 4 groups: low vs. high dose of psilocybin and compassion vs. control meditation. Physiological and activity data was acquired 1 week before and after the day of the ceremony using Fitbits. Also, fMRI scans, blood and saliva extractions were performed 1-3 of days before and after as well. Participants responded an extensive selection of scales and questionnaires, respecting personality traits, well-being, anxiety, among others one week before the ceremony. These measures are repeated a week, 1, 3 and 6 months after the ceremony. Starting the day of the ceremony we perform individual interviews, psychometric scales responses and EEG registers. After this, volunteers received the meditative practice corresponding to their group, followed by an individual interview and proceed to their ceremony with a facilitator. We did not interact with participants during their experience. After the ceremony, participants responded a set of questionnaires designed to assess their experience, and we finished by taking EEG registers, saliva samples, individual interviews and group sharing sessions. Follow-up measures consisted of the scales and questionnaires mentioned above and a blood and saliva extraction performed 2 months after the ceremony. Results: Preliminary results, show that participants in the compassion groups scored significantly higher on interoceptive constructs such as listening to body and confidence as reported by the MAIA scale (p < 0,05), and also reported to have a subjective experience with a larger influence of elementary imagery (p < 0.05). Only for the compassion group, there was a difference in the subjective construct of changed meaning between high and low dose. Reported incidents of anxiety and difficult experiences only occurred in the high dose groups and were of low incidence (<5% of participants). Interviews presented differences in verbosity amongst high and low dose groups and EEG data also tend towards differences amongst these groups. Follow-up measures are still in progress, but we expect the differences to accentuate over time and to find new indications on how this combination of practices reflects on subjective well-being markers. Conclusions: We were able to perform a very large amount of measures on a large amount of participants in a combined psychedelic and meditation study. This experiment is ground breaking for this kind of research in Latin America. We found that these ceremonies were safe for participants and the combination with meditative practices contributed to the low amount of difficult experiences observed. The ceremonial use of Psilocybin can have a positive effect in self-reported measures of psychological well-being, mood, prosocial behavior, emotional regulation and cognitive capacities. Psychological priming with a compassionate imagery practices could be a useful tool to increment the positive impact of the subjective experience, our results show a trend in that direction. Keywords: Psilocybin, Meditation, Psychological Wellbeing, EEG, Functional MRI (fMRI) Disclosure: Nothing to disclose. P471. Kappa Opioid Receptor Availability in Borderline Personality Disorder and Relationship to Suicidal Behavior: Preliminary Results From a [11 C]EKAP PET Study Margaret Davis, Olivia Wilson, Ashley Wagner, Nabeel Nabulsi, David Matuskey Yale University School of Medicine, Waterbury, Connecticut, United States Background: Borderline personality disorder (BPD) is a serious psychiatric condition associated with high morbidity and mortality and elevated risk for suicide. Despite this, treatments capable of affecting overall symptom severity and reducing suicide risk in BPD are limited. Promisingly, recent evidence has implicated the kappa opioid receptor (KOR) in both BPD and suicidal behavior. Specifically, postmortem studies have shown an association between KOR and death by suicide, and studies in both animals and humans have shown that KOR antagonists can produce antidepressant, anxiolytic, and even anti-suicidal effects. To date, however, the relationships between KOR, BPD, and suicidal behavior have not been directly investigated. The aim of this study was to examine the relationship between KOR availability, BPD, and suicidal behavior in vivo using [11 C]EKAP PET. Methods: Individuals with BPD (N = 8, 4 with a history of suicide attempt (SA), 4 without (NSA)), and healthy adults (HA; N = 8) matched for age, sex (6 women and 2 men in each group), and smoking history were recruited from the community. All participants completed 1 MRI scan and 1 PET scan with [11 C]EKAP, as well as a battery of psychiatric and cognitive assessments. The radiotracer was injected as bolus and subjects were scanned for 120 minutes. Volume of distribution (VT: the ratio of activity in tissue relative to that in blood, corrected for metabolites) was computed using arterial input function. Primary analyses focused on a circuit of 6 frontal and limbic brain regions relevant to the pathophysiology of BPD and suicidal behavior: dorsolateral prefrontal cortex (dlPFC); orbitofrontal cortex (OFC); ventromedial prefrontal cortex (vmPFC); anterior cingulate cortex (ACC); amygdala; hippocampus. Results: ANOVA analyses revealed significant group differences in KOR availability (VT; p = .009, d = 1.85, 27% lower). Notably, differences were most pronounced among BPD individuals reporting a history of SA. Specifically, BPD individuals reporting a history of SA had significantly lower KOR availability relative to both BPD-NSA (p < .001; d = 1.93; 24% difference), and HA (p < .001; d = 2.24; 35% difference). Further, frontolimbic KOR availability in BPD was negatively correlated with endophenotypic correlates of suicide risk in BPD, including difficulties in emotion regulation (r = -.83), impulsivity (r = -.80), and depressed mood (r = -.82). Conclusions: Preliminary results support a relationship between KOR, BPD, and suicidal behavior in BPD. Individuals with BPD had lower frontolimbic KOR availability in relative matched HA. Additionally, individuals with BPD who had a history of SA had lower frontolimbic KOR availability relative to both BPD-NSA and HA, suggesting a potential role for KOR as a trait marker of suicide risk in BPD. Of clinical significance, KOR availability was negatively correlated with endophenotypic correlates of suicidal behavior in BPD including difficulties in emotion regulation, impulsivity, and depressed mood, variables which have been shown to predict both future SA and suicide mortality. Taken together, these results provide the first direct evidence for a possible role for KOR in the pathophysiology of BPD and suicidal behavior. Further evaluation of KOR targets for the treatment of suicidal behavior in BPD is warranted. Keywords: Borderline Personality Disorder, Suicide Attempt, Kappa Opioid Receptor, PET Imaging Study Disclosure: Nothing to disclose. P472. MicroRNA-124 and Suicide Ideation in Borderline Personality Disorder: Possible Protective Effect of the Globus Pallidus Macarena Aloi, Guillermo Poblete, John Oldham, Michelle Patriquin, David Nielsen, Thomas Kosten, Ramiro Salas Baylor College of Medicine, Houston, Texas, United States Background: Borderline personality disorder (BPD) patients suffer from unstable affect and interpersonal relationships, and strong suicide risk. MicroRNA-124-3p (miR-124) was identified in a Genome Wide Association Study as likely associated with BPD. Methods: Using TargetScan and the Allen Atlas datasets we identified the brain regions where co-expression of genes likely modulated by miR-124 are highest and compared morphometry in those brain regions in BPD inpatients (N = 111) vs. controls matched for psychiatric comorbidities (N = 111) in the Menninger Clinic in Houston, Texas. Next, we correlated personality measures, suicidal ideation intensity, and recovery from suicidal ideation with volumetrics. Results: Gene targets of miR-124 were significantly co-expressed in the left Globus Pallidus (GP), which was smaller in BPD than psychiatric controls. In BPD but not controls, smaller GP volume was negatively correlated with agreeableness and with recovery from suicidal ideation post-treatment. In BPD but not controls, the GP was smaller in older than younger patients. Conclusions: In BPD, GP volume may be reduced through miR-124 regulation and suppression of its target genes. Importantly, we identified that reduction of the GP could serve as a potential biomarker for recovery from suicidal ideation in BPD. Keywords: Borderline Personality Disorder, Human Epigenetics/microRNA, Globus Pallidus, Morphometry, Human Brain Imaging Disclosure: Nothing to disclose. P473. Insular-Prefrontal Circuit Driving Compassionate Social Behavior Songjun Li, Pauline Gabrieli, Moeko Suzuki, Omer Zeliger, Jack Demaree, Renee Cauchon, Nicole Occidental, Ziv Williams Massachusetts General Hospital, Boston, Massachusetts, United States Background: Compassionate behavior, or the ability to help others in need, is a cornerstone of prosocial interaction in mammals and humans. To benefit others, it is necessary for individuals not only to perceive the internal states or emotions of others but also to take appropriate actions. For example, one must identify the distress of another and perform specific actions that will relieve their discomfort. Yet, how mammalian neurons precisely link social-specific information with such complex adaptive behavior has been a major challenge to understand. Methods: We recorded single-neuronal activity in the dorsal anterior cingulate cortex (dACC) and the anterior insula (AI), two brain regions involved in prosocial behaviors, while mice directly controlled the experience of a nearby conspecific partner in real-time. This novel task allowed the animal’s own actions to be dissociated from the other’s identity and experiences. Additionally, we utilized chemogenetic tools to either inhibit or stimulate the dACC-AI circuit to modulate these behaviors. Results: We found that wild-type male mice consistently chose to reduce aversive experiences of familiar but not unfamiliar partners, actions that were not observed when visual and olfactory cues were blocked. AI cells preferentially encoded task-relevant information including the social identity of partners and their specific experience, while dACC cells preferentially encoded information about the act of helping their partners. Whereas information about the experience of others could be predominantly decoded from AI activity, information about the animal’s prosocial actions could be predominantly decoded from dACC activity. Chemogenetic excitation of AI-to-dACC projectors but not dACC-to-AI projections increased compassionate behavior, while inhibition of both dACC to AI as well as AI to dACC projectors decreased compassionate behavior with familiar partners. Conclusions: Our data suggest that the mice displayed consistent compassionate behavior towards familiar partners, where there exists a partitioning of information within the insular-prefrontal circuit. Specifically, the AI appears to transmit social-specific information to the dACC while the dACC controlled compassionate decisions based on information from the AI. Taken together, these findings identify a putative insular-prefrontal circuit for driving compassionate behavior and a mechanism that could allow insular neurons to instruct social-specific actions through prefrontal control. Keywords: Social and Behavioral Deficits, Empathy, Decision Making, Mice, Single-Unit Electrophysiology In Vivo Disclosure: Nothing to disclose. P474. Activity-Based Mapping of Altered Neural Network in a Mouse Model of Social Deficits Ruixiang Wang, Zeru Peterson, Nagalakshmi Balasubramanian, Kanza Khan, Gabrielle Bierlein-De La Rosa, Thomas Nickl-Jockschat, Catherine Marcinkiewcz The University of Iowa, Iowa City, Iowa, United States Background: Deficits in social functioning are a common feature of various psychological disorders, such as schizophrenia and autism spectrum disorders. However, the neural mechanisms underlying such deficits are still unclear due to the dynamic, complex nature of social behavior. Combining the methods of immunolabeling-enabled 3D imaging (iDISCO), chemogenetics, and functional MRI (fMRI), the present study aimed to map the altered neural network associated with social deficits in a mouse model. Methods: Male C57BL/6 J mice were treated with ketamine (3 mg/kg, i.p.) or vehicle (0.9% saline) for 10 days. 24 hours after the last treatment, they underwent a three-chamber sociability test or an equivalent test with two empty enclosures. Their whole brains were then immunostained with a c-fos antibody, cleared with dibenzyl ether, and imaged under a light sheet microscope. Because a marked reduction in c-fos activation was observed in the lateral septum (LS) after chronic ketamine exposure, we tested whether reduced activity in the LS was necessary and sufficient for social deficits by chemogenetically manipulating LS neurons. Furthermore, to examine the functional connectivity originating from the LS that regulates social behavior, we performed chemogenetic fMRI mapping of the LS neural network. Results: Chronic ketamine exposure led to a significant reduction in social interaction (p < 0.001), which was associated with blunted c-fos induction in the LS (p < 0.05), shown by iDISCO imaging. As such, we transduced naïve C57BL/6 J mice with a Gq-coupled DREADD (hM3Dq-mCherry) in the LS and then treated them with chronic ketamine. 24 hours after the last ketamine administration, it was shown that activation of the Gq-DREADD in the LS mediated by clozapine-N-oxide (CNO; 3 mg/kg, i.p.) rescued the social withdrawal induced by chronic ketamine exposure (p < 0.001). To examine if inhibition of previously activated LS neurons during social interaction was sufficient to induce social deficits, we microinjected a Cre-inducible Gi-coupled DREADD (DIO-hM4Di-mCherry) in the LS of fos-iCreER mice. After undergoing a social-interaction session, these mice were administered with 4-OH-tamoxifen (10 mg/kg, i.p.) to induce Cre-mediated recombination and DREADD expression in active neuronal populations. Four weeks later, we conducted the sociability test 45 minutes after a CNO injection. It was shown that inhibition of LS neurons by the Gi-DREADD led to reduced social interaction (p < 0.001). In eGFP-L10A mice microinjected with an hSyn-Cre virus in the LS, we observed strong eGFP expression in the hippocampus, which, accordingly, constitutes a major projection area of the LS neurons. Therefore, we studied this circuitry with fMRI. Fos-iCreER mice were transduced with a Cre-inducible Gq-coupled DREADD (DIO-hM3Dq-mCherry) in the LS. They were then exposed to a social-interaction session and injected with 4-OH-tamoxifen. After four weeks, an fMRI scan was performed following CNO administration. A trend of decreased functional connectivity between the LS and the hippocampal formation was observed (p < 0.09). Conclusions: As a valid mouse model for studying social deficits, chronic ketamine exposure results in hypoactivation of the LS neurons, which is necessary for inducing social withdrawal. Moreover, inhibiting activated LS neurons in social interaction is sufficient for the expression of social deficits. In addition, previously, it has been determined that the majority of LS neurons are GABAergic and thus inhibitory in nature. As such, activating LS neurons leads to a trend reduction in connectivity with the hippocampus, a major projection area of LS neurons. Keywords: Social Deficits, Subchronic Ketamine Model, Lateral Septum, iDISCO Mapping, fMRI Disclosure: Nothing to disclose. P475. Investigating Protein Glycosylation in Schizophrenia: From Genetics to Treatment Targets Robert Mealer, Richard Cummings, Jordan Smoller Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, United States Background: Schizophrenia is a severe and highly heritable neuropsychiatric disorder. Recent studies have shed light on the complex genetic architecture of the condition, identifying over 270 common variants and 10 rare variants which confer significant changes in risk. However, treatment development remains a considerable challenge given the polygenic pattern of inheritance and clinical heterogeneity of schizophrenia. One pathway with convergent evidence for a role in schizophrenia pathogenesis is altered glycosylation, the enzymatic attachment of carbohydrates to proteins and lipids to regulate their function. Methods: To generate a broad framework for understanding the relationship between schizophrenia and glycosylation, we will review results from several recent, well powered, and peer reviewed studies. The primary objectives are to: 1) Explore publicly available schizophrenia GWAS and exome sequencing data for enzymes of glycosylation and proteins regulated by glycosylation (glycoproteins); 2) Describe post-mortem studies of schizophrenia investigating of brain glycoproteins; 3) Summarize animal models of schizophrenia risk genes involved in glycosylation. Results: Among GWAS results, the list of glycosylation enzymes unambiguously associated with schizophrenia continues to grow, with nearly a dozen prioritized genes implicated across several domains of protein glycosylation. Of the rare coding variants, protein products of the 3 genes directly involved in neurotransmission (GRIN2A, CACNA1G, and GRIA3) are all well known to be regulated through glycosylation. Post-mortem studies have identified changes in levels of glycoproteins and their transcripts within several glycosylation pathways, though these findings may result from the environment encountered while living with schizophrenia. Finally, mouse models of genetic risk for schizophrenia, including our work involving the SLC39A8-A391T common variant, display brain glycosylation changes in glycoproteins both genetically and functionally implicated in schizophrenia pathogenesis, including cell adhesion molecules and neurotransmitter receptors. Conclusions: Several lines of evidence support a role for altered protein glycosylation in the pathogenesis of schizophrenia. As congenital disorders caused by severe mutations in many of the same genes can be effectively treated through targeted supplementation of enzymatic precursors and cofactors, the glycosylation pathway may represent a novel opportunity for therapeutic development in schizophrenia. Further, we hope this information will help researchers better understand and approach protein glycosylation in their studies of schizophrenia pathogenesis. Keywords: Schizophrenia (SCZ), Genetics, Glycosylation Disclosure: Nothing to disclose. P476. OMgp Signaling as a Mechanism of Cortical Dendritic Regression Melanie Grubisha, Nicholas Lehman, Ryan Salisbury, Susan Erickson, Gregg Homanics, Matthew MacDonald, Robert Sweet University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Onset of gray matter reductions during adolescence, coupled with postmortem findings of smaller dendrites, have consistently been shown in schizophrenia (Sz). Once formed, dendrites are largely stable structures due to competing growth and regression signals. Understanding the pathways involved in this dynamic homeostasis is crucial to developing interventions targeting dendritic regression in Sz. We have recently shown that OMGp, a ligand for Nogo receptor 1 (NGR1), activates a signaling pathway that shifts this balance leading to dendritic regression. This pathway requires the active domain of KAL9. Knock-in of a KAL9 gain-of-function mutation (KAL9-PT) resulted in adolescent-onset dendritic regression, coincident with both the timing of normal developmental increase in OMGp expression and clinical symptom onset in SZ. We hypothesized that activation of OMGp/NGR1/KAL9 leads to alterations in the phosphorylation of downstream mediators of dendritic structure, some of which are Sz-risk genes. Methods: We used OMGp stimulation in dissociated cortical culture followed by phosphoenrichment and mass spectrometry to identify the course of downstream signaling events using phosphoproteomics. Crispr/Cas9 was used to create a floxed OMGp mouse for developmental studies of OMGp KO on cortical dendritic architecture. Results: Phosphorylation is a post-translational modification capable of regulating a protein’s function, either activating or inactivating it depending on the site of phosphorylation. Among the proteins with significantly increased phosphorylation downstream of OMGp treatment were Trio and Cacna1g, both of which have an excess of loss of function mutations in SZ that are genome-wide significant. Loxp sites were successfully inserted flanking the OMGp gene without disrupting OMGp expression levels prior to recombination by Cre. Studies of dendritic architecture in OMGp KO mice are ongoing. Conclusions: We identified an overlap between the adolescent-onset OMGp signaling pathway and genetic risk for Sz (ie Trio and Cacna1g). Using mouse models, we will next determine whether targeting these mediators in vivo during the normative adolescent increase in OMGp activity can be used to prevent or reverse dendritic regression. These findings provide a new opportunity for developing novel targeted therapies. Keywords: Dendrites, Schizophrenia (SCZ), Adolescence Disclosure: Nothing to disclose. P477. 12 H Rhythm Abnormalities in the Human Dorsolateral Prefrontal Cortex of Subjects With Schizophrenia Madeline Scott, Wei Zong, Kyle Ketchesin, Marianne Seney, Bokai Zhu, George Tseng, Colleen McClung University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Schizophrenia (SZ) is a chronic neuropsychiatric illness associated with cognitive dysfunction and disrupted circadian behaviors. Consistent with circadian rhythm dysfunction, studies have demonstrated abnormal peripheral gene expression of circadian clock genes and changes in rhythmic expression of hormones in individuals with SZ. Molecular rhythm patterns, however, have only just begun to be characterized in the human brain. Our lab has recently observed a distinct set of diurnally rhythmic transcripts in SZ subjects relative to a cohort with no history of psychiatric illness (non-psychiatric; NP) using rhythmic analysis of RNA-sequencing (RNA-seq) data from the dorsolateral prefrontal cortex (DLPFC), a region associated with executive dysfunction and heavily implicated in SZ. Pathway analysis demonstrated that genes that gained 24 h rhythmicity in SZ were associated with mitochondria dysfunction and GABA-ergic signaling, which is consistent with previously observed abnormal expression of transcripts associated with mitochondria and GABA-ergic signaling. A growing body of literature, however, has demonstrated that gene expression can also be regulated by ultradian rhythms (rhythms with a period < 24 h). 12 h rhythms are observed in various aspects of human behavior (sleep patterns, cognitive performance) and biology (body temperature, blood pressure, migraine onset, circulating hormone levels). 12 h transcript expression rhythms are enriched for mitochondria-associated proteins across species and tissues, suggesting 12 h rhythmicity is a conserved component of mitochondria gene expression and may have a potential role in mitochondria rhythmic disruptions in SZ DLPFC. Methods: In the current study, we performed rhythmicity analyses on RNA-seq data obtained by the CommonMind Consortium through the NIMH Repository and Genomics Resource, a centralized national biorepository for genetic studies of psychiatric disorders, in both NP (n = 104) subjects and subjects with SZ (n = 46). Both sexes were included in all analyses. Prior to either analysis, time of death (TOD) was determined for each subject and normalized to a zeitgeiber time (ZT) scale. We then applied multiple types of rhythmicity analyses to each cohort of subjects, including cosinor nonlinear regression, the eigenvaluepencil method, and a Lomb-Scargle analysis, followed by pathway (Ingenuity Pathway Analysis, Metascape) and motif enrichment (LISA Cistrome Combined) analyses. Results: Multiple types of rhythmicity analyses identified 12 h rhythms in transcript expression in the human DLPFC. These transcripts were enriched for mitochondria-associated processes and motif enrichment analyses implicated ETS domain containing transcription factors. In subjects with SZ, fewer transcripts with 12 h rhythms were observed (800 transcripts, p < 0.05), and this loss of rhythmicity was associated with the unfolded protein response. In both cohorts, two timing patterns emerged, one in which transcripts peaked in expression in the morning/evening (ZT 2-3; ~9 AM/PM) and another in which transcripts peak during the afternoon/night (ZT 9; ~3 PM/AM). Transcripts associated with neuronal structural maintenance peaked during the afternoon/night in NP subjects but were not rhythmic in SZ subjects. Mitochondria-associated transcripts peaked in expression during the morning/evening in NP subjects, but during the afternoon/night in SZ subjects. Conclusions: Overall, this study identified 12 h rhythms in transcript expression in human DLPFC. These rhythms are associated with fundamental cellular processes and had distinct timing patterns. However, in SZ, there is a strong reduction in the number of transcripts with 12 h rhythms. Additionally, in the NP cohort, transcripts associated with mitochondria peak in expression during transition periods (~9 AM/PM), in which people are likely switching from states of wake/sleep and/or fasting/eating and may require increased energy. However, in the SZ cohort these genes have an anti-phasic shift and peak during static periods (~3 PM/AM), implicating a deficit in mitochondria expression during transition periods in SZ. These data suggest alterations at multiple levels in the rhythmic regulation of mitochondria-associated genes in schizophrenia. Future work will be necessary to determine whether these changes underlie disruptions in energetic homeostasis and neuronal dysfunction in the DLPFC that could potentially contribute to symptoms of cognitive dysfunction in SZ. Keywords: DLPFC, Circadian Rhythms, Postmortem Human Brain Tissue, Mitochondria Disclosure: Nothing to disclose. P478. Alterations in RBFOX1 and its Target Transcript in Prefrontal Cortical Dysfunction in Schizophrenia Youjin Chung, Daniel Chung, Samuel Dienel, Kenneth Fish, David Lewis University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Current findings suggest that prefrontal cortical dysfunction in schizophrenia involves alterations in parvalbumin interneurons, but not calretinin interneurons. For example, molecular markers of inhibitory drive from parvalbumin interneurons are lower in the prefrontal cortex of schizophrenia subjects. In rodents, the strength of inhibitory drive from parvalbumin interneurons is partly regulated by RNA binding protein Rbfox1. Rbfox1 is alternatively spliced into isoforms that localize to either the nucleus (nuclear Rbfox1) or cytoplasm (cytoplasmic Rbfox1) of neurons. The binding of cytoplasmic Rbfox1 to its target transcripts increases the stability of, and therefore the cellular levels of these transcripts. The targets of cytoplasmic Rbfox1 are enriched for transcripts that regulate neurotransmitter release. Thus, we hypothesize that cytoplasmic Rbfox1 levels are altered in schizophrenia and could contribute to lower inhibitory drive from parvalbumin interneurons by disrupting the expression of its target transcripts. Methods: Multi-label fluorescent immunohistochemistry was performed to quantify the protein levels of Rbfox1 in parvalbumin interneurons in the prefrontal cortex of schizophrenia and unaffected comparison subjects. Next, a prior microarray study that assessed the transcriptome of parvalbumin interneurons in schizophrenia and unaffected comparison subjects was explored to identify targets of cytoplasmic Rbfox1 that might be altered in schizophrenia. Finally, multi-label in situ hybridization combined with immunohistochemistry was used to quantify the levels of cytoplasmic Rbfox1 protein and its target mRNA in parvalbumin interneurons in schizophrenia and unaffected control subjects. Results: In the prefrontal cortex of unaffected comparison subjects, levels of total Rbfox1 were significantly 4.0-fold greater in parvalbumin interneurons relative to calretinin interneurons. The levels of cytoplasmic or nuclear Rbfox1 were 9.6-fold or 3.0-fold greater in parvalbumin interneurons relative to calretinin interneurons, respectively. Levels of total Rbfox1 were significantly 27% lower in parvalbumin interneurons in schizophrenia relative to unaffected comparison subjects, with both cytoplasmic and nuclear isoforms of Rbfox1 showing similarly lower levels. Given that levels of cytoplasmic Rbfox1 are markedly enriched in parvalbumin interneurons and show a reduction in schizophrenia, we explored whether the targets of cytoplasmic Rbfox1 are altered in schizophrenia. Of the 109 transcripts that have been identified as direct targets of cytoplasmic Rbfox1, levels of 7 transcripts were positively correlated with cytoplasmic Rbfox1 levels in parvalbumin interneurons across schizophrenia subjects. Of these 7 transcripts, Vamp1 was chosen for further study given its enrichment in parvalbumin interneurons and its role in regulating the strength of inhibitory drive from these neurons. The highest densities of Vamp1-immunoreactive cells were found in layer 4 of the prefrontal cortex where parvalbumin interneurons are enriched and show lower parvalbumin levels in schizophrenia. Vamp1 mRNA levels were significantly 20% lower in parvalbumin interneurons in schizophrenia compared to unaffected comparison subjects. Finally, Vamp1 mRNA levels were positively correlated with cytoplasmic Rbfox1 levels in parvalbumin interneurons across both schizophrenia and comparison subjects. Conclusions: Our findings demonstrate a cell type-specific enrichment of Rbfox1 in parvalbumin interneurons in the human prefrontal cortex. Additionally, we show that the levels of Rbfox1 and its two isoforms are lower in parvalbumin interneurons in schizophrenia. Finally, lower levels of cytoplasmic Rbfox1 in schizophrenia predicted lower levels of its downstream presynaptic target transcript, Vamp1, in parvalbumin interneurons. Together, these findings suggest that the lower level of cytoplasmic Rbfox1 in parvalbumin interneurons in schizophrenia may contribute to reduced inhibitory synaptic drive from these neurons by disrupting the stability of, resulting in lower levels of, its downstream transcript Vamp1. Given that Rbfox1 has been implicated in the genetic risk for schizophrenia, our findings suggest that lower inhibitory drive from parvalbumin interneurons in schizophrenia might arise, at least in part, from an intrinsic deficit in these neurons via altered regulation of the Rbfox1 pathway. Keywords: Rbfox1, Parvalbumin Interneurons, Schizophrenia (SCZ), Prefrontal Cortex, Inhibitory Synaptic Transmission Disclosure: Nothing to disclose. P479. Effects of Psychiatric Disease and Aging on FKBP5/1 Expression are Specific to Cortical Supragranular Neurons Natalie Matosin, Darina Czamara, Janine Knauer-Arloth, Naguib Mechawar, Brian Dean, Gustavo Turecki, Thomas Hyde, Elisabeth Binder Illawarra Health and Medical Research Institute, Wollongong, Australia Background: Deducing genes capable of classifying biologically-distinct psychiatric subtypes, and their targets for treatment, is a priority approach for the field of psychiatry. FKBP5 is a gene with decades of evidence indicating its pathogenic role in a subset of psychiatric patients, with high potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, in which cell-types and sub-anatomical brain areas FKBP5/1 is specifically affected is not known. This knowledge is critical to propel FKBP5/1-targeted treatment development. Methods: We performed an extensive, large-scale postmortem study (n = 1024, 6 cohorts) of FKBP5/1 examining dorsolateral prefrontal/orbitofrontal cortex (BA9, BA11, BA24) samples derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease-state, aging and genotype on cortical FKBP5/1 expression. Results: Our results demonstrate that FKBP5/1 cortical expression was strikingly increased (+17-40%) in individuals with schizophrenia or depression (+24%) vs controls. We also observed a strong effect of age with heightened FKBP5/1 expression in older psychiatric subjects versus older controls (e.g., mRNA: R = 0.664, P(FDR) = 5.6E-06). Further examination of the cell-type specificity of these findings with single nucleus RNA sequencing (snRNAseq) and targeted RNAscope/immunohistochemistry demonstrated that the disease- and aging-effects on FKBP5/1 expression are specific to supragranular neurons in cortical layer 3, with +25-32% increase specifically in supragranular layer 2-3 excitatory neurons in both schizophrenia and depression. Conclusions: Our results provide a new contribution to the field, indicating that effects of psychiatric disease-state and age converge on supragranular neurons of the superficial cortical layers, thus pinpointing a clear cellular target for future drug development. Keywords: Early Life Stress (ELS), FKBP5, Schizophrenia Novel Treatment, Major Depression, Schizophrenia (SCZ) Disclosure: Nothing to disclose. P480. The Clinical Candidate Xanomeline Displays a Dual Orthosteric and Allosteric Binding Profile at the M4 mAChR Wessel A.C. Burger, Vi Pham, Alexander Powers, Ziva Vuckovic, Jesse I. Mobbs, Alisa Glukhova, Denise Wootten, Andrew B. Tobin, Patrick M. Sexton, Steven M. Paul, Christian Felder, Radostin Danev, Arthur Christopoulos, Ron O. Dror, Celine Valant, David M. Thal Karuna Therapeutics, Indianapolis, Indiana, United States Background: The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The M1 and M4 mAChR preferring investigational drug candidate, xanomeline, has demonstrated clinical efficacy in the Positive and Negative Symptom Scale (PANSS) in a Phase IIb clinical trial for the treatment of schizophrenia. Initially, xanomeline had been considered to bind only to the orthosteric acetylcholine binding site of the mAChRs. However, recent studies have shown much greater complexity as to the precise nature of ligand-receptor binding interactions including efficacy-driven selectivity, subtype-dependent wash-resistant binding, and an atypical interaction with positive allosteric modulators. Understanding how xanomeline binds to the mAChR family and the molecular mechanism behind its unique pharmacological profile are key for the design of novel antipsychotics that target the M4 mAChR. Methods: We determined a cryogenic electron microscopy (cryo-EM) structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Molecular dynamics (MD) simulations were used to corroborate the additional allosteric binding mode that was identified in the cryo-EM structure. The allosteric binding mode of xanomeline was further validated using site-directed mutagenesis. Results: Unexpectedly, two molecules of xanomeline were found to simultaneously bind to the M4 mAChR. One molecule was bound to the orthosteric acetylcholine binding site and a second molecule was bound to the canonical mAChR allosteric binding site. MD simulations support the structural findings by showing that xanomeline was able to stably bind to the allosteric binding site in a manner similar to the positive allosteric modulator LY2033298. In contrast, the agonist iperoxo does not stably bind to the allosteric site, suggesting the dual orthosteric and allosteric binding profile is specific to xanomeline. Pharmacological validation using kinetic dissociation assays confirmed xanomeline can act as an allosteric modulator and mutation of well-known key allosteric site residues to alanine abolished the allosteric effect of xanomeline. Conclusions: The clinical candidate xanomeline is capable of binding to both the orthosteric and allosteric sites of the M4 mAChR. Historically, xanomeline has shown a complex pharmacological profile, and the findings of this study provide a potential explanation and serve as the basis for future studies that will include the other mAChR subtypes. Collectively, these findings will inform future rational drug design at the M4 mAChR. Keywords: M1 and M4 Muscarinic Receptors, Schizophrenia (SCZ), Xanomeline Disclosure: Karuna Therapeutics: Employee (Self), Karuna Therapeutics: Stock / Equity (Self) P481. Higher Levels of AKT-Interacting Protein in the Frontal Pole From a Sub-Group of Schizophrenia Patients With Markedly Lower Levels of Muscarinic M1 Receptors Megan Snelleksz, Brian Dean The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia Background: We have shown there are lower levels (-23%) of AKT-interacting protein (AKTIP) RNA in the frontal pole from people with schizophrenia, a region which has a significant role in its pathophysiology. It is now argued that studying subgroups within schizophrenia is fundamental to understanding its molecular pathology. Our laboratory has defined a subgroup, termed Muscarinic Receptor Deficit Schizophrenia (MRDS), that is defined by markedly lower levels of the muscarinic M1 receptor (CHRM1) in a quarter of people with schizophrenia. We have also shown that AKTIP RNA is higher (+57%) in the cortex of CHRM1 knockout mice. Hence, we now seek to determine if levels of AKTIP protein are altered only in the cortex from patients with MRDS as a result of altered CHRM1 signaling. Methods: This study uses autoradiography to measure CHRM1 binding in the frontal pole from people with schizophrenia and controls to first establish a cohort of 19 MRDS (15 male, 4 female), 19 non-MRDS (15 male, 4 female) and 19 controls (12 male, 7 female). Based on our gene expression studies in human frontal pole and mouse cortex, this study uses Western Blotting to determine if the lower levels of AKTIP RNA translates to lower levels of protein in the frontal pole from people with schizophrenia and if such changes were limited to those with MRDS due to altered CHRM1 signalling. Results: This study found that CHRM1 binding is 90% lower in the frontal pole from MRDS, with no differences between non-MRDS and controls. This study also found higher levels (+43%, p = .02) of AKTIP protein in the frontal pole from people with schizophrenia. However, when separating schizophrenia into 2 subgroups (MRDS and non-MRDS), this increase in AKTIP protein was specific to the MRDS group (+47%, p = .05), with no differences between non-MRDS and controls. Conclusions: Our findings suggest that, as predicted, AKTIP is altered in the cortex of patients with MRDS possibly due to altered CHRM1 signalling. Significantly, CHRM1 binding in the frontal pole from MRDS was profoundly lower than any other region previously studied. The cohort used in our previous transcriptomic study was predominantly patients who were non-MRDS and therefore the lower levels of AKTIP RNA in those subjects may be an attempt to normalize levels of that protein. As AKTIP is implicated in cell signaling and vesicle trafficking, our data suggests these important functions may be particularly affected in the MRDS subtype of schizophrenia. Keywords: Schizophrenia Subtypes, M1 Muscarinic Receptors, Postmortem Human Brain Tissue, Frontal Pole (Brodmann’s Area 10), Schizophrenia Disclosure: Nothing to disclose. P482. Altered Expression of Excitatory and Inhibitory Ionotropic Receptor Subunit across the Cortical Visuospatial Working Memory Network in Schizophrenia Kirsten Schoonover, Samuel Dienel, Holly Bazmi, Nora Miller, David Lewis University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Working memory dysfunction in individuals with schizophrenia is thought to reflect altered excitatory and inhibitory neurotransmission across multiple regions of the cortical visuospatial working memory (vsWM) network. However, key ionotropic glutamatergic and GABAergic receptor subunits have only been studied in the dorsolateral prefrontal cortex (BA46). Methods: Using qPCR on total gray matter homogenate samples from BA46, posterior parietal cortex (BA7), and primary (BA17) and association (BA18) visual cortices, we quantified transcript levels of critical subunits for excitatory N-methyl-D-aspartate receptors (NMDARs), excitatory alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), and inhibitory GABAA receptors (GABRAs) in 20 matched pairs of schizophrenia (SZ) and unaffected comparison (UC) subjects. Results: In UC subjects, AMPAR and NMDAR levels generally exhibited opposite rostral-to-caudal gradients, with AMPAR GRIA1 and GRIA2 expression highest in BA46 and NMDAR GRIN1 and GRIN2A expression highest in BA17; however, the regional pattern of NMDAR GRIN2B expression was like that of AMPARs. GABRA5 and GABRA1 levels were highest in BA46 and BA17, respectively. In SZ subjects, levels of all transcripts (except GRIN2B and GABRA5) were lower in caudal regions, with no differences in BA46. Conclusions: Our analyses of transcript levels across regions of the cortical vsWM network revealed distinct regional patterns of ionotropic glutamatergic and GABAergic receptor subunits in UC subjects, suggesting that balances between excitation and inhibition are achieved in a region-specific manner. In SZ subjects, the distinct alterations in excitatory and inhibitory receptor transcripts across regions suggests differential contributions of each region to impaired WM performance in the illness. Keywords: Postmortem Brain Tissue Gene Expression, AMPA Glutamate Receptors, NMDA Glutamate Receptors, GABA-A Receptors, Schizophrenia (SCZ) Disclosure: Nothing to disclose. P483. Prefrontal D2 Dopamine Receptor Regulates Psychomotor Symptoms via Controlling Striatal Cholinergic Modulation, an Intersectional CRISPR/Cas9 Driven Circuit Mapping Clementine Quintana, Jivan Khlghatyan, Lakshmi Rajakrishna, Jean-Martin Beaulieu* University of Toronto, Toronto, Canada Background: Psychomotor abnormalities have been abundantly reported in psychiatric disorders like bipolar disorder (BD), schizophrenia and major depressive disorder. Although neglected in contemporary psychiatry, studies have focalized to psychomotor dimension, which has been recently proposed and officially included as an additional domain in the RDoC. The dopamine D2 receptor (DRD2) remains the principal target of antipsychotic drugs used for the management of psychomotor agitation. Besides the high expression of DRD2 in striatum, DRD2 is expressed into the prefrontal cortex where it has been shown its expression upregulated in BD dorsolateral prefrontal cortex (PFC) in post-mortem tissues patients. However, the function of DRD2 in PFC in psychomotor symptoms in bipolar mania remains unknown. Methods: Amphetamine-induced psychomotor excitability allowed a predictive validity model that mimicking many aspects of bipolar mania in humans. We employed intersectional CRISPR/Cas9 gene editing to investigate the role of DRD2 in PFC in adult mice and characterize the circuits that are regulated by this receptor. Results: The deletion of DRD2 in PFC induced an abolition of amphetamine-induced psychomotor behavior without inducing extrapyramidal side effects and cognitive deficit. An investigation of prefronto-striatal circuit revealed the role of PFC DRD2 projecting neurons into the regulation of the cholinergic interneuron activity. Injection of an antagonist of muscarinic receptors rescued the psychomotor behavior induced by amphetamine in mice lacking DRD2 in PFC. Conclusions: This study highlights the role of PFC DRD2 in regulation of psychomotor symptoms via the modulation of striatal cholinergic interneuron activity. This comprehensive analysis paves the way for re-examination of cortical DRD2 functions and treatments in psychomotor disorders. Keywords: Hypofrontality, D2 Dopamine Receptor, Acetylcholine, CRISPR/Cas9, RNAseq Disclosure: Nothing to disclose. P484. The Relationship Between Synaptic and Cognitive Markers in Schizophrenia: A Positron Emission Tomography Study Using [11 C]UCB-J Ellis Onwordi, Thomas Whitehurst, Ekaterina Shatalina, Ayla Mansur, Atheeshaan Arumuham, Martin Osugo, Ben Statton, Alaine Berry, Marina Quinlan, Declan O’Regan, Maria Rogdaki, Tiago Reis Marques, Eugenii Rabiner, Roger Gunn, Sridhar Natesan, Oliver Howes Bart’s and The London, Queen Mary’s School of Medicine, London, United Kingdom Background: Cognitive impairment remains an unmet clinical need in the treatment of schizophrenia, and its pathoaetiology is poorly understood. Converging lines of evidence implicate synaptic loss in schizophrenia pathogenesis. Recent studies have used positron emission tomography with novel radioligand [11 C]UCB-J to index levels of synaptic vesicle glycoprotein 2 A (SV2A) in vivo as a proxy for synaptic density, finding lower levels of [11 C]UCB-J binding in the frontal cortex and hippocampus in patients with schizophrenia compared to healthy volunteers. Using [11 C]UCB-J PET, we tested directly whether SV2A levels and markers of cognitive function are linked in schizophrenia. Methods: Thirty-two volunteers with schizophrenia and 24 healthy volunteers underwent [11 C]UCB-J PET and T1-weighted structural MRI. Volumes of distribution (VT) were estimated for two regions of interest (ROIs) (frontal cortex and hippocampus). Cognitive performance was tested using the National Adult Reading Test (NART), which evaluates premorbid intelligence, and Rey’s Auditory Verbal Learning Test (AVLT). We explored relationships between NART and AVLT scores and VT using Pearson’s correlation coefficient. Results: There was a positive relationship between NART score and [11 C]UCB-J VT in the frontal cortex (FC, r = 0.46, p = 0.01, 95% CI = 0.11 to 0.71) and hippocampus (r = 0.72, p = 0.046, 95% CI = 0.02 to 0.45) in the schizophrenia group (n = 29). There were no significant relationships between NART score and [11 C]UCB-J VT in any region of interest in the healthy volunteer group (n = 21). There was a positive relationship between Trial I AVLT score and [11 C]UCB-J VT in the frontal cortex (FC, r = 0.42, p = 0.02, 95% CI = 0.08 to 0.67) but not the hippocampus (r = 0.32 p = 0.07, 95% CI = -0.03 to 0.60) in the schizophrenia group (n = 32). There were no significant relationships between Trial I AVLT score and [11 C]UCB-J VT in either region of interest in the healthy volunteer group (n = 24). There was a positive relationship between AVLT recognition score and [11 C]UCB-J VT in the frontal cortex (FC, r = 0.46, p = 0.02, 95% CI = 0.07 to 0.73) and the hippocampus (r = 0.54 p = 0.007, 95% CI = 0.17 to 0.77) in the healthy volunteer group (n = 24). There were no significant relationships between AVLT recognition score and [11 C]UCB-J VT in either region of interest in the schizophrenia group (n = 27). There were no significant relationships between total words learned and [11 C]UCB-J VT in either region of interest in the schizophrenia (n = 32) or healthy volunteer group (n = 24). Conclusions: SV2A levels are linked to measures of premorbid intelligence and verbal learning but not recognition in schizophrenia, supporting the hypothesis that synaptic dysfunction in vivo is linked to cognitive performance in schizophrenia. Keywords: SV2A PET imaging, [11 C]UCB-J, Cognition, Schizophrenia, Synaptic Aberrations, PET Imaging Disclosure: Nothing to disclose. P485. Plasma Free Fatty Acids After Six Weeks of Antipsychotics Treatment of Schizophrenia Gregory Oxenkrug, Hans‑Gert Bernstein, Henrik Dobrowolny, Paul Summergrad, Johann Steiner Tufts University School of Medicine, Boston, Massachusetts, United States Background: Free (non-esterified) fatty acids (FFA) are partially derived from myelin, a phospholipid sheath that surrounds axons. Elevated blood FFA levels were observed in schizophrenia patients and may reflect an increased degradation of myelin [1]. The data on the effect of antipsychotics on FFA are sparse. In in vitro study, clozapine (but not haloperidol) decreased FFA content in cultured oligodendrocytes and increased the expression of the rate limiting enzyme of FFA synthesis [2]. In healthy volunteers, plasma FFA decline was observed after 3 days of administration of olanzapine [3] but not of haloperidol [4]. In schizophrenia patients, plasma FFA decline was observed after treatment with olanzapine (n = 20) and risperidone (n = 14) but not abilify (n = 16) [5]. Methods: Plasma FFA levels were evaluated in acutely ill and either antipsychotic-naive or antipsychotic-free for at least six weeks patients, before and after 6 weeks of inpatient treatment with olanzapine (n = 16), risperidone (n = 22) or quetiapine (n = 12). Positive and Negative Syndrome Scale (PANSS) total scores were previously evaluated in these patients [6]. Statistical difference between pre- and post-drug treatment were assessed by paired T-test Results: Plasma FFA were higher in patients (0.779 + 0.469 nM, n = 50) that in controls (0.427 + 0.427, n = 50). Plasma FFA levels decreased after treatments with olanzapine (by 55% to 0.345 nM, p < 0.0004), and risperidone (by 60% to 0.320, p < 0.004), but not quetiapine (0.519 nM, p < 0.418). PANSS total corrected scores before treatment were 56.895. Olanzapine treatment resulted in PANSS decrease to 25.500 (by 55%, p < 0.0001), risperidone - to 20.217 (by 65%, p < 0.001) and quetiapine - to 39.5000 (by 40%, p < 0.002). Conclusions: Plasma FFA levels decreased after olanzapine and risperidone treatment in agreement with previous reports [2,3]. Quetiapine did not change FFA levels similar to haloperidol and abilify [4,5]. Notably, the mean decrease of PANSS total scores was lower in patients treated by quetiapine than in patients treated by olanzapine and risperidone, although we did not observe correlations between FFA and PANSS scores [5]. Early (e.g., 3 days) [4] response of plasma FFA to antipsychotics may help to select an optimal medication for the treatment of a schizophrenia endophenotype characterized by cell membrane phospholipid impairments [7]. References: 1. Kaiya H, Horrobin DF, Manku MS, Fisher NM (1991) Biol Psychiatry; 30: 357–362 2. Steiner J, Martins-de-Souza D, Schiltz K, Sarnyai Z, et al (2014) Front Cell Neurosci 2014) Nov 18;8:384 3. Albaugh VL, Singareddy R, Mauger D, et al (2011) PLoS One. 6(8): e22662 4. Vidarsdottir S, Weenen JE, Frolich M, Roelfsema F, et al. (2010). J Clin Endocrinol Metab 95: 118–125. 5. Kaddurah-Daouk R, J McEvoy1, RA Baillie, D Lee, et al (2007) Molecular Psychiatry 12, 934–945 6. Martins-de-Souza D, Solari FA, Guest PC, Zahedi RP and J Steiner (2015) Schizophrenia Article number: 15050; doi:10.1038/npjschz.2015.50 7. Messamore, E. (2018) Prostaglandins Leukot. Essent. Fat. Acids 136, 95–97. Keywords: Free Fatty Acids, Antipsychotics, Schizophrenia Subtype Disclosure: Nothing to disclose. P486. A Subset of Olfactory Neurons Derived From Schizophrenia Patients Exhibit Increased Markers for Protein Aggregation And Reduced Cognitive Performance Leslie Nucifora, Koko Ishizuka Ishizuka, Gayane Yenokyan, Nicola Cascella, David Schretlen, Philip Harvey, Christopher Ross, Russell Margolis, Akira Sawa, Frederick Nucifora* Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Schizophrenia is a debilitating disorder that affects 1% of the population. Symptoms associated with schizophrenia include positive and negative symptoms as well as cognitive deficits. Significant efforts have been made towards understanding the genetic factors underlying schizophrenia, however, the mechanisms implicated in schizophrenia remain unclear. The heterogeneity in clinical symptoms and course of illness, suggest the possibility of different mechanist subtypes of the illness. Reducing the heterogeneity by subtyping patients could help identify the underlying mechanisms. One approach is to subtype patients by clinical presentation or course and determine the mechanism underlying the clinical presentation. Alternatively, pathogenic processes could be identified in a subset of patients, and then correlated with clinical phenotypes. While this biological approach has helped other medical fields advance, it has had less application to research in mental illness. Previously, we reported increased protein insolubility and ubiquitination, two markers for protein aggregation, as a pathological process in human postmortem brain from a subset of patients with schizophrenia. This is an example of biological subtype classification for schizophrenia. In the present study, we investigate whether the process of protein aggregation is observed in an independent model system using olfactory neurons derived from living patients with schizophrenia. Utilizing a complementary system to autopsy brains such as olfactory neuronal cells provides the advantages that they can be acquired from living subjects, with clinical and cognitive data obtained at the time of assessment. In the present study, we build upon our previous work from human autopsy brains and now show that a subset of living patients with schizophrenia exhibit increased markers of protein aggregation as a mechanism utilizing olfactory neurons. We further identify potential clinical and cognitive impairments that relate to this subtype of the illness. Methods: Olfactory neurons were obtained from the Johns Hopkins Schizophrenia Center. Twenty-three schizophrenia patients and nineteen controls with both sexes were included. Harvested olfactory neurons were processed utilizing a sarkosyl cellular fractionation protocol designed to isolate the insoluble protein fraction. All purified insoluble protein samples were processed by western blot and Coomassie gel analysis and insoluble protein levels and ubiquitin reactivity were quantified using ImageJ software and normalized to total homogenate protein concentrations. Clinical and cognitive testing were performed at the time of olfactory neuron obtainment. Results: A subset of living patients with schizophrenia exhibit increased protein insolubility and ubiquitination, two markers for protein aggregation, utilizing olfactory neurons. Significant reduction in cognitive performance was observed across every cognitive measure except for processing speed, in the subset of schizophrenia participants with increased protein aggregation compared to schizophrenia participants without aggregation and controls. Conclusions: These studies further help to establish protein aggregation as a novel mechanism involved in the pathogenesis of a subset of patients with schizophrenia that may relate to specific symptomatology. Further exploration of the mechanisms leading to protein insolubility could lead to novel therapeutic targets and to a reconceptualization of diagnostic categories. Keywords: Schizophrenia, Protein Aggregation, Ubiquitination, Cognitive Impairment Disclosure: Teva: Consultant (Self), Galyan Bio: Advisory Board (Self) P487. Alterations of SGK1 Expression and Activity in Schizophrenia Abdul Hammoud, Ali Imami, Consuelo Walss-Bass, Robert McCullumsmith* University of Toledo, Toledo, Ohio, United States Background: We conducted RNA-sequencing of hiPSC-derived cell lines from schizophrenia (SCZ) subjects, from the Central Valley of Costa Rica. hiPSCs, neural precursor cells, and cortical neurons derived from six healthy controls and seven SCZ subjects were generated using standard methodology. We identified 454 differentially expressed genes in hiPSC-derived neurons, enriched in pathways including phosphoinositide 3-kinase/glycogen synthase kinase 3 (PI3K/GSK3) signaling, with serum-glucocorticoid kinase 1 (SGK1), an inhibitor of glycogen synthase kinase 3β, as part of this pathway. We further found that pharmacological inhibition of downstream effectors of the PI3K/GSK3 pathway, SGK1 and GSK3, induced alterations in levels of neurite markers βIII tubulin and fibroblast growth factor 12, with differential effects in patients compared to controls. These studies support a role for disruption of PI3K/GSK3 and SGK1 signaling as a risk factor for SCZ. To further investigate the role of these kinases in SCZ we developed an in-vitro kinome array assay, to permit assessment of putative pharmacological modulators. Methods: hiPSC-derived cell lines were generated from schizophrenia (SCZ) subjects from the Central Valley of Costa Rica. RNA was isolated and RNAseq analyses performed to assess differential gene expression. We used the serine threonine PamGene kinomearray chip to develop a specific assay for SGK1 kinase activity. The kinome array assay was run in the presence of recombinant SGK1 protein (2.5 ng, 25 ng, and 250 ng). For the negative control we heat inactivated 250 ng. All assays were run in triplicate. We used our standard kinomic analyses, including KRSA and Kinopedia developed in the McCullumsmith laboratory to analyze our data. To determine protein kinase (hits) we used a Monte Carlo simulation that generates 2000 simulations of protein kinase assignments to peptides. We used this permutation analysis to determine which protein kinase hits are over or underrepresented in our dataset. A 95% confidence interval is used to determine the range of expected protein kinase hits. When an observed protein kinase falls outside the 95% confidence interval that is expected it is moved forward as a candidate for further confirmation study. All kinome array assays are run in triplicate, and the recombinant protein kinase studies are run with a heat inactivated sample as our negative control. Results: We identified 11 peptides on the array that have high affinity for SGK1 phosphorylation activity. Pathway analysis of these peptides using EnrichR confirmed several well characterized pathways associated with SGK1 signaling, as well as several novel pathways not previously attributed to this protein kinase. Based on our transcriptional profiling results from hiPSCs derived from SCZ subjects we reexamined our published data for the serine threonine kinome array in postmortem brain in SCZ with a focus on SGK1. Using our KRSA we found increased activity of SGK1 kinase in SCZ in the frontal cortex compared to control (P > 0.05). Conclusions: Converging evidence suggest the perturbations of SGK1 protein kinase activity may contribute to the pathophysiology of severe mental illness; in particular, molecular correlates of learning and memory including long-term potentiation and long-term depression may be impacted by subtle changes in protein signaling networks. In this study we have developed an assay that will allow us to interrogate drugs that modulate protein kinase implicated in SCZ including SGK1. We are currently assessing novel compounds as well as repurposed FDA approved drugs as candidates that may modulate SGK1 in a manner that could lead to improvement in neuroplastic endophenotypes. Keywords: Schizophrenia, Kinome, Protein Kinase, Bioinformatics Disclosure: Nothing to disclose. P488. Extracellular Matrix and Vasculature Dysregulation May Impair Neurogenesis in Schizophrenia Cases With Elevated Inflammation Hayley North, Christin Weissleder, Maina Bitar, Theresa Salthouse, Guy Barry, Maree Webster, Jan Fullerton, Cynthia Shannon Weickert Neuroscience Research Australia, Randwick, Australia Background: Schizophrenia is a clinically and neuropathologically heterogeneous disorder. We find a subgroup of ~40% of people with schizophrenia have elevated inflammation in various brain regions including the brain’s largest niche for neurogenesis, the subependymal zone (SEZ). This subgroup of schizophrenia cases with elevated cytokines have exaggerated changes of transcripts indicating increased stem cell quiescence/dormancy and reduced neuronal differentiation. In addition, the high inflammation schizophrenia group have suppressed microglia marker expression and increased density of macrophages. While studies show that macrophages can secrete factors that may impair neurogenesis, we do not know if the broad molecular changes are consistent with this possibility, or if other factors are key to inflammatory-related suppression of neuronal differentiation in schizophrenia. This research aimed to discover broad transcriptomic differences relating to SEZ inflammatory status within schizophrenia to elucidate disease heterogeneity. Methods: Deep total-RNA sequencing was performed on RNA extracted from the post-mortem SEZ tissue of 27 schizophrenia cases previously designated into low inflammation (n = 13) and high inflammation (n = 14) subgroups based on cluster analysis of inflammation marker gene expression (starting with IL6, IL6R, IL1β, IL1R1, CXCR8, SERPINA3 and IL6ST). Differentially expressed (DE) genes were determined by EdgeR software with a false discovery rate adjusted (FDR) p value < 0.05. The DE gene list was subsequently analysed using Ingenuity Pathway Analysis. Immunohistochemistry was conducted on 14μm thick SEZ sections from the same cohort. Results: 718 genes were DE in high compared to low inflammation schizophrenia (FDR p < 0.05). The DE genes were most significantly over-represented in the pathway ‘Hepatic Fibrosis/Hepatic Stellate-Cell Activation’. Genes in this pathway, which predominantly had increased expression in high inflammation schizophrenia, encoded proteins involved in extracellular matrix (ECM) rigidity (including ten collagens) and remodelling of the vasculature (including angiogenesis genes VEGFA, VEGFR1, EDN1). Collagen-IV was primarily localised around blood vessels and in the SEZ hypocellular gap. The results suggest novel changes to the SEZ vasculature, which may facilitate immune cell transmigration as indicated by positive correlations between markers of angiogenesis, macrophages and recruitment molecule ICAM1. Conclusions: This is the first discovery-driven comparison of the transcriptome between inflammatory subgroups in schizophrenia brain tissue. The findings of inflammation-dependent changes in the SEZ suggesting angiogenesis and ECM alterations have important implications for how inflammation contributes to heterogeneity in schizophrenia neuropathology; especially regarding reduced neurogenesis. Considering the SEZ microenvironment and vasculature intricately regulate neurogenesis, alterations to the ECM and collagens surrounding blood vessels would likely dysregulate the development and migration of newborn neurons. This exploration of disease heterogeneity may be a step towards developing more personalised treatment options for those with elevated inflammation. Keywords: Schizophrenia (SCZ), Inflammation, Subgroups, Neurogenesis Disclosure: Nothing to disclose. P489. Impaired Microtubule-Based Structural Dynamic Changes in Monocyte-Derived-Neuronal-Like Cells (MDNCs) From Patients With Schizophrenia Alonso Cortez-Resendiz, Lauren Forrest, Anjali Iyer, Janani Iyer, Alfredo Bellon Penn State Milton S. Hershey Medical Center, Hershey, Pennsylvania, United States Background: Ample evidence indicates the pathophysiology of schizophrenia (SCZ) begins in early neurodevelopment (1) but the cellular process at fault is yet to be identified. Decades of postmortem research have shown subtle abnormalities in the neuronal structure across the brain that often involve reductions in size and number of neuronal extensions (2-5). But increased number of extensions (6,7) and malformed neurites have also been found (8,9). These findings could suggest that the capacity to modify the neuronal structure is impaired in SCZ. During early human development, neurons undergo dynamic structural changes consisting of continuous formation and retraction of neurites (10). Data from cellular models of neurodevelopment support the possibility of impaired neurostructural dynamics in SCZ. Maturing neurons generated from patients’ pluripotent cells evidenced longer neuronal extensions early in development (10, 11) and shorter neuronal outgrowths in more advanced stages of differentiation (12, 13). Microtubules are an indispensable component of the cytoskeleton that play a significant role in sculpting the neuronal shape (14). In patients with SCZ, dynamic changes in microtubules are diminished in olfactory neuroepithelial cells (15), while neuronal precursors generated from the olfactory epithelium showed altered microtubule organization (16). Whether deficits in microtubule polymerization also affect neurostructural dynamic changes in SCZ remains unknown. Methods: Human monocytes were transdifferentiated following our protocol (17). After transdifferentiation, light microscopy pictures of the same group of Monocyte-Derived-Neuronal-like cells (MDNCs) were taken at time zero (T0) and then after an hour of incubation (T1hr) either under control conditions or after treatment with colchicine 0.4 µM, a compound that arrests microtubule polymerization. Each single MDNC received a numeric score based on a scoring rubric we developed (18). The sum of all structural changes present in all MDNCs in one hour is the Structural Dynamic Index (SDI). Experiments were conducted with blood samples from 12 controls and 13 patients with SCZ frequency-matched by age and gender as reported previously (19). This study was approved by IRBs from Penn State Hershey and INSERM, France. Results: We have recently developed a protocol to transdifferentiate blood circulating monocytes into neuronal-like cells in 20 days and without reprograming (17). MDNCs express several neuronal markers and conduct spontaneous action potentials as well as postsynaptic inhibitory and excitatory currents (17). Moreover, MDNCs deliver reproducible results in sequential samples from the same donors (17,18). We have also shown that these cells retract neuronal extensions when treated with colchicine similarly to human neurons (18). In addition, MDNCs from a subgroup of patients with SCZ prune more primary neurites in the presence of dopamine than cells from controls (18). Here we show, based on a mixed model analysis, that the number of differentiated cells between cohorts in comparable (CTL, 6.7 ± 0.95; SCZ, 8.0 ± 0.9; P = 0.31; N = 600 MDNCs from 12 CTL and 802 MDNCs from 13 patients). At baseline, SDI is similar among groups (CTL, 13.9 ± 1.2; SCZ, 12.0 ± 1.0; P = 0.25; N = 465 MDNCs from 8 CTL and 879 from 10 patients). But after treatment with colchicine 0.4 µM SDI in patients is higher (CTL, 7.4 ± 1.9; SCZ, 12.7 ± 1.4, P = 0.06; effect size, 1.16; N = 251 MDNCs from 4 CTL and 393 from 7 patients). Conclusions: MDNCs from patients with SCZ remain structurally dynamic after treatment with colchicine 0.4 µM but show no differences at baseline when compared with CTL cells. These results suggest that there is an impaired microtubular response in SCZ that interferes with neurostructural dynamic changes. While we have previously reported that haloperidol does not affect SDI (20), the potential influence of other antipsychotics needs to be studied. Keywords: Adult Stem Cells, Neurodevelopment, Monocytes, Schizophrenia (SCZ), Cytoskeleton Disclosure: Nothing to disclose. P490. Cognition as a Moderator of Relapse Risk Among Patients With Early Phase Schizophrenia Treated With Long-Acting Injectable Antipsychotics: Data From the Prelapse Trial Delbert Robinson, Nina Schooler, Majnu John, Patricia Marcy, John Kane Hofstra/Northwell, Glen Oaks, New York, United States Background: Cognitive deficits are a well-established component of early phase psychosis. Cognition has been found to moderate initial response to antipsychotic treatment with first episode patients. A next step question is whether cognition is also associated with relapse. Studies examining relapse following response among recent onset patients have found variable results. Relapse studies require long follow-up periods and long-term adherence to antipsychotic treatment is challenging for many recent onset patients. Variable long-term antipsychotic adherence may be one factor that has limited the ability to detect possible relationships between cognition and relapse among recent onset patients. Studies with long-acting injectable antipsychotic (LAI) formulations limit nonadherence effects and therefore may be particularly useful for detecting cognition moderator effects on relapse risks. Methods: The PRELAPSE trial was a large simple trial with cluster randomization of 39 US clinics. Nineteen clinics provided participants the LAI aripiprazole monohydrate (AM) and 20 provided treatment as usual (TAU). The primary outcome was time to first hospitalization; the observation period was 2 years. Inclusion criteria were: DSM-5 schizophrenia diagnosis; fewer than 5 years of lifetime antipsychotic treatment; age 18–35 years. Cognition was assessed with the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) at baseline and then yearly. The RBANS had the advantage of easy administration for a large simple trial. The RBANS consists of ten subtests that give five domain scores and a total score. For analysis of cognition as a moderator of hospitalization outcomes, the sample was characterized as having poorer or better cognition based upon a median split of the baseline RBANS total score. Time to first hospitalization was analyzed using a Cox Proportional Hazard model with shared random effects for sites (also known as a Shared Frailty model) accounting for clustering effects. A lognormal distribution was assumed for the frailties. Results: The examination of baseline RBANS as a moderator of time to first hospitalization included 457 of the 489 participants in the PRELAPSE trial. Mean age of the cognition sample (342 men and 115 women) was 25.2 years. Mean duration of prior antipsychotic treatment was 627 days. Poor and better cognition groups did not differ on age, duration of prior antipsychotic treatment or on being at an AOM or TAU site. Women were more likely to be in the better cognition group than men (71 of 115 women versus 159 of 342 men). The Cox Proportional Hazard analyses revealed a treatment condition by baseline cognition interaction (Wald Chi-Square = 3.3540, df =1, p = 0.0506). Subsequent analyses of participants in the poorer cognition group (N = 227) found no effect of treatment condition (Wald Chi Square = 0.6792; df=1; p = 0.2367); the hazard ratio for AM versus TAU was 0.776 (95% CI = 0.424, 1.419). In contrast, among participants with better cognition, there was a significant treatment effect (Wald Chi-Square=10.6210; df=1, p = 0.0006) favoring the AM versus the TAU condition (hazard ratio for AM versus TAU was 0.304 (95% CI = 0.149, 0.622)). Conclusions: Our data suggest that patients with early phase schizophrenia being treated with LAI antipsychotics who have better baseline cognition may have more reduction in relapse risk from treatment than those with poorer baseline cognition. These results extend the findings of cognition moderating acute response to antipsychotic treatment to suggest that cognition may also moderate antipsychotic treatment effects on relapse risk. Keywords: Cognition, Long-Acting Injectable Antipsychotics, Recent Onset Psychosis, Relapse and Hospitalization Disclosure: Teva Amylyx, C4 Innovations, Health Choice, Levo, Interactive Forums, PH Associates Limited: Consultant (Self) P491. Sustained Treatment Response With Long-Term Valbenazine in Patients With Tardive Dyskinesia Christoph Correll, Jean-Pierre Lindenmayer, Khodayar Farahmand, Scott Siegert, Eduardo Dunayevich Neurocrine Biosciences, Inc., San Diego, California, United States Background: Valbenazine is a once-daily VMAT2 inhibitor approved for the treatment of tardive dyskinesia (TD), a persistent and potentially disabling movement disorder associated with prolonged exposure to antipsychotics, antiemetics, and other dopamine receptor blocking agents. The efficacy, safety, and tolerability of valbenazine has been established in several phase 3 trials, including a long-term study (KINECT 4 [NCT02405091]) in which participants received open-label valbenazine (40 or 80 mg) for 48 weeks. Post hoc analyses of KINECT 4 data were conducted to assess patterns of treatment response. Methods: Data from KINECT 4 treatment completers (participants who reached the Week 48 visit and had the longest duration of treatment) were analyzed post hoc. TD was assessed using the Abnormal Involuntary Movement Scale (AIMS) total score (sum of items 1-7, as rated by the study investigator), the Clinical Global Impression of Change-Tardive Dyskinesia (CGI-TD), and the Patient Global Impression of Change (PGIC). Analyses were conducted at Week 8 (first study visit after the valbenazine dose-optimization period) and Week 48 using the following definitions of response: ≥50% and ≥70% improvement from baseline in AIMS total score; rating of “much improved” or “very much improved” (score ≤2) on the CGI-TD and PGIC. Results: Of the 167 participants who entered KINECT 4, 103 (62%) were treatment completers and included for analysis. Of these 103 participants, 39% and 86% met the ≥50% AIMS response threshold at Weeks 8 and 48, respectively. The percentages of participants who met the highly rigorous AIMS ≥ 70% response threshold at Weeks 8 and 48 were 17% and 52%, respectively. Of the 40 participants with AIMS ≥ 50% total score improvement at Week 8, 95% also met this threshold at Week 48 (“sustained response”). Of the 63 participants with <50% AIMS improvement at Week 8, 81% achieved the ≥50% response threshold by end of treatment at Week 48. The proportion of participants meeting the threshold for CGI-TD response also increased over time, from 50% at Week 8 to 92% at Week 48. PGIC results were similar, with response rates of 53% and 88% at Weeks 8 and 48, respectively. Conclusions: Post hoc analyses of data from a 48-week, open-label study of once-daily valbenazine showed that the proportion of participants meeting rigorous treatment response thresholds increased over time. By the end of treatment at Week 48, >80% of participants demonstrated robust improvements in TD, as assessed using the AIMS ( ≥ 50% improvement), CGI-TD (score ≤2), and PGIC (score ≤2). Keywords: Tardive Dyskinesia, Valbenazine, Long-Term Treatment Disclosure: Neurocrine Biosciences: Employee (Self) P492. The Pharmacodynamic Effects of TAAR1 Agonist Ulotaront on Metabolic Biomarkers of Glucose, C-Peptide, and Insulin Following a Meal in Patients With Schizophrenia Snezana Milanovic, Nina Dedic, Yu-Luan Chen, Kuangnan Xiong, Roberto Gomeni, Robert Lew, Gerald R. Galluppi, Seth Hopkins, Kenneth S. Koblan Sunovion Pharmaceuticals, Inc., Marlborough, Massachusetts, United States Background: Obesity, dyslipidemia, hypertension, hyperglycemia, and non-alcoholic fatty liver disease (NFLD) are highly prevalent in schizophrenia due in large part to the propensity of the current class of treatments to cause these metabolic changes. Hyperglycemia, diabetes mellitus, dyslipidemia and weight gain are highlighted in the Warnings and Precautions sections of the FDA approved labels for the current class of drugs approved for the treatment of schizophrenia. Although each antipsychotic may have its own risk profile, possibly related to pharmacological effects at histamine, serotonin and dopamine receptor subtypes, the metabolic changes associated with all drugs in the current class inform treatment decisions, benefit/risk, and result in increased public health costs. Ulotaront (SEP-363856) is a trace amine-associated receptor 1 (TAAR1) and 5-HT1A agonist currently in Phase 3 clinical trials for the treatment of schizophrenia. Recent preclinical evidence has identified TAAR1 as a novel regulator of metabolic control and a promising target for obesity and type 2 diabetes. Here we evaluated the effects of ulotaront on liquid metabolic biomarkers which were collected in Phase 1 clinical pharmacology studies. Methods: Metabolic effects of ulotaront were examined in response to a meal following a 8-12 h fast. In a study to determine the effect of ulotaront on QTc interval (NCT04369391), schizophrenia subjects (N = 60) were randomized in a 3-way crossover design to receive single doses of ulotaront, placebo and moxifloxacin. Separately, in a study to evaluate ulotaront for a drug-drug interaction (NCT04865835), schizophrenia subjects (N = 25) were randomized in a 2-way crossover design to receive single doses of ulotaront or placebo. Plasma samples were analyzed for C-peptide, insulin, and glucose. Results: Following administration of a meal, ulotaront lowered insulin and C-peptide levels compared to placebo, indicating an effect of ulotaront on glycemic control in response to feeding, with large (0.8 – 1.0) effect sizes on insulin and C-peptide levels. An integrated population PK/PD model jointly described insulin, C-peptide and glucose change, in response to a meal, as a function of ulotaront plasma concentrations. Conclusions: The effects of ulotaront on metabolic markers, derived from plasma samples collected in the course of clinical pharmacology studies, suggest that the beneficial effects observed in animal models may translate to humans. Phase 1 clinical studies are currently ongoing to test the direct effects of ulotaront on metabolic parameters in schizophrenia patients (NCT05402111, NCT05463770). The healthcare burden of hyperglycemia, diabetes mellitus, dyslipidemia, weight gain and NAFLD in the treatment of schizophrenia with the current antipsychotic class would be avoidable with the advent of a new pharmacological class demonstrating benefit on these metabolic parameters. Keywords: TAAR1, Ulotaront, Metabolic Biomarker, Schizophrenia Novel Treatment Disclosure: Sunovion Pharmaceuticals, Inc.: Employee (Self) P493. Gender Differences in the Relationship Between Trauma Exposure and Symptoms in First Episode Psychosis Julie McCarthy, Jacqueline Dow, Kim Mueser McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: A history of trauma is common in people with psychotic disorders, and it is associated with more severe symptoms. Men and women with psychosis often experience differences in their course of illness and symptom profile. Past studies have found gender differences in trauma among those with first episode psychosis, but data in this area are limited. The current project aims to examine the relationship between trauma exposure and baseline quality of life and symptom profiles by gender, as well as the association of trauma exposure and change in 12- and 24-month quality of life and symptom profiles by gender. Methods: Included in this secondary data analysis were n = 404 participants from the NIMH Recovery After an Initial Episode of Schizophrenia-Early Treatment Program (RAISE-ETP) study, which examined the effects of the NAVIGATE treatment group compared to the community care (CC) control group. The sample consisted of participants with schizophrenia, schizoaffective disorder, or other non-affective psychosis diagnoses. Trauma exposure was measured using an abbreviated version of the Traumatic Life Events Questionnaire (TLEQ); outcome measures were the Quality-of-Life Scale (QLS), Calgary Depression Scale for Schizophrenia (CDSS), and the Wallwork five-factor model for the Positive and Negative Syndrome Scale (PANSS). At baseline, individuals with TLEQ data were included for analyses (n = 400; n = 291 men, n = 109 women); at follow-up, subjects were included with baseline and 12-month data (n = 252; n = 185 men, n = 67 women) and baseline and 24-month data (n = 205; n = 151 men, n = 51 women). Spearman correlations were examined between baseline trauma exposure and baseline symptom and quality of life measures (QLS, CDSS, PANSS total and PANSS factor scores). Reported correlations survived Bonferroni correction (.05/16 tests = .003). Linear mixed effects models of baseline trauma exposure on change in QLS, CDSS, and PANSS outcomes were examined at 12 and 24 months. Covariates included treatment group, study site, low or high duration of untreated psychosis, substance use disorder history, and baseline outcome of interest. Parallel analyses were conducted by gender. Results: There was a positive correlation at baseline between trauma exposure and depression (CDSS) for men (r = .18, p = .0019) and women (r = .28, p = .0027). Positive correlations were also found at baseline between trauma exposure and PANSS depression/anxiety factor in men (r = .25, p < .001), and PANSS excited factor in women (r = .30, p = .002). A negative correlation was found at baseline between trauma exposure and PANSS negative factor for men only (r = -.20, p = .005). In the mixed model analyses, trauma exposure was associated with less decrease in the PANSS excited factor (p = .038) at 12-months and the PANSS total (p = .019) and positive symptom factor (p = .002) at 24-months in women; the remaining analyses were not significant. Conclusions: Results suggest that gender differences in the relationship between trauma exposure and symptoms for individuals with first episode psychosis depend on the timeframe of assessment. At baseline, trauma was related to depression on the CDSS in men and women, and the depression/anxiety factor on the PANSS for men. Depression/anxiety on the PANSS was also related to trauma in women (r = .19 vs r = .25 in men), but it did not survive Bonferroni correction partly due to smaller sample size. So, the association between trauma and depression was a relatively consistent finding across gender. However, during the first years of treatment, trauma exposure may have a prolonged impact on total, positive, and excited symptom severity especially for women in that they may experience less benefit than men. These results indicate that further clinical interventions may be indicated in the consideration of lifetime trauma exposure by gender for those experiencing their first episode of psychosis. Supported by: K23DA050808 Keywords: First Episode Psychosis, Gender Differences, Trauma Exposure Disclosure: Nothing to disclose. P494. The Effects of Lesion Network Guided Transcranial Electrical Stimulation on Symptoms, Behavior, and Electrophysiology in Patients With Psychosis: An Open Label Clinical Trial Paulo Lizano, Nicolas Raymond, Robert Reinhart, Matcheri Keshavan Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States Background: Transcranial electrical stimulation (tES) has been shown to improve psychotic symptoms and cognition in psychosis spectrum disorders. However, few investigations have used novel tES approaches, such as high definition tES (HD-tES) to more specifically target brain circuits. Convergent data from neuroimaging, neurophysiological, and cause-effect studies points towards the extrastriate visual cortex (V5/MT) as being causally linked to visual hallucinations and delta frequency deficits being associated with motion processing alterations in psychosis. We aimed to determine if causal lesion network guided HD-tES to the extrastriate cortex is efficacious and safe in improving psychosis symptoms, motion processing, and steady state visual evoked response potentials (ssVEP). Secondary outcomes include determining HD-tES effects on emotion processing evoked potentials, cognition, and depression symptoms. Methods: To examine the functional role of the extrastriate visual cortex a between-participants, open-label, non-randomized, cross-over design, pilot study at a single site was performed to characterize the efficacy and safety of using cathodal HD-tDCS (transcranial direct current stimulation) or delta frequency HD-tACS (transcranial alternating current stimulation) in psychosis (ClinicalTrials.gov Identifier: NCT04870710). Enrolled participants were allocated to 20 mins of tES twice daily for 5 consecutive days applied bilaterally to the extrastriate visual cortex with one month of wash out between tES conditions. Clinical (psychosis and depression symptoms), cognitive (BACS), ssVEP and emotion processing evoked potential assessments (IAPS) were performed at baseline, day 5 and day 30. Results: A total of 6 patients with a psychosis spectrum disorder were enrolled. Six individuals received cathodal HD-tDCS with 5 of them completing all follow-up visits. Three individuals received delta frequency HD-tACS and also completed all follow up assessments. HD-tDCS resulted in a significant reduction in psychosis general symptoms and emotion processing evoked potentials at 5 days, which were directly correlated. HD-tACS resulted in longer term reductions in psychosis general symptoms at 30 days, as well as cognitive improvements. Both type of tES were well tolerated and there were no adverse effects reported. Conclusions: The findings herein provide proof of concept evidence that causal lesion network mapping can be used to enhance brain targeting using tES in psychosis. We showed that bilateral stimulation of the extrastriate visual cortex resulted in short term effects with HD-tDCS, while HD-tACS resulted in longer term benefits on general psychosis symptoms. Further research is needed in in a larger and more acute psychosis population to determine the clinical efficacy and safety of this type of stimulation. Keywords: Psychosis, Transcranial Current Stimulation, Cognition, EEG, ERP Disclosure: Nothing to disclose. P495. A Phase 2, Randomized, Double-Blind, Placebo-Controlled Study of BIIB104 in Cognitive Impairment Associated With Schizophrenia (CIAS): The Tally Study Alexander Coppell, Michael Serenko, Michael Rooney, Jonathan Smith, Donna Babiar, Estibaliz Arce, Christopher L. Shaffer, Michael F. Green Biogen Inc, Maidenhead, United Kingdom Background: Certain cognitive impairments in schizophrenia are hypothesized to result from dysfunction in NMDAR glutamatergic neurotransmission. BIIB104 (formerly PF-04958242), a potent and highly selective high-impact AMPA receptor (AMPAR) positive allosteric modulator (PAM), has potential to enhance AMPAR activation and hence augment NMDAR-induced synaptic potentiation. Both preclinical (Shaffer et al., 2015; Shaffer 2018) and early clinical (Evans et al., 2016; Ranganathan et al., 2017) studies suggest the potential efficacy of BIIB104 for cognitive impairment associated with schizophrenia (CIAS). Methods: TALLY (263CS201, NCT03745820, EudraCT 2018-003825-27) was conducted in the USA, Japan, Spain, Germany, and the UK. Eligible participants were 18-55 years old with a DSM-5 diagnosis of schizophrenia of at least 2 years and selection criteria conformed to the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) recommendations (with additional criteria). Participants were randomized to receive oral doses (BID) of BIIB104 (0.15 mg or 0.5 mg) or placebo in a 1:1:1 ratio for 12 weeks. Dose selection was intended to permit the evaluation of both phasic and tonic dosing regimens relative to the preclinically predicted clinically efficacious plasma concentration. The primary endpoint was the change from baseline in MATRICS Consensus Cognitive Battery (MCCB) Working Memory (WM) Domain T-score to Week 12. The University of California, San Diego Performance-Based Skills Assessment–Brief (UPSA-B), Schizophrenia Cognition Rating Scale (SCoRS) total interviewer score, MCCB Neurocognitive Composite (NCC), and Positive and Negative Syndrome Scale (PANSS) change from baseline to Week 12 were assessed as secondary endpoints. Safety and pharmacokinetics (PK) were also assessed. A standardized effect size of 0.35, which was considered a reasonable clinically meaningful treatment effect, was targeted. Results: 195 participants were randomized from 52 centers; mean age was 39.8 years (range 21-55 years), 136 (69.7%) were male and 155 (79.5%) completed the 12-week treatment period. Demographic characteristics were well-matched across treatment groups. PK sampling demonstrated consistent and expected drug plasma exposures for both BIIB104 doses over the entire 12-week evaluation period. Dosing compliance exceeded 95% across all treatment groups. Analyses of the primary endpoint showed no statistically significant improvement for BIIB104 (0.15 mg or 0.5 mg) versus placebo at Week 12 (p = 0.85 and p = 0.81, respectively). Least squares (LS) mean differences from placebo in MCCB WM were -0.26 (95% confidence interval [CI] -2.88, 2.37) and -0.33 (95% CI -2.94, 2.29) for BIIB104 at 0.15 mg and 0.5 mg, respectively. Analyses of the secondary endpoints also showed no statistically significant improvements for either BIIB104 dose across MCCB NCC (LS mean difference from placebo -1.10 [95% CI -3.14, 0.94], p = 0.29 and 0.49 [95% CI -1.55, 2.53], p = 0.63); UPSA-B (LS mean difference from placebo 3.43 [95% CI -0.20, 7.06], p = 0.06 and 3.42 [95% CI -0.23, 7.06], p = 0.07); or SCoRS (LS mean difference from placebo 0.43 [95% CI -1.43, 2.29], p = 0.65 and -0.43 [95% CI -2.28, 1.42], p = 0.65), for BIIB104 at 0.15 mg and 0.5 mg, respectively. There was no worsening or improvement of PANSS total score. BIIB104 was generally well tolerated. Conclusions: BIIB104 did not show statistically significant efficacy in the treatment of CIAS at either tested dose. The hypothesis that the high-impact AMPAR PAM BIIB104 would demonstrate clinically meaningful efficacy in the treatment of CIAS was not supported. Keywords: BIIB104, PF-04958242, Schizophrenia, Cognitive Impairment, AMPA Disclosure: Biogen Inc.: Employee (Self), Biogen Inc.: Stock / Equity (Self) P496. Longitudinal Ecological Momentary Assessments of the Behavioral Indicators of Avolition in Schizophrenia Identify Changes that Are Correlated With Clinical Ratings of Negative Symptoms Philip Harvey, Colin Sauder, Soumya Chaturvedi, Stephen Targum Miller School of Medicine, University of Miami, Miami, Florida, United States Background: Reductions in emotional experiences in subjects with schizophrenia (avolition, asociality, and anhedonia) are associated with the real-world social deficits experienced by these individuals. The assessment of negative symptoms is more difficult than assessment of positive symptoms because it requires self-awareness and corroborative information from knowledgeable informants. Recent advances in the observational assessment of negative symptoms have employed participant reports using ecological momentary assessment (EMA) which can capture critical behavioral features of avolition and asociality, including the amount of time spent home and alone, and engagement in passive and unproductive activities. We report interim, but substantive pilot data from the first half of a 12-month longitudinal study of EMA, combined with clinical ratings of negative symptoms during an unblinded open label safety study of an antipsychotic medication in development. Methods: Stable outpatients (PANSS ≤ 80) with schizophrenia entered a 12-month open-label treatment study of the candidate drug that included monthly clinical ratings of the PANSS and negative symptom ratings with the NSA-16. Longitudinal EMA assessments were delivered in 7-day bursts, 3 surveys per day for one week intervals monthly throughout the study. The EMA surveys were delivered by a smartphone and queried location and social context (home vs. away; alone vs. with someone), positive and negative affect (PA, NA), hallucinations and delusions, and 1 of 3 targeted activity surveys that were customized for home alone, home with someone, and away from home. A total of 23 different activities were sampled across the three surveys with a sampling window of “the last hour”. Participants also wore a “fit-bit” actigraph daily during EMA sampling weeks. Three individual NSA items were selected for analysis because they most closely defined avolition (reduced activities, reduced sense of purpose, and reduced social drive). Because this is an exploratory assessment of EMA, the analyses were limited to subjects who answered at least 33% of the EMA surveys. Mixed Model Repeated-Measures Analysis of Variance (MMRM) strategies were used for EMA data analysis, including use of dynamic correlates to predict activity outcomes. Correlations (and regression analyses) with aggregated EMA variables and scores on the NSA items were also computed. Results: A total of 4138 fully completed EMA surveys have been answered to date by 54 subjects with completed clinical assessments during the sampling period. Scores on momentary PA as measured by EMA increased significantly (X2 = 47.30, p < .001). Step counts increased in concert with momentary PA (p < .001). The increases in PA also correlated with significant increases in the time-synchronized proportion of at-home productive activities and decreases in passive and unproductive activities. Further, significant decreases in the proportion of surveys answered at home were associated with increases in momentary PA (p < .001). Regression analyses indicated that more EMA surveys answered while at home, alone, and engaging in unproductive activities shared 21% of the variance with higher the NSA “reduced activity” item scores. More away from home activities, fewer EMA surveys answered home and alone, and more daily steps shared 31% of the variance with lower ratings of the NSA “reduced sense of purpose” item. Fewer EMA surveys answered home and alone and more surveys answered while engaging in productive activities shared 29% of the variance in the NSA item ratings of “reduced social drive”. In contrast to these consistent correlations with negative symptom items rated with the NSA, scores on the PANSS reduced emotional experience items manifested much smaller correlations with EMA-derived variables. Numbers of surveys answered at home and alone, and PA all shared less than 4% variance with PANSS reduced emotional experience items. The only correlation with PANSS reduced emotional experience that was substantial was the correlation between lower reduced emotional experience scores and more away from home activities (r = -.40). Conclusions: These preliminary findings are the first report to document that treatment related changes in a constellation of the behavioral indicators of avolition can be detected with EMA surveys. The results of these surveys were substantially correlated with clinician ratings of NSA items indexing avolition (i.e., reduced emotional experience) that are the primary predictors of the social deficits associated with schizophrenia. Increases in PA after baseline were robustly detected and predicted increased engagement in positive daily activities measured on a momentary basis over a 6-month period, including more surveys answered away from home that corresponded with more productive activities and reductions in at-home unproductive activities. Increased physical activity was also longitudinally associated with increased PA. These EMA findings were validated by their convergence with clinician-rated scores on negative symptoms generated with a “gold standard” instrument (NSA-16) using a clinician-based, monthly rating strategy. Keywords: Schizophrenia; Technology, Ecological Momentary Assessment, Schizophrenia Negative Symptoms Disclosure: Allermes, Bioexcel, Boehringer-Ingelheim, Karuna Therapeutics, Merck, Minerva Pharma, Sunovion Pharma, WCG Clinical: Royalties (Self), EMA Wellness: Consultant (Self), i-Function, Inc.: Owner (Self) P497. Metformin and Lorcaserin Combination Treatment for Antipsychotic-Associated Weight Gain Lars Jarskog, Thomas Stroup, Kai Xia, Anastasia Ivanova, Kim Brownley, Joseph McEvoy, Robert Millet University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: Weight gain is a common and serious side effect of antipsychotic treatment and there are few effective options to mitigate this risk. Metformin has been identified as the most effective adjunctive treatment for antipsychotic-associated weight gain, with meta-analyses demonstrating ~3 kg weight loss in studies of 12-24 weeks duration. Lorcaserin, a 5-HT2C agonist, is an effective weight loss agent in obese but otherwise healthy individuals. Some combination drug approaches for weight loss using agents with different mechanisms have demonstrated promise over monotherapy treatment but this approach had not been studied for antipsychotic-associated weight gain. Methods: This 52-week double-blind, randomized trial compared lorcaserin/metformin (LOR/MET, 10 mg BID/1,000 mg BID, n = 23) combination treatment, lorcaserin (LOR, 10 mg BID, n = 24) monotherapy and placebo (PBO, n = 24) in 71 non-diabetic outpatients with schizophrenia and schizoaffective d/o with a body mass index (BMI) > 27. All subjects also received a behavioral intervention aimed at improving diet and increasing physical activity. The primary outcome measure was change in weight at study endpoint compared to baseline. Secondary outcomes included change in measures of glucose and lipid metabolism. Results: LOR/MET combination treatment was associated with significantly greater weight loss compared to placebo, with an estimated least-squares mean change from baseline to 52 weeks of -8.4 kg (p = 0.010). The effect of LOR monotherapy on weight was not significantly different compared to placebo at 52 weeks (-0.7 kg, p = 0.817). Of the secondary outcome measures of lipid and glucose metabolism, only fasting glucose in the LOR/MET arm was significantly reduced compared to placebo by -10.1 mg/dL (p = 0.030). Neither hemoglobin A1c nor any of the markers of lipid metabolism differed significantly from placebo for LOR/MET or LOR arms. Treatments were generally well tolerated, with somewhat higher rates of diarrhea, headache, dizziness, restlessness and diaphoresis in the treatment arms compared to placebo. Notably, the study was prematurely terminated on 02/13/2020 due to withdrawal of FDA approval for lorcaserin due to evidence of a small excess in cancer incidence following long-term lorcaserin treatment (median 3.3 years) in a large Phase 4 study aimed at assessing cardiovascular safety and efficacy. There were no new cases of cancer in the current study. Conclusions: Combination treatment with LOR/MET was effective and generally well-tolerated in reducing body weight in outpatients with schizophrenia or schizoaffective disorder who were on stable antipsychotic regimens. The magnitude of weight loss for LOR/MET combination treatment was nearly 3-fold greater compared to weight loss seen for metformin monotherapy from prior studies in the same population. Among secondary outcome measures, LOR/MET appeared to improve fasting glucose levels, but hemoglobin A1c was unchanged as were markers of lipid metabolism. LOR monotherapy did not show efficacy at 52 weeks in primary or secondary outcomes. Two important limitations of the study include the current inability to use lorcaserin in clinical practice as well as reduced statistical power due to premature termination of the study. While lorcaserin is no longer available to prescribe for weight loss in the US, these data suggest viability of the 5-HT2C agonist mechanism as a generally well-tolerated and effective adjunct when used in combination with metformin to achieve clinically meaningful weight loss for antipsychotic-associated weight gain. The development of novel 5-HT2C agonists for weight loss represents a potentially important approach for addressing antipsychotic-associated weight gain. More generally, the findings also highlight the potential benefits of combining agents with distinct mechanisms for addressing antipsychotic-associated weight gain. Keywords: Metformin, Lorcaserin, Antipsychotic Induced Weight Gain Disclosure: Boehringer Ingelheim: Contracted Research (Self), Corcept Pharmaceuticals: Contracted Research (Self), UpToDate: Royalties (Self), SignantHealth: Consultant (Self), Otsuka: Contracted Research (Self) P498. Cerebellar TMS Dosing Reveals Cerebellar-Cortical and Cerebellar-Basal Ganglia Connectivity Mark Halko, Roscoe Brady, Laura Holsen, Jing Xie, Adam Beerman, Won Young Cheong, Ivy Lee, Uzma Nawaz, Emily Payne, Emma Joncas, Madelaine Nye, Michael Esterman, Simon Laganiere Harvard Medical School, McLean Hospital, Belmont, Massachusetts, United States Background: Cerebellar stimulation has a renewed interest for the treatment of psychiatric and neurological conditions. Recent discoveries of motor function within the cerebellum have made understanding the mechanics of cerebellar stimulation imperative. Critically, we have previously demonstrated that cerebellar stimulation impacts network connectivity at rest in acute studies (Halko et al 2014) and across multiple stimulation sessions (Brady et al 2019), showing promise for reduction of symptoms of schizophrenia (Brady et al 2019). Here, we examined if dosage of TMS can be titrated by network connectivity. Methods: In a repeated measures study, 26 healthy participants received 3 different intensities of intermittent theta-burst simulation determined by active motor threshold (100% aMT, 87.5% aMT, 75% aMT). Stimulation was targeted at the cerebellar vermis representation of the dorsal attention network, with a standard figure of 8 coil, handle facing upward. Functional connectivity during a continuous performance task (gradCPT) was collected before and immediately after stimulation. Results: Increasing cerebellar connectivity between default network regions of the cerebellum and default network regions of cortex following stimulation at 100% AMT regions of cortex was observed, but did not reach significance, when compared to 87% and 75% conditions (p = .21). However, cortical network connectivity change between default network and dorsal attention network was strongly linked to attentional performance improvement (r = -0.225, p = 0.002). Preliminary analysis suggests that cerebellar-basal ganglia connectivity may be intensity dependent (r = 0.19, p = 0.07). Conclusions: Overall, our study indicates that titrating the dose of TMS based on motor threshold is an ineffective strategy for cerebellar modulation. We also demonstrate that connectivity change is a required component of behavioral change, suggesting that parameters that can deterministically impact connectivity at the targeted site of intervention may be more relevant considerations for determining effective TMS intensity dosages. Keywords: Theta Burst Transcranial Magnetic Stimulation, Cerebellum, Basal Ganglia, Non-Invasive Brain Stimulation Disclosure: Nothing to disclose. P499. Cognitive Flexibility is Differently Linked to Callosal Fibre-Bundles and Functional Connectivity Between Homotopic Grey Matter Regions in Antipsychotic-Naïve Patients with First Episode Schizophrenia Tina Kristensen, Karen Ambrosen, Jayachandra Raghava, Egill Rostrup, Warda Syeda, Thijs Dhollander, Cecilie Lemvigh, Kirsten Borup Bojesen, Simon Anhøj, Birgitte Fagerlund, Mette Nielsen, Christos Pantelis, Birte Glenthoj, Bjørn Ebdrup Center for Neuropsychiatric Schizophrenia Research (CNSR), Glostrup, Denmark Background: Cognitive impairments are a core feature of schizophrenia. Abnormalities in the neuronal connectivity of the brain have been proposed as an essential biomarker of schizophrenia. Higher cognitive processes, such as executive functions, rely on an interplay between several distributed regionally based processes, and have been investigated in conjunction with a range of neuroimaging modalities. Typically, each biomarker has been analysed separately. However, due to the interconnected nature of brain processing, changes in one modality may modulate changes in other modalities. Little is known about how the structural connectivity (white matter (WM) fiber bundles connecting grey matter (GM) regions) and functional connectivity (concurrent functional activity between GM regions) associate in parallel to higher order cognitive functioning. Nonetheless, it has become increasingly clear that combining multi-modal brain imaging data is able to provide more detailed information about the neurobiological underpinnings of higher order cognitive processes. Fixel-based analysis (FBA) is a novel statistical framework to analyse fibre-specific measures of WM, such as fibre density (FD) and fibre-bundle cross-section (FC). We used FBA and resting state functional MRI to investigate associations between WM micro- and macrostructure, functional connectivity and executive functions in 64 antipsychotic-naïve patients with first-episode schizophrenia and 95 matched healthy controls. Methods: All participants underwent MRI (diffusion weighted imaging and resting state functional MRI). Executive functioning was assessed using tests from the Cambridge Neuropsychological Test Automated Battery (cognitive flexibility (IED) and planning (SOC); and the Brief Assessment of Cognition in Schizophrenia (verbal working memory (digit sequencing) and verbal fluency). Group comparisons and associations between fixel-based measures and executive functions were analysed using multivariate linear regression. Post hoc, homotopic connectivity was estimated as the correlation between the functional activity of corresponding grey matter regions of interest (ROIs) in each hemisphere connected via the isthmus, namely the left and right isthmus cingulate, precuneus, postcentral, and superior parietal regions, respectively. Associations between the homotopic connectivity between each ROI pair and cognitive flexibility, as well as to fixel-based measures were tested using partial correlation analyses. Analyses were covaried for age, sex, cohort, intracranial volume, 6 motion regressors, and the relative motion during the fMRI acquisition as appropriate, and results were corrected for multiple comparisons using FWE with a threshold of p < 0.05. There was an equal distribution of male/female sex in patients and matched controls (52% males, 48% females) Results: Patients displayed cognitive impairments on all executive functions (p < 0.001) and reduced FD in the body of corpus callosum and cingulum(p < 0.05) compared to controls. When modelling FC, we found a significant interaction between group and cognitive flexibility in the isthmus of corpus callosum (p < 0.05), i.e., larger callosal fibre-bundles of the isthmus was linked to increased cognitive flexibility in patients, but not in controls. We did not identify any significant interaction effect with planning, verbal working memory, or verbal fluency. Furthermore, when post hoc modelling homotopic connectivity, we identified a significant interaction effect between group and cognitive flexibility in the precuneus and postcentral ROIs. In patients, increased homotopic connectivity in the precuneus was associated with more cognitive flexibility (p = 0.028), while in controls, increased homotopic connectivity in the postcentral region was associated with more cognitive flexibility (p = 0.012). We did not identify any significant interactions between fixel-based measures and homotopic connectivity, but a trend level negative correlation between FC in isthmus and homotopic connectivity between postcentral ROIs in controls (p = 0.08, RHO -0.19), but not in patients (p = 0.86, RHO = -0.02). Conclusions: Cognitive flexibility is linked to macroscopical characteristics of the fibre bundle cross-section of the isthmus of corpus callosum and is differentially associated with homotopic connectivity in the precuneus and postcentral GM regions in antipsychotic naïve patients with first-episode schizophrenia compared to controls. These group specific links may reflect that the structural and functional neurobiological underpinnings of cognitive flexibility in antipsychotic-naïve patients with first episode schizophrenia differs from controls. Keywords: Antipsychotic-Naïve First-Episode Schizophrenia, Executive Function, Diffusion Weighted Imaging, Resting State Functional Connectivity Disclosure: Nothing to disclose. P500. Neurocognitive and Sensorimotor Biomarkers of Psychosis-Risk in 22q.11.2 Deletion Syndrome and Their Relationship to Clinical Symptoms David Parker, Sid Imes, Brett Henshey, Nicholas Massa, Bruce Cuthbert, Grace Lee, Elaine Walker, Erica Duncan, Joseph Cubells, Opal Ousley Emory University, Athens, Georgia, United States Background: The 22q.11.2 deletion syndrome (22q11DS) is one of the most robust genetic predictors of the development of psychosis and other psychiatric illnesses. We aimed to untangle the complex relationship between 22q11DS and mental illness by utilizing a specific battery of cognitive tests and psychophysiological biomarkers known to be associated with psychosis-risk. In this study we examined performance on neurocognitive tests, motor speed, target detection, and auditory EEG responses in 22q11DS individuals and controls to understand the relationship between cognition, sensorimotor responses, and clinical symptoms. Methods: We recruited 15 22q11DS individuals (Mean age=30, M/F = 9/6) and 19 healthy controls (HCs; Mean age=34, M/F = 5/14) from the local community. Each individual completed the MATRICS Consensus Cognitive Battery (MCCB), the Wechsler Abbreviated Scale of Intelligence, Second edition (WASI-II) Verbal IQ subtests, and the computerized Wisconsin Card Sorting Task (WCST). Sensorimotor reactivity was measured via a finger-tapping task (i.e., both dominant and non-dominant hands) and a visual oddball target detection task. For the auditory EEG task, each participant completed the “Double-Deviant” target detection paradigm, which presents a pseudorandom sequence of frequent standard tones and infrequent deviant tones (i.e., 90% of trials, 633 Hz, 50 ms duration; 10% of trials, 1000 Hz, 100 ms; respectively). Mismatch negativity (MMN) metrics were generated from this assessment. Current symptoms were assessed with the Structured Interview for Psychosis-Risk Syndromes (SIPS), including subscales that assess prodromal symptoms of schizophrenia (i.e., Positive, Negative, Disorganized, and General). Data analysis: Group comparisons were examined for all measures. Welch’s t-tests were completed for all age/sex adjusted standardized neurocognitive variables. One-Way ANOVAs were completed to examine sensorimotor and EEG results, with sex entered as a separate factor and age entered as a covariate for non-normed data (sensorimotor and EEG results). All p-values were false discovery rate (FDR)-adjusted. We tested for correlations between significant variables and clinical measures from the SIPS Subscales. Results: Significant group differences were found in 8 of the 9 neurocognitive measurements (FDR-adjusted p’s< 0.02, average Cohen’s d = 1.62, average observed power= 0.91) indicating widespread cognitive deficits in 22q11DS subjects across multiple domains. Significant differences were found for the dominant and non-dominant hands on the motor finger tapping task (Cohen’s d = [0.558, 0.716], average observed power: 0.68). Group differences were not found for accuracy or latency of detecting a visual target (FDR-adjusted p’s>0.16). The Double-Deviant MMN ERP response was significantly smaller in absolute magnitude in the 22q11DS group (FDR-adjusted p = 0.048, Cohen’s d = -0.864, observed power= 0.58). The MMN ERPs for the frequency and duration deviants were not significantly different (FDR-adjusted p’s> 0.33). No group by sex interactions were observed in any of the measures. Two neurocognitive variables and the finger-tapping for the non-dominant hand were significantly negatively correlated with the SIPS Positive symptoms (i.e., MCCB Working Memory, MCCB Verbal Learning, finger tapping non-dominant hand; Spearman’s rho = -0.473, -0.403, -0.396, respectively; FDR-adjusted p’s< 0.049). These results indicated that poorer neurocognitive and motor speed performance were associated with greater SIPS symptoms. Verbal IQ and each of the MCCB cognitive domains were negatively associated with Negative and Disorganized symptom scores on the SIPS (i.e., Spearman’s rho range = -0.436 to -0.71, FDR-adjusted p’s< 0.049), indicating that greater prodromal symptoms were associated with poorer cognition. Lastly, MCCB Reasoning and Problem-Solving score was negatively associated with the SIPS General Symptom score (Spearman’s rho = -0.486, FDR-adjusted p = 0.015), indicating that more severe General symptoms were associated with poorer cognitive performance. Conclusions: Identification and validation of psychosis-risk biomarkers in 22q11DS could provide important translational targets for future clinical trials for individuals with 22q11DS and other individuals at-risk for psychosis syndromes. Through the use of a dense battery of cognitive and psychophysiological biomarkers, these preliminary results indicate robust deficits across cognitive, motor, and auditory neural processing domains in 22q11DS that associate with clinical symptoms of the schizophrenia prodrome. Future work will build upon and attempt to verify the findings seen in this preliminary study. We plan to compare these data to individuals who are at clinical high-risk for psychosis to untangle genetic versus idiopathic risk. Furthermore, our current work in progress is examining deficits in pluripotent stem cell-derived neurons from 22q11DS and control subjects to understand abnormalities at the cellular level that could contribute to 22q11DS pathology. Keywords: 22q11.2 Deletion Syndrome, Psychosis-Risk, Biomarkers, Cognition, EEG Disclosure: Nothing to disclose. P501. Astrocyte Subtype Gene Enrichment in Psychiatric Disorders and Psychotropic Medication Datasets Sinead O’Donovan, Xiaolu Zhang, Alyssa Wolfinger, Xiaojun Wu, Anna Lundh, Vladimir Parpura, Robert McCullumsmith, Rammohan Shukla University of Toledo, Toledo, Ohio, United States Background: Astrocytes have many important functions in the brain, but their roles in psychiatric disorders and their responses to psychotropic medications are still being elucidated. Reactive astrogliosis describes a spectrum of heterogeneous changes in astrocyte gene expression, morphology, and overall function, which can have a protective or pathological effect, depending on the subtype of astrocyte involved and the type of injury sustained. Different subtypes of astrocyte are classified based on their cellular morphology, location and primary functions, and can have potentially different responses to insult. Our understanding of the different types of astrocytes in humans and their roles in psychiatric disorders is still limited. Methods: Gene and pathway enrichment of astrocyte subtypes, psychiatric disease and psychotropic medication gene-sets was assessed using hypergeometric overlap analysis with a background of 21,196 genes (GeneOverlap R v1.26.0). A density index (DI) was applied to quantitatively summarize how common or unique a biological theme is across different astrocyte subtypes and disease and drug gene-sets. Gene-sets were derived from publicly available transcriptomic datasets and gene-set size was restricted to between 10 to 500 significant (p < 0.05) differentially expressed genes. Confirmation studies included qPCR assays of astrocyte marker gene expression in chronic haloperidol- and vehicle- treated rat brain (n = 20), and exploratory kinome analysis (Pamgene12) of gliosome fraction isolated from postmortem brain tissue. Results: Bioinformatic analysis identified gene enrichment [–log10(p < 0.05)] of different astrocyte subtypes in psychiatric disorders. The highest level of enrichment (DI = 0.6) was found in schizophrenia. Astrocyte subtypes were differentially enriched in specific biological processes, including protein phosphorylation (DI = 0.25). Enrichment of protein phosphorylation in astrocytes was confirmed by increased (FC 1.22) kinome signal intensity in gliosome fraction relative to total homogenate. Common gene enrichment of different psychotropic medications and astrocyte subtypes was limited. qPCR analysis also found little effect of psychotropic medication (haloperidol-decanoate, Student’s t-test, p > 0.05) on common astrocyte marker gene expression. Conclusions: Gene enrichment analyses suggest enrichment of astrocyte subtype-specific genes in psychiatric disorders like schizophrenia, indicating unique roles for different astrocyte subtypes in these disorders. Psychotropic medication and astrocyte gene enrichment are low suggesting that astrocytes are not significant targets of these medications. Overall, this study provides a unique view of astrocyte subtypes and the effect of medications on astrocytes in disease. Keywords: Astrocyte, Schizophrenia (SCZ), Major Depression Disorder, Psychotropic Medications, Phosphorylation Disclosure: Nothing to disclose. P502. Assessing the Roles of Prefrontal Excitatory and Parvalbumin-Expressing Neurons During Working Memory Tyler Dexter, Megan Attard, Daniel Palmer, Lisa Saksida, Tim Bussey University of Western Ontario, London, Canada Background: Working memory (WM) is the process by which information is temporarily held available to aid goal-directed behavior. The prefrontal cortex (PFC) is a major modulator of WM function, and PFC disruption is known to impair WM processes across species. Synchronous neuron firing at the gamma frequency (gamma oscillations) in the PFC has been shown to increase alongside WM task difficulty, which has been implicated in stabilizing information in WM. Previous studies have demonstrated that inhibitory GABAergic neurons expressing the protein parvalbumin (PV) are critical for facilitating gamma oscillations in the cortex. Additionally, specific disruption of this population has been shown to impair PFC functions such as attention and cognitive flexibility. However, studies in mice have produced conflicting findings as to whether prefrontal PV neurons are necessary for WM task performance. Here, we combined in vivo optogenetics with a touchscreen-based paradigm of WM to 1) assess how prefrontal excitatory neurons contribute to maintaining WM over a delay period; and 2) assess how prefrontal PV neurons contribute to WM processes. Methods: We used in vivo optogenetics to inactivate either prefrontal excitatory or inhibitory PV neurons during the trial unique non-match to location (TUNL) task. The TUNL paradigm contains three phases, where mice are presented a stimulus on the screen, they must retain the location of this stimulus over a delay, and then correctly chose a novel stimulus when it is presented alongside the original stimulus. All mice were trained on the TUNL task prior to surgery. To inactivate excitatory neurons, we injected male C57BL/6 mice (n = 9) bilaterally with a CAMKii-dependent archaerhodopsin (Arch) virus targeted to the medial PFC (mPFC; AP + 1.8, ML ± 0.3, DV -2.2). Two fiber optic probes were implanted above the injection sites. To inactivate PV neurons, male and female mice (n = 7 each) were bred to selectively express Arch under the PV promoter. These mice were then implanted with bilateral fiber optics at the same coordinates described above. Following surgery, mice were re-baselined on the TUNL task prior to optogenetic manipulations. To assess the role of mPFC excitatory neurons during WM maintenance, mice received optogenetic stimulation at temporally specific points of the delay (early phase of the delay, middle of the delay, or end of the delay). For these experiments, the delay period was held fixed at 2 s. For PV neuron inactivation experiments, WM maintenance was assessed by increasing the delay duration across separate sessions (0 s, 1 s, and 2 s). Additionally, to test whether PV neuron activity protected against proactive interference, mice were assessed on an increased interference probe. Here, the intertrial interval (normally 15 s) was removed, resulting in trials occurring back-to-back and increasing the potential for memories of previous trials to interfere with the current trial configuration. Results: First, we observed that optogenetic inhibition of prefrontal excitatory neurons impairs task performance during specific temporal phases of the delay. Inhibition during the early and middle, but not late (choice phase), significantly impaired choice accuracy (p = 0.002, F(2, 12) = 29.9342). Next, two-way ANOVA revealed that inhibiting prefrontal PV neurons throughout the delay produced a significant delay-independent reduction in choice accuracy (p = 0.025, F(2, 28) = 6.344). Further, we observed a significant impairment when PV neurons were inactivated during the high interference condition (p < 0.001, F(1, 28) = 19.625). There were no significant effects of optogenetic stimulation under any conditions in a control group of PV:Arch- mice who underwent identical protocols (n = 10). Further, there were no significant effects of optogenetic stimulation on response or reward collection latencies under any conditions. Conclusions: Our results describe the involvement of relevant PFC circuitry during a touchscreen-based task of WM designed to emulate paradigms used in human testing. We show that performance on TUNL not only depends on PFC excitatory neuron functioning, but that information held in WM is stable against PFC disruption by the choice phase. While multiple recent studies have evaluated the role of prefrontal PV neurons during WM paradigms in mice, the results have been inconsistent as to whether this population is necessary for optimal task performance. With TUNL, we are able to present sample and choice stimuli across multiple locations on the touchscreen. Therefore, this paradigm provides a high number of stimuli configurations, which differs from common two-choice working memory tasks. As such, TUNL may introduce a greater degree of interfering information on a given trial, as there may be lingering memories of past trial configurations that could disrupt the current information held in WM. PV neurons are critical for the generation and maintenance of gamma oscillations, which have been implicated in holding information in WM and protecting against competing task-irrelevant information. Therefore, our data suggests that prefrontal PV neurons facilitate the maintenance of WM information during TUNL, potentially by protecting against distracting information that interferences with the target held in WM. Keywords: Visuospatial Working Memory, Medial Prefrontal Cortex, Parvalbumin Neurons, Touchscreen Cognitive Testing Disclosure: Nothing to disclose. P503. Impaired Face Emotion Recognition in Schizophrenia: Task Performance, Eye-Tracking and Neurophysiological Analyses and Comparison to Autism Spectrum Disorder Antigona Martinez, Elisa Dias, Pamela Butler, Russel Tobe, Daniel Javitt Nathan S Kline Institute for Psychiatric Research, Orangeburg, New York, United States Background: A key component of human social behavior is orientation to faces in general and eyes in particular during social interaction. In both schizophrenia (Sz) and autism spectrum disorder (ASD), deficits in social cognition have been attributed to reduced orientation to eyes while processing faces. Nevertheless, the mechanisms underlying reduced eye fixation remain incompletely understood. In the present study we performed a fine-grain analysis of fixation patterns during face viewing in Sz and ASD participants, along with electrophysiological recordings of event-related potentials (ERPs). The study builds from prior studies in significant ways. First, it includes both Sz and ASD subjects, permitting direct comparison of mechanisms underlying social cognitive deficits across the two disorders. Second, it uses incongruent as well as congruent face stimuli to better clarify the role of eye versus mouth fixation in face-emotion recognition (FER) deficits across the disorders. Third, it assesses fixation patterns relative to responses on a trial-by-trial basis in order to assess cause and effect relationships between alterations in gaze and alterations in behavior. Finally, it builds from recent studies showing differential patterns of early visual processing alterations in Sz and ASD compared to neurotypical (NT) individuals to assess potential neurophysiological contributions to altered behavior. To our knowledge, this is the first study to directly compare fixation patterns related to FER across Sz and ASD groups, to utilize trail-by-trial fixation information, and to combine these with neurophysiological analyses tied both to stimulus and fixation onset. Methods: Participants were 23 patients diagnosed with Sz (mean age 37.1 years), 22 ASD individuals (29.2 years) and 24 demographically-matched NT subjects (36.0 years). Participants were presented with 324 human face stimuli and reported the perceived emotion in each (happiness, sadness, anger, fear or neutral). The face stimuli were either congruent (top and bottom halves of faces conveyed the same emotion) or incongruent (top and bottom halves conveyed different emotions). Eye movements (gaze position) were recorded on each trial and the ongoing electroencephalogram (EEG) was recorded from 64 scalp channels. Results: As expected, the percent correct performance for congruent stimuli differed significantly across groups (p = .01). Post-hoc differences between Sz (p = .004) and ASD (p = .027) versus NT subjects were also significant. The severity of FER deficits was similar in Sz and ASD participants (p = .20), however, despite the much different level of overall socioeconomic function. Relative to NT subjects, both Sz (p < .001) and ASD (p < .001) participants showed reduced fixation on eyes and increased fixation on mouths and were more likely than NT individuals to use mouth-, rather than eye-, information to disambiguate chimeric faces (SZ: p < .001; ASD: p = .006). However, whereas ASD individuals correctly identified faces in trials where they did fixate on eyes, Sz subjects remained impaired despite their fixation location (p = .002). In the time-domain, ERP responses elicited by all face stimuli consisted of a P1 followed by N170 potential over the ventral occipital scalp. Overall, the peak amplitude of the P1 and N70 was significantly different across groups (p = .011) and in Sz compared to NT (p = .013). Lastly, as in previous reports, the onset of the N170 was delayed in ASD relative to NT subjects (p = .035). When the spectral composition of EEG responses was assessed using time-frequency analyses, a significant across-group difference in evoked power in the theta (4-7 Hz) frequency band was observed (p = .02) with a significant post-hoc difference between Sz and NT individuals (p = .016). The onset of face stimuli also modulated ongoing oscillatory activity in the alpha (8-12 Hz) band with reduced modulation in Sz (p = .024) (relative to NT) but significantly enhanced modulation in ASD (p = .026) which additionally predicted their delayed N170 latencies (p = .048). Conclusions: These findings show the potential value of the use of chimeric face stimuli, and, despite similar levels of overall dysfunction, our findings also point to significant mechanistic differences underlying FER deficits in Sz and ASD participants. In Sz patients, the deficit patterns are consistent with impaired magnocellular function and impaired bottom-up input to visual sensory systems. Deficits in ASD are consistent with hyperactivity of the retinocollicular system as indexed electrophysiologically by alpha-band activity. Abnormalities in stimulus-induced alpha modulation may also underlie the well-described N170 latency increases in ASD and may thus serve as an additional sensitive biomarker of early face-processing dysfunction in ASD. Keywords: Schizophrenia (SCZ), Autistic Spectrum Disorders, Face Emotion Processing, EEG Electrophysiology, Eye-Tracking Disclosure: Nothing to disclose. P504. Brain and Physiological Stress Responses in Early Psychosis Brandee Feola, Elizabeth Flook, Dongju Seo, Victoria Fox, Jesse Oler, Neil Woodward, Stephan Heckers, Jennifer Blackford Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: Stress is proposed to contribute to the onset and expression of psychosis. Yet, how people with psychosis differ in responses to stressors remains unclear, especially in early-stage psychosis. The current study examined whether brain or physiological stress responses differ in people with early-stage psychosis. Methods: 20 people with early psychosis (schizophrenia spectrum disorders/bipolar disorder with psychotic features) and 20 healthy control participants completed a fMRI stress task that involved viewing stress images, neutral relaxing images, and fixation baselines. Additional assessments included physiological stress responses (cortisol, heart rate), self-report of stress, and psychosis symptoms (PANSS). Region of Interest (ROI) analyses were conducted for a prior stress network (amygdala, hippocampus, striatum, hypothalamus, prefrontal cortex). Linear mixed models compared differences in ROI activation using group (psychosis/control), phase (baseline, provocation), and emotion (stress, neutral). Correlations were conducted between brain activation, stress responses, and clinical characteristics. Results: During the stress task, significant group differences in brain activation were found for the hippocampus (group x phase x emotion interaction p < 0.001) and prefrontal cortex (group x phase interaction p = 0.04). Within the psychosis group, activation in the hippocampus, amygdala, and prefrontal cortex correlated with physiological stress responses and clinical characteristics (all p < 0.05). Conclusions: Our findings suggest that people with early psychosis have altered brain responses to stress. Within the psychosis group, brain responses to stress correlated with clinical characteristics. Future studies utilizing multi-method assessment of stress may provide the most insight into the role of stress in early psychosis. Keywords: Acute Stress, Early Psychosis, Brain Imaging, fMRI Disclosure: Nothing to disclose. P505. Functional Imaging and Genetic Predictors of Future Employment in Patients With Schizophrenia Performing a Working Memory Task Rachael Blackman, Daniel Eisenberg, Dwight Dickinson, Michael Gregory, Karen Berman Clinical and Translational Neuroscience Branch, National Institute of Mental Health, Intramural Research Program, Bethesda, Maryland, United States Background: With a dearth of treatment options, cognitive deficits in patients with schizophrenia are well known to be linked to real-life functional outcomes in patient populations. There is great heterogeneity in this regard; some individuals become active members of the workforce despite their illness, while others do not have this resilience. Uncovering predictors of positive outcomes may inform the development of better treatment targets for patients with schizophrenia. Deficits in working memory have been well characterized in patients, for example using the N-Back task which activates frontal networks during task performance. Here we examine whether neural activation during this task may predict whether patients are employed or not at a future timepoint. In addition, as schizophrenia is a highly heritable illness, we test whether overall genetic risk moderates these effects of cognition on future employment. Methods: Patients with schizophrenia spectrum illness (n = 125, 31.2% female, mean age 32.5 + /- 10.5 years) participated in a research study at the National Institute of Mental Health Intramural Research Program’s Clinical Center and were recontacted at a later timepoint (8.7 + /- 4.0 years after initial visit) when outcomes information such as employment status (employed vs. unemployed) was collected. At the initial visits, patients participated in neuropsychological testing, functional neuroimaging (3 T fMRI during N-Back performance), and provided blood samples for genotyping. Cognitive measures including g (a general measure of cognition), IQ, and six cognitive subdomain scores (verbal memory, N-Back working memory, visual memory, processing speed, card sorting/executive function, and span working memory) calculated at the initial visit were tested as predictors of employment status at follow-up using logistic regressions. Covariates in the models included age at the initial visit, sex, length of time to follow-up, and initial visit employment status. In a subset of participants (n = 102, 28.4% female, mean age 32.9 + /- 10.9 years), polygenic scores for schizophrenia risk (PGSscz) were derived using summary statistics from genome wide association studies. Logistic regressions including PGSscz and the first three ancestry-related genetic principal components as covariates were added to the models and repeated. Finally, for the subset of participants who performed the N-Back working memory task during fMRI scanning at their initial visit (n = 54, 31.5% female, mean age 33.2 + /- 11.6 years), neural activation during task performance (2-Back > 0-Back) was tested using AFNI software to identify regions that differed between the individuals who were employed vs. unemployed at follow-up. To further focus on a sub-group who may benefit from targeted intervention, we repeated the fMRI analyses restricting the analyses to only those participants who were unemployed at the initial visit (n = 37) to characterize regions that predict employment status change (i.e., unemployed to employed) at follow-up. Results: 44.8% of the total participants (n = 56) were employed at follow-up, with 23.2% of participants at the initial visit having an employment change such that they were unemployed at the initial visit and became employed by the follow-up timepoint. Of the cognitive domains examined at the initial visit, g (n = 115, B = 0.61, SE = 0.30, Wald=4.08, p = 0.043), IQ (n = 116, B-0.06, SE = 0.02, Wald=7.07, p = 0.008), and verbal memory (n = 115, B = 0.43, SE = 0.21, Wald=4.23, p = 0.040) predicted employment status at follow-up such that higher scores were observed in those who were employed compared to those who were unemployed. We further found that PGSscz moderated the effect of N-Back behavioral scores at the initial visit on the ability to predict employment status at the later timepoint (n = 83, interaction N-Back score by PGSscz p = 0.022). Participants who were unemployed at follow-up and had higher PGSscz had worse performance at the initial visit, whereas the reverse was true in the employed population. Greater neural activation in right dorsolateral prefrontal cortex during the N-Back task (peak p = 0.002, uncorrected) and right cingulate cortex (peak p = 0.003, uncorrected) was observed for employed individuals at follow-up. Results from the analysis examining only participants unemployed at the initial visit were consistent. Conclusions: The results of our study demonstrate that in addition to general cognitive predictors of future employment there are other subdomains that may be relevant, specifically verbal and working memory. The N-Back behavioral effects were evident when considering PGSscz, highlighting the need to consider the heterogeneity of the schizophrenia patient population. The fMRI findings suggest avenues for further investigation into the neural underpinnings of real-world outcome measures in this population. Keywords: Cognitive Impairment Associated With Schizophrenia, Functional Outcomes, Polygenic Risk Score, Functional Neuroimaging Disclosure: Nothing to disclose. P506. The Impact of Early Risk Factors on Cognition in Children, Adolescents, and Adults With First-Episode Psychosis Cecilie Lemvigh, Birgitte Fagerlund, Merete Osler, Jens Richardt Møllegaard Jepsen, Mette Nielsen, Jacob Rydkjaer, Kirsten Borup Bojesen, Christos Pantelis, Birte Glenthoj, Anne Katrine Pagsberg, Bjørn Ebdrup Center for Neuropsychiatric Schizophrenia Research (CNSR), Mental Health Center, Glostrup, Copenhagen University Hospital, Valby, Denmark Background: Schizophrenia is associated with widespread cognitive deficits that often precede the onset of psychosis by several years. Twin studies have demonstrated that cognitive functions are heritable and show genetic overlap with schizophrenia risk. Moreover, several early risk factors for schizophrenia have been identified, and some studies report and association between low birth weight and impaired cognition. Nevertheless, the potential additive or interactive impact of multiple early risk factors on cognition and the relation to age of illness onset remains unclear. Methods: Clinical cohorts of patients with first-episode psychosis (FEP) and matched healthy controls (HC) with comparable cognitive measures (recruited from 1998 – 2017) were combined and linked with information from the medical birth registry. The final sample consisted of 608 participants, including 166 children and adolescents with FEP and 148 matched HCs (age 9-17), and 146 adults with FEP and 148 HCs (age 18-45). From the medical birth registry, we included gestational age, Apgar score, birth weight and length, parental age, maternal smoking, and winter/spring birth. Specific cognitive functions were assessed at illness onset using the Brief Assessment of Cognition in Schizophrenia, and intelligence (IQ) was estimated using selected subtests from the Wechsler child or adult intelligence scales. Multiple linear regression was used to explore if the early risk factors significantly predicted cognitive performance. Results: FEP patients performed worse than HCs on all cognitive measures (all p’s < .001). In the whole sample, children and adolescents performed worse than adults on verbal memory, working memory, planning, fluency and motor speed (all p’s < .001). Among the early risk factors, FEP patients only displayed significantly lower birth length (p = .007) and birth weight (p = .032). Linear regression with IQ as the dependent variable revealed gestational age (p = .006), birth weight (p = .047) and group (FEP vs HC) as significant independent variables, while birth length, Apgar score, paternal age, winter birth and age (child vs adult) did not contribute significantly (adjusted R2 for the model = 0.164). Only birth weight (p = .043), group (p < .001) and age (p = .015) were significant for processing speed (adjusted R2 = 0.162). We observed no interaction effects between the significant early risk factors and group (FEP vs HC) or age (child vs adult). Including sex in the models did not change the findings. The remaining cognitive measures were not significantly influenced by the included early risk factors. Only a small subsample (N = 85) including both FEP patients and HCs had information on maternal smoking status, but here individuals with a positive history of maternal smoking during pregnancy showed significant impairments in processing speed (p = .003), working memory (p < .001), and planning (p = .018). Conclusions: The FEP groups showed widespread cognitive deficits compared to HCs regardless of age of onset, consistent with the current evidence implicating cognitive deficits as a core feature of schizophrenia. However, the majority of the cognitive functions examined were not significantly influenced by the included early risk factors for schizophrenia. Only IQ and processing speed were significantly associated with gestational age and birth weight, although the observed effects were small. Low birth weight and premature birth has previously been associated with impaired cognition in both healthy individuals and schizophrenia subjects, yet the available literature is sparse. Exploratory analyses of maternal smoking status revealed a possible, but imprecisely measured association with lower cognition. More studies are needed to gain a better understanding of the complex processes leading to cognitive deficits in schizophrenia. Keywords: Cognition, First Episode Psychosis, Neurodevelopment Disclosure: Nothing to disclose. P507. EEG Correlates of Aggression in Patients With Psychotic Disorders Michael Murphy, Ricardo Carrion, Jose Rubio, Anil Malhotra Harvard Medical School McLean Hospital, Belmont, Massachusetts, United States Background: Active psychosis symptoms have been associated with an elevated acute risk of violence. However, most patients with active psychosis symptoms do not commit acts of violence. It is unclear why some patients commit violent acts and current prediction tools are insufficient. Identification of easily-measurable neurological correlates for aggression could help reduce the risk of harm to patients, caregivers, and clinicians. Electroencephalography (EEG) is a widely used and easily applied g technique that can provide millisecond-level resolution of neural activity. Literature suggests that the alpha rhythm is linked to cognitive ability and can be impaired in schizophrenia. We hypothesized that larger alpha abnormalities would be associated with more impulsivity and aggression. Since the alpha rhythm is the primary generator of EEG microstates, we hypothesized that microstate parameters would also be abnormal in patients with higher levels of aggression. Methods: Eyes-closed resting EEG data was collected from 31 patients with psychotic disorders (18 males) and 18 age matched controls (7 males) using an EEG cap with 64 Ag-AgCl electrodes. Bad channels were spline-interpolated and data was re-referenced to average reference. For individual alpha peak frequency analysis (IAPF), we estimated the power spectral density using Welch’s method with non-overlapping Hamming windows of 2 seconds in length and identified a peak in the power spectral density between 7 and 13 Hz. Microstate analysis was performed using CARTOOL. A k-means clustering algorithm was used to identify microstate topographies and a meta-criterion was used to select k. Participants completed the four questionnaires about aggression: the Barratt Impulsivity Scale (BIS), the Reactive Proactive Aggression Questionnaire (RPAQ), the Buss-Perry Scale (BPS), and the Urgency, Premeditation (lack of), Perseverance (lack of), Sensation Seeking, Positive Urgency, Impulsive Behavior Scale (UPPS-P). In addition, participants completed a questionnaire about their history of aggressive behavior, the Lifetime History of Aggression (LHA). P values from statistical tests were adjusted with the Dubey Armitage-Parmar method. Results: Patients scored significantly higher on all aggression scales compared to controls (all p < .037). There were no statistically significant relationships between delta, theta, alpha, beta, or gamma power and any of the aggression scales. After correction for multiple comparisons, in the combined group, IAPF was inversely correlated with BPS and RPAQ total scores (both Spearman’s R = -.33) and Hostility (R = -.39) and Physical Aggression (R = -.33) subscores of the BPS. These effects were stronger in women than in men. In patients only, IAPF was inversely correlated with RPAQ Proactive Aggression subscore (R = -.39). There were no statistically significant relationships between IAPF and LHA. We then repeated the analysis using only a single channel (FPz) and found that in patients there was a statistically significant relationship between IAPF and BPS. For the microstate analysis, the segmentation algorithm produced four canonical microstates that have been repeatedly demonstrated in the literature. There were no case-control differences in microstate duration, occurrence, or transition probabilities. In patients only, uncorrected exploratory analyses suggested that microstate A duration was associated with LHA, Antisocial Behavior, and Verbal Aggression (R = .39, .37, .47 respectively). Transitions between microstates A and B were inversely correlated with UPPS-P as well as subscales for Non-planning, Proactive Aggression, Perseveration, and Hostility (all R > .37). The overall deviation from a completely random transition matrix was inversely associated with Physical Aggression (R = -.37). Conclusions: We used resting state EEG frequency and microstate analyses to identify correlates of aggression in controls and patients with schizophrenia. We found that IAPF was inversely correlated with self-assessed aggression but not lifetime history of aggression. We note that these results could be robustly demonstrated with a single EE electrode. We also found that transitions between canonical microstates A and B were correlated with aggression. Keywords: Irritability/Aggression, Psychotic Disorders, Electroencephalography Disclosure: Nothing to disclose. P508. BDNF Levels as a Predictor of Cognitive Response to Antipsychotic Treatment. Preliminary Results Rodrigo Nieto, Hernan Silva, Manuel Kukuljan Universidad de Chile, Santiago, Chile Background: Cognitive symptoms are a central manifestation of schizophrenia and are significantly related to patients´ social functioning and quality of life. Several studies have linked Brain-Derived Neurotrophic Factor (BDNF) not only to the pathogenesis of schizophrenia, but also to neuronal plasticity, learning, and memory. Changes in BDNF levels have been related to improvements in cognition in schizophrenia patients during cognitive remediation. Some patients improve their cognitive function during pharmacological antipsychotic treatment, but some patients do not. A personalized medicine approach to help identify which patients will have a better cognitive outcome is necessary. We hypothesized that baseline BDNF levels could be related to cognitive response to antipsychotic treatment in schizophrenia patients. Methods: Patients with schizophrenia (n = 24) were evaluated with Montreal Cognitive Assessment (MoCA). Those with cognitive deficit (n = 18) were reassessed after 6 months of treatment with atypical antipsychotics, and 61% of them (n = 11) improved enough to reach a normal MoCA score. Plasma and serum BDNF levels were measured with ELISA at baseline evaluation. We compared baseline plasma BDNF levels in two group of patients: those that improved cognition enough to reach a normal MoCA score vs those that did not. We also searched for correlations between changes in MoCA score and baseline BDNF levels. Results: Baseline plasma BDNF levels showed a trend to be higher in the subgroup of patients that has cognitive deficit and that improve cognition enough to reach a normal MoCA score at follow-up evaluation, although this difference is not significant (U Mann Whitney; p = 0.29). A trend towards a positive correlation was found between baseline serum BDNF levels and improvement in MoCA score (Spearman Rho 0.32; p = 0.19). Conclusions: The study of BDNF as a biomarker of cognitive improvement in patients with schizophrenia requires further research, with potential clinical implications for personalized medicine in the treatment of cognitive symptoms in schizophrenia patients. Keywords: BDNF, Cognition, Schizophrenia Disclosure: Nothing to disclose. P509. Network Hub Strength as a Predictor of Working Memory Performance in Schizophrenia Hamdi Eryilmaz, Melissa Pax, Mark Vangel, Ibai Diez, Daphne Holt, Joan Camprodon, Jorge Sepulcre, Joshua Roffman Massachusetts General Hospital, Charlestown, Massachusetts, United States Background: Working memory impairment is a debilitating and treatment-resistant aspect of schizophrenia, which is also a strong predictor of functional outcome. Breakdown in brain network hubs, putatively related to altered neurodevelopment may underlie the cognitive deficits associated with this illness. However, our understanding of network hub characteristics in schizophrenia and their relationship to working memory impairment remain elusive. Methods: Here, we used weighted degree, a robust graph theory metric representing the number of weighted connections to a node based on resting state functional connectivity. In doing so, we quantified node strength across 333 cortical hubs (based on Gordon et al. parcellation) in 29 patients with schizophrenia and 29 age- and gender-matched healthy controls (including both male and female subjects). We assessed the relationship between participants’ behavioral performance (retrieval accuracy and reaction time) in a verbal working memory task (Sternberg Item Recognition Paradigm) and node strength of the regions across the cortex using a correlation analysis. We utilized bootstrapping and False Discovery Rate to correct for multiple comparisons and determine significance (p < 0.025, for two behavioral measures). In an additional analysis, we employed a connectome-based predictive modeling framework and leave-one-out cross-validation to test whether a summary degree score can be used to predict participants’ working memory performance. Results: In both patients and controls, elevated weighted degree in the default mode network (DMN) regions was generally associated with poorer performance (retrieval accuracy and reaction time, p < 0.025). Higher weighted degree in the ventral attention network (VAN) nodes along the right superior temporal cortex was associated with better performance (retrieval accuracy) in both groups (p < 0.025). Weighted degree in several prefrontal and parietal areas was also associated with behavioral performance only in patients (p < 0.025). In regions that are critical for sustained attention, these correlations were primarily driven by between-network connectivity in patients (p < 0.05). Finally, our cross-validated prediction analysis revealed that a linear model using a summary weighted degree score can be used to predict an individual’s working memory performance in patients with schizophrenia (r = 0.36 for retrieval accuracy and 0.68 for reaction time). Conclusions: Our results indicate that schizophrenia is associated with dysfunctional hubs across the cortical systems supporting internal and external cognition and highlight the importance of topological network analysis in the search of biomarkers for cognitive deficits in schizophrenia. Keywords: Working Memory, Schizophrenia (SCZ), Graph Theory, Cognitive Outcome Prediction Disclosure: Elsevier Inc.: Employee (Spouse) P510. Mice Exhibit Distinct Behavioral Signatures While Executing a Multi-Alternative Spatial Working Memory Task in an Automated Radial Arm Maze Joshua Taliaferro, Julia Greenwald, Elizabeth Pekarskaya, Alexandra Tarasenko, Clay Lacefield, Christoph Kellendonk Columbia University, New York State Psychiatric Institute, New York, New York, United States Background: Working memory (WM) is the cognitive capacity for temporarily holding information in mind for processing or use and undergirds most of our actions and activities. Despite a rich history of WM investigation, the characterization of the neural basis of WM is still emerging. In rodents, unlike in nonhuman primates (NHPs), robust neural encoding of retrospective actions or stimuli—theoretically a quintessential WM characteristic—is not regularly observed, calling into question the congruity of neural WM mechanisms across species. Spatial working memory in particular is usually tested in rodents using tasks with two spatial choices. One possibility is that such paradigms do not provide the spatial complexity and alternative choices needed to evoke explicit neural representations of retrospective actions, locations, or stimuli. Methods: Adult mice (n = 8) were trained in a novel spatial working memory task in an automated, 8-arm radial arm maze with a large, open center. Inspired by multi-alternative NHP WM tasks, the task was designed to increase choice optionality, while minimizing the possibility of behavioral subversion of the working memory maintenance requirement, likely thus necessitating clearer neural representations of WM content for successful execution. Results: Despite task difficulty, mice learn the task to proficiency, consistently operating well beyond chance. During task acquisition, mice develop distinct behavioral signatures, which help reveal the underlying cognitive frameworks mice are using to solve the task. Conclusions: Here we present a novel spatial WM paradigm for freely moving mice in an automated radial arm maze. Even amidst variability in behavioral signatures, mice perform the task well, suggesting that this multi-alternative behavioral paradigm is a promising testing ground for a more complete elucidation of the neural basis of working memory. Keywords: Working Memory, Behavioral Tasks, Behavioral Variability Disclosure: Nothing to disclose. P511. Increased Rostral Medial Frontal GABA in Early Psychosis is Obscured by Levels of Negative Affect Molly Simmonite, Beier Yao, Robert Welsh, Stephan Taylor University of Michigan, Ann Arbor, Michigan, United States Background: Evidence suggests dysfunction of gamma-aminobutyric acidergic (GABAergic) interneurons in psychosis, and prior research has linked GABAergic function with a tendency toward negative affective states and poor tolerance for stress. Magnetic resonance spectroscopy (MRS) studies measuring GABA have yielded inconsistent findings. Here we investigate GABA concentrations in young adults with attenuated psychosis syndrome (APS) and first episode psychosis (FEP), as well as testing the hypothesis that negative affect is a clinical phenotype that is associated with reduced GABA, a relationship which may confound GABA measurement in psychosis. Methods: MRS were obtained from 14 patients with FEP (9 men, 5 women), 7 patients with APS (5 men, 2 women) and 15 healthy controls (HC, 10 men, 5 women), using a MEGA-PRESS sequence on a 3 T Philips Ingenia scanner. Voxels were placed in rostral MFC and midline occipital cortex. Gannet 3.1 was used to determine GABA + and Glx (glutamate and glutamine combined) concentrations. Results: We found a trend towards increased rostral MFC GABA + concentrations in FEP (F2,28 = 3.04, p = .06), but no group differences in the occipital cortex GABA + concentrations (F2,32 = 0.49, p = .62) or Glx concentrations were found (rostral MFC Glx: F2,28 = 0.08, p = .93; midline occipital Glx: F2,32 = 0.34, p = .72). When covarying for scores on the Psychological Stress Index, rostral MFC GABA + levels in FEP were significantly greater than APS and HC (F2,28 = 4.26, p = .02). Planned comparisons also revealed a trend towards increased rostral MFC GABA + in APS relative to HC (t27 = 2.00, p = .0548). No group differences in Glx (rostral MFC Glx: F2,31 = 0.26, p = .77; midline occipital Glx: F2,32 = 1.03, p = .23) or midline occipital GABA + were found (F2,31 = 0.48, p = .62). Conclusions: These results, considered alongside previously published findings, suggest multiple factors influencing GABA + levels in psychosis. We conclude a process exists which drives up GABA + levels in early psychosis, alongside a separate process in which reduced GABA + is associated with increased negative affect. These multiple processes have resulted in contradictory findings in the literature, and their untangling is critical to the understanding of GABA + in psychosis. Keywords: Magnetic Resonance Spectroscopy, Psychosis, GABA, Negative Affect Disclosure: Nothing to disclose. P512. Medial Prefrontal Cortex Dysfunction Mediates Working Memory Deficits in Patients With Schizophrenia John Williams, Zu Jie Zheng, Philip Tubiolo, Jacob Luceno, Roberto Gil, Ragy Girgis, Mark Slifstein, Anissa Abi-Dargham, Jared Van Snellenberg Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States Background: Schizophrenia (SCZ) is marked by deficits in working memory (WM), which are strongly predictive of poor functional outcomes. WM performance has been found to depend critically on the function of circuits in prefrontal cortex (PFC) and their modulation by dopamine (DA). Considerable evidence has implicated pathology of the PFC in the etiology of SCZ, including findings of dramatically reduced cortical DA release in SCZ. Reduced WM task-evoked deactivation in regions of the default mode network (DMN), including medial PFC (mPFC), has been shown to correlate with reduced task performance in both SCZ and HC. However, the mechanisms by which cortical hypodopaminergia may produce WM deficits in SCZ, including relationships with mPFC hypodeactivation, are unclear. Methods: Patients with SCZ (N = 41) and HC (N = 40) performed a visual object n-back WM task across 4 functional magnetic resonance imaging (fMRI) runs, each comprising two 40-second 1-back and 2-back blocks. Performance was quantified as the proportion of correct 2-back trials, with the significance of group differences tested using a Mann-Whitney-Wilcoxon rank-sum test. In subject-level analysis, deactivation during 2-back blocks was quantified using generalized linear models in Statistical Parametric Mapping version 12 (SPM12). An mPFC region of interest (ROI) was produced from the Gordon (2016) parcellation by selecting DMN parcels in PFC, dilating each by 8 mm, uniting them, and eroding the result by 5 mm. Group-level analyses were conducted in Permutation Analysis of Linear Models (PALM), using threshold-free cluster enhancement (TFCE) and family-wise error rate correction. Within mPFC, we first isolated voxels significantly deactivating during 2-back blocks. We then localized 205 voxels (5.535 cm3) additionally showing associations between deactivation and performance, controlling for diagnosis. Mean mPFC deactivation was obtained for each participant by averaging across all 205 voxels; the significance of group differences was assessed by Welch’s t-test. mPFC deactivation was evaluated as a mediator of the impact of diagnosis on task performance using the CANlab M3 Mediation Toolbox (significance evaluated by bias-accelerated and corrected bootstrap procedure with 10 million resamplings). Prefrontal synaptic dopamine release was additionally quantified in a subset of 9 unmedicated patients with SCZ and 14 HC via two positron emission tomography (PET) scans with the DA D2/3 receptor antagonist [11 C]FLB457: a baseline scan, and a second scan 3 hours after oral administration of 0.5 mg/kg of amphetamine. For both PET scans, binding potential relative to the non-displaceable compartment (BPND) was quantified in ROIs drawn on each participant’s T1-weighted anatomical magnetic resonance image. DA release capacity was quantified in medial frontal cortex (MFC) as the percentage change in BPND after amphetamine challenge (∆BPND). mPFC deactivation was input as the dependent variable into a robust regression model including intercept, MFC ΔBPND, diagnosis, ΔBPND×Diagnosis interactions, and age. Results: Relative to HC (N = 41), SCZ (N = 40) exhibited worse performance (median 2-back proportion correct, SCZ: 0.781, HC: 0.914, p = 0.0015). SCZ showed significantly reduced task-evoked mPFC subregion deactivation relative to HC (p = 0.0351). Mediation analysis revealed a significant mediated (indirect) effect (p = 0.0431) of diagnosis on WM task performance, and an unmediated (direct) effect with only trend level significance (p = 0.0545). The regression model for mPFC deactivation as a function of MFC ΔBPND (in 9 SCZ and 14 HC) included: Intercept (Β = -0.430, p = 0.0269), Diagnosis (Β = 0.0703, p = 0.503), ΔBPND (Β = -0.913, p = 0.0257), ΔBPND×Diagnosis (Β = 0.888, p = 0.311) and age (Β = 0.00308, p = 0.569); greater release capacity was associated with reduced mPFC deactivation during the WM task. When evaluated in each group separately, MFC DA release capacity (ΔBPND) was associated with mPFC deactivation in HC (Β = -1.335, p = 0.0268), but not SCZ (Β = -0.280, p = 0.543). Conclusions: Failure of task-evoked mPFC deactivation is a mediator of and potential substrate for WM impairment in SCZ. HC participants show an association between mPFC deactivation magnitude and DA release capacity, which is absent in SCZ. This suggests that DA deficit in PFC in SCZ is a potential mechanism underlying failure to deactivate mPFC, thus limiting WM performance. A better understanding of how DA deficit affects microcircuitry within this region would allow better targeted therapies for cognitive impairments in SCZ. Keywords: Schizophrenia (SCZ), Working Memory, Functional Magnetic Resonance Imaging (fMRI), Task fMRI, Positron Emission Tomography (PET) Disclosure: Nothing to disclose. P513. Cerebellar Effects on Abnormal Psychomotor Function are Mediated by Processing Speed in Psychosis Spectrum Alexandra Moussa-Tooks, Anna Huang, Baxter Rogers, Julia Sheffield, Neil Woodward, Stephan Heckers Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: Psychomotor disturbance has been identified as a key feature of psychotic disorders, with motor signs observed in upwards of 66% of unmedicated, first-episode patients. Aberrations in the cerebellum have been directly linked to sensorimotor processing deficits including processing speed, which may underly psychomotor disturbance in psychosis, though these brain-behavior-symptom relationships are unclear. Methods: In a sample of 339 psychosis patients (242 schizophrenia-spectrum, 97 bipolar with psychotic features) and 217 controls, we evaluated the relationship between cerebellar grey matter volume in the Yeo sensorimotor network and processing speed (assessed via the Screen for Cognitive Impairment in Psychiatry [SCIP]). To further test the role of these variables in predicting psychomotor disturbance (mannerisms and posturing, retardation, excitement of the Positive and Negative Syndrome Scale [PANSS]), a mediation analysis was performed for an a priori model in the psychosis group: predictor=cerebellar volume; mediator=processing speed; outcome=psychomotor disturbance. Cerebellar volumes were calculated using optimized processing through the Spatially Unbiased Infratentorial (SUIT) toolbox. Statistical analyses were performed in R; all models included total intracranial volume, age, sex, and chlorpromazine equivalents as covariates. Results: As expected, sensorimotor cerebellar volume was positively associated with processing speed in the whole sample (t = 3.428, p < 0.001, d = 0.30). Effects were present in both the control sample (t = 2.345, p = 0.020, d = 0.32) and the psychosis sample (t = 2.188, p = 0.029, d = 0.24). Next, we tested whether processing speed was a significant mediator between cerebellar volume and psychomotor aberrations. Within the psychosis group, no significant direct effect (cerebellar volume --> psychomotor disturbance) was observed in this mediation model (ß=-0.035, p = 0.59, d = 0.06). Rather, the indirect path (average combined effect: ß=-0.043, p = 0.014) showed a significant partial mediation by processing speed, with a small effect of cerebellum on processing speed (ß=0.172, p = 0.029, d = 0.24) and medium effect of processing speed on psychomotor disturbance (ß=-0.254, p < 0.001, d = 0.60). Conclusions: The current findings suggest a critical role of cerebellar circuitry in a well-established sensorimotor aberration in psychosis (processing speed) and the presentation of related psychomotor phenotypes within psychosis. Due to the nature of the PANSS, our mediation model could not be evaluated in the control sample. Future work will employ more dimensional measures of psychomotor disturbance to capture normative and aberrant brain-behavior-symptom relationships. Future research may also determine the magnitude of these relationships within subtypes of psychosis (e.g., disorganized behavior or catatonia) as well as the specificity to motor, versus non-motor (e.g., cognitive, social), cerebellar functions. Keywords: Psychomotor Symptoms, Cerebellum, Brain-Behaviour Relationships, Structural MRI, Processing Speed Disclosure: Nothing to disclose. P514. Prioritization of Potential Causative Genes for Schizophrenia in Placenta Gianluca Ursini, Pasquale Di Carlo, Sreya Mukherjee, Qiang Chen, Shizhong Han, Jiyoung Kim, Maya Deyssenroth, Carmen Marsit, Jia Chen, Ke Hao, Giovanna Punzi, Daniel Weinberger Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: The placenta plays a critical role since very early phases of development, paving the path for brain development. Disruptions in placenta functioning may later result in neurodevelopmental psychiatric disorders, such as schizophrenia. Our prior findings show that genomic risk for schizophrenia converges with early-life complications (i.e., prenatal, intrapartum, and early postnatal complications, such as serious obstetric complications) and affects risk for the disorder, in a sex-biased fashion. Computational approaches such as Transcriptome Wide Association Studies (TWAS) and Summary data based Mendelian Randomization (SMR) have the ability to integrate summary statistics from GWAS data with expression mapping studies, to allow predicting gene expression in specific tissues for individuals in case-control cohorts. This makes possible to estimate, in larger cohorts, the association of predicted gene expression in a specific tissue with traits or diseases. Here, we use these methods to identify specific genes and potential mechanisms that, in placenta, may condition trajectories of risk that lead to schizophrenia. Methods: We performed TWAS in healthy term placentae (N = 147) samples to derive candidate placental causal genes that were confirmed with SMR. To search for placenta and schizophrenia-specific associations, we performed an analogous analysis in fetal brain (N = 166) samples, and additional placenta TWAS for other disorders/traits. We also analyzed if there were differences in sex-biased placental processes relevant for brain development, as prior data suggest. Finally, we performed placental TWAS for other disorders and traits, to further support that the placental transcriptome is relatively specific to schizophrenia. Results: The analyses in the whole sample and stratified by sex highlight 186 genes whose predicted expression in placenta is associated with schizophrenia (Bonferroni corrected p-value<0.05); 138 of these placenta and schizophrenia specific risk genes are SMR-validated, and many risk genes are sex-biased. The molecular mechanisms highlighted by gene ontology prediction converge on nutrient-sensing capabilities of placenta and trophoblast invasiveness. These genes do not show analogous effects on risk for other developmental disorders and traits and do not show a potentially causal role in similar analyses in fetal brain. This result suggests an etiological role in the risk for schizophrenia (versus other disorders of development) relatively specific of placenta (versus brain). Additionally, transcript-level analysis detects gene isoforms not linked with schizophrenia at the gene level, thus implicating splicing events in the placental transcriptome that do not seem biased by sex-related differences. Conclusions: Placenta and brain might converge in contributing to early, though reversible, trajectories of risk for the disorder. However, most research on brain development and schizophrenia has been exclusively focused on brain, a strategy that may miss relevant discovery opportunities. In this study we show that the investigation of placental mechanisms of risk leads to the detection of potential causal genes for schizophrenia and points to novel candidate mechanisms that may inform new venues of for prevention. Keywords: TWAS, Psychiatric Disorders, Schizophrenia (SCZ), Placenta, Mendelian Randomization Disclosure: Nothing to disclose. P515. Evaluating the Psychometric Reliability of Nonlinear Dynamics in Schizophrenia Christopher Gonzalez, Claudia Lainscsek, Juan Molina, Yash Joshi, Terrence Sejnowski, Neal Swerdlow, Gregory Light Department of Veterans Affairs, San Diego, California, United States Background: Schizophrenia (SZ) is a chronic psychiatric disorder that is highly heterogenous in clinical manifestations, and sensitivity to therapeutics such as medications or cognitive interventions. Identifying biomarkers that characterize treatment-sensitive subpopulations can match patients with treatments that improve outcomes. EEG measures such as event-related potentials (ERPs) serve as such biomarkers in SZ and demonstrate high psychometric reliability. In addition to classical ERPs, novel EEG measures of nonlinear dynamics (NLD) could similarly serve as biomarkers, though the reliability of these measures have not been assessed. This study applies a nonlinear signal processing technique, Delay Differential Analysis (DDA), to capture dynamical features inaccessible from traditional neurophysiological, linear techniques. Because brain processes are intrinsically nonlinear, DDA is well-suited to study both group differences between healthy controls and SZ patients, as well as individual differences within SZ. Here, we estimate the test-retest reliability of the NLD measure for the first time alongside traditional ERPs computed from the same trials. Methods: This study is part of an ongoing clinical trial, with N = 24 to date (SZ = 20, healthy control=4, age=46.9, M/F 14/6). Each person is administered a passive auditory oddball task with EEG for two sessions, separated by 1 week, both prior to intervention. Traditional ERP analysis follows standard pre-processing pipelines, whereas DDA pre-processing is minimal. DDA calculates NLD features in a sliding window manner by fitting a three-term delay differential equation to each time window. The estimated coefficients from this model serve as NLD features. Model parameters were defined in a previous publication and were selected for classifying SZ from healthy control EEG dynamics. At each EEG session, amplitudes for Mismatch Negativity (MMN), P3a, and a peak in the NLD feature were extracted. Reliability metrics were calculated using a frontal electrode (Fz) from 150 randomly selected standards and deviants, with the same trials for both approaches. Reliability was assessed using Pearson correlations. Results: MMN (r = 0.75,t = 5.3(22),95%CI = 0.5-0.9,p < 0.001) and P3a (r = 0.74, t = 5.1(22),95% CI = 0.47-0.88,p < 0.001) showed high reliability across a 1 week interval for all individuals. The NLD measure showed moderate reliability (r = 0.52, t = 2.87(22),95% CI = 0.15-0.76, p < 0.01). Conclusions: MMN, P3a, and NLD exhibited significant test-retest reliability, though NLD reliability was lower than that of traditional ERP measures. Notably, this study modeled subsets of random trials and likely underestimates true reliability. Future studies are needed to determine whether comparable data pre-processing improves reliability for NLD and whether this measure is associated with demographic, clinical, cognitive and functional outcomes in SZ. Keywords: Test-Retest Reliability, Auditory Mismatch Negativity, Nonlinear Analysis Disclosure: Nothing to disclose. P516. Normative Modelling of Brain Morphometry in Individuals at Clinical High-Risk for Psychosis From the ENIGMA CHR Working Group Shalaila Haas, Ruiyang Ge, René Kahn, Maria Jalbrzikowski, Dennis Hernaus, Cheryl Corcoran, Sophia Frangou Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Psychosis is characterized by substantial heterogeneity at the level of brain organization, cognitive dysfunction and clinical severity. However, group-level inferences derived from case-control differences is not representative of individual-level deviations. In this study, we used normative modeling of brain morphometry to investigate whether the normative deviations derived from harmonized MRI data from a large independent sample of healthy individuals could help inform associations with positive symptoms and general intellectual ability. Methods: T1-weighted MRI brain scans were available for 1237 healthy controls (HC; 684 [55.3%] males; mean [SD] age = 20.75 [4.74])) and 1340 CHR (709 [52.9%] males; mean [SD] age = 22.32 [4.95]) with positive symptom data from 29 sites. Structural neuroimaging data was processed within each respective site using Freesurfer analysis software using standardized ENIGMA protocols to derive measures of cortical thickness (CT), surface area (SA), and subcortical volume (SV). The dataset underwent site effect correction using ComBat-GAM harmonization. Normative modeling of neuroanatomical data was generated using Fractional Polynomial Regression separately for males and females from an independent sample of 38,050 healthy individuals (53.3% female) aged 3-90 years pooled across 81 datasets and was used to compute individual-level statistically quantified deviations from the normative range by computing a z‐score. Subsequently, we averaged the regional z-scores separately for each phenotype in each participant to generate an “average deviation score” for cortical thickness (ADS-CT), cortical surface area (ADS-SA), subcortical volume (ADS-SV), and across all measures to generate a global average deviation score (ADS-G). Mixed linear models including age were used to determine associations between ADS with positive symptoms and general intellectual ability in CHR, correcting for multiple comparisons using FDR correction. In posthoc follow-up analyses, we examined the possibility that associations between psychopathology and IQ are not linear but are restricted to most deviant groups in CHR individuals with ADS below the 5th percentile (“infranormal”) and above the 95th (“supranormal”) centiles for each of these metrics and compared them to those in between these percentiles. The three groups were compared in terms of positive symptoms and IQ using analysis of covariance with age as covariates. Results: CHR individuals showed widespread decrements in regional mean deviation of cortical thickness, particularly in superior temporal and frontal regions. Regional mean deviations in surface area also indicated decrements with the exception of visual, sensory, and motor regions. Regional mean deviation in subcortical volumes showed pronounced decrements, especially in the thalamus, putamen, and hippocampus. Within the CHR group, there was a negative association between positive symptoms and ADS-SA (β = -0.08, P-FDR = 0.02) and positive associations between IQ with ADS-SA (β = 0.08, P-FDR = 0.03) and ADS-G (β = 0.10, P-FDR = 0.03). Comparison of CHR individuals with supranormal, average, and infranormal ADS-CT, ADS-SA, ADS-SV, ADS-G did not identify significant group differences at P-FDR < 0.05 in terms of positive symptoms and IQ. At a nominal level, CHR individuals with infranormal ADS-G had higher positive symptom scores (F = 4.07, P = 0.01) and a trend towards lower IQ (F = 2.71, P = 0.07) compared to those with average or supranormal ADS-G. Conclusions: The present study identified marked neuroanatomical heterogeneity in CHR states based on the largest dataset of individual-level neuroimaging measures in CHR individuals using robust estimates of the normative range of regional cortical thickness, cortical surface area and subcortical volume. Moreover, we identified a subset of CHR individuals that showed reduced global brain morphometry, with corresponding higher positive symptom severity and lower IQ. These findings suggest normative modeling of brain morphometry may help identify individuals at extremes with both preserved and atypical brain morphometry which could help inform clinical and cognitive heterogeneity in other psychiatric disorders. Keywords: Normative Modeling, Clinical High Risk for Psychosis, FreeSurfer, Clinical Heterogeneity, Stratification Disclosure: Nothing to disclose. P517. Computer-Vision Analysis of Craniofacial Dysmorphology in 22q11.2 Deletion Syndrome and Psychosis Spectrum Disorders David Roalf, Donna McDonald-Mcginn, Joelle Jee, Mckenna Krall, T. Blaine Crowley, Paul Moberg, Christian Kohler, Alice Bailey, Monica Calkins, Ian Campbell, Kelly Clark, Andrew Crow, Nicole Fleischer, R. Sean Gallagher, Virgil Gozenbach, Ruben Gur, Emily McClellan, Daniel McGinn, Arianna Mordy, Kosha Ruparel, Russell Shinohara, Bruce Turetsky, Lauren White, Elaine Zackai, Raquel Gur University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Minor physical anomalies (MPAs) are phenotypic abnormalities of aberrant development. MPAs are common in 22q11.2 deletion syndrome (22q11DS) and psychosis spectrum disorders (PS). MPAs in these disorders include abnormalities of the eyes, ears, mouth, and head. Since MPAs likely represent a disruption of early embryologic development, in-depth study of MPAs offers a promising entry point for better understanding the neurodevelopmental neuropathology associated with 22q11DS and PS. Methods: 2D photographs of individuals using standard digital photography. Participants included 22q11DS (n = 150), PS (n = 55), and typically developing (TD; n = 93) subjects. Photographs were analyzed using two computer-vision techniques: 1) Facial Dysmorphology Novel Analysis (Face2Gene (F2G)) technology to identify the presence of genetically mediated facial disorders, and 2) Emotrics—a recently developed semi-automated machine learning technique that localizes facial landmarks and computes regional facial measurements. Results: F2G detected 22q11DS in 99% of 22q11DS individuals. F2G scores were robust and significantly higher in 22q11DS as compared to PS or TD. PCA-derived factor loadings of all F2G scores indicated unique and overlapping facial patterns that were related to both 22q11DS and PS. Regional facial measurements of the eyes (marginal reflex distance) and nose (philtrum length) were shorter in 22q11DS as compared to TD, while PS showed an intermediate pattern. Conclusions: These preliminary results indicate overlap in facial dysmorphogenesis between 22q11DS and PS. The extent to which these developmental facial markers are evident before the impairment or distress of sub-psychotic symptoms may allow us to identify at-risk youths more reliably and at an earlier stage of development. Keywords: Psychosis-Risk, 22q11.2 Deletion Syndrome, Facial Dysmorphology Disclosure: Nothing to disclose. P518. Quantifying Speech and Language Disturbance in Schizophrenia With Neural Language Models Yan Cong, Amir Nikzad, Sameer Pradhan, Sunghye Cho, Katrin Hänsel, Aarush Mehta, Sarah Berretta, Leily Behbehani, Mark Liberman, Sunny Tang Feinstein Institute for Medical Research, Glen Oaks, New York, United States Background: Disorganized speech is a hallmark of schizophrenia spectrum disorders (SSD) and related psychotic disorders and represents complex language disturbances which cannot be easily captured on a semantic or syntactic level. Since their introduction in 2017, pre-trained neural language models (nLMs) continue achieving state-of-the-art results on a variety of natural language processing tasks. We hypothesized that nLMs are promising tools for automatically quantifying disorganized speech in SSD because nLMs are effective at encoding contextual content and more complex referential information. We tested multiple validated semantic similarity strategies to examine the extent to which nLMs can improve accuracy of detecting and characterizing different types of speech and language disturbances, especially incoherent and inefficient disorganized speech among people with SSD. Methods: Healthy volunteers (HV) and participants with SSD were recruited from inpatient and outpatient facilities (SSD = 74, HV = 37). They were rated on the Scale for the Assessment of Thought Language and Communication (TLC) and two items from the Scale for the Assessment of Negative Symptoms (SANS; decreased vocal inflection and increased latency). Open-ended speech (162 ± 121 words) was recorded and underwent transcription and processing for cosine similarity of word- and sentence-level embeddings. Three levels of pre-processing were conducted incrementally. Level 1 included verbatim transcripts excluding non-speech verbalizations like laughter. Level 2 excluded disfluencies such as repetitions and filled pauses. Level 3 additionally excluded NLTK stop words. We adopted 3 primary strategies to measure semantic similarity, sometimes referred to as “semantic coherence” in other studies: (1) the word-to-word variability at K inter-word distances, with K ranging from 2 to 10 (K2:10); (2) average semantic similarity of each word in 5- or 10-words window (W5/10); (3) First Order similarity of consecutive phrase vectors and Second Order similarity between phrase separated by another intervening phrase (FO/SO). Vector embeddings were generated from a static model GloVe and 3 nLMs (BERT, T5, GPT3) to compute similarity. Median, inter-quartile range (IQR), 5th percentile (Q5), and 95th percentile (Q95) values were then derived. Welch’s t-test were used to examine group diagnosis (SSD vs. HV). Significance was two-tailed with alpha=0.05. Results: Using the K2:10 semantic similarity strategies, across all levels of pre-processing, GloVe showed statistically significant lower similarity in SSD than in HV for 7 measures with medium effect size (Cohen’s d 0.5-0.8) in median, Q5, and Q95 similarity. BERT showed lower similarity in SSD for 9 measures with medium effect size, and for 46 measures with large effect size (Cohen’s d > = 0.8) in median, Q95, and IQR values. T5 showed lower similarity in SSD for 43 measures with medium effect size, and for 37 measures with large effect size in median, Q95, and IQR values. In contrast, GPT3 showed higher similarity in SSD for 39 measures but lower IQR in SSD for 2 measures (Cohen’s d > = 0.5). For each of the models, more extensive data pre-processing generally resulted in fewer significant results. Using the W5/10 semantic similarity strategies, GloVe showed statistically significant higher similarity in SSD for 1 measure (Cohen’s d 0.41) with Level 1 pre-processing, and no significant results with more pre-processing. BERT showed lower similarity in SSD for 18 measures (Cohen’s d > = 0.5), including 13 measures with large effect size. The results were consistent across pre-processing levels. T5 showed lower similarity in SSD for 15 measures (Cohen’s d > = 0.5), including 6 measures with large effect size. In contrast, GPT3 showed higher similarity in SSD for 8 measures of median, Q5 and Q95 values but lower IQR in SSD for 3 measures (Cohen’s d > = 0.5). For both T5 and GPT3, Level 3 pre-processing resulted in fewer significant effects. Using the FO/SO strategies, GloVe showed statistically significant lower similarity in SSD for SO Q95 (Cohen’s d -0.77) and FO Q95 (Cohen’s d -0.81) with Level 1 pre-processing, but higher similarity in SSD for FO Q5 (Cohen’s d 0.43) with Level 2, and null results for Level 3. BERT showed higher similarity in SSD for 4 measures (Cohen’s d 0.2-0.5), and T5 for 2 measures with medium effects. The results improved with more extensive pre-processing for both BERT and T5. GPT3 showed higher similarity in SSD for 6 measures (Cohen’s d 0.5-0.8), including 2 measures with large effect size. The results were mostly consistent across levels of pre-processing. Conclusions: We evaluated three different types of nLMs to quantify speech and language disturbances in SSD, using static embeddings from GloVe as baseline. nLMs were, in general, more sensitive than GloVe in identifying language disturbances in SSD, reflecting that nLMs are better tools for studying SSD. Pre-processing by removing disfluencies did not greatly alter the results but removing stop words masked the SSD group difference in some cases, particularly for GPT3. GloVe, BERT and T5 all generally show lower similarity in SSD, across analysis frameworks, while GPT3 mostly shows higher similarity in similar tests. We hypothesized that the differences can be explained by how nLMs are pre-trained and what data they are trained on. Future directions include evaluating the robustness of language measures generated from nLMs across speech tasks and samples, and finetuning nLMs with domain specific data. Keywords: Schizophrenia Spectrum Disorders, Natural Language Processing (NLP), Digital Phenotyping, Computational Psychiatry, Biomarkers Disclosure: Nothing to disclose. P519. Cognitive Flexibility and Psychotic Symptoms: A Computational Analysis of Learning in a Dynamic Environment James Waltz, Adam Culbreth, Pantelis Leptourgos, Sonia Bansal, Philip Corlett, Molly Erickson, James Gold University of Maryland School of Medicine, Baltimore, Maryland, United States Background: While deficits in reinforcement learning (RL) and decision-making (DM) in psychosis are well-established, it is not clear precisely which aspects of RL and DM relate to which psychotic symptoms. We have previously shown that the ability to adaptively modulate learning rates dynamically, around contingency shifts, relates to negative symptoms in schizophrenia. However, we have also found that rates of switching in response to positive and negative outcomes also predict positive symptom severity. We hypothesized that, in a large sample of schizophrenia patients (SZs) and non-clinical voice-hearers (VHs), the ability to adaptively modulate learning rates in a dynamic learning environment would distinguish SZs and VHs from healthy controls (HCs), and that measures of the ability to adaptively modulate learning rates would relate systematically to the severity of positive symptoms. Methods: We administered 68 schizophrenia patients, 33 non-clinical voice-hearers, and 47 healthy controls an RL task where they chose from three decks of cards and were probabilistically reinforced for their choices. After the participant reached a criterion, the contingencies changed, with a new deck becoming the best. Participants achieved as many stages as possible in 240 trials. We quantified overall switch rates, as well as switch rates after rewards and punishments. Furthermore, we used a computational model to estimate learning rates (α), on a trial-wise basis, as a function of the slope of prediction errors. We estimated the impact of uncertainty (operationalized through the slope of prediction errors) on learning rate change, on a subject-wise basis. We used ANOVAs with post-hoc tests to examine differences between participant groups, and we used correlation analyses to investigate relationships between learning measures and symptom severity. We also examined differences between subgroups of SZ patients, based on their duration of illness (less than 3 years vs. greater than or equal to 3 years). Results: We found that switch rates for SZs early in their illness course (n = 19; p < 0.001) and non-clinical VHs (p = 0.028) were significantly greater than those of controls, and that uncertainty had a greater impact on learning rate change in both SZs (p = 0.029) and VHs (p = 0.003) than in HCs. However, more paranoid SZs (p = 0.002) and VHs (p = 0.008) showed lower mean learning rates (α) under greater uncertainty, and SZs and VHs with more severe overall psychosis (p = 0.024) showed less learning rate modulation around a contingency shift. Conclusions: These results provide further evidence that people with clinical psychosis are less able to adaptively use uncertainty to modulate the impact of feedback on learning and decision-making. Furthermore, these results indicate that this form of cognitive flexibility may be disrupted in people with unusual percepts and beliefs who do not seek help. Consistent with prior findings, these results suggest a particularly close relationship between learning rate and psychotic symptoms, and paranoia, in particular. Further researcher is needed to examine whether abnormalities in learning rate modulation in schizophrenia patients and non-clinical voice-hearers have the same or different neural substrates. The research was supported by NIH Grant R01 MH112887 (PIs: J. Gold, P. Corlett). Keywords: Schizophrenia (SCZ), Delusions, Decision Making, Reinforcement Learning Modelling, Uncertainty Disclosure: Nothing to disclose. P520. A Functional Connectome-Based Neural Signature for Individualized Prediction of Antipsychotic Response in First-Episode Psychosis Hengyi Cao, Todd Lencz, Juan Gallego, Anita Barber, Michael Birnbaum, Delbert Robinson, Anil Malhotra Feinstein Institute for Medical Research, Queens, New York, United States Background: Identification of robust biomarkers that predict individualized response to antipsychotic treatment at the early stage of psychotic disorders remains a challenge in precision psychiatry. This study aimed to investigate if any functional connectome-based neural traits would serve as such a biomarker. Methods: In a discovery sample, 49 first-episode patients with psychosis received multi-paradigm fMRI scans at baseline and were clinically followed up for 12 weeks under antipsychotic monotherapies. Treatment response was evaluated at the individual level based on the psychosis scores of the Brief Psychiatric Rating Scale (BPRS). Cross-Paradigm Connectivity and Connectome-based Predictive Modeling were employed to train a predictive model that uses baseline connectomic measures to predict individualized change rates of psychotic scores. The generalizability of the model performance was further examined in an independent validation sample of 24 first-episode patients with a similar design. Results: The results revealed a paradigm-independent connectomic trait that significantly predicted individualized treatment outcome in both the discovery sample (r[predicted vs observed] = 0.44, P = 0.007) and the validation sample (r[predicted vs observed] = 0.50, P = 0.005). This neural trait involved connections predominantly related to the cerebellar-cortical circuitry and spanned multiple sensory (e.g., sensorimotor, auditory, visual) and cognitive (e.g. default-mode, frontoparietal, cingular-opercular) systems. Conclusions: This study discovers and validates a connectome-based functional signature as a promising early predictor for individualized response to antipsychotic treatment in first-episode psychosis, thereby highlighting the potential clinical value of this biomarker in precision psychiatry. Keywords: Early Psychosis, Treatment Outcome Prediction, Functional MRI (fMRI), Brain Connectome, Machine Learning Disclosure: Nothing to disclose. P521. Contrasting Case-Control and Normative Reference Approaches to Capture Clinically Relevant Structural Brain Abnormalities in Antipsychotic Medication-Naïve First-Episode Psychosis Patients Natalie Remiszewski, James Edward Bryant, Saige Rutherford, Andre Marquand, Eric Nelson, Ibrahim Askar, Adrienne Lahti, Nina Kraguljac University of Alabama at Birmingham, Birmingham, Alabama, United States Background: The central premise of precision medicine is that an individual’s unique physiologic characteristics play a significant role in disease vulnerability and response to specific therapies. To make progress towards this goal, it is imperative to move beyond group-based studies that disregard individual variations as noise, and instead, interpret them in the context of normal-range of biological systems. Normative modeling (“brain growth charting”) is a statistical technique that allows characterization of disease signatures at the individual level, where positive (higher volumes compared to the normative range) and negative (lower volumes compared to the normative range) deviations can be calculated. Recent applications in psychiatry have shown that heterogeneity in structural brain abnormalities is substantial and that group averages do not accurately reflect patterns of atypicality at the individual level. However, it remains to be determined whether these structural deviations map onto clinical outcomes in psychosis spectrum disorders. We enrolled antipsychotic medication-naïve first-episode psychosis patients (FEP) in a 16-week trial with risperidone and quantified raw subcortical and ventricle volumes as well as individual deviations from a normative reference model at baseline. We chose subcortical regions because they include principal sites of dopaminergic drug action and ventricles because these are one of the hallmark structural features in schizophrenia. We hypothesized that subcortical and ventricle volume deviations would be highly heterogeneous, and that deviations derived from the normative model in subcortical regions would be superior to raw volumes in predicting treatment response. Methods: We enrolled 98 antipsychotic medication-naïve first episode psychosis patients (age 23.4 + /- 5.7, 61% male, 63% identified as racial/ ethnic minority) and treated them for sixteen weeks with risperidone. We used data from 92 controls (age 23.9 + /- 5.4, 55% male, 43% identified as racial/ ethnic minority) as comparison group for case control contrasts and calibration of normative curves. Imaging was performed on a 3 T Siemens Magnetom Prisma using an MPRAGE [TR/TE/TI: 2400/2.22/1000 ms; 0.8mm3 voxels]. Data were processed in FreeSurfer 7.1.18. We quantified raw subcortical volumes in FreeSurfer, as well as individual deviations based on a reference model (n = 58,836; age 2-100) using the PCN and braincharts toolkits. Results: We found a significant group difference in raw volume measures when variables were considered jointly on all measures (Pillai’s Trace= 0.18; F = 3.23; p < 0.01). Post hoc analyses demonstrated that FEP had lower thalamus (F = 9.36; p < 0.01), hippocampus (F = 17.23; p < 0.01), amygdala (F = 6.55; p = 0.01), ventral diencephalon (F = 4.84; p = 0.03), and brain stem volumes (F = 8.39; p < 0.01) compared to controls. Across all regions, 36% of FEP displayed extreme deviations, 22% had them in ventricles, and 22% had them in subcortical regions. Extreme deviations were shared among patients more commonly in ventricle regions (6-9% of FEP) compared to subcortical regions (0-5%). They were twice as likely to be in the negative (lower volume compared to the normative reference) than positive (higher volume) direction in subcortical regions, whereas 62% of extreme ventricle deviations were in the positive direction. While subcortical raw volumes at baseline did not predict treatment response, deviations in the caudate, putamen, and ventral diencephalon did. For these regions, correlations significantly differed between raw volume and deviation measures in the caudate (z = -2.17; p = 0.03) and putamen (z = -2.15; p = 0.03). Ventricle raw volumes or deviations did not predict response. Conclusions: Our finding that structural deviations are heterogeneous in antipsychotic-naïve FEP is consistent emerging literature reporting this in several major psychiatric disorders. We extend the literature by providing empirical evidence that greater negative deviations in key dopaminergic brain regions better predict antipsychotic treatment response in FEP compared to raw volumes. Our data suggests that this normative modeling allows to capture inter-individual heterogeneity of regional brain volumes in FEP and to characterize structural pathology in a clinically relevant fashion. This holds great promise for progress in precision medicine in psychiatry where group-level studies have failed to derive reliable maps of structural pathology. Keywords: Medication-Naive First Episode Psychosis, Predictor of Treatment Response, Normative Modelling, Subcortical Volume Disclosure: Neurocrine Biosciences, Inc.: Advisory Board (Self), American Board of Psychiatry and Neurology: Other Financial or Material Support (Self) P522. Prolonged Kynurenic Acid Elevation During the Prenatal Period Elicits Electrophysiological and Behavioral Changes in Adult Mice Robert Schwarcz, Sarah Beggiato, P. Leon Brown, Snezana Milosavljevic, Marian Thomas, Maria Piroli, Korrapati Sathyasaikumar, Francesca Notarangelo, Ana Pocivavsek Maryland Psychiatric Research Center, Baltimore, Maryland, United States Background: Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation are believed to be involved in the pathophysiology of several psychiatric diseases, including schizophrenia (SZ) and bipolar disorder (BPD). Kynurenic acid (KYNA) has received special attention in this regard since its levels are elevated in the brain and CSF of persons with SZ and may be causally related to the cognitive dysfunctions seen in the disease. Previous studies in rats had indicated that abnormally high KYNA levels in the fetal brain may play a pathophysiologically significant role in this context (“EKyn model”; Notarangelo and Pocivavsek, 2017; Beggiato et al., 2018). Methods: In anticipation of future studies with genetically altered animals, we now fed pregnant C57Bl/6 J mice either with the immediate KYNA precursor kynurenine (10 mg or 30 mg/day; EKyn) or with control mash (ECon) from embryonic day (ED) 11 to ED 18. Separate cohorts of adult, male and female offspring were then assessed in electrophysiological, behavioral, and biochemical studies on postnatal day (PD) 56-70. Electrophysiology was conducted by ex vivo recording of evoked local field potentials in coronal brain slices (N = 10 per group). Barnes maze, a spatial learning task, was used to evaluate hippocampal-dependent acquisition training (2 trials per day for 3 consecutive days) and prefrontal cortex-dependent reversal learning (2 trials on the 4th day of testing; N = 20 – 28 per group). Biochemical analyses were designed to measure the KP metabolites 3-hydroxykynurenine (3-HK) and KYNA in tissue homogenates from hippocampus and cortex (N = 7 – 8 per group) and to determine extracellular KYNA levels by in vivo microdialysis in the prefrontal cortex (N = 4 – 5 per group). Results: Ex vivo recordings revealed that prenatal treatment with 30 mg kynurenine/day promoted a longer contralateral response latency in EKyn compared to ECon mice (P < 0.05), likely reflecting a decline in white matter function. Adult male EKyn mice also showed significantly longer latency (P < 0.05) and lower speed to enter the escape box during acquisition training when spatial learning was tested in the Barnes maze. During subsequent reversal learning, the latency to escape into the goal box and the number of re-visits to the previous escape location were higher in male EKyn offspring compared to controls (P < 0.05). Of note, female EKyn mice were not impaired in the Barnes maze. Measured in the cortex or hippocampus in adulthood, the tissue levels of 3-HK and KYNA did not differ between EKyn and ECon groups. Separately, in preparation of experiments designed to investigate the role of KYNA in the adverse long-term effects seen in EKyn mice, we verified by in vivo microdialysis that inhibition of KYNA neosynthesis with the potent kynurenine aminotransferase inhibitor PF-04859989 (30 mg/kg, s.c.) induces a rapid decrease in extracellular KYNA in the prefrontal cortex of adult EKyn animals. Conclusions: The observation that physiological and behavioral abnormalities are seen in adult EKyn mice without concurrent elevations in brain KYNA levels indicates that prolonged treatment with kynurenine during the embryonic period causes permanent, functionally significant changes in brain development in these experimental animals. Elaboration of the mechanisms underlying these changes, especially of the role of cerebral KP metabolism in this context, can be expected to provide useful information regarding the etiology of neuropsychiatric illnesses. Keywords: Kynurenic Acid, Perinatal Development, Schizophrenia Disclosure: Nothing to disclose. P523. Incomplete Hippocampal Inversion Impacts Hippocampal Subfield Morphology in Schizophrenia Maxwell Roeske, Maureen McHugo, Simon Vandekar, Kristan Armstrong, Stephan Heckers Vanderbilt University School of Medicine, Nashville, Tennessee, United States Background: Hippocampal morphology is altered in schizophrenia, but it is unclear whether deficits in specific regions and subfields are linked to altered neurodevelopment. Converging shape and volumetric analyses suggest that the anterior region and cornu ammonis (CA) 1 subfield are affected in the early stages of illness. A recent study revealed that incomplete hippocampal inversion (IHI), an anatomical variant of the human hippocampus resulting from an arrest during neurodevelopment, is more prevalent and severe in patients with schizophrenia. We hypothesized that IHI contributes to hippocampal subfield differences in schizophrenia. Methods: We studied 199 schizophrenia patients and 161 healthy individuals with 3-Tesla MRI and measured the prevalence and severity of IHI in each hippocampus bilaterally using established, quantitative criteria. We generated hippocampal surface reconstructions using a combination of manual and Freesurfer automated segementation and the SPHARM-PDM toolkit. We regressed the average local shape vertex displacement onto IHI score to test the effect of IHI on hippocampal shape variation and conducted a sensitivity analysis to test for group shape differences with and without IHI included as a main effect. We completed the shape analysis using SurfStat and applied random field theory to account for multiple comparisons. We measured volumes of subfields (CA1, CA2/3, dentate gyrus, subiculum) across anterior and posterior hippocampal regions using ASHS automated segmentation in an overlapping group of participants: 86 individuals in the early stage of a psychotic disorder and 67 demographically similar healthy individuals. We completed statistical analysis of hippocampal subfield volume using linear mixed models in R. We included age, sex, and intracranial volume as covariates in all models. Results: IHI is associated with rounding of hippocampal shape. Linear models not including IHI as a main effect replicated well-known hippocampal shape differences in schizophrenia patients localized to the CA1 region of the antero-lateral hippocampus. Including IHI as a main effect in the model eliminated the bilateral significant shape differences in the CA1 subfield. The relationship between IHI score and hippocampal volume varied by region and subfield (F2,2253 = 11.76, P < 0.001). Increased IHI score was associated with lower volume in the anterior CA1 (slope = -21.46, 95% CI = [-27.32, -15.61]) and anterior dentate gyrus (slope = -14.24, 95% CI = [-20.09, -8.39]) subfields. Conclusions: IHI impacts hippocampal shape and volume with regional and subfield specificity. Our results suggest that abnormal development of the hippocampus contributes to morphologic differences, particularly in the anterior CA1 subfield, observed in patients with schizophrenia. Keywords: Hippocampal Subfields, Schizophrenia (SCZ), Human Neuroimaging, Neurodevelopment Disclosure: Nothing to disclose. P524. Altered Maturation of Functional Connectivity in Thalamocortical Networks in 22q11.2 Deletion Syndrome Charles Schleifer, Kathleen O’Hora, Amy Lin, Leila Kushan-Wells, Carrie Bearden University of California, Los Angeles, Los Angeles, California, United States Background: 22q11.2 Deletion Syndrome (22qDel) is a recurrent copy number variant (CNV) in which a hemizygous deletion of ~46 protein-coding genes from the long arm of chromosome 22 results in greatly increased risk for schizophrenia and other developmental neuropsychiatric conditions. Studying individuals with 22qDel can provide a powerful framework for linking genes to brain phenotypes and behaviors relevant to these conditions. The cortex and thalamus are anatomically and functionally organized into a set of distributed circuits that support diverse brain functions. Dysconnectivity within these thalamocortical circuits has been consistently implicated in idiopathic neuropsychiatric disorders, and in 22qDel. Specifically, resting-state functional MRI has reproducibly revealed thalamic dysconnectivity to a network of frontoparietal regions and a network of somatomotor regions as a biomarker of both schizophrenia and 22qDel. Psychosis spectrum disorders generally emerge in mid-adolescence to early adulthood, yet no study to date has investigated longitudinal development of functional connectivity in thalamocortical networks in 22qDel during this sensitive age period. Methods: In a longitudinal sample of resting-state fMRI from 22qDel (n = 100, 62% female) and typically developing (TD) youth (n = 100, 48% female) ages 6-22 y, we used an atlas-based approach to compute thalamocortical functional connectivity across a set of functionally defined networks. Thalamocortical connectivity was measured as the correlation in fMRI time-series between thalamic and cortical regions classified as belonging to the same functional network based on an a priori parcellation. For both groups, non-parametric age-related curves for thalamocortical connectivity were generated using Generalized Additive Mixed Models. Results: We report a novel finding of altered thalamocortical developmental trajectories, particularly in the frontoparietal and somatomotor networks. In both networks, TD controls exhibit a relatively flat developmental trajectory with no significant effect of age on connectivity (Somatomotor F = 0.11, p = 0.75; Frontoparietal F = 0.97, p = 0.33). However, 22qDel show significant changes across the age range in both networks (Somatomotor F = 8.13, p = 0.005; Frontoparietal F = 7.11, p = 0.0007). GAMM curves were compared for regions of non-overlap based on 95% confidence intervals. In the youngest 22qDel patients, thalamic-frontoparietal connectivity is lower than TD but switches to overconnectivity in mid-adolescence. The opposite pattern is observed for somatomotor connectivity. Conclusions: This pattern implicates dysfunction and altered maturation of circuits involved in sensory and executive function which are disrupted in 22qDel and related conditions like Autism Spectrum Disorder and schizophrenia, and broadly aligns with findings of age-related functional connectivity changes in the 22q11 mouse model. Keywords: 22q11.2 Deletion Syndrome, Thalamo-Cortical Connectivity, Longitudinal MRI, Copy Number Variants Disclosure: Nothing to disclose. P525. Cannabis Use is Associated With More Symptoms and Better Functioning but Not Psychotic Conversion in Participants at Clinical High Risk (CHR) for Psychosis in the NAPLS 3 Study Kristin Cadenhead, Jean Addington, Carrie Bearden, Tyrone Cannon, Matcheri Keshavan, Daniel Mathalon, Diana Perkins, Elaine Walker, Scott Woods University of California, San Diego, La Jolla, California, United States Background: Multiple studies have demonstrated an association between cannabis use and future risk of psychosis. In patients with established psychosis, cannabis use is also associated with worsening psychotic symptoms and better functioning in some studies. The aim of the present study was to examine cannabis use patterns across CHR and Health Comparison (HC) participants in the NAPLS 3 consortium and determine the association to symptoms, functioning and psychotic conversion. Methods: The sample included 699 participants at CHR for psychosis and 96 HCs. All participants were queried about current and past cannabis use, symptoms and functioning. A total of 71 CHR participants are known to have converted to psychosis during the course of the study. Results: The CHR participants had a higher rate of cannabis use compared to HCs (50.9% vs 32.3%, p < 0.001) and the majority used at least once per week. Within the CHR sample who had used cannabis, an earlier age of first use was significantly correlated (r = -0.15, p < 0.001) with greater use in the last 6 months. Heavier use in the last 6 months was also associated with greater positive (r = 0.20, p < 0.001) and disorganized (r = 0.11, p < 0.02) symptoms at baseline assessment. Heavier cannabis users had greater positive symptoms (F = 4.6, p < 0.01) but better global (F = 3.9, p < 0.05) and social (F = 8.8, p < 0.001) functioning than those with none or minimal cannabis use. There was no significant difference in the rate or pattern of cannabis use between the CHR sample who converted to psychosis and those who did not. Conclusions: The CHR sample in the NAPLS 3 study has a higher rate and heavier pattern of cannabis use compared to HC participants. Heavier use was associated with more positive symptoms but not conversion to psychosis in this sample. Heavier cannabis users tended to have better functioning consistent with prior studies in schizophrenia populations. Keywords: Clinical High Risk for Psychosis, Cannabis Use, Social Functioning Disclosure: Nothing to disclose. P526. Risk of Sudden Cardiac Death Among Patients With Schizophrenia, and Other Psychotic Disorders: An Exploratory Analysis Using the Million Veteran Program Dataset Rishab Gupta, Nina Schooler, Panos Roussos, Mihaela Aslan, Philip Harvey, Tim Bigdeli SUNY Downstate Medical Center, Jamaica Plain, Massachusetts, United States Background: Patients with schizophrenia typically die ~15-20 years earlier compared to those without any psychiatric illness. The two most common causes of death in patients with schizophrenia are cardiovascular disease (most commonly, myocardial infarction) and suicide. In particular, patients with schizophrenia are up to 4.5 times more likely to die from sudden cardiac death (SCD). SCD in schizophrenia may occur in the context of either a structural heart disease (coronary heart disease, congestive heart failure) or due to arrhythmia (genetic or multifactorial). Psychotropic medications also predispose to SCD by prolonging QTc interval and leading to Torsades de pointes. To date, most studies have focused on schizophrenia only to estimate the risk of SCD even though people with other psychotic disorders also share risk factors similar to schizophrenia. Methods: We conducted a cross-sectional observational study to evaluate the prevalence of SCD in patients with schizophrenia, and a broader psychosis phenotype enrolled in the VA Million Veteran Program study. We operationalized some definitions for this study: schizophrenia was defined as patients assigned phecode 295- (created using ICD-9 and ICD-10 equivalent codes) over at least two visits; a broader psychosis phenotype was defined based on two or more phecodes for psychotic disorders, 295-298 (created using ICD-9/10 codes); and SCD was defined based on phecode 427.5 (created using ICD-9/10 codes) for cardiac dysrhythmia not otherwise specified (NOS) (as a proxy). Results: Among 654,023 participants with available data, 17,900 met criteria for schizophrenia (~2.7%). There were 46,885 patients with history of cardiac dysrhythmia NOS in the total eligible sample, out of which 1,839 also had co-morbid schizophrenia (~7.25%). We calculated the odds ratio of 1.45 (p < 0.0001) for those with history of cardiac dysrhythmia NOS and co-morbid schizophrenia versus those with cardiac dysrhythmia NOS but without co-morbid schizophrenia. We next applied general linear modeling, with age, age-squared, gender, and self-identified race as covariates to examine the relationship between schizophrenia and cardiac dysrhythmia NOS. We found that cardiac dysrhythmia NOS was positively predicted by schizophrenia (β = 0.4881; p < 2×10-16), African American ancestry (β = 0.122; p < 2×10-16), and age (β = 0.1122; p < 2×10-16), while Asian ancestry (β = -0.7569; p < 2×10-16) and male gender (β = -0.1145; p = 2.48×10-9) were protective factors. Surprisingly, age-squared was also a negative predictor of cardiac dysrhythmia NOS (β = -0.6369 ×10-3; p < 2×10-16). Among more than 30,000 participants meeting criteria for the broader psychosis phenotype, 5,371 also met criteria for cardiac dysrhythmia NOS, corresponding to a 1.73-fold increased risk (p < 0.0001). Demographic covariates showed a near-identical pattern of association as observed for schizophrenia. Conclusions: Among individuals receiving health care at the VA, we observed significantly elevated odds of sudden cardiac death among individuals with diagnosed schizophrenia or a related psychotic disorder. We observed risk-increasing effects of age and African American ancestry and protective effects of Asian ancestry, male gender, and age-squared. Implications of this work include recognition of potentially modifiable mortality risk, need for a close examination of the risk factors for SCD, and possible ethnic disparities in cardiac outcomes of patients with a psychotic disorder. Ongoing analyses are leveraging available mortality records to identify confirmed cases of SCD, and evaluation of how polygenic risk for schizophrenia and heart disease interplay to moderate cardiac outcomes in our patients. This study will build on our recent work showing that polygenic risk score for schizophrenia increase the risk for a range of medical disorders. Keywords: Sudden Cardiac Death, Schizophrenia, Psychotic Disorder Disclosure: Nothing to disclose. P527. The Relationship Between Breast Cancer and Long-Term Exposure to Prolactin Sparing Anti-Psychotic Medications Among Schizophrenia Patients: A Population-Based Study Amir Krivoy, Ariel Asper, Noa Menkes-Caspi, Michael Davidson, John M Davis, Mark Weiser* Sheba Medical Center, Ramat Gan, Israel Background: Most anti-psychotics cause an increase in prolactin, a hormone hypothesized to increase risk for breast cancer. Methods: We utilized population-based data from the Clalit Health Services (CHS) database, the largest provider of health insurance in Israel (N = 5.5 million persons), followed-up between 2000 and 2020. In 15,686 female patients with schizophrenia, we examined the relationship between anti-psychotic medication and later breast cancer. Specifically, we wanted to see whether female schizophrenia patients who were exposed to at least 4 years of prolactin sparing anti-psychotic medication (clozapine, quetiapine, aripiprazole) were at a lower risk of developing breast cancer compared to female schizophrenia patients who were exposed to at least 4 years of non-prolactin sparing anti-psychotic medication and were not exposed to prolactin sparing anti-psychotic medications. Results: Female schizophrenia patients who were exposed to at least 4 years of prolactin sparing anti-psychotic medication were at a lower risk of developing breast cancer compared to female schizophrenia patients who were exposed to at least 4 years of anti-psychotic medication (HR = .44, p < .001). This trend remained significant even after adjusting for age, socioeconomic status, smoking, diabetes and obesity (HR = .5, p < .001). Conclusions: In female schizophrenia patients, prolactin sparing antipsychotic medications decrease the risk for breast cancer compared to non-sparing anti-psychotic medications. This should be taken into consideration when prescribing anti-psychotic medications to female schizophrenia patients. Keywords: Antipsychotics, Breast Cancer, Schizophrenia (SCZ), Prolactin Disclosure: Jansen, Lundeck: Advisory Board (Self), Teva, Dexcel, Msd, Orion, Clearmind: Consultant (Self), Pfizer: Honoraria (Self), Minerva: Contracted Research (Self) P528. Violent Behavior Among Young Adults Receiving Treatment for Early Psychosis Stephanie Rolin, Leah Pope, Elizabeth Ford, Michael Compton, Paul Appelbaum, Lisa Dixon Columbia University, New York State Psychiatric, New York, New York, United States Background: Overall, the absolute risk of violence for individuals with serious mental illnesses (SMI) is low. However, when compared to the general population, people with SMI have an increased absolute risk of engaging in violent behavior. This seems to be particularly true for young adults experiencing early stages of psychosis, with research suggesting that violence risk peaks during the period of early psychosis. This study analyzed the prevalence of and risk factors for violent behavior in a sample of young adults receiving treatment for non-affective psychosis in an early intervention services (EIS) setting. Methods: A total of 1726 young adults (ages 16-30) within two years of onset of non-affective psychosis were enrolled in 21 EIS sites in New York State between October 2013 and January 2020. Clinical teams completed standardized forms on all enrolled participants every 3 months, including measures of demographics, clinical outcomes, and violent behavior. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of violent behavior at time of entry into treatment in predicting future violent behavior was calculated. Results: The total sample was mostly men (n = 1263, 73%) and included 599 Black (35%), 454 Latinx (26%), 417 White (24%) and 153 Asian (9%) individuals. Approximately three-quarters (n = 1238, 73.4%) of the sample had no violent behavior at any time point. Of individuals with violent behavior at time of entry into treatment at an EIS clinic (n = 452, 26.7%), approximately half had violent behavior that persisted during treatment (n = 228, 50.4%) and half had violent behavior that resolved during treatment (n = 224, 49.6%). The sensitivity, specificity, PPV and NPV of violent behavior at time of entry for predicting future violent behavior during treatment was 32.4%, 82.5%, 18.3%, and 91.0%. Conclusions: Overall, violent behavior is relatively uncommon among a sample of young adults receiving treatment in an EIS setting for nonaffective psychosis. Among individuals who have violent behavior at time of entry into treatment, about half do not have violent behavior following entry into treatment. Overall, past violent behavior had low sensitivity in predicting future violent behavior. Keywords: Early Psychosis, Violence, Early Intervention Services Disclosure: Nothing to disclose. P529. Lifetime Consequences and Correlates of Anticholinergic Medication Burden in the VA Healthcare System Tim Bigdeli, Yash Joshi, Yuli Li, Nallakkandi Rajeevan, Fred Sayward, Ronald Przygodzki, Grant Huang, Saiju Pyarajan, Sumitra Muralidhar, J Michael Gaziano, David Braff, Gregory Light, Mihaela Aslan, Panos Roussos, Philip Harvey SUNY Downstate Medical Center, Brooklyn, New York, United States Background: Cognitive impairments are a hallmark of schizophrenia (SCZ) and can be worsened by medications with anticholinergic properties used to mitigate side-effects of commonly prescribed antipsychotics. Following recent reports from the COGS and BSNIP consortia that demonstrate the robust association of anticholinergic medication burden (ACB) across multiple cognitive domains, we replicated these findings in VA Cooperative Studies Program (#572), a cohort of 9,378 patients with confirmed diagnoses of SCZ or bipolar I disorder (BIP). We extend these findings here, hypothesizing that cumulative ACB from multiple medication classes may underlie the increased prevalence of dementia among individuals diagnosed with SCZ. Methods: Leveraging the VA’s extensive electronic health record (EHR), we constructed individual-level ACB scores from prescription records for over 700,000 participants in the Million Veteran Program (MVP). We included first- and second-generation antipsychotics, mood stabilizers, antidepressants, anticholinergics, antiparkinsonian medications, anticonvulsants, and benzodiazepines. We examined the distribution of ACB among diagnosed cases of SCZ and BIP, and in the VA patient population at large, and modeled the associations of ordinally-coded ACB (0-2 => “low”; 3-4 => “intermediate”; 5 or greater => “high”) on ~1,600 categories of ICD-9/10 billing codes (“phecodes”). Our preliminary analyses consider all inpatient and outpatient prescriptions within 90 days of study enrollment. Results: Among 16,287 diagnosed SCZ patients, and after controlling for recent inpatient hospitalization, high ACB corresponded to a 1.4-times higher risk (95% CI: 1.2,1.6; p < 10^-5) of being diagnosed with dementia (phecode 290.1). Among SCZ patients, high ACB also significantly increased risk of being diagnosed with mild cognitive impairment (phecode 292; OR = 1.44, 95% CI: 1.3,1.6; p < 10^-17), suicidal behavior or ideation (phecode 297; OR = 1.41, 95% CI: 1.3,1.6; p < 10^-14), and tobacco use disorder (phecode 318; OR = 1.36, 95% CI: 1.2,1.5; p < 10^-10). We observed comparable effects of high ACB on dementia risk in 32,022 BIP patients (OR = 1.46, 95% CI: 1.3,1.6; p < 10^-13). This effect was still detectable in 9,935 patients who had not been treated with antipsychotics or mood-stabilizers and had no record of inpatient treatment (OR = 1.53, 95% CI: 1.27,1.83; p < 10^-5). Conclusions: We have previously reported that ACB robustly influences cognitive performance in SCZ and BIP patients, even after adjusting for the influences of age, sex, gender, and IQ polygenic scores. In the current analysis, we explore the broader consequences of chronic ACB exposure in a broader cohort of participants. Planned follow-up analyses include joint modeling of ACB and polygenic scores for SCZ, Alzheimer’s Disease, and executive functioning, and comparative analyses of partial-ACB attributable to particular classes of medications. Keywords: Schizophrenia (SCZ), Anticholinergic Medication Burden, Electronic Health Record (EHR), Veterans, Polygenetic Risk Score Disclosure: Nothing to disclose. P530. Examining Mitochondrial DNA Variants in the Risk for Schizophrenia Fernanda Souza, Ana Paula Mendes-Silva, Wei Deng, Lei Sun, James Kennedy, Marquis Walter, Vanessa Gonçalves* Centre for Addiction and Mental Health, University of Toronto, Toronto, Canada Background: Schizophrenia is a complex disorder with clinical and biological heterogeneity. Despite of the recent success in identifying contributing genetic variants from the nuclear genome, the effective diagnosis, treatment and prevention strategies are largely absent. Mitochondria are crucial for the brain physiology, and mitochondrial DNA (mtDNA) variants may contribute to the etiology of mental illnesses. Previous studies of primary mitochondrial diseases have observed impairment in brain function and high prevalence of psychosis among cases. Here, we aim to identify novel mitochondrial variants associated with schizophrenia using a subset of the Psychiatric Genomics Consortium (PGC) schizophrenia samples, which had mtDNA available for analysis. Methods: In this study, we assess the contribution of mtDNA and jointly the influence of nuclear genome on the risk of SCZ using a subset of carefully curated samples (n = 37,500) with high-quality mtDNA data available in the Psychiatric Genomics Consortium. The mtDNA associations were performed individually for common single nucleotide variants (minor allele frequency > 1%) and aggregated at the gene-level for rare variants (minor allele frequency < 1%). Mitochondrial haplogroups were assigned using HaploGrep2 and were tested at macro-group level (H (H-HV-V), JT (J-T), UK and Others (W-X-I). To harmonize mtDNA variants across the genotyping platforms and to increase the number of variants for analysis, we performed imputation using a custom reference panel. Results: After standard quality control and imputation, we recovered 370 mtDNAs that were partially shared across the 37,500 samples (17,283 cases and 20,217 controls), including 144 common variants (MAF > 0.01) and 226 rare variants (MAF between 0.001 and 0.01). Among the macro-haplogroups, only JT was marginally associated with a decreased risk of SCZ (Odds ratio = 0.94, p = 0.035) while adjusting for sex. For common variants (N = 144), the SNP MT-15924 was significant after correcting for macro-haplogroup H (p = 0.00026). The gene-set analysis revealed three potentially interesting gene complexes: complex I (gene ND1-6), complex IV (cytochrome c oxidase 1-3), and complex III (cytochrome b) that deserved further investigation into individual gene component. Conclusions: To the best of our knowledge, the current analysis is the largest in major psychosis. These positive findings are expected to shed light on the roles of mitochondrial variants in schizophrenia etiology and complement discoveries from the nuclear genome. Keywords: Mitochondrial DNA, Genomics, DNA, Whole-Genome, Sequencing, Schizophrenia Disclosure: Nothing to disclose. P531. The Three Dimensional Landscape of Open Chromatin Regions in Schizophrenia Kiran Girdhar, Jaroslav Bendl, Pengfei Dong, Steven Kleopoulos, Samir Rahman, Vahram Haroutunian, Gabriel Hoffman, John Fullard, Panos Roussos Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: To translate scientific discoveries from lab to clinic for treatment requires deeper understanding of molecular mechanisms of disease. One of the ways to gain more insights in mechanism is to investigate the structure and function of human genome in affected cell type in diseases. For example, to identify how risk variants are organized in three-dimensional genome and what specific cellular functions are affected in disease. In this study, we investigate how majority of schizophrenia (SCZ) variants underneath non-coding regions are organized in three-dimensional genome in a critical cell type neurons in schizophrenia (SCZ). To answer this, we profile population scale (N = 1,394) chromatin accessibility libraries from neurons and non-neurons in two neocortical brain regions. Methods: Using frozen postmortem tissue from 469 cases with SCZ and controls, we performed ATAC-seq to profile chromatin accessibility in neuronal and glial populations of cells, isolated by FACS from two different brain regions, i.e. dorsolateral prefrontal cortex and anterior cingulate cortex. Then, we characterized epigenetics changes associated with SCZ phenotypes. The availability of RNA-seq and whole-genome data for this cohort allowed us to measure the overlap between differentially regulated transcriptome and epigenome signatures and related pathways. Finally, we determined enhancer-promoter interactions, by utilizing additional omics in the brain tissue (Hi-C and H3K27ac ChIP-seq) and jointly analyzing with ATAC-seq based on the “activity-by-contact” (ABC) approach. Results: First, multi-omic data integration associated global patterns of changes in chromatin accessibility with gene expression and identified enhancer-promoter interactions that are impaired in disease phenotypes. Second, we explored cis-regulatory domains (CRDs), a structural attribute of 3D genome architecture and demonstrated how schizophrenia specific dysregulation in CRDs contributes to nuclear topography. We found genome wide mapped CRDs delimited the cell-region specific OCRs (~35%) into active chromatin regions enriched for SCZ risk loci. Interestingly, SCZ heritability was found significantly enriched at CRD boundaries and unique to neuronal cell types indicating borders OCRs prominently represented by promoters as sites of dysregulation in SCZ patients. Third, trans interactions of diseased CRDs revealed a separate cluster of domains governing fetal development in higher order A (Active) compartments. Conclusions: This finding links the disease risk loci to the chromatin landscape that could be reconfiguring during the early stages of fetal development. Overall, this dataset provides a unique insight into molecular mechanisms underlying brain region and cell type-specific vulnerability in both schizophrenia and bipolar disorder. Keywords: Neuropsychiatric Disorders [Schizophrenia, Parkinson’s Disease, Major Depressive Disorder], Computational Genomics, Open Chromatin Regions Disclosure: Nothing to disclose. P532. Diurnal Alterations in Gene Expression across the Human Dorsal and Ventral Striatum in Psychosis and Unaffected Comparison Subjects Kyle Ketchesin, Wei Zong, Mariah Hildebrand, Madeline Scott, Marianne Seney, Kelly Cahill, Vaishnavi Shankar, Jill Glausier, David Lewis, George Tseng, Colleen McClung University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Psychosis is a defining feature of schizophrenia and highly prevalent in bipolar disorder. Individuals suffering with these illnesses also have major disruptions in sleep and circadian rhythms, and disturbances to sleep and circadian rhythms can precipitate or exacerbate psychotic symptoms. A prior study from our group used a time-of-death analysis of RNA-seq data and found that subjects with schizophrenia show altered gene expression rhythms in the human postmortem dorsolateral prefrontal cortex (dlPFC), a region associated with the cognitive symptoms of schizophrenia. Positive symptoms (i.e., psychosis) are associated with the striatum, though no study to date has directly measured molecular rhythms and determined how they are altered in the striatum of subjects with psychosis. Methods: In the current study, we performed both differential expression and rhythmicity analyses to determine diurnal alterations in gene expression across the nucleus accumbens (NAc), caudate, and putamen in subjects with psychosis relative to unaffected comparison subjects. NAc, caudate, and putamen samples were collected from male and female subjects with psychosis [n = 36; schizophrenia/schizoaffective disorder (n = 28) and bipolar disorder with psychosis (n = 8)] and unaffected comparison subjects (n = 36). Total RNA-seq was performed on the striatal tissue samples. Differential expression analyses were first performed between psychosis and comparison subjects in each region regardless of time of death (TOD). Given our previous findings in the dlPFC, we then split the cohorts into subjects who died either during the day or during the night and performed differential expression analyses. Transcripts were considered differentially expressed if p < 0.01 and log2 fold change ≤ -0.26 or ≥0.26. Circadian patterns of gene expression were detected using nonlinear regression based on individual subject TOD. Sinusoidal curves were fitted to the expressed data using the nonlinear least-squares method and coefficient of determination (R2) was used as a measure of goodness-of-fit. Transcripts with either a gain or loss of rhythmicity between psychosis and matched comparison subjects were determined using the difference in R2 between the cohorts. Results: Across the three striatal subregions, we found differential expression of immune-related transcripts and a substantial loss of rhythmicity in core circadian clock genes in subjects with psychosis. In the NAc, mitochondrial-related transcripts showed decreased expression in psychosis subjects, but only in those who died at night. Moreover, we found a loss of rhythmicity in small nucleolar RNAs and a gain of rhythmicity in glutamatergic signaling in the NAc of psychosis subjects. Between region comparisons revealed that rhythmicity in the caudate and putamen is highly similar in subjects with psychosis, suggesting synchronization in daily rhythms across the dorsal striatum in these subjects. Conclusions: Overall, we have identified multiple gene expression differences in the striatum in subjects with psychosis, as well as changes in rhythmic gene expression that could underlie disease pathology or response to treatment. Ongoing studies focused on between-region differential expression analyses in both unaffected comparison and psychosis subjects will provide insight into how the identified gene expression differences are coordinated across ventral and dorsal striatal subregions. Keywords: Human Postmortem Brain Tissue, Circadian Rhythms, Psychosis, Schizophrenia (SCZ), RNA-seq Disclosure: Nothing to disclose. P533. Pathways From Genes to Functioning: Integrating Genetic Risk, Neurophysiology, Anticholinergic Medication Burden, Cognition, and Symptom Contributions to Disability in Schizophrenia Yash Joshi, Michael Thomas, Tim Bigdeli, Juan Molina, David Braff, Neal Swerdlow, Gregory Light* University of California, San Diego, La Jolla, California, United States Background: Emerging disease-relevant measures generated from large schizophrenia (SZ) psychiatric consortium studies invite opportunities to investigate multivariate contributions to psychosocial disability. Clarification of pathways which lead to disability have substantial implications for drug development, research funding allocation, clinical care delivery, and health policy. The objective of this study was to assess the contributions of genetic risk, neurophysiologic abnormalities, medication burden, cognition, and symptoms to disability in SZ. Methods: SZ outpatients (n = 1120) were assessed via their participation in the Consortium on the Genetics of Schizophrenia-2 (COGS-2) study. Polygenic scores for SZ risk (SZ PRS) and for intelligence (IQ PS) were derived via the PsychArray and Multi-Ethnic Global Array. Neurophysiologic indices of early auditory information processing (EAIP) included mismatch negativity, P3a, and reorienting negativity. A modified Anticholinergic Cognitive Burden (ACB) Scale served to generate ratings of anticholinergic medication burden. Positive (POS) and Negative (NEG) symptoms were assessed via the Scales for the Assessment of Positive and Negative Symptoms. Cognition (COG) was measured via the Penn Neurocognitive Battery. Functional outcomes (FNX) were measured by the Role Functioning Scale. Informed by prior studies, an iterative multivariate approach testing 28 separate models using structural equation modeling (SEM) was used to generate an integrative model of SZ disability. Results: An optimized model illustrated that FNX was affected by COG (via EAIP), and separately, NEG. ACB impacted FNX (d = -0.44) via independent effects on EAIP, COG and NEG. By contrast, POS was only associated with NEG, and both SZ PRS and IQ PS did not affect FNX. Conclusions: Genetic factors and positive symptoms have little impact on disability in SZ outpatients stabilized on psychotropics. Anticholinergic medication burden reduction and remediation of information processing are predicted to have beneficial effects on cognition, symptoms and psychosocial outcomes in SZ. Iterative SEM-based modeling may be a useful tool in clarifying multivariate effects in large SZ studies. Keywords: Polygenetic Risk Score, Cognition, Neurophysiology, Schizophrenia (SCZ), Anticholinergic Medication Burden Disclosure: Astellas, NeuroSig, Sosei-Heptares: Consultant (Self) P534. Additive and Multiplicative Influence of CNVs and Common SNPs on Cognition Emma Knowles, Josephine Mollon, Laura Schultz, Guillaume Huguet, Samuel Mathias, Amanda Rodrigue, Aaron Alexander-Bloch, Zohra Saci, Laura Almasy, Sebastien Jacquemont, David Glahn* Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Numerous copy number variants (CNVs) have been identified as risk markers for neurodevelopmental and psychiatric disorders. Such disorders are characterized by decrements in cognitive performance. Yet, the influence of these CNVs, and their joint influence in interaction with known common risk variants for psychiatric illness, on neurotypical cognitive performance remains unclear. We investigate the influence of common CNVs and polygenic risk for both psychiatric illness and intelligence related phenotypes (e.g., brain morphology) on general cognitive ability (g) and reaction time in the UK Biobank (UKBB). Methods: Reaction time data were available in 491,185 individuals (mean age = 57.59, sd = 8.11, range = 39.17 - 76.54 years, 54.39% female). Reaction time data was winsorized and inverse normalized. g was calculated in a subset of the sample (N = 257,976) in line with a previously published investigation of the integrity and structure of the cognitive data from the UKBB (Lyall et al, 2016). To minimize missingness all cognitive data was collapsed across time points, where missing data at a preceding time point was directly imputed with available data from the following one. CNVS were called using PennCNV and QuantiSNP via an established pipeline from genotypic data derived by the UKBB. CNVs were annotated using Gencode for overlapping gene components. Polygenic scores were calculated using an established pipeline for White British individuals in the sample. PRS for disorders related to cognitive ability were calculated using summary statistics derived from samples with minimal overlap with the UKBB. Linear regression models, run in python (statsmodels) and R (lm), were used to test for main effects of CNV, PRS, and CNVPRS interactions on cognitive ability. Deletions or duplications with fewer than five copies were dropped prior to regression analyses. Results: Effects for overall CNV effect were small by significant for both cognitive phenotypes. CNV carriers showed lower reaction time (B = -0.10, p = 1.78×10-13) and g scores (B = -0.10, p < 2.00×10-16). Significant effects were observed for the presence of any deletion and any duplication also. Nominally significant CNVPRS interaction effects included schizophrenia (B = -0.1, p = 0.03), brain surface area (B = -0.05, p = 0.04), autism spectrum disorder (B = 0.03, p = 0.02). Top-ranked individual CNVs with small to large effect on reaction time included deletions in 3q29 (B = 1.17, p = 4.10×10-04), 7q11.23 (B = -0.52, p = 3.62×10-03), 15q13.1 (B = -0.53, 3.52×10-05), 16p13.11 (B = -0.40, p = 7.40×10-07), 15q11.2 (B = 0.19, p = 1.10×10-16), duplications in 22q11.2 (B = 0.60, p = 7.28×10-10), and deletion (B = 0.52, p = 4.29×10-09) and duplication (B = 0.54, p = 3.69×10-12) in 16p11.2. A similar profile of individual CNV associations was observed for g, though effect sizes tended to be larger. Additional effects were observed for duplication in 1q21.1 (B = 0.40, p = 8.62×10-08), 16p12.1 (B = 0.52, p = 2.87×10-06), 17q12 (B = 0.61, p = 2.81×10-04), and 1q21.1 (B = 0.39, p = 7.08×10-04) as well as duplication (B = 0.52, p = 4,49×10-04), and deletion (B = 0.23, p = 2.35×10-05) within 16p13.11. Conclusions: CNVs that are established risk factors for neurodevelopmental and psychiatric disorders have moderate effect on both reaction time and general (g) cognitive ability. When considering the overall effect of the presence of any CNV effects are small and interact with polygenic risk for various psychiatric diseases and brain morphology traits such that effects are potentiated Keywords: CNV, Polygenetic Risk Score, Cognition, IQ Disclosure: Nothing to disclose. P535. Polygenic Risk Scores for Psychiatric Disorders in a Diverse Postmortem Brain Tissue Cohort Laramie Duncan, Hanyang Shen, Anton Schulmann, Tayden Li, Bhaskar Kolachana, Ajeet Mandal, Ningping Feng, Pavan, K. Auluck, Stefano Marenco Stanford University, Stanford, California, United States Background: A new era of human postmortem tissue research has emerged thanks to the development of ‘omics technologies that measure genes, proteins, and spatial parameters in unprecedented detail. Also newly possible is the ability to construct individual-level metrics of genetic risk (polygenic risk scores, PRS, based on genome-wide association studies, GWAS). Here we report on clinical, educational, and brain gene expression correlates of polygenic scores in ancestrally diverse samples from the Human Brain Collection Core (HBCC). Methods: Genotypes from 1,418 donors, derived from cerebellum tissue, were subjected to quality control filters, imputed, and used to construct PRSs. Gene expression from dorsolateral prefrontal cortex (DLPFC), for a subset of 312 samples, was obtained from the CommonMind Consortium. Results: Polygenic scores for schizophrenia predicted schizophrenia status in donors with African ancestry (p = 1.6×10-5, 10.4% of phenotypic variance explained) and in donors of European ancestry (p = 4.7×10-8, 17.2%). This pattern of higher variance explained among European ancestry samples was also observed for other psychiatric disorders (depression and bipolar disorder) and for height, body mass index, and years of education. Schizophrenia polygenic scores also predicted an aggregate measure of schizophrenia differential gene expression (African ancestry: p = .04, European ancestry: p = .01). Conclusions: Polygenic scores performed as expected in ancestrally diverse samples, given historical biases toward use of European ancestry samples and variable predictive power of polygenic scores across phenotypes. The transcriptomic results reported here suggest that inherited schizophrenia genetic risk influences gene expression, even in adulthood. For future research, these and additional polygenic scores are available for analyses, and for selecting samples, using postmortem tissue from the Human Brain Collection Core. Keywords: GWAS, Postmortem Brain Tissue, Schizophrenia (SCZ), Schizophrenia, Bipolar Disorder, Depression, Dorsolateral Prefrontal Cortex (DLPFC) Disclosure: Nothing to disclose. P536. Differences in Alternative Splicing and Transcript Isoform Usage in Layer 3 Pyramidal Neurons Across the Visuospatial Working Memory Network Kevin Dowling, John Enwright, Dominique Arion, David Lewis University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States Background: Human visuospatial working memory (VSWM) depends on a network of functionally distinct cortical regions, including primary visual (V1) and association cortices in the dorsolateral prefrontal cortex (dlPFC) and posterior parietal cortex (PPC). Across these regions, layer 3 pyramidal neurons (L3PNs) differ in excitability, morphology, and activity during VSWM tasks, partly due to regional differences in gene expression, which are particularly pronounced between the dlPFC or PPC and V1. Alternative splicing of pre-mRNA (AS), a process that alters the composition of exons and retained introns in mature RNA transcripts, is evident in nearly all genes. AS occurs more extensively in the CNS than in other tissues, where it contributes to the regulation of neuron physiology and morphology. However, it is unclear whether AS differs between L3PNs across regions of the VSWM network. Moreover, other processes concomitant with AS further augment transcript isoform diversity (e.g., usage of alternative transcription start sites and alternative polyadenylation sites); the cumulative effects of these processes are indexed by the abundance of distinct transcript isoforms in a given tissue. However, as with AS, it is unclear whether there are regional differences in the proportion of distinct transcript isoforms (a measure known as differential transcript usage; DTU) in L3PNs of the VSWM network. Methods: Samples of 100 L3PNs were collected from the dlPFC, PPC, and V1 of neurotypical adults (n = 39, 11 female) by laser capture microdissection, sequenced to an average depth of 50 million 100-nt paired end reads, and aligned to the genome using STAR. Genome-guided de-novo transcript isoform assembly was performed using StringTie. LeafCutter and IsoformSwitchAnalyzeR were used to analyze regional differences in AS and DTU. All analyses were FDR-corrected for multiple comparisons (α = 0.05) and included age, sex, tissue pH, RNA integrity number, PMI, and sequencing batch as covariates. PANTHER was used for pathway enrichment analyses. As many AS events are evolutionarily conserved between humans and macaques, samples of 120 long-range projection L3PNs collected from the dlPFC and PPC of post-pubertal rhesus macaques (n = 5, 2 female) were sequenced and used to validate AS differences in human cortex. Results: A greater number of genes were alternatively spliced between association cortices (dlPFC or PPC) and V1 (877 and 794 genes, respectively) than between the dlPFC and PPC (144 genes); of these genes, 35%-74% were not differentially expressed across regions. Likewise, >79% of genes differentially expressed across regions did not demonstrate significant DTU or AS. Genes exhibiting AS across all three regions were enriched for pathways related to excitatory neurotransmission, synaptic plasticity, and calcium flux. Of AS events that differed between the human dlPFC and PPC, and which occurred at splice sites evolutionarily conserved between the human and the macaque (105 events), 59% of the AS differences between the dlPFC and PPC were replicated in the macaque (62/105 events). As with AS, a greater number of transcripts demonstrated significant DTU between dlPFC (4,109 transcripts) or PPC (3,643 transcripts) and V1 than between dlPFC and PPC (21 transcripts). To predict the functional consequences of DTU, transcript isoform open reading frames were annotated for protein domains, intrinsically disordered regions, coding potential, and sensitivity to nonsense mediated decay (NMD) using PFAM, IUPRED2A, and CPAT3. Transcript isoforms more abundant in V1 were significantly enriched for non-coding potential, intron retention, and sensitivity to NMD – a homeostatic mechanism that degrades mRNA with a premature termination codon prior to translation. Of genes with both expression and NMD sensitivity differences between dlPFC (180 genes) or PPC (223 genes) and V1, NMD sensitivity differences at least partially mediated regional differences in the expression of 49% and 69% of genes, respectively. Conclusions: Many exons and transcripts demonstrated significant AS or DTU between dlPFC or PPC and V1, a pattern consistent with prior gene expression findings. However, a majority of all genes with significant AS or DTU were not differentially expressed across regions. Likewise, the majority differentially expressed genes did not demonstrate differences in AS or DTU. These results are consistent with prior findings that AS and DTU capture distinct aspects of the transcriptome. Although fewer genes demonstrated significant AS between L3PNs in the dlPFC and PPC than between either region and V1, many AS events that differed across all 3 regions have previously been shown to be functionally significant. For example, splicing of GRIN1 exon 5, which may be altered in schizophrenia and autism spectrum disorder, has been experimentally shown to influence the number of pyramidal neuron spines. GRIN1 exon 5 splicing (but not GRIN1 expression) significantly differed among all three cortical regions, a pattern consistent with spine density on L3PNs being highest in dlPFC, intermediate in PPC and lowest in V1. Lastly, differences in transcript sensitivity to degradation by NMD between dlPFC or PPC and V1 suggest a mechanism by which splicing may regulate regional gene expression differences in L3PNs of the adult VSWM network. Together, the present results suggest AS and DTU may represent important processes contributing to regional differences in L3PN physiology and morphology across the VSWM network. Keywords: Visuospatial Working Memory, Alternative Splicing, Pyramidal Neuron, Genomics, Transcript Isoforms Disclosure: Nothing to disclose. P537. An Investigation of the Prevalence of Copy Number Variants Associated With Neurodevelopmental Disorders in a Multi-Ancestry Brain Biobank Rebecca Birnbaum, Joo Heon Shin, Joshua Stoltz, Joel E. Kleinman, Thomas M. Hyde, Leonardo Collado-Torres, Daniel Weinberger Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Several large, copy number variants (CNVs), deletions or duplications >1 kb, each spanning single or multiple genes, are known to be pathogenic for neurodevelopmental disorders (NDD), rare in frequency, but of relatively large effect (“NDD-CNVs”). NDD-CNVs are notable for pleiotropy, variable expressivity and penetrance, underlying multiple disorders, including autism spectrum disorders, schizophrenia, intellectual disability and seizure disorder, as well as global developmental delay. Underscoring their clinical relevance, testing for NDD-CNVs is being increasingly considered in neuropsychiatry clinics broadly, while diagnostic testing for NDD-CNVs for some childhood-onset disorders is already standard-of-care. Numerous studies over past decades have elucidated clinical, cognitive and neuroimaging outcomes of NDD-CNV carriers in clinical and population cohorts, and animal and cellular studies have attempted their functional genomic characterization. To date however, investigation of NDD-CNVs in human brain has been comparatively limited, owing to rare frequency and difficulty accessing human neural tissue, precluding direct neurobiological genomic and transcriptomic characterization of NDD-CNVs. Furthermore, despite the clinical import of NDD-CNVs (discovered in peripheral blood-based analyses), the precise mechanism of central nervous system pathogenicity remains unknown. A rigorous, functional genomic characterization of NDD-CNVs, specifically in human brain, is therefore critical, to directly investigate NDD-CNVs in neurobiological context and to elucidate potential NDD-CNV pathogenic mechanisms. From a translational perspective, such direct query may enable “precise,” genetic-based therapeutic interventions. The current analysis leveraged a cross-disorder, multi-ancestry post-mortem brain bank from the Lieber Institute for Brain Development (LIBD) to identify the prevalence of NDD-CNVs. Methods: Brain tissue collection methods, procurement and dissection by LIBD were previously described. DNA was extracted from post-mortem cerebellum and dura matter from 2,348 brain donors across multiple disorders and across ancestry and genotyped on at least one genotyping array (Human 1 M Duo, Human Hap650Y, Infinum Omni2.5-8, Infinum Omni5-4). PennCNV was applied to genotype data from each brain sample to call CNVs, genome-wide. Subsequent to QC/filtering (exclusions for waviness factor >0.05 or <0.05, B-allele frequency drift>0.01, LogR Ratio standard deviation>0.3, and CNV burden>3 sd of median), CNVs were evaluated for overlap with 121 published NDD-CNVs, a threshold of at least 40% overlap for multigenic NDD-CNVs and overlap of at least one exon for single gene NDD-CNVs. Results: Initial QC/filtering resulted in the exclusion of 547 samples, yielding 1,801 samples for downstream analyses, multi-ancestry (75% European, 21% African, 2% Hispanic) and cross-disorder, including: 414 neurotypical controls, 522 major depressive disorder, 292 bipolar disorder, 234 schizophrenia, 130 post-traumatic stress disorder, 58 preclinical Alzheimer’s, 38 Alzheimer’s, 29 autism spectrum disorder, 15 substance use disorder, 14 eating disorders, 12 obsessive compulsive disorder, 10 alcohol use disorder, 6 bipolar disorder NOS, 5 Depression NOS, 4 Attention Deficit Hyperactivity Disorder, 3 Tic Disorder, 1 generalized anxiety disorder and 1 Williams Syndrome. Overall, NDD-CNVs across 16 loci were identified in a total of ~1.8% of samples, and excluding the common 15q11.2 duplication, in 1.3% of brain samples. NDD-CNV prevalence was comparable among major diagnostic groups (1.5% of major depressive disorder, 1.3% schizophrenia, 1.0% of bipolar disorder) but was not enriched compared to neurotypical controls, with 1.4% of neurotypical controls harboring at least one NDD-CNV. Among 29 ASD samples, there was one NDD-CNV carrier, a NRXN1 deletion, and the current analysis confirmed the 7q11.23 deletion in Williams syndrome. Interestingly, there appeared to be ancestry bias in prevalence, as nearly all NDD-CNV carriers, 31 of the 33 identified, were of European ancestry, and only two NDD-CNV carriers of African ancestry. There was no NDD-CNV sex bias, with females found to constitute 1/3 of NDD-CNV carriers proportionate with their representation in the brain biobank. Conclusions: The current analysis identified NDD-CNV prevalence in a cross-disorder, post-mortem brain bank, indicating a lack of enrichment among major diagnostic groups, confirming past reports of variable penetrance and expressivity. Given the relatively small sample, this may not deviate from expected probability. Future analyses will query NDD-CNVs in the LIBD post-mortem brain cohort using alternative and additional genotype-based calling algorithms for increased sensitivity and specificity, as well as expand sample size of NDD-CNV analyses to additional genotyped post-mortem brain cohorts. The polygenic risk scores of samples will be considered for multiple disorders, as well as background CNV burden in influencing penetrance. Future analyses will investigate transcriptomic and epigenetic effects of a subset of specific NDD-CNVs and also query potential cell-specificity and mosaic characteristics. Keywords: Copy Number Variant, Human Post-Mortem Brain, Neurodevelopmental Disorders Disclosure: Nothing to disclose. P538. Association Between Schizophrenia Polygenic Risk Score, Treatment Resistance, and Symptomatic Remission in Schizophrenia Max Lam, Keane Lim, Todd Lencz, Jimmy Lee Institute of Mental Health, Singapore, Singapore Background: Treatment-resistant schizophrenia (TRS) occurs in approximately one-third of individuals who fail to show adequate response to antipsychotic medications, with variability in symptoms severity. This study aims to examine the association between the genetic liability of schizophrenia, TRS status, and symptomatic remission status. Methods: Individuals diagnosed with schizophrenia (N = 876) were categorised as TRS (n = 150) and non-TRS (n = 726), using well-defined TRS criteria. Schizophrenia polygenic risk score (PRS) was calculated, and logistic regression analyses were conducted to discriminate TRS and non-TRS. The TRS status was further stratified based on symptomatic remission criterion, as indexed by the Positive and Negative Syndrome Scale (PANSS), and regression analyses were conducted to examine the association of PRS with the following symptomatic remission subtypes – antipsychotic remitter; antipsychotic non-remitter; clozapine remitter; clozapine non-remitter. Results: Schizophrenia PRS discriminated TRS and non-TRS (R2 = 3.04%, p = 2.27 ×10-5, 0 R = 1.55), with higher PRS observed in the TRS group, compared with non-TRS. Stratification of TRS status based on symptomatic remission criterion revealed significant difference in PRS across subtypes (R2 = 1.62%, p = 2.68 ×10-4, 0 R = 1.27). Within TRS, higher schizophrenia PRS was observed in clozapine remitter subtype; while in non-TRS, higher schizophrenia PRS was observed in antipsychotic non-remitter subtype. Compared to the lowest decile, the odds ratio of being classified as TRS in the top decile is 3.29 (95% CI: 1.21-8.94). Conclusions: The common risk variants for schizophrenia are associated with TRS status, and symptomatic remission. Findings suggest that the genetic burden of schizophrenia may in part explain treatment resistance status in schizophrenia, and indexes treatment outcomes. Keywords: Schizophrenia (SCZ), Treatment-Resistance, Polygenic Risk Scores, Antipsychotic Treatment, Remission Disclosure: Nothing to disclose. P539. Excess Homozygosity in Neuropsychiatric Patients From the Ashkenazi Jewish Populations Implicates TUBGCP4 as a Recessive Locus for Schizophrenia Todd Lencz, Jin Yu, Max Lam, Anil Malhotra, Itsik Pe’er Zucker Hillside Hospital, Glen Oaks, New York, United States Background: To date, rare variant research in schizophrenia (SZ) has focused on dominant exonic variants; there has been limited research examining the recessive mode of inheritance. The Ashkenazi Jewish (AJ) population is marked by an excess of homozygosity, due to a history of endogamy and a small founder population and may provide enhanced power to detect recessive loci. Methods: We examined homozygous loci in exome data derived from high-depth (>30x) whole genome sequencing performed on 786 schizophrenia cases and 463 controls drawn from the AJ population. To enhance power, we merged our dataset with 1934 AJ neuropsychiatric cases from gnomAD and 3106 AJ controls from gnomAD (non-neuro). We compared cases and controls for recessive effects across three types of alleles: synonymous, loss-of-function (LoF), and damaging missense plus loss-of-function (MisLoF). Analagous to our prior work in rare variant discovery (Lencz et al. 2021, Neuron), we hypothesized that significant enrichment would occur if variants observed homozygous even once in gnomAD (non-neuro) and TOPMED databases (total N > 158 K) were filtered out. We compared cases and controls for recessive effects across three allele frequency bins (0.1%<MAF < 1%; MAF < 0.1%; and MAF < 0.1% plus never observed as homozygous in healthy gnomAD and TOPMed samples), and across three types of alleles: synonymous, loss-of-function, and damaging (MPC > 2) missense plus loss-of-function. Results: Cases were enriched for ultra-rare recessive alleles in the LoF (OR~2) and LoF+Mis (OR~4) categories. Recessive synonymous variants were not enriched in cases relative to controls. While most of the homozygous LoF+Mis variants were observed in poorly annotated genes, a homozygous LoF splice-donor variant (chr15:43696752:T/C) in TUBGCP4 was observed in one patient with schizophrenia. Recessive mutations in TUBGCP4 have been associated with intellectual disability with microcephaly (Scheidecker et al., 2015, Am J Hum Gen). Conclusions: Results suggest that a small, but detectable, fraction of risk for schizophrenia derives from recessive effects of ultra-rare variants. Unique founder populations, such as AJ, are enriched for such variants and may be worthy of further study. Recessive mutations in TUBGCP4 have been previously associated with intellectual disability with microcephaly, consistent with research showing overlap in dominant rare variants between SZ and neurodevelopmental disorders. This gene encodes a protein that is part of the gamma-tubulin ring complex, which is required for microtubule nucleation, an essential step in the process of neuronal morphogenesis. Keywords: Human Genetics, Schizophrenia (SCZ), Neurodevelopment Disclosure: Nothing to disclose. P540. Investigating Trait Variability of Gene Co-Expression Network Architecture in Brain by Controlling for Genomic Risk of Schizophrenia Eugenia Radulescu, Qiang Chen, Giulio Pergola, Pasquale Di Carlo, Joo Heon Shin, Thomas Hyde, Joel Kleinman, Daniel Weinberger Lieber Institute for Brain Development, Baltimore, Maryland, United States Background: Schizophrenia (SCZ) genetic risk is linked with variation in gene expression in brain, but how this affects gene co-expression networks is unclear. To better understand this issue, we implemented a novel approach to network analysis by adjusting gene expression input to account for the effects of SCZ genomic risk score (GRS). Methods: Postmortem brain samples, RNA-Seq preparation and calculation of variables to enter the selection for gene expression adjustment: data was acquired from assays of postmortem human brain tissue from the LIBD Human Brain Repository, collected under a standard protocol of brain acquisition, processing, and curation. After pre-processing, a final gene expression (N = 18,980 genes) dataset of 78 DLPFC samples (age of death: 43 ± 15.8; M/F: 64/14) from neurotypical adults of European ancestry was retained for further analysis. The gene expression was further adjusted by linear regression models to remove variance explained by RNA quality, cell type proportion, age, sex and genetic ancestry. WGCNA was then used to create DLPFC brain co-expression networks after preserving or removing GRS effects and to calculate consensus networks. The consensus (i.e., background) networks- representative of gene co-expression architecture free of genomic scores effects- were used as baseline for differential network analysis. Specifically, the differential network analysis consisted of calculating the overlap between modules of GRS preserved networks and background modules. Differential modules were considered preserved modules with weak or no overlap in background; they were subsequently tested for biological significance, i.e., correlations with GRS, enrichment in gene ontology (GO) biological processes and enrichment in PGC3 SCZ GWAS priority loci genes. From the same dataset, we created also analogous co-expression networks based on the genomic score of height, as a control normative trait. Results: We identify key aspects of SCZ GRS effects on brain co-expression networks: 1) Co-expression networks derived from expression input adjusted to account for the genomic scores of SCZ and height traits share a similar architecture (i.e., module composition) with subtle differences between preserved and removed GRS effects. 2) For each trait- SCZ risk and height, modules of networks that preserve GRS effects have a pattern of overlap with the corresponding background modules characterized by strong correspondence for half of the modules and weaker correspondence for the other half. 3) Several SCZ risk and height GRS preserved modules with weak correspondence in the background modules had module eigengenes (MEs) correlated with GRS SCZ or GRS height. Preserved modules with MEs negatively correlated with GRS SCZ were enriched for brain specific functional ontologies and priority PGC3 SCZ GWAS loci genes. Preserved modules with MEs correlated with GRS height were mainly enriched for general cellular processes of transcription, translation and metabolism. Conclusions: Overall, our results indicate that SCZ GRS is associated with brain gene co-expression networks and creates a molecular background for gene-gene interactions that affect diverse biological pathways. Furthermore, consistent with the pleiotropy phenomenon, SCZ risk genetic influence on gene co-expression architecture shares a part of signal with other complex traits (i.e., height), but has also specific contributions convergent toward brain functionality. Keywords: Schizophrenia (SCZ), Dorsolateral Prefrontal Cortex, Co-Expression Network Analysis Disclosure: Nothing to disclose.	36456694	PMC9714399	NO-CC CODE	2022-12-06 23:15:03	no	Neuropsychopharmacology. 2022 Dec 1; 47(Suppl 1):220-370	utf-8	Neuropsychopharmacology	2,022	10.1038/s41386-022-01485-0	oa_other
==== Front Bull Malays Math Sci Soc Bull Malays Math Sci Soc Bulletin of the Malaysian Mathematical Sciences Society 0126-6705 2180-4206 Springer Nature Singapore Singapore 1435 10.1007/s40840-022-01435-5 Article Objective Bayesian Estimation for the Differential Entropy Measure Under Generalized Half-Normal Distribution http://orcid.org/0000-0001-7600-6407 Ahmadi Kambiz [email protected] 1 Akbari Masoumeh [email protected] 2 Raqab Mohammad Z. [email protected] 3 1 grid.440800.8 0000 0004 0382 5622 Department of Computer Science, Shahrekord University, Shahrekord, Iran 2 grid.411622.2 0000 0000 9618 7703 Department of Statistics, University of Mazandaran, Babolsar, Iran 3 grid.411196.a 0000 0001 1240 3921 Department of Statistics and OR, Kuwait University, Al-Shadadiyah, Kuwait Communicated by Rosihan M. Ali. 1 12 2022 2023 46 1 393 5 2022 13 11 2022 21 11 2022 © The Author(s), under exclusive licence to Malaysian Mathematical Sciences Society and Penerbit Universiti Sains Malaysia 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The evaluation of the information entropy content in the data analysis is an effective role in the assessment of fatigue damage. Due to the connection between the generalized half-normal distribution and fatigue extension, the objective inference for the differential entropy of the generalized half-normal distribution is considered in this paper. The Bayesian estimates and associated credible intervals are discussed based on different non-informative priors including Jeffery, reference, probability matching, and maximal data information priors for the differential entropy measure. The Metropolis–Hastings samplers data sets are used to estimate the posterior densities and then compute the Bayesian estimates. For comparison purposes, the maximum likelihood estimators and asymptotic confidence intervals of the differential entropy are derived. An intensive simulation study is conducted to evaluate the performance of the proposed statistical inference methods. Two real data sets are analyzed by the proposed methodology for illustrative purposes as well. Finally, non-informative priors for the original parameters of generalized half-normal distribution based on the direct and transformation of the entropy measure are also proposed and compared. Keywords Differential entropy Generalized half-normal distribution Maximum likelihood estimation Metropolis–Hastings algorithm Monte Carlo simulation Objective Bayesian estimation Mathematics Subject Classification 62F10 62F15 issue-copyright-statement© The Author(s), under exclusive licence to Malaysian Mathematical Sciences Society and Penerbit Universiti Sains Malaysia 2023 ==== Body pmcIntroduction The differential entropy HX(f) of a continuous random variable X with the probability density function (PDF) f(x) is defined (see, Shannon [1]) by1 HX(f)=E[-lnf(x)]=-∫-∞∞f(x)lnf(x)dx. In fact, it measures the uniformity of a distribution such that it increases when f(x) approaches uniform distribution that means the prediction of an outcome from f(x) gets more difficult. Additionally, for sharply picked distributions, the differential entropy HX(f) is low. In this sense HX(f) can be interpreted as a measure of uncertainty relative to f(x). For this, the parametric estimation of differential entropy under various statistical models was brought to the attention of a significant number of researchers [2–8]. Generally, most researchers have focused their attention on either classical or Bayesian estimation of entropy by adopting prior distributions for parameters involved in the underlying model. In contrast, adopting the prior distribution for the entropy itself has not received much attention and we, therefore, focus on this problem. In this context, Shakhatreh et al. [9] and Ramos et al. [10] presented some Bayesian estimates (BEs) of differential entropy under Weibull and gamma distributions, respectively. They considered the issue of estimation based on some non-informative prior distributions of differential entropy. Their results show satisfactory performance of recommended BEs relative to maximum likelihood estimate (MLE) of differential entropy. The generalized half-normal (GHN) distribution, proposed by Cooray and Ananda [11], is one of the most popular models used to describe the lifetime process under fatigue. Let X be a random variable from a GHN distribution with shape parameter α>0 and scale parameter θ>0, the associated PDF and the cumulative distribution function (CDF) can be expressed asf(x;α,θ)=2παxxθαexp-12xθ2α,x≥0,F(x;α,θ)=1-2Φ-xθα,x≥0, respectively, where Φ(·) is the CDF of the standard normal distribution. Hereafter, we denote the GHN model with parameters α and θ as GHN(α,θ). It is widely used in many practical applications because of its flexibility to fit various data sets. It is observed that the two-parameter GHN distribution can be monotonically increasing or decreasing and bathtub hazard rate shape depending upon the value of its parameters. It can be both positively and negatively skewed. Because of these properties, it is shown that the GHN distribution can be an alternative to the exponential, Weibull and gamma distributions, among others. It is shown in Cooray and Ananda [11] that the GHN model is followed from considerations of the relationship between static fatigue crack extension and the failure time of a certain specimen. Fatigue is a structural damage which occurs when a material is exposed to stress and tension fluctuations. Recently the GHN distribution has attracted the attention of many authors working on theory and methods as well as in various fields of applied statistics. For example, Kang et al.[12] discussed some non-informative priors for the GHN distribution when shape and scale parameters are of interest, respectively. They developed the first and second-order probability matching priors for the shape and scale parameters of GHN distribution when one of two parameters is the parameter of interest. They also discussed the reference priors for the shape and scale parameters of GHN distribution. Ahmadi et al. [13] derived estimation of parameters and reliability function under GHN distribution based on progressive type-II censoring. They considered both classical and Bayesian methods for estimation while the expectation-maximization (EM) algorithm and Lindley’s approximation were proposed to approximate suggested estimators. Wang and Shi [14] discussed the estimation issues in the constant-stress accelerated life test for GHN distribution. They just used classical methods and bootstrap technique for constructing confidence intervals. Ahmadi and Ghafouri [15] presented classical and Bayesian estimation of multi-component stress-strength reliability for GHN distribution in terms of progressive type-II censoring. From (1), the differential entropy of a random variable X∼GHN(α,θ) can be easily obtained byH=HX(f)=12-ln2π-1-1αψ(12)22-lnαθ, where ψ(·) is the digamma function. So, the joint differential entropy based on independent and identically distributed (iid) random variables X1,X2,…,Xn from GHN distribution is HX1,X2,…,Xn(f)=nH. To this end, it well known that entropic-based measures like differential entropy can be examined and demonstrated for application to fatigue failures. They have been used to predict damage and failure [16]. So, the differential entropy can play an efficient role in the assessment of fatigue damage. Due to the connection between the GHN distribution and fatigue crack extension explored by Cooray and Ananda [11], it is quite natural to quantify the entropy measure under the GHN lifetime data as no attempt has been made previously. The main aim of this paper is to develop some point and interval estimates for the differential entropy measure of GHN from a Bayesian perspective. In some practical situations, it is difficult to specify appropriate subjective prior information and selecting priors is like the other modeling in science that is always facing criticism. For this reason, we develop the Bayesian set-up by considering different objective priors to reflect prior knowledge on the parameters. There are some literature on objective Bayesian perspective, see for example [17–19]. Obviously, the differential entropy H is a function of the model parameters α and θ. Although Kang et al.[12], based on some non-informative priors, have obtained different posterior distributions for the parameters of interest, the obtained posterior means cannot be directly plugging into H. Moreover, the BE of H using non-informative prior distributions of the original model parameters may lead to inappropriate results unless these prior distributions are invariant under one-to-one transformations, see [9]. Therefore, this paper focuses on the BEs of H based on different non-informative priors. This paper is organized as follows. In Sect. 2, we first present some useful results on the objective priors which will be used in the next sections. In Sect. 3, we describe the Metropolis–Hastings (M–H) sampler approach used for computing the BEs of H. In Sect. 4, we discuss frequentist estimation methods including the MLE and asymptotic confidence interval of H. An extensive Monte Carlo simulation study is performed to assess how the proposed methods of estimation behave and two real data sets taken from practical fields are analyzed to illustrate the results developed here. Finally, some objective priors for the original parameters of GHN distribution are briefly discussed and some conclusions are made in Sect. 5. Objective Bayesian Estimation Procedures Let X1,X2,…,Xn be a complete sample from GHN(α,θ). The likelihood function (LF) of this sample isL(α,θ;x)=2πnαθαn∏i=1nxiα-1exp-12∑i=1nxiθ2α. Under the reparameterization α=1W andθ=1WexpH-12+ln2π+(1-W)ψ(12)22, the LF of H and W can be given by2 L(H,W;x)=2πnWnW-nδH,Wn∏i=1nxi1W-1exp-12δH,W2∑i=1nWxi2W, whereδH,W=exp-1WH-12+ln2π+(1-W)ψ(12)22. From Wang and Shi [14], the expected Fisher information (FI) matrix of α and θ is given by3 I(α,θ)=I11I12I21I22=12α2π22-2+(2-ln2-γ)2-1θ(2-ln2-γ)-1θ(2-ln2-γ)2αθ2, where γ is Euler’s constant. The FI matrix of H and W can be obtained easily by I(α,θ) and the Jacobian transformation J, whereJ=0-1W21WδH,WW-1+ψ(12)22W1W2δH,WW. Therefore, the FI matrix of H and W is readily derived as4 Σ(H,W)=JTIJ=ΣHHΣHWΣWHΣWW, whereΣHH=2W2,ΣHW=ΣWH=2W2η2-1W,ΣWW=1W2π24-1+2η2-1W2, and5 η=2-ln2-γ-ψ(12)2. In this section, four types of non-informative Bayesian estimation of H, are discussed in further detail. For this purpose, posterior distributions of H and its Bayesian estimation under squared error loss (SEL) function under Jeffreys, reference, matching probability and maximal data information priors are investigated below. Jeffreys Prior The following non-informative prior, known as Jeffreys prior [20] is defined in terms of the FI, πJ(H,W)∝detΣ(H,W) where6 detΣ(H,W)=1W4π22-2, is the determinant of Σ(H,W). From (6), we have7 πJ(H,W)∝1W2,H∈(-∞,∞),W>0. Generally, this type of prior can be useful for three reasons. First, it is reparameterization-invariant; see [20]. Second, Jeffreys prior is a uniform density on the space of probability distributions in the sense that it assigns equal mass to each different distribution; see [21]. Third, Jeffreys prior maximizes the amount of information about the parameter of interest, in the Kullback–Leibler sense, see for example [22]. From (2) and (7), the Jeffreys posterior is concluded by8 πJ(H,W\|x)∝WnW-n-2δH,Wn∏i=1nxi1W×exp-12δH,W2∑i=1nWxi2W,H∈(-∞,∞),W>0. Since πJ(H,W) is not proper, we cannot be able to say that πJ(H,W\|x) is definitely a density. In the following theorem, the conditions that πJ(H,W\|x) can be a density are verified. Theorem 1 For all n≥2, the posterior density πJ(H,W\|x) is proper. Proof Using the change of variable u=δH,W2, we have9 A(x)=∫0+∞∫-∞+∞πJ(H,W\|x)dHdW=12∫0+∞∫0+∞WnW-n-1∏i=1nxi1Wun2-1exp-12u∑i=1nWxi2WdudW=121-n2Γn2∫0+∞1Wn+1∏i=1nxi1W∑i=1nxi2Wn2dW. If n=1, then we have A(x)∝∫0+∞1W2dW=∞. For n≥2, let x(1),x(2),…,x(n) be the order observations such that x(1)≤x(2)≤…≤x(n). From Cauchy–Schwarz inequality, it follows that10 1n∑i=1nx(i)1W2≤∑i=1nx(i)2W. There exists ℓ∈{1,2,…,n}, such that xℓ<x(n). Since xi1W∑i=1nxi1W<1, we have11 ∏i=1nxi1W∑i=1nxi1Wn<xℓx(n)1W<1. From (10) and (11), it follows thatA(x)<nn221-n2Γn2∫0+∞1Wn+1∏i=1nxi1W∑i=1nxi1WndW<nn221-n2Γn2∫0+∞1Wn+1exp-1Wlnx(n)xℓdW<∞. □ Theorem 2 The posterior mean of H relative to πJ(H,W\|x) is finite for any n≥2. Proof Using the change of variable u=δH,W2, we can write\|E(H\|x)\|=\|∫0∞∫-∞∞HπJ(H,W\|x)dHdW\|=\|12-ln2π-ψ(12)22A(x)+ψ(12)42B(x)-14C(x)\|≤12-ln2π-ψ(12)22A(x)+\|ψ(12)42\|B(x)+14\|C(x)\|, where A(x) is given in (9) and12 B(x)=∫0∞∫0∞WnW-n∏i=1nxi1Wun2-1exp-12u∑i=1nWxi2WdudW, 13 C(x)=∫0∞∫0∞WnW-n∏i=1nxi1Wun2-1(lnu)exp-12u∑i=1nWxi2WdudW. From Theorem 1, A(x) is finite for all n≥2. Moreover, B(x) is finite for all n≥2 (The proof is very similar to the proof of Theorem 1 and is therefore omitted for the sake of brevity). We shall show that \|C(x)\| is finite for all n≥2. Using the fact that14 ∫0∞tz-1e-atlntdt=Γ(z)azψ(z)-ln(a), we can write C(x) in the following formC(x)=2n2Γn2ψn2+ln2C1(x)-2C2(x)-C3(x), where15 C1(x)=∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2dW,C2(x)=∫0∞1Wn+1∏i=1nxi1W∑i=1nxi2Wn2lnWdW,C3(x)=∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2ln∑i=1nxi2WdW. Clearly, C1(x) is finite for all n≥2. Let16 C4(x)=∫0∞1Wn+2∏i=1nxi1W∑i=1nxi2Wn2dW. Since for W>0, \|lnW\|≤12W+1W, then17 \|C2(x)\|≤12C1(x)+12C4(x)<∞, for all n≥2. It remains to prove that \|C3(x)\| is finite for all n≥2. Note that for ∑i=1nxi2W<1, we have \|ln∑i=1nxi2W\|<2W\|lnx(n)\| and also for ∑i=1nxi2W>1, we have \|ln∑i=1nxi2W\|<lnn+2W\|lnx(n)\|. Therefore for W>0, we immediately have18 ln∑i=1nxi2W<lnn+2Wlnx(n). Let19 C5(x)=∫0∞1Wn+1∏i=1nxi1W∑i=1nxi2Wn2dW. From (18), it follows that\|C3(x)\|<(lnn)C1(x)+2\|lnx(n)\|C5(x)<∞. The proof of Theorem 2 is readily complete. □ The marginal posterior distribution of W is given by20 πJ(W\|x)∝1Wn+1∏i=1nxi1W∑i=1nxi2Wn2,W>0. Moreover, the conditional posterior distribution of H is given by21 πJ(H\|W,x)∝exp-nHW-12δH,W2∑i=1nWxi2W,H∈(-∞,∞). Reference Prior Despite of useful features and simple calculations of Jeffreys prior, it does not have good performance in multi-parameter problems. Hence, other non-informative priors like reference prior were introduced in the literature. Reference prior was originally defined by Bernardo [22], but its further results were found by Bernardo [23] and Berger and Bernardo [24–27]. Suppose a model described by density f(x\|ϕ,λ), where ϕ is the parameter of interest and λ is a nuisance parameter. Bernardo and Smith [28] obtained an effective formula for finding a reference prior based on Berger and Bernardo’s algorithm that is given below. DefineDϕ(ϕ,λ)=det(I(ϕ,λ))Iλλ(ϕ,λ), where Iλλ(ϕ,λ) is the (λ,λ)-entry of that matrix. If ϕ and λ are independent and ifDϕ12(ϕ,λ)=f1(ϕ)g1(λ)andIλλ12(ϕ,λ)=f2(ϕ)g2(λ), then the reference prior is equal to22 πR(ϕ,λ)=f1(ϕ)g2(λ). In our study, H is the parameter of interest and W is a nuisance parameter. In the following theorem the reference prior of (H, W) is obtained. Theorem 3 The improper reference prior of the parameter (H, W) is given by23 πR(H,W)∝1Wπ24-1+2η2-1W212,H∈(-∞,∞),W>0. Proof It can be easily shown thatDH12(H,W)=f1(H)g1(W), wheref1(H)=π22-2,g1(W)=1Wπ24-1+2η2-1W2-12. On the other hand, we haveΣWW12=f2(H)g2(W), wheref2(H)=1,g2(W)=1Wπ24-1+2η2-1W212. Therefore from (22), the reference prior πR(H,W)=f1(H)g2(W). This completes the proof. □ From (2) and (23), the reference posterior of (H, W) can be expressed as follows:πR(H,W\|x)∝WnW-n-1δH,Wn∏i=1nxi1Wexp-12δH,W2∑i=1nWxi2W×π24-1+2η2-1W212,H∈(-∞,∞),W>0. Theorem 4 For all n≥2, the posterior density πR(H,W\|x) is proper. Proof 24 D(x)=∫0∞∫-∞∞πR(H,W\|x)dHdW=12∫0∞∫0∞WnW-n∏i=1nxi1Wπ24-1+2η2-1W212×un2-1exp-12u∑i=1nWxi2WdudW=121-n2Γn2∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW. Since π24-1>1, for n=1, we haveD(x)>12Γ(12)∫0∞dWW=∞. For n≥2, we have25 D(x)<121-n2Γn2π24-1+η22C1(x)+2C4(x)-2ηC5(x), where C1(x),C4(x) and C5(x) are given in (15), (16) and (19), respectively. Using the fact that C1(x),C4(x) and C5(x) are finite for all n≥2, the proof of Theorem 4 is thus complete. □ In what follows, we show the posterior mean of H under reference prior is finite for any n≥3. Theorem 5 The posterior mean of H relative to πR(H,W\|x) is finite for any n≥3. Proof E(H\|x)=∫0∞∫-∞∞HπR(H,W\|x)dHdW=14∫0∞∫0∞WnW-n+1∏i=1nxi1Wπ24-1+2η2-1W212un2-1×ψ(12)2-lnuexp-12u∑i=1nWxi2WdudW+12-ln2π-ψ(12)22D(x), where D(x) is defined in (24). Using (14), we getE(H\|x)=2n2-2Γn2ψ(12)2-ψ(n2)-ln2G1(x)+2G2(x)+G3(x)+12-ln2π-ψ(12)22D(x), whereG1(x)=∫0∞1Wn-1∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW,G2(x)=∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2(lnW)π24-1+2η2-1W212dW,G3(x)=∫0∞1Wn-1∏i=1nxi1W∑i=1nxi2Wn2ln∑i=1nxi2Wπ24-1+2η2-1W212dW. Now, it is shown that G1(x) is finite for n≥3.26 G1(x)≤π24-1+η22K(x)+22-n2Γ(n2)A(x)-2ηC1(x), where A(x) and C1(x) are defined in (9) and (15) respectively, andK(x)=∫0∞1Wn-1∏i=1nxi1W∑i=1nxi2Wn2dW. By similar arguments to the proof of Theorem 1, K(x) is finite for n≥3. Moreover, A(x) and C1(x) are finite for n≥2. Therefore, G1(x) is finite for n≥3. We have27 \|G2(x)\|≤12∫0∞1Wn+1∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW+12G1(x), and using (18)28 \|G3(x)\|≤2\|lnx(n)\|∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW+(lnn)G1(x). Using arguments similar to the ones applied in the proof of Theorem 4, it follows that integrals given in (27) and (28) are finite for n≥3. Since G1(x),\|G2(x)\|,\|G3(x)\| and D(x) are finite for n≥3, we conclude that\|E(H\|x)\|≤2n2-2Γn2\|ψ(12)2-ψ(n2)-ln2\|G1(x)+2\|G2(x)\|+\|G3(x)\|+\|12-ln2π-ψ(12)22\|D(x)<∞. For n=2, the expected value of \|H\| relative to posterior πR(H,W\|x) is given by E(\|H\|\|x)=G11(x)+G12(x), whereG11(x)=-∫0∞∫-∞0HW2W-3δH,W2∏i=12xi1Wπ24-1+2η2-1W212×exp-12δH,W2∑i=12(Wxi)2WdHdW,G12(x)=∫0∞∫0∞HW2W-3δH,W2∏i=12xi1Wπ24-1+2η2-1W212×exp-12δH,W2∑i=12(Wxi)2WdHdW. On the other hand, by Definition A.4 of Ramos et al. [10], we have δH,W∝expψ(12)22-HW, W1W∝1 and xi1W∝1,i=1,2, as W tends to infinity. Therefore, from Proposition A.5 of Ramos et al. [10], we deduce the following assertion:29 G11(x)>-∫1∞∫-∞0HW2W-3δH,W2∏i=12xi1Wexp-12δH,W2∑i=12(Wxi)2WdHdW∝-expψ(12)2∫1∞∫-∞0HW3exp-2HW-expψ(12)2-2HWdHdW=14expψ(12)2∫1∞∫1∞lnuWexp-expψ(12)2ududW>14expψ(12)2(∫1∞dWW)(∫exp{1}∞exp-expψ(12)2udu)=∞. Similarly, we also conclude30 G12(x)>-14expψ(12)2(∫1∞dWW)(∫0exp{-1}exp-expψ(12)2udu)=∞. From (29) and (30), the posterior mean of H relative to πR(H,W\|x) does not exist for n=2. □ The marginal reference posterior of W and conditional reference posterior of H can be derived, respectively, as follows:πR(W\|x)∝1Wn∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212,W>0, andπR(H\|W,x)∝exp-nHW-12δH,W2∑i=1nWxi2W,H∈(-∞,∞). Probability Matching Prior Another type of non-informative prior is called probability matching prior which was introduced by Welch and Peers [29]. It is mainly based on a possible agreement between frequentist and Bayesian inferences. Based on methods presented in Welch and Peers [29], the probability matching prior of (H, W) is derived in the following theorem: Theorem 6 The probability matching prior of (H, W) is proportional to31 πPM(H,W)∝12-ηWπ24-1+2η2-1W2120<W<2η1ηW-2π24-1+2η2-1W212W>2η, where H∈(-∞,∞) and η defined in (5). Proof The second-order probability matching prior of (H, W) when H is the parameter of interest and W is a nuisance parameter can be obtained by solving following partial differential equation in terms of πPM(H,W).32 ∂∂W(ΣHWπPM(H,W)ΣWWK)-∂∂H(πPM(H,W)K)=0, where K=ΣWW-1detΣHW. From (4), it is obvious that the FI matrix of (H, W) does not depend on H. So, (32) can be reduced to33 ∂∂W(ΣHWπPM(H,W)ΣWWK)=0. Utilizing (4), and after some simplifications, (33) is equivalent to∂∂WηW-2πPM(H,W)π22-2π24-1+2η2-1W212=0, and thus (31) is readily obtained by solving the above equation in terms of πPM(H,W). This completes the proof. □ Therefore, the probability matching posterior is as follows34 πPM(H,W\|x)∝WnW-n\|ηW-2\|δH,Wn∏i=1nxi1Wπ24-1+2η2-1W212×exp-12δH,W2∑i=1nWxi2W, where H∈(-∞,∞) and W>0. Theorem 7 For all n≥2, the posterior density πPM(H,W\|x) is proper. Proof Let’s take35 R(x)=∫0∞∫-∞∞πPM(H,W\|x)dHdW=121-n2Γ(n2)∫0∞1Wn-1\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW, where η defined in (5). For n=1,R(x)>12Γ(12)∫0∞dW\|ηW-2\|=∞. For n≥2, R(x) can be rewritten asR(x)=121-n2Γ(n2)R1(x)+R2(x), whereR1(x)=∫02η1Wn-12-ηW∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW, andR2(x)=∫2η∞1Wn-1ηW-2∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW. From Definition A.4 of Ramos et al. [10], we get36 2-ηW∝W→02,andπ24-1+2η2-1W212∝W→01W. According to Proposition A.5 of Ramos et al. [10] and the results in (36), we concludeR1(x)∝12∫02η1Wn∏i=1nxi1W∑i=1nxi2Wn2dW<12C1(x), where C1(x) defined in (15) and it is finite for n≥2. So, R1(x) is finite for each n≥2. Arguments similar to the previous ones, we obtain37 ηW-2∝W→∞ηW,π24-1+2η2-1W212∝W→∞π24-1+η2212. andR2(x)<1ηπ24-1+η22∫0∞1Wn∏i=1nxi1W∑i=1nxi2Wn2dW=1ηπ24-1+η22C1(x), where C1(x) is the same quantity defined in (15). So, it follows that R2(x) is finite for any n≥2. This completes the proof. □ Theorem 8 The posterior mean of H relative to πPM(H,W\|x) is finite for all n≥3. Proof E(H\|x)=∫0∞∫-∞∞HπPM(H,W\|x)dHdW=14∫0∞∫0∞WnW-n+2\|ηW-2\|∏i=1nxi1Wπ24-1+2η2-1W212un2-1×ψ(12)2-lnuexp-12u∑i=1nWxi2WdudW+12-ln2π-ψ(12)22R(x), where R(x) is defined in (35). From (14), it follows thatE(H\|x)=2n2-2Γn2ψ(12)2-ψ(n2)-ln2S1(x)+2S2(x)+S3(x)+12-ln2π-ψ(12)22R(x), whereS1(x)=∫0∞1Wn-2\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW,S2(x)=∫0∞1Wn-1\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2(lnW)π24-1+2η2-1W212dW,S3(x)=∫0∞1Wn-2\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2ln∑i=1nxi2Wπ24-1+2η2-1W212dW. Since, for W>0,\|lnW\|≤12(W+1W), it is readily seen that38 \|S2(x)\|≤12∫0∞1Wn\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW+12S1(x). From (18), it follows that39 \|S3(x)\|≤2\|lnx(n)\|∫0∞1Wn-1\|ηW-2\|∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212dW+(lnn)S1(x). From the proof of Theorem 7, R(x) is finite for n≥2. Also, by adopting similar arguments to the one used in the proof of Theorem 7, it can be easily shown that S1(x) and integrals given in (38) and (39) and consequently S2(x) and S3(x) are finite for n≥3. Thus for n≥3,\|E(H\|x)\|≤2n2-2Γn2\|ψ(12)2-ψ(n2)-ln2\|S1(x)+2\|S2(x)\|+\|S3(x)\|+\|12-ln2π-ψ(12)22\|R(x)<∞. It has to be noticed that for n=2, the posterior mean of H relative to πPM(H,W\|x) does not exist. The proof is similar to that of Theorem 5 and is therefore omitted for brevity. □ From (34), the marginal probability matching posterior of W and conditional probability matching posterior of H, can be expressed, respectively, asπPM(W\|x)∝1\|ηW-2\|Wn∏i=1nxi1W∑i=1nxi2Wn2π24-1+2η2-1W212,W>0 andπPM(H\|W,x)∝exp-nHW-12δH,W2∑i=1nWxi2W,H∈(-∞,∞). Maximal Data Information (MDI) Prior Zellner [30] introduced an entropy-based method for finding a prior distribution that is called MDI prior. For a density f(x\|θ) with parameter of interest θ, the MDI prior of θ is defined as40 πMDI(θ)∝exp{E[ln(f(x\|θ))]}. Now, from (40), one can easily conclude that the MDI prior of (H, W) is as followsπMDI(H,W)∝exp{-H},H∈(-∞,∞),W>0. Notice that the MDI prior is completely different with all prior distributions that discussed before. Because, it is the only prior distribution that contains information about H. But, this prior distribution leads to improper posterior of (H, W). Hence, its modified version is considered asπMDI(H,W)∝exp{-\|H\|},H∈(-∞,∞),W>0. So, for H∈(-∞,∞) and W>0 the MDI posterior of (H, W) can be expressed as41 πMDI(H,W\|x)∝WnW-nδH,Wn∏i=1nxi1Wexp-12δH,W2∑i=1nWxi2W-\|H\|. Theorem 9 The posterior πMDI(H,W\|x) is proper for all n≥3. Proof In order to simplify notation, let G(x)=∫0∞∫-∞∞πMDI(H,W\|x)dHdW. Sinceexp-12u∑i=1nWxi2W-\|H\|≤exp-12u∑i=1nWxi2W, we have42 G(x)≤12∫0∞∫0∞WnW-n+1∏i=1nxi1Wun2-1exp-12u∑i=1nWxi2WdudW. It can be shown that for n≥3, quantity (42) is finite by similar proof to the A(x) in (9). But, for n=1,2, the MDI posterior can be shown to be improper by an argument similar to that of Theorem 5. □ Theorem 10 The posterior mean H relative to πMDI(H,W\|x) is finite for all n≥4. Proof Using the change of variable δH,W2=u, we have\|E(H\|x)\|=\|12-ln2π-ψ(12)22V1(x)+ψ(12)42V2(x)-14V3(x)\|≤12-ln2π-ψ(12)22V1(x)+\|ψ(12)42\|V2(x)+14V3(x), whereV1(x)=12∫0∞∫0∞WnW-n+1∏i=1nxi1Wun2-1exp-u2∑i=1nWxi2W-\|12-ln2π-(1-W)ψ(12)42-W2lnu\|dudW,V2(x)=∫0∞∫0∞WnW-n+2∏i=1nxi1Wun2-1exp-u2∑i=1nWxi2W-\|12-ln2π-(1-W)ψ(12)42-W2lnu\|dudW, andV3(x)=∫0∞∫0∞WnW-n+2∏i=1nxi1Wun2-1\|lnu\|exp-u2∑i=1nWxi2W-\|12-ln2π-(1-W)ψ(12)42-W2lnu\|dudW. SinceV1(x)≤12∫0∞∫0∞WnW-n+1∏i=1nxi1Wun2-1exp-u2∑i=1nWxi2WdudW,V2(x)≤∫0∞∫0∞WnW-n+2∏i=1nxi1Wun2-1exp-u2∑i=1nWxi2WdudW,V3(x)≤∫0∞∫0∞WnW-n+2∏i=1nxi1Wun2-1\|lnu\|exp-u2∑i=1nWxi2WdudW, one can easily show that V1(x) and V2(x) are finite, respectively for n≥3 and n≥4 by the same procedures of B(x) in (12). Also, similar to C(x) from (13), the quantity V3(x) is finite for n≥4. So, the proof is completed. □ By (41), it can be shown that the marginal MDI posterior of W can be rewritten as43 πMDI(W\|x)∝1Wn∏i=1nxi1W2∑i=1nxi2W12(n+W)exp{ψ(12)2(1-W)}×W22W∑i=1nxi2WWΓn-W2,12∑i=1n(Wxi)2Wexp{-ψ(12)2(1-W)}+Γn+W2FG1,12(n+W),12∑i=1n(Wxi)2W,W>0, where Γ(a,b) is the upper incomplete gamma function defined as Γ(a,b)=∫b∞ta-1e-tdt. Also, FG(a,b,c) is the CDF of gamma distribution with shape parameter b and rate parameter c, at point a. Moreover, the conditional MDI posterior of H can be obtained as44 πMDI(H\|W,x)∝exp-nHW-12δH,W2∑i=1nWxi2W-\|H\|,H∈(-∞,∞). Metropolis–Hastings Algorithm For the various non-informative priors discussed in the previous section, the joint posterior distributions of (H, W) are not tractable and the computation of the BEs will be impossible. Consequently, we opt for stochastic simulation procedures, namely, the Metropolis–Hastings (M–H) samplers, to generate samples from the posterior distributions. It is worth mentioning that the BEs of W are not interesting because it is used as an auxiliary parameter to conduct the Jacobian transformation. For each non-informative priors discussed early, the joint posterior distribution of (H, W) is decomposed into the marginal posterior distribution of W and the conditional posterior distribution of H given W=w. Based on this decomposition, the M–H algorithm is adopted to generate samples from the joint posterior distribution of (H, W). A non-negative random variable U is inverse gamma (IG) distributed if its PDF is given byg(u;β,λ)=λβΓ(β)1uβ+1exp{-λu}, where β>0 is the shape parameter and λ>0 is the scale parameter. Hereafter, it will be denoted by IG(β,λ). Let v=lnx(n)/x(1). In the ℓ-th iteration of M–H algorithm we choose IG(v/W(ℓ-1)+1,v) as a proposal distribution to generate random sample from the marginal posterior distribution of W. It is noteworthy that the shape and scale parameters of IG distribution are selected in a such way that the mean of IG is W(ℓ-1). We also choose the normal distribution, N(H(ℓ-1),σ2), as a proposal distribution to generate sample from the conditional posterior distribution of H. The mode of the marginal posterior distribution of W is used as initial value, say W(0). For example, let us consider the case of Jeffreys prior. On taking the natural logarithm of (20), differentiating with respect to W and equating to zero, we have the following non-linear equation:45 (n+1)W-n∑i=1nxi2Wlnxi∑i=1nxi2W+∑i=1nlnxi=0. The mode of posterior πJ(W\|x), say W~ can be computed by solving equation (45). Moreover, on taking the natural logarithm of (21), differentiating with respect to H and equating to zero, the initial value for H can be obtained asH~=12-ln2π-ψ(12)22(1-W~)-W~2lnn∑i=1nW~xi2W~. The initial values for W and H in the cases of reference, probability matching and MDI priors can be obtained in a similar way. The M–H algorithm needed to generates samples from (8) can be described as follows:Algorithm 1: M–H algorithm within Gibbs Step 1. Set W(0)=W~,H(0)=H~ and v=lnx(n)x(1). Step 2. Set ℓ=1. Step 3. Generate W∗ from the proposal distribution IGv/W(ℓ-1)+1,v. Step 4. Calculate the acceptance probability τ1=min{1,πJ(W∗\|x)gW(ℓ-1);v/W∗+1,vπJ(W(ℓ-1)\|x)gW∗;v/W(ℓ-1)+1,v}. Step 5. Generate U1 from the standard uniform distribution. Step 6. If U1≤τ1, accept W∗ and set W(ℓ)=W∗. Otherwise reject W∗ and set W(ℓ)=W(ℓ-1). Step 7. Generate H∗ from the proposal distribution N(H(ℓ-1),σ2). Step 8. Calculate the acceptance probability τ2=min{1,πJH∗\|W(ℓ),xπJH(ℓ-1)\|W(ℓ),x}. Step 9. Generate U2 from the standard uniform distribution. Step 10. If U2≤τ2, accept H∗ and set H(ℓ)=H∗. Otherwise reject H∗ and set H(ℓ)=H(ℓ-1). Step 11. Set ℓ=ℓ+1. Step 12. Repeat Steps 3-11, N times. Based on the resulting generated samples obtained using Algorithm 1, the BE of H under SEL function can be computed asH^J=1N-M∑ℓ=M+1NH(ℓ), where M is burn-in period. In addition, for constructing the credible interval (CrI) of H, we sort all the H(ℓ),ℓ=M+1,M+2,…,N, in an ascending sequence, as H(1),H(2),…,H(N-M). Then, for 0<ϵ<1, a 100(1-ϵ)% CrI of H is specified by(H(k),H(k+N-M-⌊(N-M)ϵ+1⌋)),k=1,2,…,⌊(N-M)ϵ⌋, where ⌊x⌋ is the largest integer less than or equal to x. Therefore, the 100(1-ϵ)% HPD CrI of H can be obtained as the k∗-th one satisfyingH(k∗+N-M-⌊(N-M)ϵ+1⌋)-H(k∗)≤H(k+N-M-⌊(N-M)ϵ+1⌋)-H(k), for all k=1,2,…,⌊(N-M)ϵ⌋. For the reference prior, probability matching prior and MDI prior, the BEs and HPD CrIs of H can be obtained in a similar way. Numerical Comparisons and Data Analysis Here we perform a comprehensive simulation study to examine the performances of the sample-based estimates developed in the previous sections and conduct the analysis of two practical data sets with GHN fitting distribution. All the computations are performed using R Software package (R x64 4.0.3) and the R code can be obtained on request from the authors. Numerical Comparisons Here, a simulation study is mainly conducted to compare the performance of MLEs and BEs of the differential entropy measure H under the different non-informative priors discussed in previous sections. For this purpose, we first briefly mention how to obtain the MLE of H. The natural logarithm of LF given in (2) may be written asℓ(H,W;x)∝nW-nlnW-nWH-12+ln2π+(1-W)ψ(12)22+1W∑i=1nlnxi-12δH,W2∑i=1nWxi2W. By setting the derivatives of ℓ(H,W;x) with respective to H and W to zero, the MLE of W, say W^, can be obtained from the equation U(W)=0, whereU(W)=W-∑i=1nxi2W(lnxi)∑i=1nxi2W+1n∑i=1nlnxi. Since U(W)=0 does not admit an explicit solution, we propose a simple iterative scheme to compute W^. Start with an initial guess of W, say W(0). Then, obtain W(1)=UW(0). Continue this process until convergence is achieved. Once W^ is obtained, H^ can be obtained as follows:46 H^=H(W^)=12-ln2π-(1-W^)ψ(12)22+lnW^-W^2lnn-ln∑i=1nxi2W^. Since the MLEs of H and W are not obtained in closed form, it is not possible to derive the exact distributions of the MLEs. Now, the asymptotic confidence interval (CI) for H is obtained based on the asymptotic distributions of the MLEs H^ and W^. Therefore, by applying the property of the asymptotic normality of MLEs, we have((H^-H),(W^-W))T→N2(0,V), where V is the asymptotic variance-covariance matrix of the MLEs H^ and W^. That is V=1nΣ-1(H^,W^). Consequently, the approximate 100(1-ϵ)% two sided CI for H is H^-zϵ/2V11,H^+zϵ/2V11, where 0<ϵ<1, zϵ/2 is the upper (ϵ/2)th percentile of the standard normal distribution andV11=W2nπ22-2π24-1+2η2-1W2. It is clearly noticed that as α changes for a fixed θ, the PDF and hazard rate function of GHN have different properties. Hence, a simulation study based on various shapes of the PDF and hazard rate function of GHN distribution was carried out. We consider α=0.5,0.75,1.5,2.17,3 and θ=0.25,2,5. According to Cooray and Ananda [11], for 0<α<2.17, GHN distribution is positively skewed and for α=2.17 and α>2.17 it is symmetric and negatively skewed, respectively. The hazard rate function of GHN decreases monotonically, concave up and approaches to 1/(2 scale parameter) as lifetime goes to ∞, for α=0.5. Also, it is bathtub shape for α=0.75. It increases monotonically, concave down and approaches ∞ as lifetime goes to ∞, for α=1.5. Moreover, for α=2.17,3 it increases monotonically, concave up and approaches ∞ as lifetime goes to ∞. To compute the BEs of the differential entropy measure H using the M–H algorithm, we considered σ=0.25 and also ran the iterative process up to N=5500 iterations by discarding the first M=500 iterations as burn-in-period. It was considered a thin parameter of 5 to diminish significant auto-correlation among sample draws. Moreover, to solve equation (45), Broyden Secant method of nleqslv package was used. The biases and mean squared errors (MSEs) of MLEs and BEs of the differential entropy measure H are computed for sample sizes n=10,20,30,40,50,70 using 5000 simulated samples for different combinations of α and θ and are given in Tables 1, 2, 3, 4 and 5. The 95% asymptotic CIs and HPD CIs for the differential entropy measure H were also obtained. The average lengths (ALs) and coverage probabilities (CPs) of the so obtained CrIs are presented in Tables 6, 7, 8, 9, and 10. These results for MLEs and BEs under priors, Jeffreys, reference, probability matching and maximal data information are shown in tables with labels H^, H^J, H^R, H^PM and H^MDI, respectively. Since the CI is constructed based on the asymptotic normality of the MLE H^, the corresponding AILs and CPs are reported for sample sizes greater than n=10. By taking θ to be fixed and changing the shape parameter α, we can observed from these tables that the BEs perform well when compared to the MLEs in the sense of bias and MSE criteria. The performance of all estimates behaves the same for large sample sizes. It is also observed that for α≤1, the BEs based on Jeffreys prior compete other BEs for small sample sizes. In this case, the BEs under MDI prior are the worse one. For α>1, all BEs perform similarly. It is evident that for α≤1, the HPDs under PM prior work well in terms of the AIL and for α>1, the CIs are shorter. By considering the CP criterion, the asymptotic CIs and HPDs are most valid intervals in the sense that the corresponding CPs tend to be high and close to the true confidence level 1-ϵ=0.95, especially for α>1. In Fig. 1, the performance of the proposed M–H algorithm under different posterior distributions of the differential entropy is monitoring by inspecting the value of average acceptance rate(AAR). Figure 1 provides a limited summary when the parameters are α=0.5,0.75,1.5,2.17,3 and θ=5. In the cases where α=0.5,0.75, the ARRs are very good and high for all sample sizes. In the cases of α=1.5,2.17,3 the ARRs lie within the intervals of 0.28-0.61, 0.27-0.53 and 0.21-0.45, respectively. For α=0.5,2.17,3, there is no a significant difference between the AARs of M–H algorithm when sample size n is greater than 10. Figure 1 also demonstrates how the ARR changes by changing sample size n. We observe that AAR decreases when sample size n increases. Three such plots would be required to consider full collections of AARs for all combinations of the parameters α and θ(θ=0.25,2) that are omitted for the brevity’s sake, but it is worth mentioning that we get similar results for them.Table 1 Biases and MSEs (between parentheses) of the MLE and BEs of H for α=0.5 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.6615 10 -0.0912 (0.2924) -0.0108 (0.2640) -0.0888 (0.2886) 0.0722 (0.2758) 0.0308 (0.1570) 20 -0.0423 (0.1433) -0.0129 (0.1372) -0.0580 (0.1460) 0.0226 (0.1420) -0.0162 (0.1201) 30 -0.0247 (0.0952) -0.0100 (0.0932) -0.0415 (0.0973) 0.0091 (0.0946) -0.0018 (0.0832) 40 -0.0145 (0.0726) -0.0061 (0.0718) -0.0300 (0.0744) 0.0072 (0.0724) 0.0067 (0.0654) 50 -0.0194 (0.0568) -0.0138 (0.0563) -0.0332 (0.0583) -0.0038 (0.0564) 0.0010 (0.0520) 70 -0.0129 (0.0423) -0.0101 (0.0422) -0.0243 (0.0432) -0.0032 (0.0423) 0.0037 (0.0402) 2 1.4179 10 -0.0966 (0.3032) -0.0148 (0.2709) -0.0932 (0.2978) 0.0659 (0.2860) -0.7666 (1.0992) 20 -0.0542 (0.1486) -0.0248 (0.1414) -0.0700 (0.1517) 0.0106 (0.1455) -0.2978 (0.2734) 30 -0.0321 (0.0967) -0.0174 (0.0944) -0.0490 (0.0990) 0.0021 (0.0951) -0.1948 (0.1533) 40 -0.0226 (0.0721) -0.0141 (0.0711) -0.0379 (0.0739) -0.0011 (0.0714) -0.1440 (0.1039) 50 -0.0173 (0.0571) -0.0121 (0.0566) -0.0313 (0.0586) -0.0019 (0.0569) -0.1138 (0.0772) 70 -0.0072 (0.0418) -0.0046 (0.0417) -0.0183 (0.0426) 0.0025 (0.0418) -0.0760 (0.0516) 5 2.3342 10 -0.0946 (0.3035) -0.0142 (0.2731) -0.0916 (0.2998) 0.0679 (0.2864) -1.0915 (2.2555) 20 -0.0448 (0.1498) -0.0154 (0.1431) -0.0601 (0.1520) 0.0195 (0.1478) -0.3055 (0.3104) 30 -0.0176 (0.0952) -0.0031 (0.0933) -0.0343 (0.0972) 0.0159 (0.0949) -0.1804 (0.1501) 40 -0.0221 (0.0707) -0.0134 (0.0696) -0.0377 (0.0725) -0.0008 (0.0699) -0.1442 (0.1030) 50 -0.0126 (0.0577) -0.0072 (0.0573) -0.0263 (0.0588) 0.0028 (0.0574) -0.1088 (0.0769) 70 -0.0113 (0.0404) -0.0087 (0.0403) -0.0224 (0.0413) -0.0015 (0.0402) -0.0798 (0.0504) Table 2 Biases and MSEs (between parentheses) of the MLE and BEs of H for α=0.75 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.6042 10 -0.1004 (0.1173) -0.0616 (0.1085) -0.0902 (0.1168) 0.0045 (0.1028) 0.0008 (0.0840) 20 -0.0459 (0.0551) -0.0314 (0.0536) -0.0478 (0.0565) 0.0528 (0.0631) -0.0036 (0.0496) 30 -0.0302 (0.0360) -0.0219 (0.0356) -0.0335 (0.0370) 0.0125 (0.0357) -0.0033 (0.0341) 40 -0.0259 (0.0267) -0.0203 (0.0265) -0.0294 (0.0274) 0.0065 (0.0267) -0.0076 (0.0259) 50 -0.0176 (0.0204) -0.0133 (0.0203) -0.0205 (0.0208) 0.0079 (0.0208) -0.0039 (0.0200) 70 -0.0141 (0.0147) -0.0113 (0.0147) -0.0164 (0.0151) -0.0029 (0.015) -0.0052 (0.0145) 2 1.4752 10 0.1100 (0.1237) -0.0705 (0.1137) -0.1007 (0.1234) -0.0048 (0.1070) -0.3476 (0.3350) 20 -0.0470 (0.0541) -0.0323 (0.0526) -0.0492 (0.0556) 0.0141 (0.0513) -0.1322 (0.0792) 30 -0.0346 (0.0359) -0.0264 (0.0354) -0.0382 (0.0369) 0.0080 (0.0352) -0.0896 (0.0467) 40 -0.0234 (0.0264) -0.0177 (0.0261) -0.0268 (0.0271) 0.0090 (0.0265) -0.0640 (0.0322) 50 -0.0219 (0.0218) -0.0174 (0.0217) -0.0247 (0.0223) 0.0036 (0.0220) -0.0542 (0.0258) 70 -0.0133 (0.0139) -0.0104 (0.0138) -0.0156 (0.0142) 0.0039 (0.0142) -0.0361 (0.0157) 5 2.3915 10 -0.1061 (0.1242) -0.0672 (0.1143) -0.0957 (0.1236) -0.0010 (0.1072) -0.4153 (0.5462) 20 -0.0479 (0.0563) -0.0336 (0.0549) -0.0505 (0.0578) 0.0127 (0.0535) -0.1353 (0.0863) 30 -0.0280 (0.0339) -0.0198 (0.0335) -0.0317 (0.0348) 0.0144 (0.0337) -0.0825 (0.0451) 40 -0.0237 (0.0263) -0.0180 (0.0261) -0.0270 (0.0269) 0.0086 (0.0264) -0.0641 (0.0320) 50 -0.0203 (0.0209) -0.0161 (0.0208) -0.0233 (0.0214) 0.0053 (0.0211) -0.0529 (0.0247) 70 -0.0147 (0.0144) -0.0119 (0.0144) -0.0170 (0.0147) 0.0022 (0.0146) -0.0373 (0.0163) Table 3 Biases and MSEs (between parentheses) of the MLE and BEs of H for α=1.5 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.8346 10 -0.1163 (0.0553) -0.0807 (0.0468) -0.0787 (0.0478) 0.0144 (0.0385) 0.0311 (0.0347) 20 -0.0533 (0.0201) -0.0345 (0.0181) -0.0325 (0.0183) 0.0227 (0.0175) 0.0120 (0.0158) 30 -0.0349 (0.0119) -0.0219 (0.0110) -0.0204 (0.0110) 0.0194 (0.0115) 0.0073 (0.0101) 40 -0.0263 (0.0086) -0.0164 (0.0080) -0.0153 (0.0081) 0.0149 (0.0086) 0.0049 (0.0075) 50 -0.0217 (0.0065) -0.0138 (0.0062) -0.0128 (0.0062) 0.0111 (0.0067) 0.0030 (0.0058) 70 -0.0159 (0.0045) -0.0103 (0.0043) -0.0094 (0.0043) 0.0054 (0.0047) 0.0017 (0.0041) 2 1.2449 10 -0.1191 (0.0567) -0.0834 (0.0480) -0.0817 (0.0491) 0.0111 (0.0397) -0.0772 (0.0392) 20 -0.0506 (0.0202) -0.0316 (0.0182) -0.0297 (0.0185) 0.0248 (0.0179) -0.0274 (0.0168) 30 -0.0335 (0.0121) -0.0205 (0.0112) -0.0191 (0.0113) 0.0208 (0.0118) -0.0172 (0.0109) 40 -0.0266 (0.0085) -0.0167 (0.0079) -0.0156 (0.0080) 0.0148 (0.0085) -0.0139 (0.0078) 50 -0.0196 (0.0063) -0.0118 (0.0060) -0.0107 (0.0060) 0.0133 (0.0066) -0.0090 (0.0060) 70 -0.0147 (0.0045) -0.0090 (0.0043) -0.0082 (0.0044) 0.0070 (0.0047) -0.0066 (0.0043) 5 2.1612 10 -0.1107 (0.0558) -0.0754 (0.0477) -0.0733 (0.0487) 0.0200 (0.0402) -0.0691 (0.0472) 20 -0.0557 (0.0208) -0.0369 (0.0187) -0.0347 (0.0188) 0.0203 (0.0178) -0.0279 (0.0167) 30 -0.0356 (0.0120) -0.0227 (0.0111) -0.0212 (0.0111) 0.019 (0.0115) -0.0155 (0.0102) 40 -0.0254 (0.0082) -0.0156 (0.0077) -0.0142 (0.0077) 0.0161 (0.0083) -0.0101 (0.0072) 50 -0.0218 (0.0067) -0.0140 (0.0063) -0.0129 (0.0063) 0.0108 (0.0068) -0.0092 (0.0060) 70 -0.0140 (0.0045) -0.0084 (0.0043) -0.0076 (0.0043) 0.0075 (0.0047) -0.0053 (0.0042) Table 4 Biases and MSEs (between parentheses) of the MLE and BEs of H for α=2.17 θ H n H^ H^J H^R H^PM H^MDI 0.25 -1.0609 10 -0.1186 (0.0568) -0.0751 (0.0460) -0.0796 (0.0487) 0.0321 (0.0566) 0.0779 (0.0335) 20 -0.0552 (0.0205) -0.0304 (0.0177) -0.0332 (0.0184) 0.0095 (0.0225) 0.0370 (0.0151) 30 -0.0346 (0.0123) -0.0169 (0.0111) -0.0190 (0.0114) 0.0009 (0.0127) 0.0261 (0.0102) 40 -0.0278 (0.0088) -0.0140 (0.0080) -0.0155 (0.0081) -0.0041 (0.0085) 0.0181 (0.0074) 50 -0.0218 (0.0068) -0.0104 (0.0062) -0.0117 (0.0064) -0.0037 (0.0065) 0.0150 (0.0059) 70 -0.0174 (0.0047) -0.0088 (0.0044) -0.0099 (0.0044) -0.0047 (0.0044) 0.0092 (0.0041) 2 1.0185 10 -0.1178 (0.0568) -0.0742 (0.0462) -0.0787 (0.0489) 0.0338 (0.0564) -0.0114 (0.0256) 20 -0.0567 (0.0209) -0.0320 (0.0179) -0.0347 (0.0187) 0.0085 (0.0230) -0.0041 (0.0127) 30 -0.0371 (0.0126) -0.0193 (0.0112) -0.0214 (0.0115) -0.0015 (0.0129) -0.0027 (0.0086) 40 -0.0259 (0.0086) -0.0120 (0.0079) -0.0136 (0.0080) -0.0023 (0.0084) -0.0005 (0.0062) 50 -0.023 (0.0069) -0.0116 (0.0063) -0.0129 (0.0064) -0.0049 (0.0065) -0.0026 (0.0050) 70 -0.0156 (0.0048) -0.0071 (0.0045) -0.0081 (0.0046) -0.0031 (0.0046) -0.0013 (0.0037) 5 1.9348 10 -0.1141 (0.055) -0.0706 (0.0446) -0.0752 (0.0474) 0.0367 (0.0557) -0.0278 (0.0407) 20 -0.0526 (0.0205) -0.0279 (0.0178) -0.0306 (0.0185) 0.0136 (0.0232) -0.0018 (0.0167) 30 -0.0396 (0.0126) -0.0219 (0.0111) -0.0240 (0.0114) -0.0046 (0.0126) -0.0037 (0.0105) 40 -0.0288 (0.0086) -0.0149 (0.0078) -0.0166 (0.0080) -0.0052 (0.0083) -0.0007 (0.0075) 50 -0.0233 (0.0068) -0.0118 (0.0062) -0.0132 (0.0063) 0.0052 (0.0064) -0.0004 (0.0060) 70 -0.0146 (0.0045) -0.0061 (0.0043) -0.0072 (0.0043) -0.0018 (0.0044) 0.0022 (0.0042) Table 5 Biases and MSEs (between parentheses) of the MLE and BEs of H for α=3 θ H n H^ H^J H^R H^PM H^MDI 0.25 -1.2963 10 -0.1139 (0.0644) -0.0628 (0.0523) -0.0730 (0.0547) 0.0015 (0.0702) 0.1439 (0.0524) 20 -0.0537 (0.0232) -0.0227 (0.0198) -0.0289 (0.0202) -0.0113 (0.0215) 0.0735 (0.0201) 30 -0.0360 (0.0138) -0.0128 (0.0121) -0.0171 (0.0123) -0.0077 (0.0124) 0.0503 (0.0124) 40 -0.0274 (0.0098) -0.0088 (0.0088) -0.0119 (0.0088) -0.0054 (0.0089) 0.0392 (0.0091) 50 0.0071 (0.0079) -0.0063 (0.0071) -0.0088 (0.0072) -0.0037 (0.0072) 0.0325 (0.0074) 70 -0.0155 (0.0053) -0.0035 (0.0049) -0.0053 (0.0050) -0.0017 (0.0050) 0.0247 (0.0051) 2 0.7831 10 -0.1194 (0.0632) -0.068 (0.0506) -0.0783 (0.0531) -0.0064 (0.0664) 0.0530 (0.0301) 20 -0.0564 (0.0234) -0.0254 (0.0198) -0.0314 (0.0204) -0.0141 (0.0216) 0.0391 (0.0156) 30 -0.0372 (0.0139) -0.0140 (0.0122) -0.0181 (0.0124) -0.0089 (0.0125) 0.0317 (0.0108) 40 -0.0274 (0.0104) -0.0089 (0.0094) -0.0118 (0.0095) -0.0053 (0.0095) 0.0270 (0.0089) 50 -0.0225 (0.0079) -0.0068 (0.0072) -0.0093 (0.0073) -0.0042 (0.0073) 0.0226 (0.0070) 70 -0.0156 (0.0054) -0.0035 (0.0050) -0.0053 (0.0051) -0.0016 (0.0051) 0.0180 (0.0051) 5 1.6994 10 -0.1179 (0.0618) -0.0665 (0.0495) -0.0771 (0.0520) -0.0032 (0.0660) -0.0201 (0.0470) 20 -0.0554 (0.0230) -0.0245 (0.0195) -0.0304 (0.0200) -0.0131 (0.0212) 0.0020 (0.0204) 30 -0.0410 (0.0147) -0.0178 (0.0127) -0.0219 (0.0129) -0.0127 (0.0130) 0.0015 (0.0128) 40 -0.0271 (0.0101) -0.0085 (0.0091) -0.0116 (0.0092) -0.0049 (0.0092) 0.0065 (0.0090) 50 -0.0223 (0.0080) -0.0066 (0.0073) -0.0092 (0.0074) -0.0040 (0.0074) 0.0054 (0.0072) 70 -0.0164 (0.0056) -0.0043 (0.0051) -0.0062 (0.0052) -0.0025 (0.0052) 0.0040 (0.0051) Table 6 AILs and CPs (between parentheses) of 95% CI/HPD of H for α=0.5 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.6615 10 1.8939 (90.66) 2.0878 (93.30) 1.6845 (82.82) 2.6418 (96.96) 20 1.4129 (92.62) 1.3747 (92.42) 1.4420 (93.34) 1.3229 (89.16) 1.5007 (95.62) 30 1.1637 (93.24) 1.1283 (92.56) 1.1649 (92.96) 1.1076 (91.12) 1.1820 (94.68) 40 1.019 (93.54) 0.9884 (92.66) 1.0127 (93.48) 0.9774 (91.80) 1.0159 (94.20) 50 0.9173 (94.32) 0.8912 (93.36) 0.9080 (93.84) 0.8844 (93.20) 0.9073 (94.76) 70 0.7818 (94.10) 0.7641 (93.46) 0.7737 (93.60) 0.7595 (93.16) 0.7694 (93.92) 2 1.4179 10 1.9039 (90.84) 2.0952 (93.02) 1.6946 (83.08) 3.1302 (91.98) 20 1.413 (91.76) 1.3737 (91.78) 1.4410 (92.72) 1.3221 (88.98) 1.5090 (87.46) 30 1.1774 (93.58) 1.1386 (93.02) 1.1774 (93.80) 1.1200 (91.80) 1.2191 (89.74) 40 1.0244 (93.60) 0.9924 (93.10) 1.0170 (93.26) 0.9825 (92.20) 1.0491 (90.74) 50 0.9175 (94.14) 0.8926 (93.24) 0.9084 (93.66) 0.8846 (93.06) 0.9340 (91.76) 70 0.7809 (94.36) 0.7630 (93.66) 0.7730 (93.58) 0.7589 (93.12) 0.7891 (92.34) 5 2.3342 10 1.8947 (90.40) 2.0885 (92.82) 1.6823 (83.20) 3.4601 (79.92) 20 1.4132 (91.66) 1.3735 (91.40) 1.4414 (92.60) 1.3226 (88.58) 1.5468 (87.08) 30 1.1676 (93.32) 1.1320 (92.62) 1.1682 (93.46) 1.1131 (91.72) 1.2197 (90.74) 40 1.0239 (94.04) 0.9931 (93.40) 1.0171 (93.90) 0.9806 (92.52) 1.0512 (91.64) 50 0.9163 (94.14) 0.8914 (93.48) 0.9081 (93.88) 0.8829 (92.96) 0.9330 (92.02) 70 0.7788 (94.20) 0.761 (93.52) 0.7717 (93.78) 0.7571 (93.56) 0.7876 (92.46) Table 7 AILs and CPs (between parentheses) of 95% CI/HPD of H for α=0.75 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.6042 10 1.2684 (92.22) 1.4025 (93.90) 1.0404 (87.16) 1.6319 (97.06) 20 0.8413 (91.88) 0.8884 (93.44) 0.9339 (93.92) 0.7625 (88.34) 0.9587 (95.24) 30 0.6980 (92.68) 0.7243 (93.44) 0.7481 (93.90) 0.6459 (88.86) 0.7596 (94.88) 40 0.6118 (93.28) 0.6289 (94.04) 0.6452 (94.34) 0.5768 (90.22) 0.6517 (94.90) 50 0.5474 (94.06) 0.5595 (94.22) 0.5713 (94.44) 0.5239 (91.52) 0.5762 (95.08) 70 0.4652 (94.18) 0.4727 (94.20) 0.4796 (94.78) 0.4532 (92.20) 0.4827 (94.90) 2 1.4752 10 1.2776 (92.18) 1.4142 (93.92) 1.0479 (86.78) 1.7182 (90.82) 20 0.8461 (92.74) 0.8927 (94.38) 0.9390 (95.10) 0.7681 (89.00) 0.9778 (92.82) 30 0.6991 (93.10) 0.7258 (94.02) 0.7500 (94.62) 0.6469 (89.12) 0.7718 (93.18) 40 0.6091 (93.48) 0.6254 (93.84) 0.6421 (94.32) 0.5741 (90.32) 0.6569 (93.56) 50 0.5485 (93.66) 0.5607 (94.06) 0.5718 (94.40) 0.5253 (91.02) 0.5835 (93.70) 70 0.4650 (94.90) 0.4725 (95.00) 0.4795 (95.42) 0.4534 (93.24) 0.4865 (95.04) 5 2.3915 10 1.2687 (91.52) 1.4047 (93.68) 1.0432 (87.30) 1.8608 (90.20) 20 0.849 (91.72) 0.8956 (93.20) 0.9427 (93.94) 0.7722 (88.00) 0.9830 (92.48) 30 0.6975 (93.86) 0.7236 (94.74) 0.7490 (95.38) 0.6455 (90.50) 0.7689 (93.58) 40 0.6086 (93.26) 0.6254 (93.82) 0.6421 (94.24) 0.5732 (90.10) 0.6557 (93.40) 50 0.5498 (94.18) 0.5622 (94.40) 0.5737 (94.34) 0.5264 (91.56) 0.5845 (93.94) 70 0.4644 (94.46) 0.4723 (94.64) 0.4785 (94.98) 0.4530 (93.08) 0.4856 (94.56) Table 8 AILs and CPs (between parentheses) of 95% CI/HPD of H for α=1.5 θ H n H^ H^J H^R H^PM H^MDI 0.25 -0.8346 10 0.7421 (90.78) 0.7779 (91.50) 0.8559 (97.08) 0.8714 (97.56) 20 0.4754 (90.62) 0.4997 (93.84) 0.5092 (94.14) 0.5772 (96.80) 0.5342 (96.48) 30 0.3847 (92.26) 0.4007 (94.76) 0.4053 (95.04) 0.4593 (96.46) 0.4184 (96.38) 40 0.3320 (92.60) 0.3443 (94.88) 0.3470 (94.94) 0.3892 (96.78) 0.3555 (96.22) 50 0.2965 (92.90) 0.3062 (94.64) 0.3084 (94.88) 0.3419 (96.08) 0.3145 (96.10) 70 0.2496 (93.44) 0.2568 (94.74) 0.2579 (95.20) 0.2781 (95.84) 0.2617 (95.72) 2 1.2449 10 0.7438 (90.68) 0.7793 (90.90) 0.8547 (96.90) 0.7684 (92.54) 20 0.4756 (90.62) 0.4998 (93.58) 0.5092 (93.62) 0.5769 (96.74) 0.5019 (93.76) 30 0.3850 (91.42) 0.4006 (94.18) 0.4054 (94.40) 0.4597 (96.50) 0.4017 (94.16) 40 0.3322 (92.56) 0.3443 (94.84) 0.3469 (94.76) 0.3896 (96.74) 0.3451 (94.66) 50 -0.2966 (93.48) 0.3065 (94.72) 0.3086 (94.92) 0.3423 (96.42) 0.3074 (94.84) 70 0.2498 (93.58) 0.2572 (94.72) 0.2584 (94.76) 0.2791 (95.52) 0.2578 (94.80) 5 2.1612 10 0.7431 (91.44) 0.7793 (91.52) 0.8551 (96.88) 0.7869 (92.80) 20 0.4751 (89.68) 0.4994 (93.04) 0.5089 (93.44) 0.5776 (96.76) 0.5033 (94.20) 30 0.3848 (91.66) 0.4008 (94.28) 0.4050 (94.48) 0.4599 (96.78) 0.4019 (95.20) 40 0.3323 (92.88) 0.3442 (94.72) 0.3472 (94.96) 0.3898 (96.48) 0.3440 (95.16) 50 0.2965 (92.82) 0.3060 (94.78) 0.3079 (94.68) 0.3418 (96.06) 0.3063 (95.12) 70 0.2501 (93.54) 0.2570 (95.06) 0.2584 (94.92) 0.2789 (96.02) 0.2569 (95.18) Table 9 AILs and CPs (between parentheses) of 95% CI/HPD of H for α=2.17 θ H n H^ H^J H^R H^PM H^MDI 0.25 -1.0609 10 0.7082 (91.74) 0.7181 (91.30) 0.8747 (93.90) 0.7533 (97.06) 20 0.4790 (91.76) 0.4981 (94.64) 0.5011 (94.14) 0.5645 (94.10) 0.4790 (91.76) 30 0.3879 (92.84) 0.4099 (95.42) 0.4113 (95.00) 0.4375 (95.00) 0.4112 (95.62) 40 0.3349 (93.32) 0.3578 (96.06) 0.3586 (96.02) 0.3701 (95.68) 0.3582 (96.02) 50 -0.2986 (93.48) 0.3209 (96.00) 0.3217 (96.02) -0.3281 (96.24) 0.3214 (96.50) 70 0.2520 (094.00) 0.2734 (96.38) 0.2741 (96.20) 0.2774 (96.32) 0.2736 (96.38) 2 1.0185 10 0.7081 (91.44) 0.7191 (90.98) 0.8772 (93.84) 0.6543 (96.80) 20 0.4795 (91.50) 0.4990 (94.28) 0.5014 (94.16) 0.5656 (93.98) 0.4664 (96.80) 30 0.3883 (92.46) 0.4105 (95.68) 0.4115 (95.18) 0.4375 (95.18) 0.3881 (96.90) 40 0.3349 (93.68) 0.3577 (96.06) 0.3578 (95.88) 0.3698 (96.14) 0.3399 (97.10) 50 0.2988 (93.66) 0.3212 (95.88) 0.3219 (96.06) 0.3282 (96.20) 0.3063 (97.22) 70 0.2519 (93.56) 0.2731 (96.08) 0.2737 (96.08) 0.2767 (96.00) 0.2620 (97.22) 5 1.9348 10 0.7093 (92.28) 0.7194 (91.20) 0.8753 (93.60) 0.6782 (93.18) 20 0.4788 (91.82) 0.4983 (94.08) 0.5014 (93.82) 0.5663 (93.68) 0.4802 (94.22) 30 0.3885 (92.52) 0.4107 (95.10) 0.4115 (94.76) 0.4371 (95.10) 0.3995 (95.10) 40 0.3350 (93.72) 0.3578 (96.02) 0.3588 (95.96) 0.3703 (96.06) 0.3504 (95.72) 50 0.2989 (93.50) 0.3218 (95.98) 0.3224 (95.76) 0.3285 (96.10) 0.3159 (95.84) 70 0.2517 (93.96) 0.2730 (96.10) 0.2737 (95.74) 0.2766 (96.02) 0.2697 (95.84) Table 10 AILs and CPs (between parentheses) of 95% CI/HPD of H for α=3 θ H n H^ H^J H^R H^PM H^MDI 0.25 -1.2963 10 0.7391 (91.14) 0.7379 (90.62) 0.8660 (88.98) 0.7561 (90.30) 20 0.5202 (92.46) 0.5365 (94.68) 0.5353 (94.10) 0.5558 (94.48) 0.5295 (93.08) 30 0.4224 (93.40) 0.4496 (96.02) 0.4486 (95.98) 0.4552 (96.08) 0.4449 (95.08) 40 0.3649 (93.76) 0.3959 (96.36) 0.3949 (95.98) 0.3990 (96.20) 0.3930 (95.58) 50 0.326 (93.98) 0.3585 (96.38) 0.3583 (96.50) 0.3608 (96.30) 0.3563 (95.66) 70 0.2749 (94.16) 0.3071 (96.98) 0.3068 (96.80) 0.3085 (96.90) 0.3057 (96.36) 2 0.7831 10 0.7397 (91.58) 0.7393 (91.16) 0.8645 (90.32) 0.6616 (94.56) 20 0.5206 (92.46) 0.5368 (94.66) 0.5359 (94.32) 0.5557 (94.56) 0.5019 (94.64) 30 0.4227 (93.42) 0.4496 (95.86) 0.4488 (95.76) 0.4549 (96.06) 0.4300 (95.46) 40 0.3650 (93.16) 0.3957 (95.96) 0.3951 (96.18) 0.3991 (95.78) 0.3842 (95.36) 50 0.3259 (94.10) 0.3581 (96.68) 0.3576 (96.56) 0.3599 (96.74) 0.3509 (95.88) 70 0.2749 (93.86) 0.3071 (96.52) 0.3069 (96.60) 0.3083 (96.36) 0.3037 (95.90) 5 1.6994 10 0.7384 (92.04) 0.7383 (91.32) 0.8644 (90.56) 0.6835 (88.22) 20 0.5205 (92.60) 0.5362 (94.52) 0.5355 (94.66) 0.5554 (94.70) 0.5150 (92.36) 30 0.4233 (92.86) 0.4508 (95.40) 0.4501 (95.40) 0.4564 (95.76) 0.4369 (94.58) 40 0.3651 (93.78) 0.3965 (96.60) 0.3957 (96.24) 0.3994 (96.46) 0.3854 (95.62) 50 0.3260 (93.84) 0.3579 (96.24) 0.3577 (96.44) 0.3610 (96.20) 0.3501 (95.86) 70 0.2750 (93.92) 0.3073 (96.66) 0.3073 (96.64) 0.3087 (96.56) 0.3017 (96.14) Data Analysis In this subsection, the proposed methods are illustrated by two real data sets. For both data sets, we apply Algorithm 1 to generate samples from the posterior densities and then compute the BEs. The simulation process is repeated 5500 iterations and initial 500 iterations are discarded as a burn-in period. Data Set 1: First we discuss the analysis of real life data representing the lifetimes of Kevlar 49/epoxy strands which were subjected to constant sustained pressure at the 90% stress level until all had failed. The data set was initially reported by Barlow et al. [31]. It has been analyzed by many authors, among them, Cooray and Ananda [11] and Ahmadi et al. [13]. The failure times in hours are as follows:Fig. 1 Average acceptance rate of M–H algorithm under different posterior distributions of the differential entropy: πJ(H\|x),πR(H\|x),πPM(H\|x),πMDI(H\|x) Fig. 2 Auto-correlation graphs and trace plots of M–H algorithm under different posterior distributions of the differential entropy: πJ(H\|x),πR(H\|x),πPM(H\|x),πMDI(H\|x) Based on the above observed data, we obtain the MLE of H as well as the corresponding BEs under Jeffreys, Reference, probability matching and maximal data information priors. The MLEs of H is computed numerically using Broyden Secant method to be H^=0.9867. The asymptotic 95% CI of H is (0.777, 1.196). We have generated 5500 observations to compute the BEs of H based on M–H samplers after discarding the initial 500 burn-in samples. Note that, for computing BEs and CrIs, we assume that the above four objective priors. The M–H algorithm with inverse gamma and normal proposal distributions(see Algorithm 1) is used to generate numbers from the target probability distribution. Here, we assume the initial value of H to be its MLE, H^. To check the convergence of M–H algorithm, graphical diagnostics tools involving the auto-correlation function (ACF) and trace plots are used. Figure 2 displays ACF and trace plots of the simulated draws of H, under different posterior distributions. It is checked that the ACF plots show that chains are dissipating rapidly from sampling lag 8, for all posterior distributions. Hence, to eliminate high auto-correlations, we considered a thinning interval equal to 8 iterations. It is evident that the chains have very low autocorrelations. From the trace plots, we can easily observe a random scatter about some mean value represented by a solid line with a fine mixing of the chains for the simulated values of H. As a result, these plots indicate the rapid convergence of the M–H algorithm based on the proposed normal distribution of H. The results of the BEs of H using M–H samplers under different objective priors are computed to beH^J=0.9815,H^R=0.9871,H^PM=0.9997,H^MDI=0.9745. and the corresponding 95% HPD CrIs of H under Jeffreys, reference, PM and MDI priors are also obtained and computed, respectively, as(0.769,1.183),(0.789,1.199),(0.777,1.211),(0.745,1.210). Further, Geweke diagnostics (Geweke [32]) are used to confirm the convergence of chains under a confidence level of 95%. Its values under the difference priors are, respectively,GJ=0.8352,GR=1.9351,GPM=0.4349,GMDI=0.3041. Since the differential entropy of a system shows the degree of irregularity and randomness of that system, the small values of Bayesian point estimation of entropy H, H^J=0.9815 and H^PM=0.9997 with 95% HPD CrIs (0.769, 1.183) and (0.777, 1.211) demonstrate good performance and stability in the production system. Data Set 2: The COVID-19 pandemic has affected almost every country in the world. The monthly mean number of deaths from the pandemic of coronavirus disease in Iran since March, 1, 2020 till August, 29, 2021, based on the National Vital Statistics System (https://ourworldindata.org/coronavirus/country/iran) are as Fig. 3 Auto-correlation graphs and trace plots of M–H algorithm under different posterior distributions of the differential entropy: πJ(H\|x),πR(H\|x),πPM(H\|x),πMDI(H\|x) The Ljung-Box test is used to ensure that observations over time are independent. The values of its chi-squared statistic with one degree of freedom and p-value measure are 1.4709 and 0.2252, respectively. The p-value indicates that observations over time are independent. Here, we show that the GHN distribution is a correct fitting distribution. The MLEs of the unknown parameters α and θ are computed numerically to be α^=1.22019 and θ^=251.704. The Kolmogorov-Smirnov (K-S) distance between the fitted and the empirical distribution functions is K-S=0.1355, and the corresponding p value is 0.8527. Therefore, these values indicate that the two-parameter GHN distribution fits the data set well. The MLE of differential entropy measure is H^=6.1803 and the corresponding asymptotic 95% CI of H is (5.908, 6.453). Based on 5500 iterations with discarding a burn-in period of 500 iterations, the ACF and trace plots of the generated samples using M–H algorithm are displayed in Figure 3. Under four posterior distributions, a thinning interval equal to 11 iterations was considered to obtain independent samples. From these plots, a rapid convergence of the M–H algorithm based on the proposed normal distribution can be observed clearly. For this, the BEs and 95% HPD CrIs under Jeffreys, reference, PM and MDI priors using M–H algorithm are given byH^J=6.2011,H^R=6.2038,H^PM=6.2165,H^MDI=6.1797. and(5.892,6.442),(5.877,6.487),(5.966,6.564),(5.710,6.561). The Geweke diagnostics are also computed, respectively, 0.4354, 0.2235, 1.2793, 1.2982. It worth mentioning that the point BEs H and corresponding CrIs tend to be relatively high. This shows that the available information content in this data is low. Therefore, it is suggested to further monitor the daily deaths for necessary decisions and actions. Discussion and Conclusion In the previous sections, we obtained the objective priors when H was the parameter of interest and W was a nuisance parameter. Now, by transforming back (H, W) into original parameters (α,θ), the Jeffreys prior (7), reference prior (23) and probability matching prior (31) are respectively equivalent to47 π∗J(α,θ)∝1θ, 48 π∗R(α,θ)∝1αθπ24-1+212-α212, 49 π∗PM(α,θ)∝1αθ(2α-η)π24-1+2η2-α212α>2η1αθ(η-2α)π24-1+2η2-α2120<α<η2. A natural question is : “Are the priors (47)–(49) respectively Jeffreys, reference and probability matching priors for the original parameters (α,θ) of GHN distribution?” To answer this question, we first consider the following theorem. Theorem 11 Assume that α and θ are the parameters of interest. The Jeffreys prior for the parameters of GHN distribution is π∗∗J(α,θ)∝1θ. The probability matching prior for the parameters of GHN distribution is π∗∗PM(α,θ)∝1αθ. The reference prior for the parameters of GHN distribution is π∗∗R(α,θ)∝1αθ. Proof (i): It is sufficient to calculate the square root of the determinant of I(α,θ) as is reported in (3). (ii): Assume α is the parameter of interest and θ is a nuisance parameter. The second-order probability matching prior παPM(α,θ) can be obtained by solving the partial differential equation50 ∂∂θI12παPM(α,θ)I22Q-∂∂απαPM(α,θ)Q=0, whereQ=I22-1det(I(α,θ))=12α2π22-2. Equation (50) becomes51 ∂∂θθπαPM(α,θ)+2α2-ln2-γ∂∂ααπαPM(α,θ)=0. Then, by applying Lemma A.1 of Sun [33], it follows that52 παPM(α,θ)=1αθh12lnθ2-ln2-γ+1α, where h1(.) is some continuously differentiable function. Similarly, when θ is the parameter of interest and α is a nuisance parameter, the second-order probability matching prior πθPM(α,θ) is the solution of the partial differential equation53 ∂∂θθπθPM(α,θ)+2(2-ln2-γ)π22-2+(2-ln2-γ)2∂∂ααπθPM(α,θ)=0. It follows that54 πθPM(α,θ)=1αθh22(2-ln2-γ)lnθπ22-2+(2-ln2-γ)2+1α, where h2(.) is some continuously differentiable function. Now, we assume that α and θ are simultaneously the parameters of interest. Since second-order jointly matching prior πPM(α,θ) should satisfy two Eqs. (51) and (53), it follows that h1(.)=h2(.)≡1 in (52) and (54). Now, let I-1(α,θ)=(κij) be the inverse of the expected Fisher information I(α,θ). Define M=(mij), where mij=κijκiiκjj,i,j=1,2. We get m11=m22=1 andm12=m21=(2-ln2-γ)π22-2+(2-ln2-γ)2-12. According to Datta [34], since matrix M does not depend on α and θ it follows that π∗∗PM(α,θ)∝1αθ is the probability matching prior for the parameters of GHN distribution. (iii) See Remark 3 of Kang et al. [12]. □ It is observed that the prior in (47) is Jefferys prior for the original parameters (α,θ), i.e. π∗J(α,θ)=π∗∗J(α,θ). This is to be expected, because the Jefferys prior is invariant with respect to one-to-one transformation. Part (ii) of Theorem 11 implies that π∗PM in (49) is not the probability matching prior for the GHN distribution parameters. For sufficient small and large α, π∗PM are only equivalent to π∗∗PM. According to part (iii) of Theorem 11, it is obvious that π∗R in (48) is not the reference prior for the original parameters (α,θ) of GHN distribution. For sufficient large α, π∗R and π∗∗R are approximately similar and for sufficient small α, π∗R converges to π∗J. Thus, it is recommended to adopt the objective priors which obtained directly instead of non-informative priors based on the original parameters of GHN distribution in order to compute the BEs of the differential entropy H. In addition, based on the simulation results in Sect. 5, it can be said that such BEs are better than MLE in terms of biases and MSEs. In this paper, we have discussed the estimation of differential entropy of the GHN distribution in a complete sample case. However in many practical situations, due to time constraint and cost reduction, censored samples may arise naturally. There are various schemes of censoring such as Type-I censoring, Type-II censoring, hybrid or progressive censoring. It may be mentioned, although we have provided the results mainly for complete samples but our method can be applied for other censoring mechanism also. More work is needed along these directions. Additionally, one may extend the work to include other non-informative priors such as copula prior, moment matching prior, and etc. Acknowledgements The authors are grateful to the editor and two referees for providing helpful comments and suggestions which led to the improvement of this paper. Funding The authors advise no direct funding is associated with the research reported on this article. Declarations Conflict of interest No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Shannon CE A mathematical theory of communication Bell Syst. Tech. 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==== Front J Cancer Surviv J Cancer Surviv Journal of Cancer Survivorship 1932-2259 1932-2267 Springer US New York 36454519 1301 10.1007/s11764-022-01301-0 Article Associations among hearing loss, multiple co-occurring symptoms, and quality of life outcomes in cancer survivors Miaskowski Christine [email protected] 12 Mastick Judy 2 Paul Steven 2 Wallhagen Margaret 2 Abrams Gary 1 Levine Jon D. 1 1 grid.266102.1 0000 0001 2297 6811 School of Medicine, University of California, San Francisco, CA USA 2 grid.266102.1 0000 0001 2297 6811 School of Nursing, University of California, 2 Koret Way – N631Y, San Francisco, CA 94143-0610 USA 1 12 2022 110 9 9 2022 11 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose Evaluate for differences in demographic and clinical characteristics, occurrence of common symptoms, symptom severity scores, and quality of life (QOL) outcomes in survivors with (n = 155) and without (n = 118) audiometrically confirmed hearing loss. Methods Survivors, who were recruited from throughout the San Francisco Bay area, completed the self-report questionnaires to obtain the information of demographic and clinical characteristics; the occurrence and severity of depression, anxiety, fatigue, decrements in energy, sleep disturbance, pain, and cognitive impairment; and the general and cancer-specific QOL outcomes. Parametric and non-parametric tests were used to evaluate for differences between the two survivor groups. Results Survivors with audiometrically confirmed hearing loss were older, more likely to be male, were more likely to be unemployed, report a lower annual household income, and had a higher comorbidity burden. Except for the severity of worst pain, no between-group differences were found in the occurrence rates for or severity of any of the symptoms. Survivors with hearing loss reported worse physical function and general health scores. Conclusions While no between-group differences in symptom occurrence rates and severity scores were found, across the total sample, a relatively high percentage of survivors who were over 6 years from their cancer diagnosis reported clinically meaningful levels of depression (25%), anxiety (50%), fatigue (40%), decrements in energy (70%), sleep disturbance (58%), cognitive impairment (57%), and pain (60%). Implications for Cancer Survivors Clinicians need to perform routine assessments of hearing loss, as well as common co-occurring symptoms and initiate individualized symptom management interventions. Keywords Cancer Chemotherapy Depression Fatigue Hearing loss Patient-reported outcomes Quality of life Sleep disturbance Symptoms http://dx.doi.org/10.13039/100007316 Division of Cancer Prevention, National Cancer Institute CA212064 Miaskowski Christine ==== Body pmcIntroduction With an estimated 18 million cancer survivors living in the USA [1], an evaluation of symptoms that can effect these individuals’ ability to work, engage in social activities, and experience optimal levels of physical and psychological functioning is of paramount importance. For the majority of survivors who received chemotherapy for breast, gastrointestinal, gynecological, or lung cancer, neurotoxic agents (i.e., platinum and/or taxane compounds) were administered. While chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse effect of neurotoxic chemotherapy [2], emerging evidence suggests that hearing loss is equally problematic. Studies of hearing loss in oncology patients have focused primarily on children who received platinum [3]. The limited amount of research in adults has evaluated for hearing loss in patients treated with platinum compounds for testicular [4–9] and head and neck [10–12] cancers. However, in our first cross-sectional study of cancer survivors with breast, gastrointestinal, gynecologic, or lung cancers [13], of the 371 survivors who had objectively confirmed CIPN, 41.5% self-reported hearing loss. Compared to the survivors with only CIPN, those with hearing loss had higher state and trait anxiety scores. Given the underestimation of hearing loss by self-report [14], in our recent study of survivors with breast, gastrointestinal, gynecologic, or lung cancer who received either a platinum- and/or a taxane-containing chemotherapy regimen [15], hearing loss was confirmed audiometrically. While only 32.9% of the 273 survivors in this study self-reported hearing loss, between 52.3 and 71.4% had hearing loss confirmed with an audiogram. Of note, no statistically significant differences in the occurrence rates for and effects of hearing loss were found among the three chemotherapy regimens (i.e., only platinum, only taxane, both platinum and taxane). While our findings warrant confirmation, this study is the first to provide evidence that audiometrically confirmed hearing loss occurs in a large percentage of survivors with four of the most common solid tumors. On average, cancer survivors report nine co-occurring symptoms [16]. Some of the most common symptoms include depression, anxiety, sleep disturbance, fatigue, cognitive impairment, and pain [17–19]. While not studied in oncology patients, in the general population, recent evidence suggests that hearing loss is associated with higher levels of depression [20, 21], anxiety [22], cognitive impairment [23], sleep disturbance [24], and fatigue [25, 26]. Therefore, it is reasonable to hypothesize that compared to survivors without hearing loss, cancer survivors with hearing loss would report higher levels of these common symptoms. Given the paucity of research on associations between hearing loss and symptoms and QOL outcomes in cancer survivors, the purposes of this study were to evaluate for differences in demographic and clinical characteristics, occurrence of common symptoms, symptom severity scores, and QOL outcomes in survivors with (n = 155) and without (n = 118) audiometrically confirmed hearing loss. Patients and methods Survivors and settings This study is part of a larger study that evaluated for hearing loss, tinnitus, and CIPN in cancer survivors who received neurotoxic chemotherapy. Survivors were recruited from throughout the San Francisco Bay area using a variety of recruitment strategies (e.g., investigator registry, clinician referral, medical record review, emails to participants in the Dr. Susan Love Foundation’s Love Research Army® Program). Survivors with and without CIPN were ≥ 18 years of age; had received a platinum and/or a taxane compound; had a Karnofsky Performance Status (KPS) score of ≥ 50 [27]; were able to read, write, and understand English; and were willing to complete questionnaires that took 90 to 150 min over 2 weeks and travel to UCSF for a 3-h study visit. For the CIPN evaluation, survivors with and without CIPN were excluded if they had peripheral vascular disease, vitamin B12 deficiency, thyroid dysfunction, HIV neuropathy, another condition that was difficult for them to distinguish from their CIPN, a hereditary sensory or autonomic neuropathy [28], and/or a hereditary mitochondrial disorder [29]. For the hearing and tinnitus evaluation, survivors were excluded if they had tinnitus of > 8 on a 0 to 10 numeric rating scale prior to chemotherapy; had hearing loss prior to chemotherapy that prevented understanding a one-to-one conversation; had a history of vestibular schwannoma; had radiation to head or neck; or had diagnosis of cancer to the brain. A detailed history was obtained to evaluate for the presence of these conditions. Of the 1012 survivors who were screened (primary reason for ineligibility was not meeting the inclusion criteria for the CIPN portion of the study), 365 were enrolled and 273 completed the self-report questionnaires and the study visit. Visit completions were interrupted by the COVID-19 pandemic. Study procedures Survivors communicated their willingness to participate in the study by phone or email. Research staff phoned survivors and determined their eligibility to participate. For survivors who met our inclusion criteria, the research nurse or audiologist obtained consent over the phone; asked the survivors to complete the self-report questionnaires prior to their study visit either electronically or by hard copy; and scheduled the study visit. During the study visit, the research staff obtained written informed consent, reviewed the study questionnaires for completeness, and performed the audiometric testing. The study visit was conducted by research nurses and audiologists in a large, dedicated research space that contained all the necessary equipment to conduct the study procedures including a double-walled sound-treated unit for hearing testing. Measures Demographic and clinical characteristics Survivors completed a demographic questionnaire, the KPS scale [27], and the Self-Administered Comorbidity Questionnaire (SCQ) [30]. Survivors were interviewed to obtain information on their cancer diagnosis, previous and current cancer treatments, and chemotherapy regimens. Medical records were reviewed for detailed information on cancer diagnosis, previous cancer treatments, and chemotherapy regimens. Audiometric testing Prior to the audiometric assessment, survivors underwent video otoscopy (Teslong, Irvine, CA) and tympanometry (Titan, Interacoustics, Eden Prairie, MN). Pure tone air conduction thresholds were obtained bilaterally at frequencies of between 0.25 and 16.0 kHz covering the speech frequency range. An audiometer (Pello Interacoustics, Eden Prairie, MN), with insert earphones, that utilized the GSI-AMTAS automated threshold assessment (Grayson-Sadler, Eden Prairie, MN) was used to perform the audiometric assessment [31]. A bone oscillator, insert earphones, and circumaural high-frequency earphones were used to assess air and bone conduction hearing thresholds. Co-occurring symptom measures An evaluation of other common symptoms was done using valid and reliable instruments. The symptoms and their respective measures were depressive symptoms (Center for Epidemiological Studies-Depression scale (CES-D) [32]); state and trait anxiety (Spielberger State-Trait Anxiety Inventories [33]); morning and evening fatigue and morning and evening energy (Lee Fatigue Scale (LFS) [34]); sleep disturbance (General Sleep Disturbance Scale (GSDS) [35]); cognitive impairment (Attentional Function Index (AFI) [36]); and pain (Brief Pain Inventory (BPI) [37]). QOL measures QOL was evaluated using generic (i.e., Medical Outcomes Study-Short Form-12 (SF-12) [38]) and disease-specific (i.e., QOL-Patient Version (QOL-PV) [39]) measures. QOL-PV measures four dimensions of QOL (i.e., physical, psychological, social, and spiritual well-being), as well as a total QOL score. The individual items on the SF-12 were evaluated and the instrument was scored into two component scores (i.e., physical component summary (PCS) and mental component summary (MCS)). For both measures, higher scores indicate a better QOL. Analysis Determination of audiometrically confirmed hearing loss Following the audiogram, to adjust for age- and gender-related changes in hearing, each survivor’s audiogram was evaluated using the National Health and Nutrition Examination Survey (NHANES)-modified Occupational Safety and Health Administration (OSHA) age adjustment standards [40, 41]. A survivor was classified as having hearing loss if at any frequency they scored poorer than the 50th percentile for their age and gender. Data analysis Study data were collected and managed using the Research Electronic Data Capture (REDCap) system hosted at UCSF [42]. REDCap is a secure, web-based software platform designed to support data capture for research studies. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) Version 28 (IBM Corporation, Armonk, NY). Differences between the survivors with and without hearing loss in demographic and clinical characteristics, symptom occurrence rates, symptom severity scores, and QOL outcomes were evaluated using parametric and non-parametric tests. A p-value of < 0.05 was considered statistically significant. Results In this study that evaluated 273 survivors, 56.8% and 43.2% did and did not have audiometrically confirmed hearing loss, respectively. Demographic and clinical characteristics As shown in Table 1, compared to survivors without hearing loss, survivors with hearing loss were older, more likely to be male, less likely to be employed, more likely to have a lower annual household income, and less likely to report child care responsibilities. In addition, survivors with hearing loss had a higher number of comorbidities and a higher comorbidity burden, were a longer time since their cancer diagnosis, were less likely to report breast cancer, were more likely to report gastrointestinal cancer, had a higher number of metastatic sites, and were more likely to self-report diagnoses of osteoarthritis and lung disease.Table 1 Differences in demographic and clinical characteristics between survivors with and without audiometrically confirmed hearing loss Characteristic No hearing loss 43.2% (n = 118) Hearing loss 56.8% (n = 155) Statistic, p-value Mean (SD) Mean (SD) Age (years) 54.6 (11.7) 66.1 (9.4) t = -8.79, p < .001 Education (years) 16.4 (2.3) 16.2 (2.4) t = 0.91, p = .364 Body mass index (kg/m2) 27.4 (7.2) 27.6 (6.4) t = -0.77, p = .783 Karnofsky Performance Status score 88.0 (10.4) 86.4 (10.6) t = 1.28, p = .203 Number of comorbidities 1.4 (1.5) 2.1 (1.4) t = -3.65, p < .001 Self-Administered Comorbidity Questionnaire score 3.2 (3.7) 4.3 (3.3) t = -2.54, p = .012 Years since cancer diagnosis 6.6 (5.6) 8.7 (7.4) t = -2.59, p = .010 Number of prior cancer treatments 3.1 (0.7) 3.1 (0.9) t = -0.11, p = .916 Number of current cancer treatments 0.5 (0.6) 0.4 (0.6) t = 1.53, p = .128 Number of metastatic sites (out of 7) 0.7 (0.8) 0.9 (0.9) t = -2.53, p = .012 Number of metastatic sites without lymph node involvement (out of 6) 0.2 (0.6) 0.3 (0.7) t = -2.15, p = .033 Dose of platinum compounds for patients who received only a platinum (mg/m2) 1092.9 (598.5) 1534.9 (1071.4) t = -1.22, p = .231 Dose of taxane compounds for patients who received only a taxane (mg/m2) 1194.7 (540.7) 1390.2 (1783.9) t = -0.85, p = .395 Dose of drugs for patients who received both a platinum and a taxane compound Platinum dose (mg/m2) 3502.29 (1071.69) 3534.9 (1613.53) t = -0.10, p = .923 Taxane dose (mg/m2) 1331.1 (791.36) 1887.1 (2444.4) t = -1.25, p = .217 % (n) % (n) Female (% yes) 96.6 (114) 87.1 (135) FE, p = .008 Married/partnered (% yes) 65.0 (76) 66.9 (103) FE p = .796 Lives alone (% yes) 25.6 (30) 26.1 (40) FE, p = 1.000 Employed 62.7 (74) 38.8 (59) FE, p < .001 Ethnicity White 65.3 (77) 76.0 (117) Χ2 = 9.11, p = .058 Black 5.1 (6) 1.9 (3) Asian or Pacific Islander 15.3 (18) 9.7 (15) Hispanic 4.2 (5) 7.8 (12) Mixed or other 10.2 (12) 4.5 (7) Annual household income < $20,000 5.3 (6) 6.1 (9) U, p = .003 $20,000–$59,999 18.4 (21) 27.9 (41) $60,000–$99,999 14.0 (16) 25.2 (37) > $100,000 10.2 (12) 4.5 (7) Child care responsibilities (% yes) 26.4 (29) 10.1 (15) FE, p < .001 Adult care responsibilities (% yes) 10.0 (11) 6.8 (10) FE, p = .367 Smoker (ever) 30.1 (34) 39.9 (59) FE, p = .118 Comorbid conditions (% yes) Osteoarthritis 22.9 (27) 41.6 (62) FE, p = .002 Back pain 28.4 (33) 33.8 (50) FE, p = .423 Depression 14.8 (17) 18.8 (28) FE, p = .414 High blood pressure 22.4 (26) 29.6 (45) FE, p = .210 Heart disease 1.7 (2) 6.6 (10) FE, p = .074 Diabetes 3.4 (4) 6.8 (10) FE, p = .274 Lung disease 1.7 (2) 7.2 (11) FE, p = .044 Anemia or blood disease 5.1 (6) 4.6 (7) FE, p = 1.000 Ulcer or stomach disease 2.5 (3) 4.0 (6) FE, p = .735 Kidney disease 0.0 (0) 0.7 (1) n/a Liver disease 0.9 (1) 1.3 (2) FE, p = 1.000 Rheumatoid arthritis 1.8 (2) 6.8 (10) FE, p = .074 Type of cancer Χ2 = 12.82, p = .013 Breast 80.5 (95) 61.9 (96) 1 > 2 Gastrointestinal 5.1 (6) 14.8 (23) 1 < 2 Gynecological 10.2 (12) 14.2 (22) NS Lung 0.8 (1) 3.2 (5) NS Other 3.4 (4) 5.8 (9) NS Any metastatic disease (% yes) 53.8 (54) 67.1 (50) FE, p = .032 Type of prior cancer treatment Only CTX, surgery, or RT 1.7 (2) 1.3 (2) Χ2 = 0.89, p = .640 CTX and surgery, or CTX and RT, or surgery and RT 33.9 (40) 39.4 (61) CTX, surgery, and RT 64.4 (76) 59.4 (92 Type of CTX Only taxane 62.7 (74) 52.3 (81) Χ2 = 4.52, p = .104 Only platinum 8.5 (10) 16.1 (25) Both taxane and platinum 28.8 (34) 31.6 (49) Abbreviations: CTX chemotherapy, FE Fisher’s exact, kg kilograms, mg milligrams, m2 meters squared, n/a not applicable, NS not significant, RT radiation therapy, SD standard deviation, U Mann–Whitney U test Co-occurring symptoms As shown in Table 2, no between-group differences were found in the occurrence rates for clinically meaningful levels of depression, state anxiety, morning and evening fatigue, decrements in morning and evening energy, sleep disturbance, or cognitive impairment. No between-group differences were found in the occurrence rates for cancer pain, non-cancer pain, and both cancer and non-cancer pain.Table 2 Differences in symptom occurrence rates between survivors with and without audiometrically confirmed hearing loss Symptom* No hearing loss 43.2% (n = 118) Hearing loss 56.8% (n = 155) Statistic, p-value % (n) % (n) Depression (≥ 16.0) 22.6 (26) 26.8 (41) FE, p = .478 State anxiety (≥ 32.2) 54.8 (63) 44.1 (67) FE, p = .086 Morning fatigue (≥ 3.2) 49.6 (57) 39.9 (61) FE, p = .136 Evening fatigue (≥ 5.6) 44.6 (50) 37.7 (57) FE, p = .310 Morning energy (≤ 6.2) 78.4 (91) 67.3 (103) FE, p = .054 Evening energy (≤ 3.5) 71.2 (79) 65.1 (99) FE, p = .351 Sleep disturbance total score (≥ 43.0) 59.5 (69) 57.5 (88) FE, p = .803 Attentional function total score (< 7.5) 62.5 (65) 52.5 (73) FE, p = .150 Type of pain None 25.2 (29) 15.1 (23) Χ2 = 6.11, p = .106 Only non-cancer pain 20.9 (24) 23.0 (35) Only cancer pain 27.8 (32) 25.0 (38) Both cancer and non-cancer pain 26.1 (30) 36.8 (56) *Percentage of survivors who scored above the clinically meaningful cut point for each of the symptoms. Clinically meaningful cut point for each symptom measure is listed in parentheses As shown in Table 3, except for worst pain scores, no between-group differences were found in depression, state anxiety, fatigue, energy, sleep disturbance, or cognitive impairment scores. Compared to the survivors without hearing loss, survivors with hearing loss reported higher worst pain intensity scores.Table 3 Differences in symptom severity scores between survivors with and without audiometrically confirmed hearing loss Symptom No hearing loss 43.2% (n = 118) Hearing loss 56.8% (n = 155) Statistic, p-value Mean (SD) Mean (SD) Depression (≥ 16.0) 10.9 (8.8) 10.0 (8.7) t = .078, p = .434 State anxiety (≥ 32.2) 35.4 (10.5) 33.7 (12.4) t = 1.21, p = .228 Morning fatigue (≥ 3.2) 3.2 (2.3) 2.8 (2.3) t = 1.22, p = .225 Evening fatigue (≥ 5.6) 5.0 (1.9) 4.9 (2.1) t = 0.62, p = .534 Morning energy (6.2) 4.2 (2.2) 4.7 (2.4) t = -1.78, p = .076 Evening energy (≤ 3.5) 2.8 (2.1) 2.8 (2.2) t = -0.09, p = .933 Sleep disturbance total score (≥ 43.0) 49.9 (18.5) 47.8 (20.6) t = 0.84, p = .401 Quantity of sleep (≥ 3.0) 5.0 (1.4) 4.9 (1.4) t = 0.57, p = .570 Quality of sleep (≥ 3.0) 3.6 (1.8) 3.3 (1.9) t = 1.27, p = .205 Sleep onset latency (≥ 3.0) 2.6 (2.1) 2.7 (2.3) t = -0.24, p = .811 Mid-sleep awakenings (≥ 3.0) 5.0 (2.1) 4.9 (2.4) t = 0.58, p = .560 Early awakenings (≥ 3.0) 3.6 (2.5) 3.3 (2.4) t = 0.86, p = .393 Excessive daytime sleepiness (≥ 3.0) 2.1 (1.3) 2.0 (1.3) t = 0.73, p = .468 Use of sleep medications (≥ 3.0) 0.5 (0.6) 0.6 (0.7) t = -0.66, p = .510 Attentional function total score (< 5.0 = low, 5.0 to 7.5 = moderate, > 7.5 = high) 6.9 (1.6) 7.1 (1.7) t = -1.28, p = .200 Effective action 6.9 (1.7) 7.1 (1.9) t = -1.14, p = .257 Attentional lapses 6.8 (2.0) 7.0 (2.0) t = -0.57, p = .567 Interpersonal effectiveness 7.0 (1.7) 7.4 (1.9) t = -1.61, p = .109 For patients with pain Pain now 2.4 (2.1) 2.6 (2.1) t = -0.98, p = .327 Average pain 2.9 (1.8) 3.4 (1.9) t = -1.94, p = .053 Worst pain 5.7 (2.4) 6.4 (2.2) t = -2.17, p = .031 Number of days in pain 2.5 (2.4) 2.9 (2.8) t = -1.25, p = .212 Hours per day in pain 6.1 (7.4) 7.7 (8.1) t = -1.41, p = .162 Percent relief from pain medication 5.4 (3.8) 5.1 (3.3) t = 0.52, p = .606 Satisfaction with pain management 5.7 (3.4) 5.3 (3.0) t = 0.76, p = .447 Abbreviation: SD standard deviation Clinically meaningful cut point for each symptom measure is listed in parentheses QOL outcomes As shown in Table 4, no between-group differences were found for any of the QOL-PV subscale or total scores. In terms of the SF-12, survivors with hearing loss had lower physical functioning, role physical, general health, and PCS scores.Table 4 Differences in quality of life scores between survivors with and without audiometrically confirmed hearing loss Symptom No hearing loss 43.2% (n = 118) Hearing loss 56.8% (n = 155) Statistic, p-value Mean (SD) Mean (SD) Multidimensional quality of life scale–cancer Physical well-being 7.6 (1.7) 7.7 (1.7) t = − 0.45, p = .651 Psychological well-being 5.7 (1.7) 5.9 (1.7) t = − 0.97, p = .334 Social well-being 6.2 (2.0) 6.5 (2.0) t = − 1.13, p = .261 Spiritual well-being 5.5 (1.8) 5.7 (2.0) t = − 0.88, p = .379 Total quality of life score 6.1 (1.3) 6.3 (1.4) t = − 1.14, p = .256 Medical Outcomes Study–Short Form 12 Physical functioning 74.8 (28.6) 61.7 (34.5) t = 3.39, p < .001 Role physical 70.0 (27.9) 62.8 (28.3) t = 2.08, p = .038 Bodily pain 78.4 (24.4) 73.0 (26.1) t = 1.73, p = .085 General health 73.3 (19.9) 65.4 (23.6) t = 2.78, p = .006 Vitality 52.2 (21.3) 53.6 (24.5) t = -0.50, p = .615 Social functioning 80.4 (24.3) 79.9 (25.1) t = 0.16, p = .870 Role emotional 79.8 (21.8) 78.0 (23.6) t = 0.66, p = .508 Mental health 67.7 (17.2) 70.0 (19.6) t = − 1.04, p = .298 Physical component summary score 48.4 (9.9) 44.0 (10.9) t = 3.28, p = .001 Mental component summary score 48.0 (9.2) 50.0 (9.4) t = − 1.66, p = .098 Abbreviation: SD standard deviation Discussion This study is the first to evaluate for differences in demographic and clinical characteristics, occurrence and severity of common co-occurring symptoms, and QOL outcomes in cancer survivors with and without audiometrically confirmed hearing loss. Contrary to our a priori hypothesis, except for worst pain scores, no between-group differences in the symptom occurrence rates and severity scores were found. These findings are somewhat surprising given the between-group differences in some of the demographic and clinical characteristics. While our evaluation of hearing loss accounted for age and gender, consistent with findings in the general population, survivors with hearing loss were older [43] and more likely to be male [44]. Equally important, survivors with hearing loss were less likely to be employed and to report a lower annual household income. While not reported in cancer patients, this later finding warrants additional investigation given that a recent meta-analysis found a positive association between adult onset hearing loss and unemployment [45]. This relationship is attributed to high levels of fatigue associated with an increased requirement for intense listening efforts in both work and social situations [46]. In terms of clinical characteristics, survivors with hearing loss had a longer time since their cancer diagnosis, had a higher comorbidity burden, and were more likely to have metastatic disease. However, no between-group differences were found in the types of chemotherapy regimens or doses of neurotoxic chemotherapy. The positive association between hearing loss and a higher comorbidity burden is consistent with studies in the general population [47]. While no between-group differences were found in the occurrence and severity of common symptoms, the findings from this study provide important and clinically useful information on depression, anxiety, fatigue, decrements in energy, cognitive impairment, sleep disturbance, and pain in a relatively large sample of cancer survivors who were over 6 years since their cancer diagnosis. As noted in one review [48], estimates suggest that while 30 to 60% of patients with cancer have psychological problems, only 10% are referred for treatment. In the current study, approximately 25% of the survivors reported clinically meaningful levels of depressive symptoms; 15% reported a diagnosis of depression on the SCQ; and almost 50% reported clinically meaningful levels of anxiety. Possible reasons for these high levels of psychological symptoms, particularly anxiety, include fear of recurrence [49] and/or financial toxicity associated with the costs of medical management of cancer and other chronic conditions [50–54]. Equally important findings in this study are the high occurrence rates for sleep disturbance and both morning and evening fatigue, as well as decrements in both morning and evening energy. In fact, clinically meaningful decrements in morning and evening energy were reported by 72.1% and 67.7% of the sample, respectively. While research on fatigue in oncology patients and survivors is relatively common [55–57], a growing body of evidence suggests that energy is a distinct symptom from fatigue [58–60]. Energy can be defined as an individual’s potential to perform physical and mental activities [60]. Adequate amounts of energy are required to perform routine, as well as work-related activities. One potential explanation for the low levels of morning and evening energy in our sample of cancer survivors is the high rate of sleep disturbance. In the current study, almost 60% of our survivors reported clinically meaningful levels of sleep disturbance. An evaluation of the subscale scores of the GSDS (Table 3) indicates that survivors rated the quantity of their sleep as inadequate on 5 out of 7 days each week. While this sample does not appear to have a problem with sleep initiation (i.e., sleep onset latency scores were < 3.0), findings suggest that they have problems with sleep maintenance (i.e., scores of > 3.0 for mid-sleep awakenings and early awakenings). Given that sleep disturbance, fatigue, and decrements in energy are inter-related, survivors with these symptoms would benefit from education on the benefits of regular exercise, cognitive-behavioral interventions (e.g., mindfulness, yoga), and routine sleep management interventions [61–63]. Consistent with previous reports [64, 65], almost 60% of the survivors in this study had AFI scores that suggest moderate to high levels of cognitive impairment. The AFI assesses an individual’s perceived effectiveness in performing daily activities that are supported by attention and working memory [66]. This finding has clinical implications particularly in terms of survivors’ work performance; ability to carry out child and/or elder care responsibilities; and ability to adhere with a therapeutic regimen and/or survivorship care plan. Equally important, given the mounting evidence of the occurrence of a neuropsychological symptom cluster that consists of pain, fatigue, sleep disturbance, and depression during and following cancer treatment [67–69], cancer survivors need to be assessed for multiple co-occurring symptoms and have individualized interventions initiated to decrease symptom burden and improve QOL. While no clinically meaningful cutoff scores exist for QOL-PV, both groups of survivors’ total QOL scores were in the moderate range (i.e., 6.1 to 6.3 on a 0 (extremely poo) to 10 (excellent) scale). However, consistent with the higher comorbidity burden in the hearing loss group, our survivors reported not only statistically significant but clinically meaningful decrements [70, 71] in the physical functioning (Cohen’s d = 0.40), role physical (Cohen’s d = 0.25), and general health (Cohen’s d = 0.35) scales of the SF-12. Of note, both groups of survivors reported PCS and MCS scores that were at or below the normative score of 50 for the general population of the USA. Taken together with the relatively high symptom burden, interventions are needed to decrease symptoms and improve survivors’ overall QOL. Several limitations warrant consideration. Due to the cross-sectional design, future studies need to evaluate for changes in symptoms and QOL outcomes in survivors with and without audiometrically confirmed hearing loss. In addition, future studies need to evaluate for differences in symptom burden among survivors with and without multiple types of chemotherapy-induced neurotoxicities (e.g., hearing loss, tinnitus, CIPN). Given that this study did not collect data on pharmacologic and non-pharmacologic interventions, the impact of symptom management interventions warrant evaluation in future studies. Despite these limitations, this study provides new information on the relatively high symptom burden associated with cancer survivorship. Clinicians can use this information to guide their ongoing assessment of these individuals and to initiate pharmacologic and non-pharmacologic interventions to reduce symptom burden and improve survivors’ QOL. Acknowledgements Recruitment was facilitated by Dr. Susan Love Foundation’s Love Research Army® Program. Author contribution Dr. Miaskowski designed the study, did the data analysis, and wrote the paper. All of the authors contributed to writing the manuscript and approving the final version of the paper. Funding This study was funded by the National Cancer Institute (NCI, CA151692) and the American Cancer Society (ACS). Dr. Miaskowski is an American Cancer Society Clinical Research Professor. This project was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through UCSF-CTSI Grant Number UL1 TR000004. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Data availability Data are available from Dr. Miaskowski following the completion of a material transfer agreement with the University of California, San Francisco. Declarations Ethics approval This study was approved by the Institutional Review Board at the University of California, San Francisco. Consent to participate Written informed consent was obtained from all of the survivors who participated in this study. Competing interests The authors declare no competing interests. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. 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==== Front Neuropsychopharmacology Neuropsychopharmacology Neuropsychopharmacology 0893-133X 1740-634X Springer International Publishing Cham 36456695 1486 10.1038/s41386-022-01486-z Abstracts Collection ACNP 61st Annual Meeting: Poster Abstracts P541 – P809 1 12 2022 12 2022 47 Suppl 1 371520 © The Author(s), under exclusive licence to American College of Neuropsychopharmacology 2022 issue-copyright-statement© American College of Neuropsychopharmacology 2022 ==== Body pmc December 4-7, 2022 Phoenix Arizona Sponsorship Statement: Publication of this supplement is sponsored by the ACNP. All content was reviewed and selected by the Program Committee, which held full responsibility for the abstract selections. Only disclosures for presenting authors are listed. Asterisks in the author lists indicate presenter of the abstract at the annual meeting. Abstract numbers do not correlate to poster number assigned for presentation at the Annual Meeting. P541. Measuring Dopamine Synthesis Capacity: A Comparison of Compartment Modelling and Graphical Methods Egill Rostrup, Dan Fuglø, Anne Sigvard, Karen Tangmose, Albert Gjedde, Bjørn Ebdrup, Lars Thorbjørn Jensen, Birte Glenthoj Center for Neuropsychiatric Schizophrenia Research (CNSR), Center for Clinical Intervention and Neuropsychiatric Schizophrenia Research (CINS), Denmark Background: The human dopamine synthesis capacity can be analyzed in vivo using 18F-FDOPA PET. This tracer kinetic model is known to be complex due to the presence of multiple compartments, as well as prominent metabolites that also enter the brain. Several methods have been proposed to analyze the data, but there is no general consensus of an optimal method. Classical compartment modeling is complicated by the need for sequential arterial samples that may be difficult to obtain in a clinical setting. Replacing the arterial input function (AIF) with a population based input function has been suggested to avoid the need for invasive samples. Graphical analysis on the other hand is computationally simpler, and can be performed with or, in a modified version, without arterial input. In the present study we compare non-linear compartment modeling to two graphical methods, using both simulated and real data. We further evaluate different strategies for estimating and scaling a population based arterial input function. Methods: We collected experimental data in a study of 62 subjects (first episode psychosis and controls (ref 1), using 18F-FDOPA and arterial blood sampling with estimation of peripheral metabolites. Values of the clearance parameters (K1,k2,k3,k4) of the compartment model were estimated using non-linear fitting (in-house software, Matlab v. 2020a). We used either the true plasma input function, or population based AIF, estimated by scaling the averaged AIF of all other subjects. Further, values of the influx parameter (Ki) were estimated from striatal regions using Gjedde-Patlak graphical analysis with input functions based either on arterial plasma or a cerebellar reference region. The true value of Ki depends on the underlying clearance parameters of the compartment model (K1,k2,k3,k4). For reference region method they also depend on the values of (K1,k2) in the reference region. Finally, simulated data were constructed using known values of the variables and analyzed using the same methods. Results: Simulated data revealed that the graphical analysis estimate of Ki is highly informative of K1 with plasma input values (r > 0.8), but only modestly so (r < 0.46) when using cerebellum reference. Graphical analysis was not informative of any other kinetic parameters. For real data Ki values of the graphical method correlated significantly (r = 0.64, p < 0.001) with K1 based on the compartment model, but only with the plasma input function. Results based on populations based AIF’s were generally only weakly correlated with those based on the true plasma AIF. A maximum correlation of 0.54 (p < 0.001) was found between Ki based on a population based AIF scaled with injected dose, and Ki based on the true AIF. Conclusions: With real data from an arterially derived input function, the graphical tissue reference method outcome parameter Ki is suitable as a proxy for the vascular clearance rate K1, although with a rather low correlation. The method is not informative of the remaining underlying kinetic parameters. Population based AIFs were not suitable to replace plasma samples with sufficient accuracy. The current data therefore suggest that an individual arterial input function may not be left out without significant loss of precision. Furthermore, studies to elucidate the relationship between the graphical reference tissue method and the underlying tracer kinetic parameters seems warranted. Reference: 1. Sigvard AK, et al. Dopaminergic Activity in Antipsychotic-Naïve Patients Assessed With Positron Emission Tomography Before and After Partial Dopamine D2 Receptor Agonist Treatment: Association With Psychotic Symptoms and Treatment Response. Biol Psychiat. 2022;91(2):236–45. Keywords: Dopaminergic System, Psychosis, Imaging Disclosure: Nothing to disclose. P542. Toward an Understanding of the Role of the Cholinergic System in Schizophrenia: A Pilot Study Using 18F-VAT PET Jodi Weinstein, Scott Moeller, Greg Perlman, Roberto Gil, Kenneth Wengler, Mark Slifstein, Anissa Abi-Dargham Stony Brook University, Stony Brook, New York, United States Background: The cholinergic system has been of longstanding interest in schizophrenia (SCZ) and its potential treatments. However, to date the lack of imaging tools has limited in vivo investigation of the cholinergic system in SCZ, particularly as it relates to labeling presynaptic targets. Filling this void, an in vivo PET imaging probe of cholinergic tone ([18 F]VAT), which targets the vesicular cholinergic transporter (VAChT) with high affinity and selectivity, was recently validated for use in human research. As VAChT regulates the trafficking of acetylcholine into synaptic vesicles, it sets how much acetylcholine is available for activity, and [18 F]VAT binding (PET signal) can serve as a proxy for cholinergic synaptic integrity and capacity for transmission. Cholinergic system alteration in SCZ has been implicated across multiple domains: from higher prevalence of cigarette smoking among patients, to postmortem and genetic studies suggesting deficits in the cholinergic nicotinic system and models of auditory sensory processing suggesting disrupted cholinergic modulation may underly abnormal gamma rhythms in SCZ. In light of this, as well as the essential role of the cholinergic system for cognition and modulation of dopamine signaling, which is also abnormal in SCZ, we conducted a proof-of-concept neuroimaging study using [18 F]VAT PET to measure cholinergic integrity across the brain in patients with SCZ. Methods: Eighteen patients with SCZ and 14 demographically-matched healthy controls (HC), aged 18-60, underwent clinical assessment, neuropsychological testing, and a PET/MR scan with [18 F]VAT. All participants were assessed and rated for psychosis-related signs and symptoms using the Positive and Negative Syndrome Scale (PANSS) and completed a computerized test battery (PennCNP) which consisted of 13 neurocognitive tasks, including the letter n-back (0-, 1-, and 2-back); short visual object learning tasks (sVOLT); and the abstraction, inhibition and working memory task (AIM). Participants were scanned on a combined PET/MR scanner, a Siemens Biograph 3 T mMR (Knoxville TN, USA), at Stony Brook University Hospital, equipped with a 12-channel head-and-neck coil. After a brief transmission scan, participants received an intravenous bolus injection (over 30 s) of ≤5 mCi (total mass ≤1.18 μg) of radiotracer [18 F]VAT while undergoing 150-min PET/MR scan with arterial blood sampling. There were no adverse reactions to the radioactive drug or study procedures. Time-activity curves were formed as the mean activity in each region of interest (ROI) in each PET frame. The main PET outcome measure of VAChT availability was derived for each ROI in each subject by calculation of the total distribution volume (VT) for [18 F]VAT using pharmacokinetic modeling of the PET data with the arterial plasma input function. Simultaneous MR acquisitions included ultra-short echo time image for PET attenuation correction, followed by T1-weighted (T1w) and T2-weighted images with 1 mm isotropic voxels and whole-brain coverage for ROI delineation. ROIs of multiple cortical and subcortical brain regions were manually drawn on each subject’s T1w MRI (1 mm isotropic voxels) and transferred to the coregistered PET images. For each ROI, we performed two-sample t-tests of VT between groups. PANSS items scores were summed for total, positive, negative, and general items subscale scores. Scores on the PennCNP were assessed as total correct score or d’ (for tasks having a true/false positive/negative structure). Pearson correlations were conducted between the VT for each ROI and PANSS subscale scores and each of the PennCNP outcome scores. Results: [18 F]VAT VT did not significantly differ between patients with SCZ compared to HC in any of the striatal or extrastriatal regions. Similarly, regional [18 F]VAT VT did not differ significantly when patients were subgrouped based on whether they were currently taking antipsychotic medication (n = 7) or not (n = 11). Despite non-significant group differences, we observed relationships between [18 F]VAT VT and key clinical and neuropsychological variables within the patient group. Most notably, patients’ psychosis symptoms, measured by the PANSS Positive subscale score, were positively correlated with [18 F]VAT VT in multiple ROIs (strongest effects in the thalamus, cerebellum, and several cortical regions). Also, within patients with SCZ, performance on the 2-back task was negatively associated with VAChT availability in several cortical regions: the higher the VT, the worse the performance (e.g. strong negative association between performance on the 2-back task and [18 F]VAT VT in the dlPFC in SCZ: R = −0.63). These correlations should be considered preliminary, in that these were exploratory analyses in a small pilot study, but suggest the need for further investigations of the cholinergic system in SCZ. Conclusions: These findings suggest the cholinergic system may be mediating a relationship between working memory deficits and psychosis severity in individuals with SCZ. An improved understanding of these modulatory systems could lead to a better mechanistic understanding of the disease. Keywords: PET, Cholinergic System, Cognitive Impairment Associated With Schizophrenia, Psychosis and Memory Disclosure: Nothing to disclose. P543. Exploring Sex Differences (and Lack Thereof) in Schizophrenia: From Cognitive Control Brain Activity to Clinical Phenotypes Tyler Lesh, Andrea Perrottelli, Jason Smucny, Joshua Rhilinger, Skylar Lin, Marina Albuquerque, J. Daniel Ragland, Cameron Carter University of California - Davis, Sacramento, California, United States Background: Sex differences in schizophrenia represent an important aspect of schizophrenia in need of further investigation. Male sex has previously been associated with earlier age of onset, higher rates of substance abuse, and in some studies a higher incidence of the disorder. Several studies have also identified differences in symptom profiles, with female patients presenting with lower levels of negative symptoms and higher depressive symptoms. However, the literature is largely mixed in terms of sex effects on cognition and brain function in patients with schizophrenia. Several studies have identified disproportionately worse performance in male patients on measures of verbal learning and memory and inhibition/switching tasks, while others suggest worse performance in female patients in visualspatial attention and spatial memory. Notably, the modest number of functional magnetic resonance imaging (fMRI) studies suggest altered activity and connectivity in male versus female patients on tasks of mental rotation and emotional memory that involve the frontal-parietal and limbic networks. The current study seeks to further expand these findings by examining a large sample of patients with schizophrenia and healthy controls who performed a cognitive control task during fMRI and completed thorough symptom and functioning evaluations. Methods: A total of 148 (116 male, 32 female) individuals with schizophrenia-spectrum diagnoses were recruited, assessed using the Structured Clinical Interview for DSM-IV, Global Social/Role Functioning Scales (GFS: R/S), Scale for the Assessment of Positive Symptoms (SAPS), Scale for the Assessment of Negative Symptoms (SANS), and Brief Psychiatric Rating Scale (BPRS). A sample of 132 control participants (78 male, 54 female) were also recruited from the community. All participants performed the AX-CPT while undergoing fMRI on a 1.5 Tesla General Electric or 3 Tesla Siemens Trio scanner. fMRI data were processed using SPM8, including slice timing correction, realignment, normalization to the MNI EPI template, and smoothing. General linear models (GLM) were used to test for the effect of sex, group and sex by group interactions. Protocol type (i.e. 1.5 T or 3 T scanner) and age were included as covariates. T-tests and Mann-Whitney U tests were used for assessing sex effects within the patient group on symptom and functioning measures. Results: Analyses of demographic data revealed a higher proportion of males in the patient group (p < 0.001) along with lower education in the patient group compared to controls (p < 0.001). In terms of global functioning, male patients showed significantly lower social functioning (p = 0.002) and a trend for lower role functioning (p = 0.062). Analyses of symptom syndrome scores revealed significantly lower poverty symptoms in female patients (p = 0.018) while reality distortion and disorganization syndrome scores did not differ between the sexes. Male patients were also significantly more likely to endorse a history of cannabis use compared to females (p = 0.027, phi = 0.159). In terms of cognitive control, healthy controls showed higher d’-context performance compared to patients (p < 0.001), but no sex or group by sex interaction emerged (all p > 0.05). Similarly, on a measure of attention lapsing (i.e., average of AX and BY error rates), patients showed higher rates of lapsing compared to controls (p < 0.001) in the absence of a significant sex or sex by group interaction (both p > 0.05). Bilateral regions of interest in the DLPFC and superior parietal cortex were evaluated on the CueB-CueA contrast. Both regions revealed significantly increased activation in controls compared to patients (p = 0.002 in DLPFC and p < 0.001 in parietal) with no significant effect of sex. Interestingly, within the patient group, females showed significantly higher activity in the right DLPFC (p = 0.042) and a similar but nonsignificant pattern of activity in the right superior parietal cortex (p = 0.243). Conclusions: These data suggest that sex differences in patients with schizophrenia are relatively circumscribed. In agreement with other published findings, female patients show a relatively more favorable clinical and functional profile, with lower negative symptoms and higher social functioning being the most pronounced in this sample. The higher proportion of males in the schizophrenia sample is also consistent with the literature. Notably, one of the few functional differences that was identified was increased prefrontal activity in female versus male schizophrenia patients. This difference is counterpointed by a lack of evidence for sex differences in terms of cognition (d’-context, attention lapsing). It is not readily apparent why sex differences in brain activity emerged without concomitant behavioral differences, although differences in laterality between the sexes might underlie this effect. Given some evidence of the impact of illness chronicity on sex effects, future analyses will also focus on age of onset and illness duration. Keywords: Functional MRI (fMRI), Schizophrenia (SCZ), Gender Differences, Sex Differences, Cognition Disclosure: Nothing to disclose. P544. Sleep and Wake Markers of Thalamocortical Circuit Functioning are Abnormal in Early-Course Psychotic Disorders Bengi Baran, Dan Denis, Dimitrios Mylonas, Courtney Spitzer, Nicolas Raymond, Christine Talbot, Erin Kohnke, Robert Stickgold, Matcheri Keshavan, Dara Manoach University of Iowa, Iowa City, Iowa, United States Background: Recent advances in psychiatric genetics and studies in rodent models reveal that abnormalities in thalamocortical circuitry contribute to the pathogenesis of psychotic disorders. Sleep spindles, rapid bursts of 12-15 Hz EEG oscillations characteristic of Stage 2 non-rapid eye movement (NREM) sleep, are initiated by the thalamic reticular nucleus (TRN) and propagated to the cortex and coordinated with other NREM oscillations by thalamocortical circuitry. Spindles mediate memory consolidation, and are associated with impaired sleep-dependent memory consolidation and symptom severity in psychotic disorders. During wakefulness, TRN gates information flow from the thalamus to the cortex to attenuate the transmission of redundant and irrelevant sensory stimuli. The goal of the present study was to investigate thalamocortical circuit functioning in early course, minimally treated patients with psychotic disorders, their young, first-degree relatives (familial high risk: FHR) and matched controls. We utilized resting-state functional connectivity MRI to determine if thalamocortical circuitry is abnormal in psychosis and FHR, and sleep and wake EEG to evaluate the functional implications of thalamocortical circuit dysfunction. This unique sample allows for the identification of trait-related alterations in thalamocortical circuitry. Methods: Seventy-one participants (13-35 yrs, n-psychosis=19, n-controls=28, n-FHR = 24) completed an overnight sleep study, a sensory gating event-related-potentials (ERP) experiment, resting-state fMRI scans and clinical interviews. Sleep was monitored with a 64 channel high-density EEG device and NREM sleep oscillations were identified using validated automated detectors. Sensory gating was calculated as the suppression of the auditory P50 component for the second of a pair of identical clicks. The thalamus was defined using the FSL Oxford atlas and thalamocortical functional connectivity was quantified using a seed-based approach. Results: For thalamocortical connectivity, the main effect of Group (psychosis, FHR or control) was significant in a cluster in the right Heschl’s gyrus (F(2,64)MIN = 7.7, 131 voxels, MNI peak: [40,-20,12], BA41). This reflected that compared to controls, both the psychosis (p < 0.001) and FHR (p < 0.001) groups exhibited hyperconnectivity of the thalamus with the primary auditory cortex. When directly compared with controls, individuals with psychosis also exhibited hyperconnectivity of the thalamus with the right motor cortex ([34,4,54], BA6) and hypoconnectivity with the left cerebellum ([-22,-80,-50], Crus II). Psychosis patients (but not FHR) showed significant reductions in spindle density (all electrodes, Fsum=356.8, p-corrected = 0.002) and spindle amplitude (37 electrodes, Fsum=207.4, p-corrected = 0.005), altered temporal coordination between spindles and slow oscillations (43 electrodes, Fsum=207.6, p-corrected = 0.009), and abnormal P50 sensory gating (F(2,54)=4.2, p = 0.02). Spindles and P50 gating were correlated (r = −0.29, p = 0.03) reflecting that individuals with fewer spindles also failed to gate repetitive auditory information. In both psychosis and FHR groups, intensity of psychotic-like experiences correlated with decreased sleep spindle activity (r = −.47, p = 0.003) and abnormal sensory gating (r = 0.31, p = 0.08). Conclusions: In young early-course patients with psychotic disorders and their first-degree relatives, we observed abnormally increased connectivity of the thalamus with the primary auditory cortex, which extended to motor and premotor regions only in patients. The function of this circuitry was impaired in patients, as evidenced by a reduction in spindle activity during sleep and abnormal gating of auditory sensory information during wakefulness. First-degree relatives of patients did not exhibit significant deficits in sleep spindles or sensory gating, perhaps due to heterogeneity of the sample, but these markers predicted psychotic-like experiences in both groups suggesting that altered thalamocortical circuit functioning contributes to the emergence of psychotic symptoms. Keywords: Sleep Spindles, Sensory Gating, Thalamo-Cortical Connectivity, Psychotic Disorders, Psychosis-Risk Disclosure: Nothing to disclose. P545. Associations Between Structural Covariance Network and Antipsychotic Treatment Response in Schizophrenia Sakiko Tsugawa, Eric Plitman, Cassandra Wannan, Andrew Zalesky, Yoshihiro Noda, Ryosuke Tarumi, Shiori Honda, Yusuke Iwata, Kamiyu Ogyu, Fumihiko Ueno, Karin Matsushita, Hiroyuki Uchida, Masaru Mimura, Mallar Chakravarty, Ariel Graff-Guerrero, Shinichiro Nakajima Keio University, Tokyo, Japan Background: Approximately 30% of patients with schizophrenia do not respond to antipsychotics and are termed treatment-resistant schizophrenia (TRS). Several studies showed that patients with TRS have widespread strong reductions in cortical thickness compared to healthy controls (HC) as well as patients with non-TRS. Recently, to evaluate these structural changes in terms of brain networks, structural covariance has been used to assess the associations of structural measures between two regions. Structural covariance increases when similar structural changes occur in two independent regions, suggesting that those regions share a common pathophysiology. A previous study reported that structural covariance between the two regions with strongly reduced cortical thickness increased in patients with schizophrenia. In this study, we examined the association between treatment resistance and structural covariance changes in this disorder. Methods: We used international multi-site cross-sectional neuroimaging datasets comprising 102 patients with TRS, 77 patients with non-TRS, and 79 HC. Eighty-nine, seventy, and ninety-nine participants were enrolled at Komagino Hospital (Tokyo, Japan), Shimofusa Hospital (Chiba, Japan), and the Centre for Addiction and Mental Health (Toronto, Canada). Antipsychotic treatment resistance was defined by the modified Treatment Response and Resistance in Psychosis Working Group Consensus criteria. T1-weighted structural images were preprocessed using the bpipe pipelines, then cortical thickness was calculated using CIVET for each of 68 regions of the Desikan-Killiany-Tourville atlas. We used a CovBat harmonization method to control for the site differences in the cortical parameters while considering disease status, age, and sex as biological variables. We calculated the residuals of cortical thickness controlling for age and sex. Analysis of variance and post-hoc pairwise t tests were used to examine group differences in residuals of cortical thickness for the 62 cortical regions. Structural connectivity was quantified with partial correlation coefficient between all pairs of regions, controling for age and sex. This approach yielded a separate connectivity matrix of dimension 62×62 for each group. Based on the Fisher method for comparing correlation coefficients, thickness correlations were r-to-z transformed. From the difference in the resulting r-to-z values between the 2 groups, we estimated a z-score that tested the null hypothesis of equality in thickness correlations between the 2 groups. Calculated z-score in each edge was normalized using the standardized deviation of z-scores in 1,000 bootstrap samples. The network-based statistic with 50,000 permutations was used to obtain the structural covariance network with significant difference, controlling the 1,891 multiple comparisons. A primary z-score threshold of 3 was used, with a family-wise error rate threshold of 5%. We estimated the degree centrality for each node of the obtained structural covariance network. This study was conducted after obtaining approval from the respective ethics review committees. Results: Group differences in residuals of cortical thickness were found in 58 out of 62 cortical regions. A total of 59 and 31 regions were found to show cortical thickness reductions in TRS and non-TRS compared with HC, respectively. Also, cortical thickness in 26 regions was reduced in the TRS group than in the non-TRS group. Both In the TRS and non-TRS groups, regions with cortical thinning were located mainly in the frontal and temporal lobes and the cingulate cortex. No significant difference was found in the variances in residuals of cortical thickness among the three groups. Structural covariances across 1,891 pairs of cortical regions were higher in the non-TRS group compared with the HC and TRS groups. The null hypothesis of equality in structural covariance between the non-TRS and HC groups was rejected using network-based statistics. We found a single network comprising connections with elevated structural covariance in patients with non-TRS compared with HC. The inferior temporal gyrus and insula had high degree centrality in the structural covariance network. The null hypothesis could not be rejected for structural covariance between the TRS group and the nonTRS or HC groups. Conclusions: While patients with TRS showed stronger cortical thinning in the frontal and temporal lobes compared to HC and patients with non-TRS, there was no structural covariance network with significant difference between the TRS group and the HC or non-TRS groups. On the other hand, the non-TRS group had a brain network with increased structural covariance compared to the HC group. Our findings suggest that coordinated cortical thinning in the brain network is related to treatment response in schizophrenia while TRS may have greater heterogeneity in the pathophysiology of cortical thinning than non-TRS, resulting in lower structural covariance. Another possibility is that cortical thinning was severe or had reached the plateau long ago in patients with TRS such that the elevation of structural covariance may not be detected. Further longitudinal study is warranted to confirm the link between pathophysiology of structural alteration and treatment resistance in schizophrenia. Keywords: Schizophrenia (SCZ), Treatment-Response, Structural Covariance Analyses Disclosure: Nothing to disclose. P546. Salience Detection Abnormalities in First Episode Psychosis: An MEG, Whole Brain 1H-MRS and Midbrain Neuromelanin Study Juan Bustillo, Crystal Garcia, Haiyang Zhu, Nicholas Shaff, Sephira Ryman, Mauricio Tohen, Julia Stephen, Rhoshel Lenroot University of New Mexico, Albuquerque, New Mexico, United States Background: In psychotic disorders, the processing of irrelevant stimuli as important may instill percepts and thoughts with abnormal salience, leading to inappropriate associations and causal attributions. In an auditory oddball paradigm, both Sz and BP-I (the best exemplars of psychotic disorders) have reduced P300a (novelty) and P300b (target) amplitudes compared to healthy volunteers (HVs). Furthermore, subjects at-risk for psychosis also have reductions in both P300 subcomponents. However, in the largest study of at-risk subjects, only the P300b reductions predicted subsequent conversion to psychosis. This suggests that a specific salience detection deficit may be critical to the development of psychosis. However, the underlying neurobiology of salience detection deficits in psychotic disorders remains unclear. In this pilot study we used MEG, 3 dimensional 1H-MRS and midbrain neuromelanin MR, to provide unprecedented spatial resolution and localization of evoked potentials and their underlying tissue characteristics (glutamate and N-acetylaspartate), as well as their relationship to dopamine function in the substantia nigra (SN). Methods: We studied 13 first episode psychosis (FEP) subjects and 12 HV with MEG, 3D-1H-MRS and MR neuromelanin. During MEG, subjects performed an auditory oddball evoked response task, pressing a button to infrequent target stimuli (10%). Also frequent standard (80%) and infrequent novel (10 %) stimuli were presented but the subject was instructed to ignore these. Magnetic fields were recorded using a Neuromag 306-channel whole-head system. Continuous data was collected at a digitization rate of 1000 Hz with filters set at 0.03–130 Hz. Epochs for each stimulus type were extracted from 200 ms pre-stimulus to 1500 ms post-stimulus and analyzed with BRAINSTORM. MRS data was acquired with Siemens PRISMA at 3 T using EPSI: TR/TE = 1551/17.6 msec; spatial array of 50 ×50 x 18, FOV of 280 ×280 x 180 mm3 (corresponding to a nominal voxel size of 5.6 ×5.6 ×10 mm3). Spectral data was processed with MIDAS. After source localization MEG regions that showed reduced amplitudes to the target in FEP (p < 0.05, non-corrected FWE) were examined for MRS group differences with MINT. MINT allows the selection of all spectral voxels in a particular region and integrates the data to generate one better quality spectrum that is fitted for metabolite quantification (eg: glutamate and N-acetylaspartate). Finally, neuromelanin MR was acquired at 3 T from a midbrain slab containing the SN: TR/TE = 444/4.11 msec; spatial array of 50 ×50 x 18, FOV 220 mm (corresponding to a nominal voxel size of 0.4 ×0.4 ×1.5 mm3). Results: FEP had reduced amplitudes to the target minus standard stimuli at the 250 msec interval over the left hemisphere cortical area involving the posterior superior temporal gyrus (STG) and middle temporal gyrus (MTG; p < 0.05). FEP also had reduced target minus standard amplitudes at 100 msec over the left and right posterior STG and MTG (p < 0.05). HV and FEP had similar low error in performance though reaction times to targets were slower in FEP. Tissue neurochemistry group contrasts over the left and right STG plus MTG will be presented. Also the relationships between target evoked amplitudes, glutamate concentrations and SN neuromelanin will be examined. Conclusions: Reduced amplitudes to targets at 250 msec in left temporal cortex in patients early in the illness, suggest specific abnormalities in the processing of relevant stimuli in psychosis. Speculations about the relationships between salience detection, glutamate and dopaminergic functions are abundant in the field of psychosis, but convergent data is limited. In rats, cue-reward learning is proportional to the strength of glutamatergic synapses onto dopamine midbrain neurons. Also in rats, NMDA hypofunction induced by acute systemic ketamine increases extracellular prefrontal glutamate and dopamine release. In humans, one study used PET and single-voxel 1H-MRS to measure striatal dopamine synthesis-capacity and medial frontal glutamate respectively, in first-episode psychosis. Glutamate and dopamine were inversely correlated in psychotic but not in healthy volunteers, supporting the importance of measuring both systems in this population. Ours is the first investigation to examine the relationships between glutamate and dopamine with electrophysiological measures of salience detection. Results from this study will have two significant specific implications. First, identifying the location of glutamate and P300 deficits may inform probe placement for future neuromodulation studies, aimed to improve cognitive deficits and persistent psychotic and negative symptoms. Second, these studies may also inform anatomical regional selection for future postmortem studies of Sz and BP-I aimed at examining the molecular underpinnings of the illness. Keywords: P300, Disorders of Glutamate, Transition to Broad Psychosis Spectrum Psychopathology, MEG, Proton Magnetic Resonance Spectroscopy Disclosure: UpToDate, Other Financial or Material Support(Self) P547. Brain Functional Segregation, Psychotic Symptoms, and Treatment Response Fei Du, Xiaopeng Song, Tao Song, Chenyanwen Zhu, Mark Halko, Ann Shinn, Brent Forester, Diego Pizzagalli, Dost Ongur McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Functional integration and segregation are the two guiding principles in human brain functional mapping. These two principles reflect the integrated and distributed nature of neuronal processing and brain organization. In brain imaging studies with fMRI, functional integration and segregation are indicated by the correlation and anticorrelation of time course of spontaneous blood oxygen level-dependent (BOLD) signal, respectively. To date, most research has focused on functional integration, with less attention paid to the role of functional segregation. It is unclear whether functional segregation is impaired only in some specifical brain regions such as between default mode networks (DMN) and the executive network, or if it is a global impairment. It is also unclear whether various psychotic symptoms have different neuropathological substrates that are related to functional segregation impairments in different circuits. Moreover, there is considerable biological and clinical heterogeneity across psychosis syndromes and in the longitudinal course of psychotic disorders. Conventional approaches investigating treatment outcomes based on clinical categorization and diagnoses are difficult to capture fundamental underlying neurobiological mechanisms of dysfunction and can be recall or responder biased. Thus, an objective neuroimaging biomarker for classifying patients and tracking the changes of psychotic symptoms would be crucial. Methods: To address the above issues, we introduce the Negative Degree Centrality (NDC) method to quantify the degree of functional segregation between a specific region and all the other parts of the brain. A whole-brain data-driven analysis was applied for a comprehensive evaluation of impaired functional segregation in psychosis. We examined the difference of NDC among early-phase patients with schizophrenia (SZ, N = 123), bipolar disorder (BD, N = 156), and healthy controls (HC, N = 139); and monitored NDC changes in a subset of patients (31 SZ and 56 BD) with disease progression one year later. We adopted an unsupervised machine learning classifier to subdivide patients based on distinct NDC patterns of neuroimaging data at baseline and one-year follow-up and investigated whether these subgroups of patients would have different behavioral and treatment outcomes. We hypothesized that: 1) various psychotic symptoms will be associated with functional segregation impairments in different neural circuits; 2) the combination of functional segregation and a machine learning method will outperform traditional diagnostic categories in tracking psychotic symptom changes and predicting treatment outcomes. Results: Compared to HC, both SZ and BD subjects showed significantly decreased NDC at baseline. Positive, negative, and general psychotic symptoms were correlated with impaired NDC in the salience, frontal-parietal, and DMN, respectively. Using a machine learning approach, we identified two transdiagnostic patient subgroups with distinct recovery trajectories: one subgroup with more NDC impairments at baseline showed significantly improved NDC and psychotic symptoms, however, the other subgroup with less NDC impairments at baseline showed only slightly improved NDC and symptom severity at one-year follow-up. Conclusions: Our study indicated that various psychotic symptoms were linked to impaired functional segregation in different brain circuits. NDC combined with machine learning might provide a helpful biomarker in identifying the neural underpinnings of outcome heterogeneity, and for psychotic symptom severity and treatment response prediction, which could not have been revealed by categorizing them based on conventional clinical diagnoses. Our approach may help to develop precision medicine and personalized interventions in psychiatry. Keywords: Psychotic Disorders, Functional Segregation, Functional Magnetic Resonance Imaging, Machine Learning, Brain Circuit Disclosure: Nothing to disclose. P548. Nanoscale Probing of Synaptic Architecture in Human Prefrontal Cortex With Expansion Microscopy Daniel Chung, Brendan Gallagher, Tyler Tarr, Alan Watson, Aleksandra Klimas, Kenneth Fish, Simon Watkins, Stephen Meriney, Yongxin Zhao, David Lewis University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Schizophrenia is associated with alterations in excitatory neurotransmission in the prefrontal cortex (PFC). The strength of this signaling depends, in part, on the spatial organization of synaptic proteins. For example, presynaptic regulators of vesicle release (e.g., Rab3-interacting molecule [RIM]), postsynaptic scaffolding proteins (e.g., PSD95) and AMPA receptors organize into nanoscale clusters that align with each other trans-synaptically. The alignment of these clusters is thought to facilitate efficient neurotransmitter movement from presynaptic terminals to AMPA receptors. Visualizing these nanoclusters requires imaging techniques with resolution higher than the light diffraction limit. Expansion microscopy physically expands biological specimens isotopically, providing nanoscale-resolution imaging of synaptic molecules with conventional confocal microscopy. Here, we explored the utility of expansion microscopy for assessing spatial organization of synaptic proteins in human PFC. Methods: Human PFC sections were incubated with methacrolein and a gelling solution to anchor the endogenous proteins to hydrogel matrix, and then incubated with a denaturant-rich solution to homogenize the contents of hydrogel while retaining endogenous proteins. Gels were then stained with antibodies for RIM1/2, PSD95 and Pan-GluA. Stained hydrogels were expanded in water and imaged with a confocal spinning-disk microscope under 40x lens. Peak-to-peak analysis was performed to quantify the axial distance between the clusters formed by RIM, PSD95 and GluA. The relative size of these clusters was assessed by auto-correlation analysis. Finally, the spatial alignment between pre- and postsynaptic clusters across synaptic cleft was assessed by enrichment analysis. Results: At 9-fold expansion, multiple clusters formed by RIM, PSD95 and GluA within individual synapses were visualized in human PFC. The axial distance between PSD95 and GluA, GluA and RIM, or PSD95 and RIM clusters was 27.5 nm, 51.8 nm or 64.8 nm, respectively. Auto-correlation analysis showed that PSD95, GluA and RIM proteins are organized in local clusters with an average radius of 80 nm. Finally, enrichment analysis showed that the expression level of each of these proteins is enriched within 40 nm radius from the center of clusters formed by the protein localized at the opposite side of synaptic terminals. Conclusions: Our findings demonstrate an optical resolution for the spatial organization of key synaptic proteins that regulate the strength of excitatory neurotransmission in human PFC by expansion microscopy. This approach may provide a novel strategy to identify the role of synaptic nanoarchitecture alterations underlying PFC circuitry dysfunction in schizophrenia. Keywords: Dorsolateral Prefrontal Cortex (DLPFC), Expansion Microscopy, Synaptic Organization Disclosure: Nothing to disclose. P549. Meta-Analytic Evidence of Elevated Choline, Reduced NAA and Normal Creatine in Schizophrenia and Their Moderation by Measurement Quality, Field Strength, TE, and Medication Status Yvonne Yang, Jason Smucny, Huailin Zhang, Richard Maddock VA Greater Los Angeles Healthcare System, UCLA, Los Angeles, California, United States Background: Magnetic resonance spectroscopy (MRS) provides insights into the pathology of schizophrenia via measurement of molecules involved in neuronal mitochondrial function (n-acetylaspartate, NAA), phospholipid metabolism (choline), and cellular energy metabolism (creatine). Decreased NAA is consistently found in MRS studies of schizophrenia. However, recent investigations suggest that T2 relaxation effects may significantly moderate these findings. The field lacks consensus regarding choline and/or creatine abnormalities in schizophrenia. To address these knowledge gaps, we performed a new meta-analysis of NAA, choline, and creatine in patients across all phases of schizophrenia, compared to healthy control subjects. In addition, recent work has demonstrated a strong influence of data measurement quality on estimates of glutamate levels with MRS. We assessed potential moderating effects of measurement quality on the three singlet peaks – NAA, choline, and creatine. Other technical and clinical moderators were also examined. Methods: All studies cited in three recent MRS meta-analyses were identified. A search of the PubMed database from December 1, 2019, and March 12, 2021 for MRS studies published after the most recent meta-analysis was conducted to identify newer studies for this updated meta-analysis. A total of 116 studies met our inclusion criteria. Pooled effect sizes (Hedges’ g) of patient-control differences were calculated whenever ten or more studies reported data on a metabolite from a specific brain area. Medial prefrontal cortex (MPFC), dorsolateral prefrontal cortex, frontal white matter, hippocampus, thalamus, and basal ganglia met this threshold and were included in the meta-analysis. To examine moderating effects of data measurement quality on results, we performed moving sub-meta-analyses akin to a moving average from the lowest to highest quality studies based on the coefficient of variation of metabolite values (standard deviation / mean). A best-fitting, 4-parameter, logistic function was fit to the pooled effect sizes and the resulting equation used to identify the inflection point. This inflection point was then used to test for a moderating effect of quality on meta-analytical results. The potential moderating effects of field strength, echo time, creatine versus water normalization, medication status, phase of illness, patient age and patient sex were also examined. Results: Choline was significantly elevated in medial and dorsolateral prefrontal cortices and basal ganglia in schizophrenia (p = 0.008, p = 0.007, p < 0.00006, respectively). Elevation in MPFC was significantly stronger in studies with better measurement quality and tended to be stronger in studies with more medicated patients. The hippocampal choline effect size was significantly moderated by field strength, with greater elevation in studies conducted at ≥ 3 Tesla. NAA was reduced in schizophrenia in medial and dorsolateral prefrontal cortices, frontal white matter, hippocampus, and thalamus, but not in basal ganglia (p = 0.00007, p = 0.0059, p = 0.0015, p = 0.0088, p = 0.313, respectively). The reduction in MPFC was significantly stronger in studies with higher signal-to-noise ratios and tended to be stronger in studies with more medicated patients. Hippocampal NAA was significantly more reduced in studies with more medicated patients. When outlier studies were excluded, hippocampal NAA was more reduced in studies using longer echo times. This effect was only robust in studies reporting water-normalized NAA values. No effect of echo time was seen in other regions or in creatine-normalized hippocampal NAA datasets. There was no meta-analytic evidence that creatine levels differed between patients and control subjects in any brain region investigated. Creatine effect sizes were not moderated by measurement quality or any other technical or clinical factors examined. Conclusions: Elevated choline in prefrontal cortex and basal ganglia may reflect greater membrane turnover in these brain regions or a disturbance involving the role of choline-containing compounds in lipid metabolism, lipid signaling, or DNA methylation. Our finding of reduction in NAA is consistent with prior meta-analyses. This may reflect a diminished synthesis of NAA by neuronal mitochondria or an abnormality involving NAA catabolism by oligodendrocytes in schizophrenia. Prior evidence that increased dopaminergic tone can stimulate NAA synthesis in the basal ganglia may account for the selective absence of reduced NAA in this region. Although prior studies have reported more reduced NAA at longer echo times, this was not seen at the meta-analytic level except for water-normalized hippocampal studies. The positive association between antipsychotic medication use and both reduced NAA and elevated choline may have clinical significance and merits further investigation. The absence of evidence for abnormal creatine levels suggests that creatine normalization may be a valid option in 1H-MRS studies of schizophrenia patients. Moderation of higher choline and lower NAA by measurement quality may account for elevated choline not being consistently observed in prior meta-analyses and indicates a need for more stringent quality measurement thresholds to improve validity of, and consensus among, future studies. Keywords: Schizophrenia (SCZ), Magnetic Resonance Spectroscopy, Meta-Analysis, Choline, N-acetylaspartate Disclosure: Nothing to disclose. P550. Neurochemical Alterations in Occipital and Prefrontal Cortex Measured With 7 T MRS as Part of the Psychosis Human Connectome Project Michael-Paul Schallmo, Kyle Killebrew, Caroline Demro, Scott Sponheim, Malgorzata Marjanska University of Minnesota, Minneapolis, Minnesota, United States Background: Psychosis spectrum disorders such as schizophrenia involve disrupted cognitive and perceptual functioning, but the neural basis of these deficits remains unclear. Neurochemical changes in people with psychotic psychopathology (PwPP) have been observed across a number of brain regions using methods including magnetic resonance spectroscopy (MRS). In particular, differences in glutamate and GABA have received attention, as an imbalance in excitatory and inhibitory neural processes has been hypothesized in PwPP. However, methodological issues including small sample sizes and difficulty isolating signals from low concentration metabolites have limited previous studies. We sought to address these limitations in the current study by acquiring 7 tesla MRS data as part of the Psychosis Human Connectome Project. We acquired data from healthy controls and PwPP as well as unaffected biological relatives, in order to explore the role of genetic liability for psychosis in brain chemistry. Our data will be made publicly available in order to facilitate further studies from other research groups. Methods: We acquired 7 T MRS data using a STEAM sequence with an ultra short (8 ms) echo time in 44 healthy controls, 43 relatives, and 65 PwPP. We also obtained re-scan data from a subset of 44 participants (10 controls, 34 PwPP; median time between scans = 132 days). Data were acquired in two volumes of interest (VOIs) within the medial occipital and dorsomedial prefrontal cortex. Scan parameters were as follows: TR = 5 s, TE = 8 ms, TM = 32 ms, VOI size = 30 ×18 x 18 mm3 (occipital) or 30 ×30 x 15 mm3 (prefrontal), # data points = 2048, spectral bandwidth = 6000 Hz, VAPOR water suppression. After frequency and phase correction, spectra were fit using LCModel to quantify concentrations for the following metabolites: ascorbic acid, aspartic acid, total choline, total creatine, GABA, glucose, glutamate, glutamine, glutathione, lactate, myo-inositol, N-acetyl aspartate (NAA), N-acetyl aspartylglutamate (NAAG), phosphorylethanolamine, scyllo-inositol, taurine, lipids, and macromolecules. Metabolite concentrations were scaled relative to the unsuppressed water signal, after correcting for relaxation time and differences in tissue composition within the VOIs across participants. We established quality metrics (linewidth of water, spectrum linewidth, and spectrum SNR) which we used to exclude low quality data. 92% of our data sets passed all quality checks. Results: We first examined differences in 6 metabolites of interest (GABA, glutamate, glutamine, glutathione, NAA, and NAAG) which were identified a priori based on previously reported differences between PwPP and controls in occipital and prefrontal regions. Group differences for these 6 a priori metabolites were examined without corrections for multiple comparisons. We observed significantly lower NAA in occipital cortex among PwPP and relatives versus controls (X2(2) = 7.03, p = 0.030). In prefrontal cortex, NAAG was significantly lower among PwPP versus controls and relatives (X2(2) = 7.66, p = 0.022). Longitudinal stability for these metabolites was fair (ICC = 0.58 for prefrontal NAAG) to good (ICC = 0.82 for occipital NAA). None of the other metabolites examined a priori (including GABA and glutamate) showed significant group differences in either VOI. We also performed an exploratory analysis of group differences in the remaining 11 metabolites (excluding lipids), which included corrections for multiple comparisons. Only glucose in prefrontal cortex showed a significant group difference in this exploratory analysis, and was higher among PwPP versus controls and relatives (X2(2) = 12.3, FDR-corrected p = 0.024). Interestingly, a similar trend was observed for glucose in occipital cortex, but did not survive correction for multiple comparisons (X2(2) = 7.67, uncorrected p = 0.022, FDR-corrected p = 0.24). We saw fair stability over time for glucose (ICC = 0.59 in occipital cortex, ICC = 0.54 in prefrontal cortex). Preliminary results from a hierarchical clustering analysis suggested that glucose levels in both occipital and prefrontal VOIs were associated with individual differences in measures of psychopathology (e.g., Brief Psychiatric Rating Scale, Schizotypal Personality Questionnaire) across all groups (Spearman’s r-values = 0.14 to 0.28, uncorrected p-values = 0.12 to 5 ×10^-4). Conclusions: People with psychosis showed lower NAA and NAAG (in occipital and prefrontal cortex, respectively), consistent with findings from previous studies. Lower occipital NAA levels were also observed among first-degree biological relatives compared to controls, suggesting a link to genetic liability for psychosis. To the best of our knowledge, our study is the first to observe higher brain glucose levels among PwPP, which may be associated with more severe psychopathology. Together, our findings indicate a unique profile of neurometabolic disruption across brain regions in PwPP, which may be associated with both genetic factors and individual differences in psychopathology. Keywords: MR Spectroscopy, Psychosis, Schizophrenia (SCZ), Occipital Cortex, Prefrontal Cortex Disclosure: Nothing to disclose. P551. Metabolomic Signatures Associated With Antipsychotic -Induced Weight Gain and Psychosis Spectrum Diagnoses Jiwon Lee, Kenya Costa-Dookhan, Araba Chintoh, Gary Remington, Daniel Mueller, Philip Gerretsen, Sri Mahavir Agarwal, Vicki Ellingrod, Kristen Ward, Margaret Hahn* Centre for Addiction and Mental Health, Toronto, Canada Background: Psychosis spectrum disorders (PSDs) are associated with intrinsic metabolic abnormalities. Beyond the intrinsic metabolic risk, antipsychotics (APs), the cornerstone of treatment for PSDs, incur additional metabolic adversities including weight gain. Currently, major gaps exist in understanding psychosis illness biomarkers, as well as risk factors and mechanisms for AP-induced weight gain. Metabolomic profiles may provide insight into biomarkers of PSDs and antipsychotic-induced weight gain. Methods: In this 12-week prospective naturalistic study, we compared serum metabolomic profiles of AP-naïve cases to healthy controls at baseline to examine biomarkers of intrinsic metabolic dysfunction in PSDs. We then examined changes in serum metabolomic profiles over 12 weeks of antipsychotic treatment in AP-naïve cases to identify metabolites that may predict or associate with AP-induced weight gain. Statistical analyses of the metabolomic datasets were conducted using Metaboanalyst 5.0. T-tests were used to compare mean baseline metabolite concentrations between 1) AP-naïve cases and controls at baseline, and 2) AP-naïve cases who do and do not develop ≥5% body weight gain at 12 weeks. Changes from baseline to endpoint between AP-naïve cases who do and do not develop significant (i.e. ≥5%) body weight gain were compared with two-way repeated measures ANOVA testing. Pearson correlations were calculated between change in weight gain and change in metabolite concentrations from week 1 to week 12 for cases. To control for multiple comparisons, a false discovery rate (FDR) of the resulting post hoc P values was calculated. Results: Twenty-five AP-naïve cases were enrolled, and 17 cases completed both baseline and 12-week follow up visits, while the remaining 8 cases had baseline visits only. Six healthy controls completed baseline visits. Following 12 weeks of AP exposure, cases experienced increases in body weight (p < 0.001), BMI (p < 0.001), waist circumference (p = 0.010), LDL cholesterol (p = 0.026), and total cholesterol (p < 0.001). Additionally, a subgroup of cases (N = 11) experienced clinically significant increases (≥5%) in body weight. Overall, 20 amino acids, 20 bile acids, 30 fatty acids, and 29 acylcarnitines were identified and quantified. AP-naïve cases were distinguished from controls by six fatty acids when compared at baseline (FDR of <0.05), namely reduced levels of palmitoleic acid, lauric acid, and heneicosylic acid and elevated levels of behenic acid, arachidonic acid, and myristoleic acid. Baseline levels of the fatty acid adrenic acid was increased in individuals who experienced a clinically significant body weight gain (≥5%) following 12 weeks of AP exposure as compared to those who did not (FDR = 0.0408). Among quantified metabolites, none met the threshold for significance when examining associations between changes in metabolite concentrations and body weight over 12 weeks. Conclusions: Specific fatty acids may represent illness biomarkers of PSDs and early predictors of AP-induced weight gain. The findings hold important clinical implications for early identification of individuals who could benefit from prevention strategies to reduce future cardiometabolic risk, and may lead to targeted treatments to counteract metabolic dysfunction in PSDs. Keywords: First Episode Psychosis, Metabolomics, Intrinsic Metabolic Risk, Antipsychotic-Induced Weight Gain Disclosure: Alkermes: Consultant (Self). P552. Proteomic Plasma Profiling of Patients With First-Episode Psychosis Identifies Inflammatory Markers Associated With Illness Severity Sophie Erhardt, Lilly Schwieler, Carl M Sellgren, Feride Eren, Simon Cervenka, Fredrik Piehl, Helena Faturos Bergman, Funda Orhan, Goran Engberg Karolinska Institutet, Stockholm, Sweden Background: Over the last 20 years numerous studies depict that the immune system and inflammatory responses may play an important role in psychiatric disorders. Thus, enhanced secretion of inflammatory cytokines and chemokines have been found in patients with depression, schizophrenia and bipolar disorder. Increased levels of immune markers have also been found to correlate with impaired cognitive ability. In the present study, Olink Proximitiy Extension Assay (PEA) technology was used to investigate immune activation in first-episode psychosis (FEP) patients and healthy controls (HC) and to further analyze if the immune profile differs between FEP patients later diagnosed with schizophrenia and those not receiving such diagnosis. Furthermore, since correlations of disease severity and neurocognitive decline has been shown previously, we aim to explore patterns of correlations with altered inflammation markers and psychopathology and cognitive scores. Methods: Somatically HC (n = 55) with no prior drug addiction and FEP patients (n = 73) were recruited in the Karolinska Schizophrenia Project (KaSP) in Stockholm, Sweden. The existence of neurologic illnesses or severe somatic illness, a history of illegal substance addiction, and the presence of co-existing neurodevelopmental abnormalities were exclusion criteria. Global Assessment of Functioning (GAF, the Positive and Negative Syndrome Scale (PANSS), Clinical Global Impression (CGI) were performed to assess clinical characteristics of the patients. 46.6% of the patients (n = 34 of 73) were treated with antipsychotics at the time of serum sampling. Maximum number of days of antipsychotic treatment was 26 days, mean number of days was 9.6. Thirty-eight patients have not used any kind of antipsychotics prior, or at the time of serum sampling. Venous blood samples were collected using standard venipuncture techniques. 92 inflammatory proteins in the serum were analyzed by Olink Bioscience, Uppsala, Sweden using the multiplex proximity extension assay technology (PEA) inflammation panel. Results: No significant differences were observed between HC and FEP patients regarding age, gender, or body mass index. Total PANSS score for patients was 71.9 ± 2.4. DUP was 8.5 ± 1.6 (mean ± s.e.m.) months for FEP patients. Differential expression analysis showed 12 proteins upregulated in FEP patients. AXIN1 was identified to be the most differentially abundant (logFC = 1.48, adj.p.val = 0.00002). The second most differentially abundant protein, with highest log fold change, was STAMBP (logFC = 1.01, adj.p.val = 0.00002). CASP-8 and SIRT2 are other top differentially abundant inflammation markers with high log fold changes found in patients. The chemokines CXCL1, CXCL5, CXCL6, IL7 and TNFSF14, playing important roles in ERK signaling pathways and neutrophil activation, were also found to be elevated. The levels of the anti-inflammatory cytokine, IL-10RA, was found to be decreased in patients. Differential expression analysis between HC and disease groups (SCZ and Non-SCZ) showed that 15 proteins are upregulated in patients diagnosed with schizophrenia compared to HC. Here, AXIN1 was identified to be the most differentially expressed with an elevated log fold change. (logFC = 1.87, adj.p.val = 4.54E-07). Ten out of 12 of the inflammation proteins (namely AXIN1, STAMBP, CASP-8, 4E-BP1, CXCL1, CXCL5, IL7, TNFSF14, CXCL6 and IL-10RA) showed a significant correlation with PANSS positive scores. The NPX values for AXIN1 (rs = 0.36), STAMBP (rs = 0.38), IL-7 (rs = 0.50) and IL-10RA (rs = −0.7) showed a high correlation with PANSS positive score. Correlations between differentially expressed proteins and symptom ratings were also performed in the subgroup of patients diagnosed with SCZ. The NPX values for AXIN1 (rs = 0.45), STAMBP (rs = 0.4), CASP-8 (rs = 0.67), SIRT2 (rs = 0.53), CXCL5 (rs = 0.35), IL-7 (rs = 0.56), TNFSF14 (rs = 0.33), CXCL6 (rs = 0.43), CD40 (rs = 0.34) and MCP-2 (rs = 0.50) showed a high correlation with PANSS positive score. PANSS Negative was found to correlate to the NPX values for CXCL6 (rs = 0.33), CCL11 (rs = 0.36), CXCL11 (rs = 0.32), PANSS General was found to correlate to the NPX values for AXIN1 (rs = 0.35) and PANSS total scores was found to correlate to the NPX values for AXIN1 (rs = 0.34), STAMBP (rs = 0.32), CASP-8 (rs = 0.5), IL-7 (rs = 0.35), CXCL6 (rs = 0.39) and MCP-2 (rs = 0.34). Correlations were not significant after correction for multiple testing. Conclusions: The current study’s findings demonstrate that peripheral immunological markers are significantly elevated in FEP patients. Interestingly, several of the markers had previously been proposed to be implicated in psychosis. We also discovered that the markers significantly associated to illness severity and cognition. This is consistent with our discovery that immunological activation was even stronger in a subset of patients eventually diagnosed with schizophrenia. Keywords: First Episode Psychosis, Chemokines, Cytokines, Schizophrenia (SCZ) Disclosure: Nothing to disclose. P553. Associations of Longitudinal Inflammatory Biomarker Changes With Cognitive, Mental, and Physical Health in Schizophrenia: Using a Mixed Models Approach to Compare 4-Year Changes in a Cohort of People With and Without Schizophrenia David H. Adamowicz, Morris Wu, Rebecca Daly, Xin M. Tu, Michael Irwin, Dilip V. Jeste, Lisa Eyler, Ellen E. Lee UCSD, San Diego, California, United States Background: Schizophrenia has been linked to a chronic inflammatory state, with previous research demonstrating elevated plasma biomarkers reflective of aberrant immune functioning. Pro-inflammatory cytokines are thought to have a direct effect on neurotransmission and are associated with severity of psychopathology. People with schizophrenia (PwS) who have elevated inflammatory biomarker levels may benefit from anti-inflammatory treatments during the early stages of the illness. Postmortem brain samples from PwS have shown increased expression of pro-inflammatory proteins, indicating persistence of some aspects of inflammation throughout the course of the illness. However longitudinal data evaluating the change in immune dynamics over time among PwS remain sparse. The current study addresses the limitations of previous cross-sectional studies by examining the rate of change of key inflammatory biomarkers from baseline to subsequent follow up, as well as their association with measures of cognitive, mental, and physical health. We hypothesized that 1) PwS would have greater increases in pro-inflammatory biomarkers over time compared to non-psychiatric comparison subjects (NCs), and 2) these increased rates of change will predict worsening cognition over time. We conducted further exploratory analyses using various measures of physical and mental functioning. Methods: The cohort included 123 PwS and 108 NCs (mean age 49.1, SD 10.2, range 26 to 66 years, 49.4% female), with no significant difference in age or sex between diagnostic groups. Plasma biomarker levels were quantified using Meso Scale Discovery MULTI-SPOT® Assay System and analyzed on a SECTOR Imager 2400 instrument. Plasma high sensitivity C-reactive protein (hs-CRP) levels were measured by enzyme-linked immunosorbent assay at UCSD. Interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha were assayed at UCLA. Executive functioning measures included a composite score of subtests from the Delis-Kaplan Executive Function System (D-KEFS): Trail Making, Color Word Inhibition, and the Letter Fluency task. Other health measures included waist-to-hip ratio, BMI, HgbA1c (assessed using a standard clinical assay), medical comorbidity (Cumulative Illness Rating Scale), depression (Patient Health Questionnaire-9 item), anxiety (Brief Symptom Inventory-Anxiety), and positive and negative symptoms (Scales for Assessments of Positive and Negative Symptoms). Rates of change of inflammatory biomarker levels, cognition, and other health measures were calculated as the difference between level/score at the final visit with level/score at baseline and converted to a yearly rate. Spearman’s correlation was used to examine the relationship between biomarker rates of change and cognitive measures. Mixed effect linear models analyzed the association of inflammatory biomarker rates of change with diagnostic group (PwS vs. NCs) as well as with cognitive/health outcomes, controlling for age, sex, baseline inflammation, and baseline cognition. Results: Over the mean 4.4-year follow-up period, PwS showed increases in IL-8 (mean yearly rate 0.496, SE .104) and IL-6 (mean yearly rate .0694, SE .0680) levels, whereas these inflammatory biomarkers remained unchanged in NCs. Primary analyses indicated elevated rate of change for IL-8 in PwS as compared to NCs (t(152.7) = −3.178, p = 0.002). D-KEFS scores improved over time in both diagnostic groups, likely due to practice effects. While D-KEFS score was not associated with biomarkers in PwS, there was a positive correlation between change in IFN-gamma levels and Color Word Inhibition (rs = 0.295, p = 0.005), indicating worse performance with decreasing biomarker levels, and a negative correlation between change in IL-10 levels and Letter Fluency (rs = −.242, p = 0.024), indicating better performance with decreasing biomarker levels. In NCs, there was a negative correlation between rate of change in IL-6 and that of D-KEFS score (rs = −.264, p = 0.041), with no other significant correlations. General linear models controlling for sex, age, diagnostic group, baseline inflammation, and baseline cognition indicated increases in baseline IL-8 level (Z = 0.249, SE = 0.081, p = 0.0024) and rate of change of IL-8 levels (Z = 0.517, SE = 0.184, p = 0.0056) as predictors of improved D-KEFS score over time. Secondary analyses demonstrated a greater number of significant correlations between rates of change of inflammatory biomarkers with physical functioning among PwS compared to NCs. This relationship was especially notable with significant positive correlations between changes in waist-to-hip ratio and changes in IFN-gamma, IL-10 and 8, and TNF-alpha (rs = 0.382, .250, .266, .400 respectively) only among PwS. Conversely, NCs showed stronger correlations than PwS between rates of change of inflammatory biomarkers with mood, as illustrated by a significant positive correlation between change in depression and hs-CRP (rs = 0.244), as well as negative correlations between change in depression with IL-10 and TNF-alpha (rs = −.475 and -.311 respectively). Conclusions: Collectively, these results indicate distinct trajectories in plasma inflammatory biomarkers in PwS when compared to NCs. Notably, increased IL-8 levels were found to be predictive of improvements in executive function over time, regardless of diagnostic group. This study is ongoing and updated results will be presented at the meeting. Keywords: Psychosis, Cytokines, Cognition Disclosure: Nothing to disclose. P554. Tumor Necrosis Factor is Associated With Altered Activation in Ventral Striatal and Anterior Insula in Response to Reward and Effort in Patients With Schizophrenia David Goldsmith, Courtney Ning, Robin Gross, Jessica Cooper, Elaine Walker, Michael Treadway, Andrew Miller Emory University, Atlanta, Georgia, United States Background: Increasing data implicates inflammation as a driver of negative symptoms in patients with schizophrenia. Relative to the mechanism by which inflammation effects negative symptoms, inflammation has been shown to decrease VS activation in response to reward in healthy controls and depressed patients. Negative symptoms, specifically motivational deficits, have been associated with decreased activation of the ventral striatum in response to reward anticipation. Previous data demonstrated that administration of inflammatory stimuli is reliably associated with decreased activation in the ventral striatum in the context of reward anticipation, whereas inflammation increases activation in the anterior insula in association with punishment prediction errors. Thus, we hypothesized that inflammation would be associated with motivational deficits as well as altered signaling in reward-relevant regions. Methods: 37 patients with schizophrenia, on medications, were recruited from Grady Hospital in Atlanta, Georgia. Patients were excluded if they had evidence of unstable medical conditions, inflammatory illness, use of anti-inflammatory medications, or active substance use. Negative symptoms were measured using the Brief Negative Symptom Scale (BNSS), with subscores for a motivated behavior factor as well as an expressivity factor. Functional connectivity analyses (n = 30) were used with an a priori seed placed in the nucleus accumbens. A subset of subjects (n = 22) performed the Monetary Incentive Delay Task (MID) and the Effort Based Decision Making Task (EBDM) in a 3 T fMRI scanner. A predefined nucleus accumbens mask was used given the a priori hypothesis regarding the ventral striatum in response to reward anticipation. A whole brain analysis was used for the EBDM task to look at the effect of increasing effort (using a parametric modulator). Linear regression models were tested to determine the relationship between inflammation and brain activation, controlling for age and sex. Results: Increases in CRP, a non-specific marker of inflammation, were significantly correlated with decreases in the BNSS motivated behavior factor (r = −0.340, p = 0.042) and specifically the avolition subscale (r = −0.438, p = 0.029). No association was found for the BNSS expressivity factor (p > 0.8). These relationships remained significant after controlling for depression. Using resting state functional connectivity analyses, increasing CRP was associated with greater connectivity between a seed in the right nucleus accumbens and activation in a cluster that included the right insula (peak [42, -14, 2] T = 3.41, p(uncorrected)<0.001). Regarding more specific inflammatory mediators, tumor necrosis factor (TNF) at higher concentrations were associated with lower activation in the nucleus accumbens in response to reward anticipation in the MID task (Win > Neutral contrast; = β= 0.462, p = 0.039) [both the left (peak left [-14, 22, -8], T = 3.48, p (uncorrected) = 0.001) and right (peak right [4, 22, -6], T = 2.73, p (uncorrected) = 0.003)]. Higher concentrations of TNF were also associated with increased activation in the right anterior insula (β = 0.690, p < 0.001) in response to increasing effort on the EBDM task (peak [38, 20, 6], T = 4.35, p (uncorrected) < 0.001).Of note, activation in the nucleus accumbens in respons to reward was significantly correlated with activation in the anterior insula in response to effort (r = −0.512, p = 0.015). Conclusions: These data support a relationship between inflammation and negative symptoms, and specifically motivational deficits. Increased TNF may lead to negative symptoms through reduced ventral striatum activation in response to reward and increased anterior insula activation in response to increasing effort. The ventral striatum is involved in the regulation of reward and previous data has demonstrated that inflammation may target brain reward circuitry, including the striatum. The anterior insula is involved in interoceptive processing and punishment prediction errors. Previous data has shown that the anterior insula may be sensitive to inflammation in response to punishment prediction errors. The anterior insula may control motivational vigor and influence downstream dopaminergic signaling in the nucleus accumbens, suggesting a coordinated circuit between the insula and basal ganglia that may be sensitive to inflammation. These findings support the hypothesis that inflammation may target the ventral striatum and anterior insula to lead to reward and effort processing deficits that may underlie negative symptom severity. Keywords: Schizophrenia (SCZ), Inflammation, TNF-Alpha, Effort Based Decision Making Task, Monetary Incentive Delay Task Disclosure: Nothing to disclose. P555. Peripheral Inflammation is Associated With Impairments of Inhibitory Behavioral Control and Visual Sensorimotor Function in Psychotic Disorders Lusi Zhang, Paulo Lizano, Yanxun Xu, Leah Rubin, Adam Lee, Rebekka Lencer, James Reilly, Richard Keefe, Sarah Keedy, Godfrey Pearlson, Brett Clementz, Matcheri Keshavan, Elliot Gershon, Carol Tamminga, John A. Sweeney, Scot Hill, Jeffrey Bishop University of Minnesota, Minneapolis, Minnesota, United States Background: Elevated markers of peripheral inflammation are common in psychosis spectrum disorders and have been associated with brain anatomy, and physiology as well as clinical outcomes. Preliminary evidence suggests a link between inflammatory cytokines and C-reactive protein (CRP) with generalized cognitive impairments in a subgroup of individuals with psychosis. Whether these patients with elevated peripheral inflammation demonstrate deficits in specific cognitive domains remains unclear. Methods: Seventeen neuropsychological and sensorimotor tasks and thirteen peripheral inflammatory and microvascular markers were quantified in a subset of participants (129 psychosis, 55 healthy controls) recruited from the first wave of the Bipolar-Schizophrenia Network on Intermediate Phenotypes consortium (B-SNIP1). Principal component analysis was conducted across the inflammatory markers, resulting in five inflammation factors. Three discrete latent cognitive domains (Visual Sensorimotor, General Cognitive Ability, and Inhibitory Behavioral Control) were characterized based on the neurobehavioral battery and examined in association with inflammation factors. Hierarchical clustering analysis identified cognition-sensitive high/low inflammation subgroups. Results: Among persons with psychotic disorders but not healthy controls, higher scores on an inflammation factor representing elevations of CRP, IL6, and C4a and reduction of IFNγ, IL8, IL10, and VEGFD had significant associations with impairments of Inhibitory Control (R2 = 0.100, p-value=2.69e-4, q-value=0.004) and suggestive associations with Visual Sensorimotor function (R2 = 0.039, p-value=0.024, q-value=0.180), but not with General Cognitive Ability (R2 = 0.015, p-value=0.162). Greater deficits in Inhibitory Control were observed in the high inflammation patient subgroup, which represented 30.2% of persons with psychotic disorders, as compared to the low inflammation psychosis subgroup. These relationships were not significantly impacted by the use of psychotropic medications, cardiometabolic diagnoses, cardiometabolic medications, or non-steroidal anti-inflammatory drugs (NSAIDs). Conclusions: These findings indicate that inflammation dysregulation may differentially impact specific neurobehavioral domains across psychotic disorders, particularly performance on tasks requiring ongoing behavioral monitoring and control. The present findings provide further supporting evidence that one important underlying mechanistic contributor to the disruption of cognition in persons with psychotic disorders is immune/inflammation dysregulation and that targeting this dysregulation may be an avenue for novel therapeutic interventions to improve cognitive outcomes in these patients. Keywords: Cognition, Psychosis, Inflammation Disclosure: OptumRx: Consultant (Self). P556. Levels of Matrix Metalloproteinase 9 (MMP-9) are Elevated in Persons With Serious Mental Illness Faith Dickerson, Dhananjay Vaidya, Yisi Liu, Robert Yolken Sheppard Pratt Health System, Baltimore, Maryland, United States Background: Matrix metalloproteinases constitute a diverse set of calcium-dependent endopeptidases with a wide range of biological functions. While there are many human metalloproteinases, MMP-9, also known as gelatinase B, has been of particular interest since it modulates several arms of the immune system, alters blood-brain barrier functioning, and can affect synaptic functioning. Increased levels of MMP-9 have also been associated with increased rates of mortality caused by impaired cardiopulmonary functioning. Due to these CNS and systemic properties, there has been interest in defining the relationship between MMP-9 and serious mental illnesses and in developing methods for lowering MMP-9 levels. Individual levels of MM9 are determined by both genetic and environmental factors. Environmental factors associated with elevated MMP-9 levels include tobacco smoking and obesity, which are common in individuals with serious mental illness and are potentially modifiable. We measured circulating MMP-9 levels in a cohort of individuals with serious mental illness and without a psychiatric disorder and determined the effects of diagnosis, tobacco smoking, obesity, and other factors on MMP-9 levels. Methods: Participants were enrolled in the period Jan 1, 2008 - Sept 1, 2021 and assessed on demographic and clinical characteristics; diagnoses were verified by SCID interview. Participants included three sub-cohorts consisting of individuals with schizophrenia, bipolar disorder, or depressive disorder enrolled from Sheppard Pratt clinical programs. A group of comparison persons without any history of psychiatric disorder was recruited from posted announcements. Levels of MMP-9 were measured in blood samples by means of sensitive solid phase immunoassays. Statistical methods: We performed separate analyses for the three psychiatric sub-cohorts, comparing them each with the common comparison group. In each analysis, the psychiatric vs. non-psychiatric condition was used as the dependent variable in logistic regression. In preliminary analyses we determined that log-transformed MMP-9 was appropriate as an independent variable for the analyses, consistent with its linear relationship with the logit of the dependent variables. In addition, age as a continuous variable, male vs female and White vs other race as dichotomous variables and the variables of tobacco smoking, and obesity (BMI ≥ 30 kg/m2) and the interaction between these two terms were included as covariates. We estimated the contributions of various independent variables to the variance of each psychiatric diagnosis vs the non-psychiatric comparison group as a continuous variable as the squared partial correlation. We employed a linear regression model with log (MMP-9) and demographic covariates as well as an additional model adding tobacco smoking, obesity and the interaction between these two. We noted the change in the partial r2 of log-MMP-9 in these sequential models. Additional models examined the effects of psychiatric medications in individuals with psychiatric diagnoses. Results: A total of 1256 individuals were enrolled in the study: 440 with schizophrenia, 399 with bipolar disorder, 135 with major depressive disorder, and 282 without any current or past psychiatric disorder. The mean age of persons in the larger cohort was 35.3 (±13.0); a total of 667 (53%) were female, 700 (56%) White; 470 (37%) were current tobacco smokers and 490 (39%) were obese. Logistic models which included current smoking, obesity and their interaction along with demographic variables indicated significant associations between MMP-9 levels (per 1 natural log-unit higher levels) and the diagnosis of schizophrenia (OR 4.71, 95% CI 2.41, 9.21, p < 0.001), bipolar disorder (OR 2.99, 95% CI 1.79, 5.01, p < 0.001) and major depressive disorder (OR 9.63, 95% CI 4.59, 20.19, p < 0.001). We also examined the fractional effects of smoking, obesity and their interaction on reducing the partial correlation of MMP-9 levels for each psychiatric group. In the case of schizophrenia, 59.6% of the partial r2 attributable to MMP-9 with age, sex and race adjustment alone was lost when smoking, obesity and their interaction were added to the model, while the remaining 40.4% or the original partial r2 remained attributable to log(MMP-9) and thus not be explained by these factors. In the case of bipolar disorder, 39.9% of the age-sex-race adjusted explanatory fraction of MMP-9 was lost when explained by smoking and obesity and their interaction, while in the case of major depressive disorder only 19.3% of the original age-sex-race adjusted explanatory fraction of MMP-9 elevation could be explained by these factors. Among persons in the psychiatric groups, levels of MMP-9 were not significantly associated with the receipt of specific antipsychotic, anti-depressant, or mood-stabilizing medications suggesting that the differences in MMP-9 levels among the diagnostic groups are not explained by medications. Conclusions: Levels of MMP-9 are significantly elevated in persons with schizophrenia, bipolar disorder, and major depressive disorder. Some of the increases are related to tobacco smoking and obesity, particularly in individuals with schizophrenia where these factors are highly prevalent. In light of the potential adverse effects of MMP-9, efforts should be directed at lowering MMP-9 levels by means of smoking cessation and weight loss interventions as well as specific pharmacological interventions. Keywords: Immune Biomarkers, Schizophrenia (SCZ), Matrix Metalloproteinase-9 (MMP-9) Disclosure: Nothing to disclose. P557. A Disease-Informed, Network-Targeted Neuromodulation Intervention Affects Craving in Individuals With Schizophrenia and Nicotine-Dependence Heather Ward, Gulcan Yildiz, Jing Xie, Adam Beermann, Amy Janes, Lauren Moran, Matcheri Keshavan, Mark Halko, Roscoe Brady Vanderbilt University Medical Center, Boston, Massachusetts, United States Background: Tobacco use is the top cause of early preventable mortality in schizophrenia, leading to a 25-year decreased life expectancy. The prevalence of nicotine use in schizophrenia is more than three times that of the general population. There is a significant gap in our understanding of nicotine dependence in schizophrenia and how this diagnosis affects treatment. Treatments for nicotine dependence are significantly less effective for people with schizophrenia compared to controls. This may be because current interventions for nicotine dependence in this population do not target schizophrenia-specific pathophysiology. We therefore sought to identify and test a neuromodulation intervention on a schizophrenia-specific circuit of nicotine dependence. Methods: This study consisted of 3 phases: Network Discovery, Network Validation, and Network-Targeted Intervention. In Network Discovery, we used a data-driven approach to identify a schizophrenia-specific circuit of nicotine dependence. We reanalyzed existing data from 35 smokers (18 schizophrenia, 17 controls) who underwent resting-state fMRI and clinical characterization. We conducted a transdiagnostic assessment to identify shared and diagnosis-specific circuits of nicotine use. A multi-variate pattern analysis of whole-connectome data was used to identify the strongest links between daily cigarette consumption and functional connectivity. In Network Validation, 12 participants with schizophrenia and 12 controls participated in a randomized, controlled crossover study of nicotine patch with resting-state fMRI. Based on the results from Network Discovery, we calculated mean change in default mode network (DMN) connectivity (FCnicotine – FCplacebo) and correlated change in whole-network functional connectivity with nicotine dose. We then determined the effect of acute nicotine administration on this network. In Network-Targeted Intervention, 10 individuals with schizophrenia and nicotine dependence were enrolled in a randomized, sham-controlled crossover study of single session DMN-targeted repetitive transcranial magnetic stimulation (rTMS) with fMRI immediately before and after each rTMS session. Individuals underwent baseline resting state fMRI where the DMN was mapped. The point of maximal connectivity of the left parietal DMN node was selected as the TMS target for all 3 sessions. Individuals received 1 session of intermittent theta-burst stimulation (iTBS, 100% active motor threshold, AMT), 1 session of continuous theta-burst stimulation (cTBS, 80% AMT), and 1 session of sham rTMS (100% AMT, coil flipped 180 degrees away from participant). Craving was assessed before and after each rTMS session using a Visual Analog Scale. Each rTMS session was separated by at least 48 hours in order to minimize any carryover effect of the previous stimulation. Using a within-subjects design, we compared changes in craving between each type of rTMS using a mixed ANOVA. Results: In Network Discovery, the strongest (p < 0.001) correlate between functional connectivity and daily cigarette consumption was driven by individual variation in the topography of the DMN. This effect was entirely driven by participants with schizophrenia despite the fact that groups were matched for severity of nicotine dependence. Specifically, with greater severity of nicotine use in schizophrenia, the DMN was pathologically expanded into territory normally occupied by the dorsal attention network. This relationship between network organization and nicotine use was specific to schizophrenia and not observed in control smokers. In Network Validation, we tested the causality of nicotine-network interactions in an independent cohort. We we observed that acute nicotine administration reverses DMN hyperconnectivity in schizophrenia in a dose-dependent relationship (R = −0.50; 95% CI -0.75 to -0.12, p < 0.05). In Network-Targeted Intervention, we observed a significant treatment x time relationship (p = 0.017) where craving was significantly increased by iTBS but not by cTBS or sham. Conclusions: We identified a schizophrenia-specific network of nicotine dependence, validated the effect of acute nicotine administration on that network, and then applied a neuromodulation intervention on that network. This disease-informed, network-targeted intervention significantly affected nicotine craving in schizophrenia. This is the first evidence for a circuit-based intervention for substance use in schizophrenia that was empirically derived, unique to schizophrenia, and affected clinical outcomes. Future experiments should test repeated sessions of rTMS in clinical trials. Keywords: Schizophrenia (SCZ), Nicotine Dependence, Resting State Networks, Default Mode Network (DMN), Repetitive Transcranial Magnetic Stimulation (rTMS) Disclosure: Nothing to disclose. P558. Taking AIMS: Characterization of Abnormal Involuntary Movement Scale Utilization in the VA Healthcare System Conner Polet, Rishab Gupta, Yash Joshi, Nina Schooler, Mihaela Aslan, Panos Roussos, Philip Harvey, Tim Bigdeli State University of New York Downstate Medical Center, Brooklyn, New York, United States Background: Tardive dyskinesia (TD) is a medication-induced movement disorder that is classically precipitated by long-term exposure to dopamine receptor blocking agents. TD is often irreversible, is debilitating, and has been associated with a poorer quality of life and increased mortality. TD surveillance is typically carried out via routine documentation through the Abnormal Involuntary Movement Scale (AIMS), especially in those prescribed medications with elevated TD risk, including antipsychotics. Methods: We reviewed electronic health records (EHRs) for 700,000 participants in Cooperative Studies Program (CSP) #572 and the Million Veteran Program, including ICD-9/10 billing codes, prescription records, and AIMS, and spanning more than 20 years. We used “phecodes”, which are groupings of conceptually related ICD codes, to assign putative diagnoses of schizophrenia (295.1), bipolar disorder (296.1), psychosis NOS (295.3), depression, and extrapyramidal symptoms (EPS; 333). We intersected these data to characterize the clinical correlates of antipsychotics-induced EPS assessed using the AIMS and corroborated by a diagnosis of TD. Results: The AIMS was administered 126,766 times to 38,364 individuals (median = 12, mean=27.3, SD = 43). AIMS scores were documented in X% of all individuals who were taking antipsychotic medications. Of those with any AIMS data, n = 18,591 had AIMS score >0; most had no incapacitation (n = 13,100;70%), followed by minimal (n = 3,3403;18%), mild (n = 1,390;7%), moderate (n = 533;3%), and severe (n = 129,1%). Symptoms most commonly affected the jaw, and least commonly affected the trunk. Schizophrenia (n = 9,033;48.6%), bipolar disorder (n = 4,775;25.7%), psychosis NOS (n = 1,123;6%) and depression-related diagnoses (n = 3,221;17.3%) were the most common diagnoses in those with AIMS > 0. AIMS scores were greater for those with a recorded diagnosis of EPS or abnormal movements compared to those without (5.4,SD = 4.8 vs 3.2, SD = 3.2; t = 30.6, df=7120.2, p < 10^-193). Consistent with expectation, patients who received treatment with a first-generation antipsychotic medication (44.8%) had higher AIMS than those treated with second-generation agents alone (55.2%; unpaired t-test, t = 20.8, df=14585, p < 10-93). Conclusions: We have undertaken a preliminary survey of extrapyramidal side-effects of antipsychotic medications in the VHA, and describe the clinical correlates of EPS and TD with respect to medication classes, specific agents, lifetime psychiatric diagnoses. Available AIMS data revealed appreciable individual differences in risk and severity of EPS/TD among patients with comparable treatment histories, and support an AIMS score of 2 or more as a relevant clinical cutoff for TD. Ongoing analyses include more granular investigations of particular medications based on receptor affinity and with consideration to dosage, and genome-wide association studies (GWAS) to identify novel TD/EPS susceptibility (or resilience) loci. Keywords: Tardive Dyskinesia, Electronic Health Record (EHR), Extrapyramidal Symptoms, Abnormal Involuntary Movement Scale (AIMS) Disclosure: Nothing to disclose. P559. Where is the Reliable Signal in the Total and New Short Forms of the PANSS in an Outpatient RCT Eric Youngstrom, Joan Busner, David Daniel, Joshua Langfus, Robert Findling University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: The Positive and Negative Symptom Scale (PANSS) is a venerable interview for rating severity of psychotic symptoms, and it is the most common outcome measure in clinical trials with youths and adults. It has 30 items that are usually summed or averaged to produce a total severity score. However, factor analyses consistently indicate that the PANSS items do not reflect a single construct. Instead, five factor solutions appear most favored in prior work, usually with low correlations between them. These findings call into question the use of a total score as a main outcome, as well as invalidating Cronbach’s alpha as a measure of reliability. If the group factors are sufficiently reliable and distinct from a general factor, that would support using them as distinct targets for intervention development or outcome. The goal of the present investigation is to use omega reliability estimates, which model the number of factors underlying the items to produce estimates of total variance in the scores related to all factors (omega total), versus related only to the general factor (total psychosis), or distinct to each factor subscale (supporting interpretation of the subscale). We use a well-characterized pediatric samples, and we calculate the omega values for the 30 item, 20 and 10 item versions we developed, and also the 19 item and two 6 item short forms proposed by others. Methods: Secondary analyses of the TEOSS study (Sikich et al., N = 180). Analyses were performed using the mirt, lavaan, BifactorIndicesCalculator, and psych packages in R. Omega estimates specified 5 factors (2 factors for the 6 item forms), and calculated alpha and omegaTotal, omegaGeneral, and omegaGroup. Alpha estimates reliability for a single underlying factor; omegaTotal combines reliability for the general (psychosis total) and 5 group factors; omegaGeneral estimates the variance attributable to the general factor alone (with values > 0.80 indicating a strong general factor), and OmegaHS is the variance in subscales attributable to the specific factors (with values > 0.40 indicating reliable specific scores, likely to support independent interpretation and replication)(Rodriguez, Reise, and Haviland, 2015; Revelle and Condon, 2019). Results: Every analysis in both samples indicated that a multi-factor model fit better than a unidimensional one across all item sets. OmegaTotal was always substantially larger than alpha. The OmegaGeneral was .45 (poor) for the 30 item TEOSS PANSS, and .17 to .39 for the short forms. In contrast, all of the subscales on the 10 item TEOSS form has OmegaHS ranging from .45 to .76, supporting their individual interpretation. The general factor only explained 16% to 26% of the variance in items, whereas subscales based on the factors were more reliable and interpretable, correlating with their respective factor scores .71 to .97 for the 10-item version. Conclusions: Results strongly indicate that the PANSS is better considered a “composite” containing different factors, rather than treating it as a single score (Revelle and Condon, 2019). Existing datasets could be reanalyzed focusing on the subscale scores. New studies and clinical applications could use shortened versions, perhaps omitting scales (e.g., internalizing) that may be redundant with other parts of a battery (e.g., CDRS-R or HDRS). Keywords: Clinical Assessment, Psychosis, Pediatric Disclosures: Signant: Consultant (Self), Guilford Press, American Psychological Association: Royalties (Self). P560. Content Validation of a Functional Co-Primary Measure for Clinical Trials Targeting Cognitive Impairment Associated With Schizophrenia (CIAS): An FDA-Funded Study Bill Horan, Colin Depp, Samantha Hurst, Hans Klein, Philip Harvey, Richard Keefe WCG VeraSci, Durham, North Carolina, United States Background: The FDA requires clinical trials for new drugs targeting CIAS to demonstrate the functional relevance of any cognitive improvements with a co-primary measure. The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is a performance-based measure that uses a realistic simulated environment to assess skills required for independent activities of daily living, including planning a meal, using transportation, using currency, and shopping. The FDA has accepted the VRFCAT into its Clinical Outcome Assessment (COA) Qualification Program for use as a co-primary measure in CIAS trials. For a full Qualification Package submission, the FDA COA Program requires data supporting content validity, including qualitative evidence that key stakeholders view the measure as relevant and important in the target population. We present findings from an FDA-funded study designed to obtain qualitative data on the VRFCAT. Methods: First, we convened a panel of academic, industry, and regulatory experts to develop a semi-structured interview to elicit key stakeholders’ views about (a) the definition of independence and associated behaviors, and (b) the functional relevance of the four domains assessed by the VRFCAT (meal planning, using transportation, using currency, shopping). Second, three stakeholder groups were interviewed: outpatients with schizophrenia (n = 24), family members (n = 12), and peer support specialists (n = 12). These interviews contained questions about general perspectives on functional independence and related activities. Participants reviewed representative audiovisual VRFCAT clips depicting the four domains assessed and responded to questions regarding each task’s relevance for functional independence. Third, a codebook was developed for thematic analyses of transcripts from recorded interviews. Results: Interviews were acquired from each of the targeted stakeholder subsamples (total N = 48). Qualitative analyses revealed that key stakeholders view the VRFCAT as assessing four functional capacity domains that are important for everyday functioning and independence. There was, however, some divergence among stakeholders in their perceptions of how the domains relate to independence. Caregivers saw these domains as important for minimizing dependence on the caregiver, whereas peer clinicians saw these functional domains as essential for pursuing higher-order goals (e.g., work) and patients emphasized practical aspects of functional skills, such as the avoidance of theft or loss. Regarding the specific skills assessed by the VRFCAT, high proportions of participants in each group rated the VRFCAT tasks as highly functionally relevant. Content analyses revealed similar themes across groups regarding why the four VRFCAT skill areas are important for functional independence. Conclusions: This qualitative study provides confirmatory support that key stakeholders view the VRFCAT as assessing four domains that are important for independent functioning. Further, the specific tasks used in the VRFCAT to assess these domains were uniformly viewed as functionally meaningful and important. The results also demonstrate that incorporating views from multiple stakeholder groups can provide complementary confirmatory evidence when assessing the content validity of a functional capacity measure in an indication like schizophrenia. This qualitative content validation evidence addresses a key requirement for measures evaluated in the FDA COA Qualification Program. Overall, the current qualitative results, along with quantitative evidence, provide convergent support that the VRFCAT is a functionally relevant, valid, and reliable co-primary to determine whether new drugs have a meaningful impact on cognition and on the lives of people with schizophrenia. Keywords: Cognitive Impairment Associated With Schizophrenia, Functional Capacity, Clinical Trial Methodology Disclosure: WCG VeraSci: Employee (Self). P561. Generalized Slowing of Resting State Neural Oscillations in People With Schizophrenia Scott Sponheim, Peter Lynn, Sophia Vinogradov, Ian Ramsay Minneapolis VA Medical Center, Minneapolis, Minnesota, United States Background: Neural oscillations are thought to reflect the flow of information through the brain. Recently, there has been interest in partitioning electroencephalography (EEG) recordings into periodic and aperiodic components. While both periodic and aperiodic components contribute to conventional measures of power within EEG bands (e.g., delta, theta, alpha, beta), the periodic aspect of EEG is thought to reflect true oscillatory behavior within neural systems while aperiodic activity captures sporadic brain activity. Given past evidence of resting state EEG power abnormalities in schizophrenia, we sought to determine if the periodic aspect of neural activity was aberrant in people with schizophrenia (PSZ) after removal of aperiodic activity. Methods: EEGs were gathered during a resting state from 104 PSZ and 105 healthy control participants (HCs). We used the fitting-oscillations-and-one-over-f (FOOOF) toolbox was to remove aperiodic neural activity, and computed the cross-correlation between power spectra for individual participants and the mean power spectrum for HCs to quantify the relative speed of neural oscillations. Results: Periodic activity in PSZ was shifted toward lower frequencies compared to HCs during eyes closed rest (t(187.86)=-3.67, p < 0.001). PSZ on average had a .50 hz shift toward oscillatory slowing compared to HCs across the frequency spectrum. Greater lag was associated with more symptoms of psychopathology and worse cognitive functioning. Conclusions: Slowed periodic activity at rest is evident in schizophrenia. A slower pace of neural oscillations may limit the transmission of information within and across brain systems. Slowed neural oscillations may contribute to poor integration of low-level perceptual and high-level cognitive functions in people with the illness. Keywords: Schizophrenia (SCZ), EEG/ERP Electrophysiology, Oscillations Disclosure: Nothing to disclose. P562. Striatal Dopamine Synthesis Capacity and Genetic Liability for Treatment-Resistant Schizophrenia in Healthy Adults Daniel Eisenberg, Philip D. Kohn, Michael D. Gregory, Jasmin B. Czarapata, Dwight Dickinson, Rachael K. Blackman, Christina A. Recto, Madeleine N. Goldberg, Bhaskar S. Kolachana, Karen F. Berman National Institute of Mental Health, Bethesda, Maryland, United States Background: Pharmacotherapy with first-line dopamine D2¬ receptor antagonists remains the primary biological treatment modality in schizophrenia, yet treatment response varies considerably across patients. Among individuals presenting with first episode schizophrenia, a subgroup representing nearly 25% will prove to be considered treatment resistant, often requiring multiple antipsychotic trials for lack of efficacy and, ultimately, treatment with clozapine, a unique agent that is helpful in this subgroup. Accumulating evidence from molecular neuroimaging studies has suggested that the abnormally increased striatal dopamine synthesis capacity observed in some patients with schizophrenia is often not observed in those with treatment-resistant schizophrenia, who, in fact, tend to show the opposite pattern (i.e., abnormally reduced striatal dopamine synthesis). This has raised the possibility that treatment-resistant schizophrenia may not simply denote one extreme of a unitary dimension of illness severity in schizophrenia, but rather is characterized by distinct underlying neurochemical mechanisms that merit dedicated investigation. The neurobiological causes of treatment resistance remain unknown; however, recent work has identified an array of common genetic variants that appear to be associated with treatment resistance in schizophrenia, though the impact of these variants on the living human brain, even in health, remains unclear, and biological validation studies are still needed. Methods: One-hundred and eighty-seven healthy individuals without psychiatric illness provided blood for genotyping and were studied with [18 F]-FDOPA positron emission tomography (PET; mean age 36 + /-11, 98 women) at the NIH Clinical Center in Bethesda, Maryland. For each individual, genotyping was conducted across the genome using Illumina SNP chips, and polygenic scores representing cumulative genetic risk burden were generated for schizophrenia generally and also for treatment-resistant schizophrenia specifically, based on genome-wide association study literature summary statistics. For neuroimaging, a 6-hour fast and 4-hour abstinence for caffeine and nicotine were required, and an oral dose of carbidopa was given one hour before injection of the tracer. Each individual’s separately-collected T1-weighted anatomical MRI scan was segmented for a cerebellar reference region and was co-registered to each participant’s native space, attenuation-corrected, and realigned PET data. After extraction of the reference region’s time activity curve, PET data were normalized to MNI-space using ANTS software. Gaussian smoothing was implemented to improve signal-to-noise ratios. PMOD software was used to implement standard graphical linear modeling to estimate the specific uptake parameter, Ki. Mean specific uptake was calculated for each of three bilateral subdivisions of the striatum (associative, sensorimotor, ventral), which were then interrogated for association with polygenic scores for schizophrenia generally and, separately, with polygenic scores for treatment-resistant schizophrenia. These analyses were performed using R software and employed general linear models that controlled for age, sex, and, to account for subtle population stratification effects, genetic principal components. Results: A significant positive association between tracer specific uptake and general schizophrenia polygenic risk score was found in the sensorimotor striatum (p = 0.04) though not in other striatal subdivisions. In contrast, a significant negative association between tracer specific uptake and treatment resistance polygenic risk score was found in all regions studied: associative striatum (p = 0.04), sensorimotor striatum (p = 0.04), and ventral striatum (p = 0.02). Conclusions: Presynaptic dopamine synthesis capacity in the striatum shows normative variability that is linked to mental health-related genetic risk burden, even in healthy individuals. Cumulative genetic risk for schizophrenia generally may predispose to elevated dopaminergic tone, whereas cumulative genetic risk for treatment-resistant schizophrenia may negatively bias dopaminergic tone. These divergent contributions to dopaminergic system variability follow expected directional effects based on observations from prior [18 F]-FDOPA PET imaging experiments in clinical cohorts. In conjunction with those prior experiments, this study provides novel insights into the unique biology of treatment-resistant schizophrenia genetic risk and has relevance for modern formulations of the dopamine hypotheses of schizophrenia. Future studies in patient populations will be important to understand the clinical implications of these associations, and further investigation of the distinct molecular mechanisms underlying risk for treatment resistance in schizophrenia is needed. Keywords: Schizophrenia, Dopamine, Genetics, Treatment Resistance Disclosure: Nothing to disclose. P563. Clozapine Therapeutic Drug Monitoring: Investigating Tolerability and Redefining Thresholds for Safety Araba Chintoh, Selai Akseer, Yukiko Mihashi, Gary Remington Centre for Addiction and Mental Health, University of Toronto, Toronto, Canada Background: One third of schizophrenia patients do not respond to standard antipsychotic medication and are classed as treatment-resistant schizophrenia (TRS). Clozapine is the only evidence-based treatment for TRS, yet clozapine is underutilized, in part, because clinicians fear serious side effects. Laboratory alerts caution that serum total clozapine and norclozapine levels above 1200 ng/mL put patients at risk of adverse effects. In response to high levels, clinicians often decrease dose or discontinue treatment altogether overvaluing the risks compared to the treatment benefits for patients. The Glasgow Antipsychotic Side Effects Scale for Clozapine (GASS-C) is a psychometric instrument used by clinicians to measure clozapine-related side effects. The current study aimed to evaluate within-subject and between-subject changes in clozapine levels and GASS-C severity. Methods: The GASS was administered to TRS outpatients prescribed clozapine, on a stable dose for a minimum of three months. All patients were followed at monthly intervals over three visits (n = 152). We applied 1200 ng/mL as the upper limit for clozapine values. Our sample included patients with clozapine levels crossing the threshold over the three follow up visits (n = 20) (mean age, 41.0 ± 10.9). We completed a secondary sensitivity analysis between patients with consistently below threshold values (n = 106) (mean age, 42.0 ± 12.6) versus patients with consistently above threshold values (n = 26) (mean age, 51.0 ± 16.2). We completed baseline descriptive analyses and mixed logistic regression models to assess changes in clozapine levels and side effect severity. Results: Amongst all the patients whose levels crossed the threshold over the three visits, no changes in side effects were reported in 66.7%, improvement of side effects was reported in 22.2%, and a worsening of side effects was reported in 11.1%. Results indicate no significant association (p = 0.8463) between changes in clozapine levels and side effects. At each visit when a patient’s level was below 1200 ng/mL, 50% reported no side effects and 50% reported moderate side effects. When a patient’s level was above 1200 ng/mL, 42.9% reported no side effects and 57.1% reported moderate side effects. The sensitivity analysis mixed logistic regression model found higher clozapine levels were associated with increased odds of side effect severity (Odds ratio [OR] = 1.72, 95% Confidence Interval[CI] = 0.48-6.22, p = 0.408) within subjects. Higher clozapine levels were associated with increased odds of side effects severity between high and low subjects (Adjusted OR = 1.48, 95%CI = 0.253-8.686, p = 0.662). Conclusions: In patients whose total serum clozapine levels cross the safety threshold of 1200 ng/mL, no significant differences in side effects are reported. Though reports of increased side effects were associated with higher levels, the results were not significant. Future studies are needed to confirm these results in larger cohorts to inform guidelines for clozapine safety thresholds and optimize clinicians’ use of clozapine to improve the outcomes for patients with TRS clozapine. Keywords: Clozapine, Treatment-Resistant Schizophrenia, Therapeutic Drug Monitoring Disclosure: Nothing to disclose. P564. Glutamatergic Target Engagement Biomarker in Schizophrenia Jack Grinband, Pejman Sehatpour, Daniel Javitt, Dan Iosifescu, Lawrence Kegeles, Tse Hwei Choo, Tarek Sobieh, Megan Mayer, Preetika Govil, Joshua Kantrowitz Columbia University, New York, New York, United States Background: N-methyl-D-aspartate-type glutamate receptors (NMDAR) antagonists reproduce both the symptoms and the cognitive deficits of schizophrenia (Sz), suggesting that NMDAR modulators may be beneficial. These psychotomimetic effects in turn are mediated, at least in part, by stimulation of presynaptic glutamate release in frontal brain regions. In animal models, both the behavioral and neurochemical effects of ketamine administration and especially its effect on glutamate release in medial prefrontal glutamate/dorsal anterior cingulate cortex (mPFC/dACC) are blocked by agonists of metabotropic (mGluR2/3) glutamate receptors, which are localized to presynaptic glutamate terminals. However, several glutamate-targeted medications predicated on these theories, such as the mGluR2/3 agonist POMA and the NMDAR modulator bitopertin, have failed in pivotal clinical trials despite robust effectiveness in preclinical models. A major barrier to effective glutamatergic treatment development is the absence of validated measures for target engagement that can identify effective compounds and guide dose selection. We present two ongoing target engagement studies. Methods: In Study 1, using the R61 mechanism, we conducted a study to assess target engagement and the optimal dose (80 vs.100 vs. 120 mg/kg) of the NMDAR modulator D-serine combined with 3 sessions of a neuroplasticity-based auditory remediation program in Sz. Milestones were designed to confirm target engagement, and were assessed with three preplanned outcomes: plasticity, mismatch negativity (MMN) and theta oscillations, requiring at least a moderate effect size difference (d = 0.5) between D-serine and placebo groups. In Study 2, we investigate the dose-response of ketamine in healthy volunteers, using ketamine-induced pharmacoBOLD (phBOLD) as a prelude to potential future target engagement studies with mGluR2/3 agonists in Sz. In our previous work, we chose a single dose of ketamine that produced a highly robust (0.23 mg/kg, d = 5.4) which have been near the peak of the dynamic range for BOLD activity. In Study 2, we assessed lower ketamine doses, predicting that doses that produce a more moderate phBOLD response may be optimally sensitive to the effects of glutamatergic agents. Results: In Study 1, 45 Sz subjects were randomized, meeting our preplanned “n.” The target engagement milestone required a moderate effect size difference (d ≥ 0.5) D-serine vs. placebo treatment. Across all 3 visits, there was a statistically significant treatment effect for plasticity improvement (p = 0.014). Significant plasticity improvement was seen in the ≤100 mg/kg dose-cohorts, starting with the 1st visit (10-13.9%, all p < 0.001, all d > 0.67). By contrast, placebo-treated participants showed non-significant changes across all visits (4-5%, n.s.). Target engagement was demonstrated by a larger MMN pitch (p = 0.049, d = 1.0) and larger ▯-ITC (d = 0.5) for the 100 mg/kg dose-cohort without safety concerns. A significant correlation was seen between plasticity improvement and auditory cognition (r = 0.46, p = 0.036), but not with other MCCB domains, suggesting specificity. In Study 2, we present a preliminary analysis of the phBOLD response for the first 30 healthy volunteer subjects in the 0.04 mg/kg to 0.08 mg/kg bolus cohorts. As expected, ketamine produced a phBOLD response of ~1% signal change peaking at ~3 minutes. The preliminary effect size for scan 1 (d = 1.1), shows the expected drop off from our previous study using a 0.23 mg/kg bolus (d = 5.4). Similarly, the preliminary analysis of the available behavioral results shows the expected dose response. The BPRS increase in the present study remains significant (p = 0.02, d = 0.61). Conclusions: Study 1 findings strongly support engagement of the NMDAR system by D-serine, as measured by behavioral plasticity and MMN. Sustained effects will be assessed in an ongoing R33. In Study 2, preliminary results support a ketamine dose response for pharmacoBOLD. Keywords: Target Engagement, Auditory Mismatch Negativity, Glutamate Receptors, Schizophrenia, Clinical Trial Disclosures: Guidepoint, Otsuka, Wedbush, Jefferies: Consultant (Self), Medincell, Karuna, Leal, Merck: Advisory Board (Self), Roche, Cerevance, Neurocrine, Click, Boehringer Ingelheim: Contracted Research (Self), Taisho: Other Financial or Material Support (Self). P565. Dopamine D2 Receptor Blockade and the Risk of Death Due to Choking Jari Tiihonen, Antti Tanskanen, Markku Lähteenvuo, Heidi Taipale Karolinska Institutet, Stockholm, Sweden Background: Several studies have reported that patients with schizophrenia have an increased risk of death due to choking, and up to 3% of deaths in this patient population may be attributable to this cause of death. However, the role of antipsychotic medication has remained unknown. Methods: We studied risk of death due to choking (adjusted Hazard Ratio, aHR) associated with schizophrenia, and with antipsychotics with differential degree of dopamine 2 receptor blockade during years 1998–2017 in a nationwide cohort of patient diagnosed with schizophrenia in Finland (n = 59,916, mean age 46.2 years, SD 15.8 years). Medication exposure periods were obtained by using data from national register of filled prescriptions. We compared with Cox model the risk of death due to choking during the use of weak D2-blocker antipsychotic (clozapine, quetiapine, aripiprazole), moderate D2-blocker antipsychotic (olanzapine), and strong D2-blocker antipsychotic (other antipsychotics) with no use of antipsychotic (reference). The hazard ratios were adjusted for age, sex, stroke, substance abuse, any neurological disorder, and time since schizophrenia diagnosis. Results: A total of 287 choking deaths occurred during 817,000 patient-years, corresponding to standardized mortality ratio 20.5 (95% CI 17.1–23.9) compared with general population. A total of 268 deaths occurred during outpatient care (162 during use of strong D2-blocker antipsychotic, 30 during olanzapine, 21 during weak D2-blocker antipsychotic, and 53 during no antipsychotic use). The corresponding adjusted Hazard Ratio (aHRs) were 1.74 (95% CI 1.19–2.55) for strong D2-blockers, 1.59 (0.96–2.64) for olanzapine, and 1.04 (0.73–1.63) for weak D2-blockers. Conclusions: Schizophrenia diagnosis is associated with a 20-fold risk of death due to choking. This risk is modified by the strength of D2-blockade of the used medication. While no incremental risk increase in addition to illness per se is observed for weak D2-blockers, the risk is substantially elevated during the use of strong D2-blockers. Keywords: Schizophrenia (SCZ), Antipsychotic Treatment, D2 Dopamine Receptor, Choking Disclosures: Eli Lilly, Janssen-Cilag: Grant (Self), HLS Therapeutics, Orion, WebMed Global: Consultant (Self), Eli Lilly, Evidera, Janssen-Cilag, Lundbeck, Mediuutiset, Otsuka, Sidera, Sunovion: Honoraria (Self). P566. Increasing Endocannabinoid 2-AG Levels Reverses Amphetamine-Induced Prepulse Inhibition Deficits in Rats Alexius Lampkin, Carla Zuniga, Jessica Gottlieb, Brian Baldo, Vaishali Bakshi University of Wisconsin-Madison, Madison, Wisconsin, United States Background: There is much interest in the cannabinoid system as a potential therapeutic target for multiple psychiatric illnesses. The active component of marijuana, THC, has been studied for its ability to modulate behavioral functions relevant to psychiatric illness through binding of the cannabinoid receptors. There are two endogenous cannabinoid ligands - anandamide (AEA) and 2-arachidonoylglycerol (2-AG), termed endocannabinoids (eCB). AEA and 2-AG have separate synthesis and breakdown mechanisms, allowing for distinct targeting and alterations in the tone of each endocannabinoid individually. Multiple studies have focused on the functional effects of AEA; very little is known about the role of 2-AG. Hence, our study focused on the behavioral sequelae of elevating endogenous levels of 2-AG. Methods: The overall design of this study was to test the compound JZL184, a potent inhibitor of monoacylglycerol lipase (MAGL). MAGL is the primary breakdown enzyme for 2-AG, and JZL184 administration increases 2-AG levels in the brain. We tested a range of JZL184 doses (administered intraperitoneally 30 min. prior to testing) in three behavioral paradigms that assess various core functions known to be disrupted in psychiatric disorders such as schizophrenia and post-traumatic stress disorder. These were prepulse inhibition of startle (PPI; assesses sensorimotor gating), elevated plus-maze (EPM; assesses anxiety-like states), and a behavioral observation paradigm (BOP; assesses ingestive, motor, and motivational responses). Separate cohorts of male and female rats were used for PPI (Ns = 7), EPM (Ns = 22), and BOP (Ns = 6). PPI and BOP experiments were within-subjects’ designs, with each animal receiving all drug treatments in a counterbalanced order over multiple test days (four days between consecutive tests). PPI experiments contained the following experimental conditions: vehicle/vehicle; vehicle/AMPH; 8 mg JZL/vehicle; 16 mg JZL/vehicle; 8 mg JZL/AMPH; 16 mg JZL/AMPH. In BOP experiments, food-deprived rats were given JZL184 (vehicle, 8 mg/kg, or 16 mg/kg) and placed in test cages with sucrose pellets. Initially, a screen blocking access to sucrose pellets was present; after 15 min of recording rats’ behaviors, the screen was removed (allowing access to the sucrose pellets), and behaviors were scored for an additional 45 min. Scored behaviors were duration and total bouts of: locomotion, rearing, eating, drinking, grooming, and screen approaches. For the EPM experiments, separate groups of rats received either vehicle (VEH) or 16 mg/kg JZL184. before being placed in the EPM for 5 min. The experimenter (blind to treatment condition) scored open arm entries and time, closed arm entries and time, center entries and time, head dips, stretch-attends, and grooming. For all experiments, males and females were housed and tested separately to avoid exposure to olfactory cues of the opposite sex. Results: To determine if 2-AG had modulatory effects on PPI, we first tested if JZL184 alone could alter PPI. When no deviations from basal PPI levels were seen with any dose of JZL184, we tested if JZL184 would reverse deficient PPI. We administered amphetamine (AMPH, 0 or 1.75 mg/kg), a psychotomimetic agent known to disrupt PPI, either with or without JZL184 pre-treatment. When JZL184 (at either dose) was co-administered with AMPH, males exhibited a reversal in the AMPH-induced PPI deficit; however, no reversal effect was seen in the females. The specificity of the AMPH-induced PPI deficit reversal in the males was further supported when no reversal in AMPH-induced startle was observed contrast to PPI, no measures in the EPM, ingestive, motor, and motivated approach behavioral paradigms were affected by the administration of JZL184 at any dose. Conclusions: This set of findings indicates that the PPI- restorative effects of JZL184 were not due to non-specific alterations on baseline startle, mood, motor, or motivated behaviors. Thus, JZL184 shows targeted restoration of disrupted sensorimotor gating (as assessed by PPI). Moreover, this PPI-restorative effect of JZL184 appears to be sex-specific, as only males have normalized PPI levels after JZL184 pre-treatment. Together, these results demonstrate that eCBs, specifically 2- AG, can improve cognitive processes related to psychiatric disorders such as schizophrenia and post-traumatic stress disorder, where startle abnormalities like disrupted PPI are seen. To our knowledge, we are the first to show reversal in AMPH- induced PPI deficits using JZL184. Previous findings show that cannabinoid receptors are localized on norepinephrine-producing neurons and that 2-AG potently stimulates these receptors; moreover, stimulation of these cannabinoid receptors inhibits norepinephrine release. Given that AMPH is known to disrupt PPI at least in part by increasing norepinephrine release, one potential mechanism by which JZL184 reverses AMPH- induced PPI deficits could be the blockade of AMPH-induced norepinephrine release through enhanced levels of 2-AG. Keywords: Cannabinoid, Endocannabinoids, Acoustic Startle Response, Prepulse Inhibition, JZL184 Disclosure: Nothing to disclose. P567. Lack of Tolerance With Repeat Dosage of Dexmedetomidine in Rat for 21 Days as Measured by EEG Friso Postma, Ronit Gupta, Frank Yocca* Bioxcel Therapeutics, BioXcel Corporation, Clinton, Connecticut, United States Background: Igalmi is an FDA-approved treatment for agitation associated with schizophrenia or bipolar I or II disorder. Igalmi is a sublingual film formulation of dexmedetomidine (Dex), a selective, full agonist at alpha-2-adrenergic receptors (ADRA2A) expressed widely in the central nervous system. Dex activates the ADRA2A receptor and modulates norepinephrine release from locus coeruleus (LC) neurons, thereby reducing sympathetic hyper-arousal. However, chronic activation of ADRA2A receptors, could also lead to tolerance, thereby reducing drug efficacy whereas discontinuation of repeated Dex dosing could result in withdrawal symptoms. In this study, we report on a repeat-dosage study with Dex in rats to examine and quantify the putative development of tolerance. To this end we characterized a response to Dex using electroencephalogram (EEG) in conjunction with locomotor activity. We used this putative Dex EEG biomarker to follow and quantify Dex activity over time in the central nervous system (CNS). Methods: Studies were carried out at a CRO (SynapCell, France). 24 Sprague-Dawley male rats aged 3 months were implanted with electrodes in the frontal and parietal cortex and connected to an intraperitoneal telemetry transmitter. Rats were dosed acutely with 5 or 10 µg/kg of Dex intramuscularly (IM) followed by a washout (1 week), repeat dosing for 22 days, 2-day washout and dosing with yohimbine. EEG signal was recorded for 3 hours and included a baseline (1 h), response following injection with compound (1 hr) and response to food administration (1 hr). EEG responses were quantified with time-frequency (TF) decompositions and fast Fourier transform (FFT) power spectra plotted relative to baseline values. Treatment groups are n = 16, and follow a balanced cross-over protocol with a 7 day washout. Averaged time points were tested for significance using 1-sided ANOVA. Results: Acute dosing had several effects on overall behavior and EEG activity in rats. Vehicle (Veh) injection rapidly caused hyperlocomotion combined with a shift in EEG intensity (power) from the lower to higher EEG frequency bands. Dosing with Dex initially elicited a similar response but withing minutes significantly attenuated hyperlocomotion and the shift in EEG power. Instead, whereas EEG signals following Veh injections returned to baseline, Dex treated animals showed significant increases in the FFT power distribution between 1-30 Hz and decreases between 31 – 140 Hz. This shift in EEG power was accompanied by a significant decrease in locomotion. Animals were food restricted. Following the response to Dex or Veh, food was added to the cage which caused excitement (locomotor activity) in the rats with significant increases in power between 100-140 Hz. In contrast to Veh, Dex treated rats did not show any increases in locomotion in response to food (although movement was not absent). Thus, responses to Dex, the injection, and food could be quantified and followed over time by the relative EEG FFT power distributions between 1-30 Hz, 31-99 Hz, and 100-140 Hz. During repeat dosing, the Dex induced EEG-signal stayed robust and no significant changes were found overtime in the response-profiles between treatment groups. Similarly following the 2-day washout period, no significant differences were found between the 2 treatment groups. Thus, no evidence of increased locomotor activity or high frequency EEG activity was observed following discontinuation of Dex dosing. These results indicate that under our conditions Dex does not induce tolerance nor does discontinuation of Dex lead to hyperlocomotion or hyperexcitability. We next investigated the effects of yohimbine, an ADRA2A receptor antagonist, with many effects opposite to Dex. If chronic dosing with Dex would build tolerance by downregulating ADRA2A receptor levels, responses to yohimbine would be attenuated. We quantified the EEG and locomotion response-profiles to yohimbine in the two treatment groups after the 2 day washout and again found no significant differences. Conclusions: IM injection of Dex elicits rapid characteristic changes in the rat EEG power distributions and locomotion. Dex also differentially affects the EEG and locomotion-response profiles to evoked events such as the IM injection itself and addition of food to the cage. The EEG and locomotion response-profiles are quantitative, can be monitored over time and may represent a putative Dex CNS biomarker. Repeat dosing of animals for 22 days with Dex, did not induce any significant changes in the EEG and locomotion response-profiles over time, both to the injection as well as the excitability induced by food. Taken together these findings conclude that in rats, repeat dosing of Dex for 22 days does not cause tolerance as measured in CNS. Consistent with these observations we also found no evidence of hyperlocomotion or EEG excitability following Dex treatment cessation. Assuming ADRA2A regulation is similar in rats and humans, these results suggest that Dex efficacy in reducing agitation should not diminish after multiple, consecutive doses. Keywords: Dexmedetomidine, Agitation, Locus Coeruleus, alpha2-adrenergic, EEG Biomarkers, Acute Agitation Disclosure: Nothing to disclose. P568. Discovery and Evaluation of Allosteric Agonists for the Schizophrenia Risk Gene GPR52 John Allen, Ryan Murphy, Pingyuan Wang, Nolan M. Dvorak, Fernanda Laezza, Kathryn Cunningham, Jia Zhou University of Texas Medical Branch, Galveston, Texas, United States Background: GPR52 is an orphan G protein-coupled receptor and recently identified GWAS schizophrenia risk gene. This orphan is a Gs/olf class receptor that activates cAMP signaling and is primarily expressed in the ventral striatum of the human brain in dopamine D2 medium spiny neurons. The unique brain expression profile of GPR52 suggests the receptor may functionally regulate cAMP signaling to oppose the neural signaling of dopamine D2 receptors. This distinguishes GPR52 as an attractive, druggable target for psychiatric disorders including schizophrenia and substance use disorders. Here we report our efforts to elucidate GPR52 neuronal signaling and to create and evaluate selective activators for this receptor. Methods: To elucidate the molecular signaling of GPR52, the human GPR52 receptor sequence was expressed in HEK293 cells, or cells lacking Gs/olf G proteins, and signaling was assessed using the Glosensor cAMP assay or Beta-arrestin TANGO assay. Medicinal chemistry and pharmacology were used in a small molecule discovery campaign to create novel GPR52 activators. GPR52 selectivity and off-target profiling was accomplished using competition radioligand binding at >30 CNS off targets and druglike pharmacokinetics and brain penetration was determined in rats. Molecular docking of novel ligands into a solved GPR52 crystal structure (PBD:6LI0) was done to explore receptor-ligand binding modes. A whole cell patch clamp study was performed by measuring evoked action potentials (AP) using mouse striatal slices (n = 3-6 cells analyzed per treatment group). A behavioral study tested vehicle or increasing doses of GPR52 agonists for antipsychotic-like activity in an amphetamine-induced locomotion study using c57BL6J mice (n = 12 animals for each treatment group). Results: In molecular signaling studies, expression of human GPR52 in wildtype HEK293 cells elevated basal cAMP levels by over 100 fold, with further elevation of cAMP in response to the activator PW0787, which was eliminated by stable knockout of Gs/olf G proteins using CRISPR/Cas9 genome editing. Medicinal chemistry and pharmacology were subsequently used to modify an indoline carboxamide ligand and determine compound features crucial for GPR52 activation. Surprisingly, when the nitrogen containing ring of the indoline system was broken into more flexible variants, this greatly increased compound potency (EC50: ~40 nM) and efficacy, while retaining excellent target selectivity, plasma exposure and serum concentration. Unexpectedly conversion of the indoline to analine derivatives greatly reduced GPR52 activation of the Beta-arrestin pathway, resulting in the creation of several G protein biased activators. Molecular docking of the balanced compound PW0787 into the GPR52 crystal structure suggested compound binding with extracellular loop 2 (ECL2) and an allosteric site of action. PW0787 did not occupy a typical orthosteric site seen within GPCRs, but instead interacted within a narrow side pocket near ECL2, further suggesting an allosteric site binding mechanism. To assess the necessity of the ECL2 allosteric interaction for PW0787 activity, ECL2 was replaced with an equivalent span of alanine residues, which eliminated compound activity entirely. Notably, treatment of striatal slices with PW0787 increased the frequency and number of evoked action potentials in D2, but not D1, medium spiny neurons of the nucleus accumbens. Dose dependent testing of the optimized agonist PW0787 revealed 3 and 10 mg/kg treatments of mice significantly reduced amphetamine-induced hyperlocomotion, indicating antipsychotic-like activity. Conclusions: Taken together, these findings indicate the schizophrenia risk gene GPR52, via Gs/Golf cAMP signaling, is an excitatory receptor selectively expressed in D2 medium spiny neurons. Our drug discovery effort has resulted in novel GPR52 activators with optimized potency and efficacy, including both balanced and functionally selective G protein biased activators. The novel balanced GPR52 activator PW0787 is a selective, orally bioavailable, brain penetrant allosteric agonist that excites D2 medium spiny neurons and also shows antipsychotic-like activity. Keywords: Orphan Receptors, Drug Discovery/Development, cAMP Signalling, Schizophrenia- Novel Treatment, D2 Medium Spiny Neuron Disclosures: New Atlas Biotechnology: Contracted Research (Self), PSYLO: Consultant (Self). P569. Pimavanserin Treatment Increases Plasma Brain-Derived Neurotrophic Factor Levels in Rats Ashutosh Tripathi, Henry Nasrallah, Anilkumar Pillai The University of Texas Health Science Center at Houston, Houston, Texas, United States Background: Pimavanserin, a serotonin 5HT-2A receptor inverse agonist, with no appreciable affinity to dopamine D2 receptors, is the first-line, FDA-approved treatment of hallucinations and delusions associated with Parkinson’s Disease psychosis (PDP), which occurs in up to 50% of PD patients. The neurobiological mechanism underlying the therapeutic effectiveness of Pimavanserin in PDP remains unknown. A number of earlier studies, including reports from our laboratory, have shown that treatment with 5HT-2A antagonists and other drugs acting on the serotonergic system like SSRIs increase Brain derived neurotrophic factor (BDNF) levels in rodents. BDNF is synthesized as the precursor proBDNF, that undergoes cleavage intra or extracellularly to produce a mature BDNF (mBDNF) protein. mBDNF is believed to play an important role in neuroplasticity and neurogenesis. The present study tested the hypothesis that treatment with Pimavanserin is associated with higher and sustained elevations of mBDNF. PDP is a progressive neurodegenerative disorder and an increase in mBDNF may contribute to a neuroprotective effect. Methods: Male Sprague–Dawley rats, weighing 280–320 g (6–8 weeks old; N = 10/group) were treated with Vehicle (0.9% saline), Pimavanserin (1 mg/kg/d) or Fluoxentine (10 mg/kg/d). Drugs were administered subcutaneously for 4 weeks (chronic) or 2 hours (acute). We have analyzed proBDNF and mBDNF in plasma, serum and key brain regions [prefrontal cortex (PFC) and hippocampus] by ELISA. Data were analyzed by One-way ANOVA followed by posthoc-Tukey’s multiple comparisons test. Results: In chronic treatment studies described above, both Pimavanserin and Fluoxetine significantly increased plasma levels of proBDNF as compared to vehicle group. However, a significant increase in plasma mBDNF was found in Pimavanserin-treated rats, but not in Fluoxetine-treated group. Both Pimavanserin and Fluoxetine significantly increased proBDNF levels in the PFC as compared vehicle group. No significant change in mBDNF levels was found in the PFC and hippocampus in any of the chronic treatment groups as compared to vehicle group. In acute treatment studies, Pimavanserin significantly increased proBDNF levels in plasma and serum as compared to vehicle group. Fluoxetine treatment increased proBDNF levels in serum as compared to vehicle group. No significant difference was observed in peripheral mBDNF levels in any of the acute treatment groups as compared to vehicle group. Conclusions: The observed increases in plasma mBDNF levels following chronic Pimavanserin treatment suggest the neuroprotective potential of Pimavanserin in the long-term treatment management of PDP in addition to its antipsychotic effects. Keywords: Pimavanserin, BDNF, Parkinson’s Disease Psychosis, Neuroprotection Disclosure: Acadia Pharmaceuticals: Contracted Research (Self). P570. Projection Specific Effects of mGlu3 Receptor Activation in Regulation of Schizophrenia-Like Nucleus Accumbens Pathophysiology and Sociability Deficits Shalini Dogra, Jason Putnam, P. Jeffrey Conn Vanderbilt University, Nashville, Tennessee, United States Background: Genetic variants in GRM3 (the gene encoding for the mGlu3 receptor) are associated with altered glutamatergic neurotransmission in schizophrenia and can predict improvement in symptoms following drug treatment. However, the mechanisms through which mGlu3 receptors modulate glutamatergic neurotransmission and pathophysiological changes that may be relevant for schizophrenia are still not clear. Methods: The mGlu3 receptor-induced long-term depression (mGlu3-LTD) of excitatory neurotransmission in the nucleus accumbens was tested using whole-cell patch-clamp electrophysiology from the slices prepared from D1-TdTomato mice (5-6 mice/group). The mGlu3 receptor signaling was activated by bath application of mGlu2/3 receptor agonist, LY379268 in the presence of mGlu2 negative allosteric modulators. The projection-specific effects were evaluated using slice optogenetics experiments after virally expressing channelrhodopsin-2 in different brain areas. Schizophrenia-like symptoms were induced by treating mice (10-15/group) with phencyclidine (PCP, 10 mg/kg; s.c.) for 7 days followed by a 7-day washout. Sociability deficits in the mice were accessed using a 3-chamber social behavior test. Both male and female mice were used in the study. The effects of treatments on the excitatory transmission and behavior test were evaluated using one-way ANOVA followed by the Newman-Keuls post hoc test. Results: Activation of mGlu3 receptors induces long-term depression (LTD) of excitatory neurotransmission onto D1 medium spiny neurons (MSNs) in the nucleus accumbens (NAc). Further, the mGlu3-LTD is expressed at the synapses from medial thalamus (mThal) afferents to the D1 MSNs of the NAc shell (Thal-D1 NAc shell), but not at synapses from basolateral amygdala inputs to the D1 MSNs of the NAc shell, indicating synapse-specific effects of mGlu3 receptor activation in the NAc. Interestingly, mGlu3 receptor activation normalized augmented glutamatergic signaling in the NAc slices of mice treated with PCP. It also decreased the PCP-induced potentiation of activity of thalamic afferents to the D1 MSNs of the NAc shell. Further, in vivo potentiation of mGlu3 receptor activation function rescued the PCP-induced impairments in the social novelty. Conclusions: These data demonstrate that activation of mGlu3 receptors can rescue schizophrenia-like physiological and behavioral deficits and provide a mechanism by which polymorphisms in the GRM3 can contribute to the pathophysiology of schizophrenia. Therefore, targeting these receptors can provide a viable approach for developing new therapeutics for treating patients with schizophrenia. Keywords: Metabotropic glutamate receptor 3 (mGluR3), Schizophrenia novel treatment, schizophrenia negative symptoms, Excitatory Synapses, Medium Spiny Neuron Disclosure: Nothing to disclose. P571. Targeting the FGF12:Nav1.2 Complex for CNS Probe Discovery Timothy B. Baumgartner, Zahra Haghighijoo, Nana Goode, Danielle Jamison, Aditya K. Singh, Fernanda Laezza University of Texas Medical Branch, Galveston, Texas, United States Background: Voltage-gated sodium (Navs) channels play a critical role in action potential initiation and propagation. Structurally, the Nav channel features a pore-forming α subunit, of which nine isoforms have been described (Nav1.1-Nav1.9), that is tightly regulated through protein:protein interactions (PPI) with auxiliary proteins. One of the alpha subunits, Nav1.2, is of particular interest. Nav1.2’s PPI with its auxiliary protein fibroblast growth factor 12 (FGF12), part of the intracellular fibroblast growth factor family (FGF11-14), occurs through interactions at its C-terminal domain (CTD). Specifically, FGF12 has been shown to interact with Nav1.2 to increase its fast inactivation kinetics and slow its recovery from fast inactivation. Notably, co-expression of FGF12 and Nav1.2 is enriched in the cerebral cortex, suggesting that FGF12:Nav1.2 interaction is a potential determinant of activity in this region. Various mutations in FGF12 have been implicated in either gain-of-function (GOF) or loss-of-function (LOF) regulatory effects on Nav1.2 channel activity in the context of autism spectrum disorder (ASD), and FGF12 has been identified as dysregulated in schizophrenia (SCZ) and major depression disorder (MDD). Thus, FGF12 ligands that could modulate the interaction of FGF12 with its binding partner, Nav1.2, could be therapeutically valuable for ASD, SCZ and MDD, disorders that have been associated with changes in FGF12 function. Methods: We employed medicinal chemistry, chemoinformatics, the split-luciferase assay (LCA), surface plasmon resonance (SPR), patch-clamp electrophysiology in heterologous expression systems to validate the activity of hits and leads in in vitro and in-cell assays, and in cortical neurons derived from human-induced pluripotent stem cells (hiPSCs). Results: Intracellular FGFs and Nav channels form PPI complex pairs that are sufficiently heterogeneous in critical binding regions to allow for specific pharmacological modulation. Based on this premise, we conducted an in-cell high-throughput screening against FGF14:Nav1.6, a PPI complex closely related to FGF12:Nav1.2, but functionally distinct in its ability to regulate Nav channels. Following development of a double-stable HEK293 cell line expressing split-luciferase complementation assay (LCA) constructs and assay optimization in 384-well plates, we screened ~50,000 small molecules, peptides and rationally-designed drug-like analogues against FGF14:Nav1.6. Compounds were ranked by a combination of % maximal luminescence and individual Z-scores, which were calculated based on the mean and SD of its respective plate controls (0.3% DMSO). Using cut-offs of Z ≤ -5 and ≥60% reduction in complex formation for inhibitors and Z ≥ 3 and ≥150% increase in complex formation for enhancers, we identified 960 primary hits. Of these, 640 compounds failed to achieve significance during triplicate validation screening, and counter-screening against full-length luciferase in transiently transfected HEK293 cells resulted in the exclusion of an additional 149 compounds due to significant inhibitory effects (Z ≤ -3). Thus, the primary set of hits was reduced to 151 inhibitors and 20 enhancers, which were then stratified by structural and chemical parameters including predicted permeability (logP). From these compounds, we derived a sub-class of 50 molecules with ideal drug-like properties. We then counter screened these 50 hit compounds against the FGF12:Nav1.2 complex, with the expectation that a unique set of ligands would exhibit specificity for FGF12. We have identified 3 lead compounds 7350, 0801, and 7827 that share common chemical scaffolds targeting the FGF12:Nav1.2 PPI interface. Ongoing studies are focusing on determining the potency, specificity, biophysical mechanisms and functional effects of these compounds in hiPSCs-derived cortical neurons. Conclusions: This evidence suggests that pharmacologically targeting FGF12 represents a promising method of modulating Nav1.2 activity for a wide spectrum of neuropsychiatric disorders. Keywords: Ion Channels, Action Potentials, Growth Factors Disclosure: Nothing to disclose. P572. 2,5-Dimethoxy-4-Propylamphetamine (DOPR) Increased Effortful Motivation in Mice Michael Noback, Johnny Kenton, Adam Klein, Zoe Hughes, Andrew Kruegel, Jared Young University of California - San Diego, San Diego, California, United States Background: There is a significant gap in available pharmacological treatments for many neuropsychiatric disorders, including schizophrenia and depression. Identifying novel treatments and targets remains paramount. The lack of efficacy of many compounds in human trials despite positive preclinical data however, signals a clear need for improved translatability of preclinical studies of behavioral disorders. The progressive ratio breakpoint task (PRBT) measures effortful motivation and exhibits pharmacological predictive validity between humans and mice. 2,5-Dimethoxy-4-propylamphetamine (DOPR) is a relatively poorly understood compound with psychedelic properties. It is closely related to 2,5-Dimethoxy-4-iodoamphetamine (DOI), another psychedelic drug that may improve anhedonia-relevant symptoms. The psychoactive effects of DOI are well documented, particularly its ability to elicit the head twitch response (HTR), a response to hallucinogenic compounds seen in mice that is highly predictive of hallucinogenic effects in humans. Here, we investigated the effects of DOPR on effortful motivation in the PRBT and HTR. As positive controls, amphetamine was used for the PRBT and DOI in the HTR assay. Methods: 40 C57BL/6 N mice (50% female) were trained in operant chambers. After initial training stages, mice were tested in the PRBT which required mice to increase their nosepokes in a progressive ratio for the same 40 µL strawberry milkshake reward. The point at which they stopped was defined as their breakpoint and was the primary outcome measure. After baseline assessment, mice were dosed with DOPR (0.0106, 0.032, 0.106, or 0.32 mg/kg, i.p.), saline, or amphetamine (0.3 mg/kg) in a within-subjects design. Breakpoint was analyzed using dose as a within-subjects factor, while sex and baseline performance (median-split) were between-subjects factors. C57BL/6 N male mice were assessed for HTR in response to either DOPR or DOI. Mice were administered with a single dose of DOPR or DOI (0.1, 0.32, 1, 3.2, or 10 mg/kg, s.c.) or vehicle (n = 6 per dose), and placed in an open field. Head twitches were counted manually by an observer blinded to the treatment condition. Binding of DOPR and DOI to the 5-HT2A receptor was also measured, as well as functional activity at the 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A receptors. Receptor binding was measured by displacement of [3H]Ketanserin in HEK293 cells expressing receptors. Functional activity was measured via Ca ++ flux FLIPR assays. Results: DOPR did not exert a main effect on breakpoint (F(4,132)=1.3, p = 0.261), but when analyzed by baseline, DOPR did not affect those with a high breakpoint (F(4,68)=1.0, p = 0.410), but increased the breakpoint of mice that had a low baseline (F(4,64)=3.3, p = 0.016) at 0.0106, 0.106, and 0.32 mg/kg doses, with a trend towards increased breakpoint seen at 0.032 mg/kg vs. vehicle. These effects were consistent with amphetamine, with no main effect (F(1,33)=0.5, p = 0.495), driven by a lack of effect in high performers (F(1,17)=0.3, p = 0.620), but improvement in low performers (F(1,16)=8.9, p = 0.009). No effect of sex was seen in response to either DOPR or amphetamine. DOPR caused a dose-dependent increase in HTR (F(5,30)=60.0, p < 0.0001), with doses of 3.2 and 10 mg/kg passing a Bonferroni post hoc correction relative to vehicle (p < 0.002), and peak effect at 3.2 mg/kg. These results closely matched the effects of DOI (F(5,30)=75.4, p < 0.0001), with 1, 3.2, and 10 mg/kg passing a Bonferroni post hoc correction relative to vehicle (p < 0.002), and peak effect at 3.2 mg/kg. The binding affinity at 5-HT2A of the two compounds was similar, with DOPR having a Ki of 17.56 nM and DOI having a Ki of 14.51 nM. DOPR and DOI also show agonist activity at 5-HT2A (EC50 of 0.12 and 0.19 nM, respectively) and 5-HT2C (EC50 of 0.27 and 0.82 nM, respectively) receptors, with >25-fold lower potency at 5-HT2B receptors and no significant activity at 5-HT1A (both > 1,000 nM EC50). Conclusions: The positive effect of DOPR on effortful motivation points to possible therapeutic applications in psychiatric illness states characterized by reduced effortful motivation as measured by the PRBT. The similarity of effects of DOPR to well-studied drugs such as DOI and amphetamine provides a useful reference point to interpret its pharmacological effects. Importantly, the doses needed to increase breakpoint in the PRBT were as low as 0.0106 mg/kg. While 0.1 mg/kg increased HTR, this effect was not significant, and maximal effect at 3.2 mg/kg, supporting the premise that low doses of DOPR may be therapeutic in anhedonia states without causing unwanted hallucinogenic side effects. Keywords: Motivation, Psychedelics, Schizophrenia (SCZ) Disclosure: Nothing to disclose. P573. Evaluation of Selective Muscarinic M4 Agonists on Cortical Oscillations: A Sleep-EEG Study in Rats Steven Leiser, Hanh Nguyen, Polina Stolyar, Srinivas Chakilam, Philip Iredale Cerevel Therapeutics, Cambridge, Massachusetts, United States Background: One of the leading theories on the pathophysiology of schizophrenia is that an overactivity of dopamine in certain brain regions is closely associated with the prevailing psychotic symptoms. Current antipsychotics target a direct blockade of dopamine receptors, yet this can lead to significant side effects. An alternate approach is to indirectly modulate levels of dopamine. The presynaptic localization of the M4 receptors differentially expressed in the striatum may serve to balance acetylcholine and dopamine levels in the region of the brain primarily responsible for psychotic symptoms. Activation of M4 receptors decreases acetylcholine, which in turn decreases levels of dopamine without the direct receptor blockade Thus, selective activation of M4 may be an effective treatment of the neurobehavioral components such as psychosis, agitation, and cognitive deficits, that are associated with schizophrenia and other neuropsychiatric disorders. Methods: A polysomnographic electroencephalography (sleep-EEG) study was carried out using radiotelemetry in unanesthetized Sprague Dawley rats following subcutaneous compound administration (n = 8 rats per treatment per crossover study). EEG was recorded and analyzed using conventional methods: spectral power was determined using NeuroScore (DSI) for each 1 Hz bin for every 10 seconds and averaged into frequency bands, per temporal epoch, per sleep-stage, and by treatment group. Results: Full and partial M4 agonists showed similar profiles on cortical oscillations. Both increased low frequency (delta) and suppressed high frequency (alpha, beta and gamma) activity. Conclusions: These data confirm dose-dependent effects on cortical oscillations following M4 activation. Interestingly, whereas activation of dopaminergic receptors results in increases in higher frequencies such as alpha, beta and gamma oscillations, antipsychotics commonly decrease alpha and gamma oscillations. Data generated with these M4 activators align with an antipsychotic profile and suggest a putative role of M4 activation in mitigating psychotic symptoms. Keywords: EEG, EEG Biomarkers, M4 Muscarinic Receptors, M1 and M4 Muscarinic Receptors, Antipsychotic Disclosure: Cerevel Therapeutics: Employee (Self). P574. TAAR1 Dependent and Independent Actions of the Potential Antipsychotic and Dual TAAR1/5HT1A Receptor Agonist SEP-383856 Per Svenningson, Marcus Saarinen, Ioannis Mantas, Ivana Flais, Richard Ågren, Kristoffer Sahlholm, Mark J. Millan Karolinska Institutet, Stockholm, Sweden Background: SEP-383856 (SEP-856) is a novel antipsychotic under clinical development. It displays a unique pattern of receptor interaction, with only weak (partial agonist) activity at dopamine D2 receptors, yet more potent agonist activity at the trace amine associated receptor (TAAR1) and 5-hydroxytryptamine 1A receptor (5HT1A). Nonetheless, these observations await independent confirmation and more detailed characterization of the in vitro and in vivo actions of SEP-856 at TAAR1 and 5HT1A receptors would be instructive. Methods: We employed luminescence complementation technology in heterologous live cell systems, confocal microscopy, voltage clamp electrophysiology, behavioral readouts and TAAR1 knockout (KO) mice to study SEP-856 in further detail. Results: We provide evidence for the ability of SEP-856 to activate TAAR1 at the surface plasma membrane, and show that this interaction results in Gαs recruitment (pEC50: 6.08 ± 0.22 EMAX: 96.41% ±15.26) and by extension, to G-protein inward rectifying potassium (GIRK) channel activation. Using TAAR1-KO mice, we find TAAR1 to be indispensable for SEP-856 control of body temperature, baseline locomotion reduction and for “antipsychotic-like” efficacy as characterized by a reversal of dizocilipine (MK-801) mediated disruption of pre-pulse inhibition. Conversely, the inhibition by SEP-856 of MK-801 induced locomotion was unaffected in TAAR1 KO mice. SEP-856 behaved as a low-potency, partial agonist at the 5HT1A receptor, while it partially inhibited recruitment of D2 receptor-coupled Gα and GIRK by DA and acted as a weak partial agonist with low potency at the same receptor when applied alone. Conclusions: Our findings corroborate and extend previous observations on the molecular substrates engaged by potential antipsychotic, SEP-856, that could prove to have major advantages in the treatment of schizophrenia and other psychotic disorders. Keywords: TAAR 1, Trace Amines, TAAR1 Knockout Mice Disclosure: Nothing to disclose. P575. Modulation of D1 Receptor-Expressing Spiny-Projection Neurons is Indicative of Antipsychotic Effect Seongsik Yun, Ben Yang, Justin Anair, Madison Martin, Stefan Fleps, Nai-Hsing Yeh, Anis Contractor, Jones Parker Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States Background: Overactive dopamine transmission in psychosis is predicted to unbalance striatal output via D1- and D2-dopamine receptor-expressing spiny-projection neurons (SPNs). Antipsychotic drugs are thought to re-balance this output by blocking D2-receptors. Methods: We used in vivo imaging to record D1- and D2-SPN Ca2+ dynamics in mice (N = 7–11 per group) under normal and hyperdopaminergic conditions using amphetamine treatment. We examined these conditions in the presence or absence of treatment with 10 different antipsychotic drugs or candidates. We evaluated the effects of drug treatment combinations on neural activity, open field locomotion, sensorimotor gating, and hallucination-like perception in both males and females. We also used chemogenetics to causally link drug effects on D1-SPN activity to the normalization of these behaviors. Results: Initially we compared effective (clozapine, olanzapine, and haloperidol) antipsychotics to a candidate drug that failed in clinical trials (MP-10). All drugs normalized amphetamine-driven hyperlocomotion, but only MP-10 normalized D2-SPN hypo-activity. By contrast, the effective antipsychotics all normalized hyperdopaminergic D1-SPN dynamics. Chemogenetic inhibition of D1-SPNs was sufficient to normalize locomotion, sensorimotor gating, and hallucination-like perception under hyperdopaminergic conditions. Given the surprising correlation between clinical efficacy and D1-SPN modulation, we next evaluated compounds selectively targeted to D1-SPNs. D1R partial agonism, antagonism, or M4 cholinergic receptor modulation all normalized the levels of D1-SPN activity, locomotion, and sensorimotor gating. The antipsychotic drug candidate xanomeline (an M1/M4 muscarinic agonist) also normalized D1-SPN activity and behavior. Remarkably, SEP-363856, a trace amine-associated receptor 1 (TAAR1) agonist with antipsychotic efficacy, also normalized D1-SPN activity, even though it does not principally target the striatum or normalize behavior under hyperdopaminergic conditions. Conclusions: In contrast to the prevailing view, our results show that modulation of D1 rather than D2-SPNs is sufficient and critical for antipsychotic drug efficacy. These findings underscore the utility of using neural dynamics to readout drug efficacy and suggest that D1-SPN activity is a more relevant therapeutic target than D2-SPN activity for the development of effective antipsychotics. Keywords: Antipsychotic Drugs, M1 and M4 Muscarinic Receptors, PDE10A, D1 dopamine Receptors, D1 Agonist, D1-PAM Disclosure: Nothing to disclose. P576. Impaired Auditory Cortex Dynamic Range in First Episode Psychosis and Associated Disease Burden Alfredo Sklar, Xi Ren, Lydia Chlpka, Mark Curtis, Brain Coffman, Dean Salisbury University of Pittsburgh Western Psychiatric Institute and Clinic, Pittsburgh, Pennsylvania, United States Background: There is a growing appreciation for the contribution of sensory disruptions to disease morbidity in psychosis. However, auditory dynamic range, the scaling of neurophysiological responses to stimulus intensity, is an attribute of high-fidelity sensory systems that remains understudied in psychosis. The goad of the present study was to assess auditory cortex (AC) dynamic range among individuals with a schizophrenia spectrum illness during their first psychotic episode (FESz) and examine its relationship to clinical outcomes. Methods: Thirty-five FESz and 40 healthy controls (HC) matched for age, sex, parental socioeconomic status, and premorbid IQ participated in the study. Magnetoencephalography (MEG) was recorded during binaural presentation of 1KHz tones at 3 intensities (75 dB, 80 dB, and 85 dB). Structural MRIs were obtained to enhance cortical localization of MEG sensor activity. All participants completed the Wechsler Abbreviated Scale of Intelligence (WASI), the MATRICS cognitive battery (MCCB), and the Global Functioning: Role and Social scales (GFR/GFS). Patients were administered the Positive and Negative Syndrome Scale (PANSS). Primary outcomes included AC activity elicited by tone stimuli as well the dynamic range of this activity, defined as the increased AC activity from 75 dB to 85 dB, and its correlations with clinical assessment scores. Results: FESz exhibited an overall reduced AC response to tones relative to HC (p = 0.002, partial ƞ2 = 0.13). Importantly, the enhancement of AC activity to tones of increasing intensity observed across groups (p < 0.001, partial ƞ2 = 0.35) was blunted in FESz relative to HC (p = 0.03, partial ƞ2 = 0.05). Reduced dynamic range among FESz was associated with lower GFS (r = 0.62, p < 0.001) and GFR (r = 0.45, p = 0.006) scores, worse MCCB performance (r = 0.49, p = 0.003), and increased PANSS Negative symptom subscale scores (r = −.53, p < 0.001). Conclusions: Beyond an impaired sensory response to pure tones, FESz exhibit reduced AC dynamic range relative to HC. This impairment was correlated with various markers of disease morbidity including poorer community functioning as well as cognitive and negative symptoms, though the most robust association was observed with social functioning scores. The relationship with impaired social functioning may reflect the role of AC dynamic range in decoding the emotional content of language and highlights its importance to future therapeutic sensory remediation protocols. Keywords: First Episode Schizophrenia, Magnetoencephalography, Auditory Cortex, Auditory Processing, Social Functioning Disclosure: Nothing to disclose. P577. Shared and Unique Abnormalities in Sleep and Rest-Activity Rhythms in Residential and Outpatient Schizophrenia Spectrum Disorder Patients Ahmad Mayeli, Alice LaGoy, Stephen Smagula, James Wilson, Cristina Zarbo, Matteo Rocchetti, Fabrizio Starace, Manuel Zamparini, Letizia Casiraghi, Stefano Calza, Matteo Rota, Giovanni de Girolamo, DiAPAson consortium, Fabio Ferrarelli University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: Sleep and rest-activity-rhythm (RAR) abnormalities are commonly reported in schizophrenia spectrum disorder (SSD) patients. However, an extensive characterization of RAR alterations in SSD patients relative to healthy control subjects is currently lacking. Furthermore, differences in RAR parameters between residential and outpatient SSD individuals, including their relationships with the SSD clinical symptoms, have not been thoroughly examined. Methods: Two hundred and fifty participants, including one hundred and thirty-seven patients diagnosed with Schizophrenia Spectrum Disorders (SSD, seventy-nine residential patients, and fifty-eight outpatients) and one hundred and thirteen healthy comparison (HC) subjects, were recruited at ten different mental health centers in Northern Italy Ras as part of the DiAPAson project. To monitor habitual sleep-wake patterns, study participants were instructed to wear an ActiGraph GT9X on the non-dominant wrist for seven consecutive days. Data from 20 participants were excluded due to having either less than 3 days of actigraphy data or being detected as an outlier. Therefore, 68 residential SSD patients, 54 SSD outpatients, and 108 HC individuals were included in further analyses. RAR parameters, including M10, L5 relative amplitude (RA), intra-daily variability (IV), inter-daily stability (IS), alpha, beta, F-statistic (F-stat), and sleep parameters (i.e., total sleep time [TST], wake after sleep onset [WASO]) were computed for each study participant. Moreover, negative symptoms were assessed in residential and outpatient SSD patients with the Brief Negative Symptom Scale (BNSS). Analysis of covariance (ANCOVA) was performed to identify differences in RAR and sleep parameters between HC, outpatient SSD, and residential SSD groups after controlling for age and sex. Statistical significance was determined by applying Bonferroni’s correction for multiple comparisons. For RAR/sleep parameters showing significant ANCOVA differences across the three groups, the Tukey HSD test was used for pairwise comparison, including differences between each SSD population with HC and between the two SSD samples. Finally, correlation analyses between BNSS scores and RAR parameters were performed. Results: Among sleep parameters, TST (F(2, 225) = 79.43, p < 0.001), but not WASO, was different between groups after Bonferroni’s correction for multiple comparisons. Furthermore, except RA and F-stat, all RAR parameters, including IV (F(2, 225) = 8.35, p = 0.003), M10 (F(2, 225) = 31.13, p < 0.001), L5 (F(2, 225) = 7.91, p = 0.005), alpha (F(2, 225) = 46.092, p < 0.001), beta (F(2, 225) = 27.68, p < 0.001), and IS (F(2, 225) = 14.33, p < 0.001) were significantly different across the three groups. Specifically, TST was higher in both SSD groups compared to HC (t = 11.18, p < 0.001, t = 9.28, p < 0.001; for residential and outpatients SSD vs HC, respectively). Both SSD groups showed also lower M10 (residential vs control: t = −7.71 and p < 0.001; outpatients vs control: t = −4.2 and p < 0.001) and L5 (residential vs control: t = −3.79 and p < 0.001; outpatients vs control: t = −2.43 and p = 0.048), along with higher alpha (residential vs control: t = 7.43 and p < 0.001; outpatients vs control: t = 8.25 and p < 0.001) compared to HC. Residential SSD patients had higher IV (residential vs control: t = 2.98 and p = 0.010), IS (residential vs control: t = 5.35 and p < 0.001), and beta (residential vs control: t = 7.16 and p < 0.001) relative to HC. In contrast, SSD outpatients showed no differences in any of those three measures compared to HC. We also observed that M10 (t = 2.67, p = 0.024) was higher in SSD outpatients compared to residential patients, whereas IV (t = −3.95, p < 0.001), beta (t = −5.51, p < 0.001), and IS (t = −2.73, p = 0.020) were higher in residential compared to SSD outpatients. Furthermore, residential patients had worse negative symptoms compared to outpatients (t = 2.6299, p = 0.010), and IS correlated with the severity of negative symptoms across all SSD patients (R = 0.248, p = 0.024). Conclusions: In this study, we found that compared to healthy controls, residential and outpatient SSD individuals had both unique (IV, beta, IS) and shared (e.g., TST, M10, L5, alpha) abnormalities in RAR/sleep measures, and IS was associated with the severity of the SSD clinical symptoms. Keywords: Actigraphy, Schizophrenia (SCZ), Circadian Rhythm, Brief Negative Symptom Scale (BNSS) Disclosure: Nothing to disclose. P578. Odor Discrimination and Identification in Schizophrenia: Relationship to MRNA in Lymphocytes and MATRCIS Battery Scores Robert Smith, Henry Sershen, Abel Lajtha, Mary Yousef, Mumei Zhang, John M. Davis New York University School of Medicine and NKI, Woodmere, New York, United States Background: Patients with schizophrenic have been reported to show deficits in various measures odor perception but odor discrimination has not been standardly assessed. DNA methylation and GABAergic input have been implicated in biochemical process controlling odor in animal studies, but this has not been investigated in human studies. Some studies have related cognitive deficits in schizophrenia to odor deficits but none have used the MATRICS battery to investigate this question. Methods: In a study of DNA methylation and GABAergic mRNAs in lymphocytes we also measured odor identification and discrimination with the Sniff and Sticks battery in 58 patients with chronic schizophrenia (CSZ) and 48 non-psychiatric controls (NPC). mRNAs in lymphocytes were assessed by qPCR using TaQMan probes. Cognition was assessed by the MATRICS battery in CSZ and NPC and symptoms in CSZ were assessed by PANSS scale. The relationship of odor deficits to mRNA levels and MATRICS scores and symptoms was explored by correlation analysis. Results: CSZ showed significant deficits compared to NPC in odor identification (P = 0.011, Cohen’s d = 0.50)), but much larger deficits in discrimination (P < 0.001, d = 1.01). In step down regression analysis odor discrimination but not odor identification had significant β weight for classify patients into the CSZ vs NPC group. There were significant negative correlations (r = −33 to -.68) of odor identification with DNMT1 mRNAs, and significant negative correlations with odor discrimination and GABAergic mRNAs in CSZ subjects (-.38 to -.42). Odor discrimination scores correlated significantly (P = 0.02 to P = 0.009) with several Matrics Domain scores in CSZ subjects but not NPC; there was a sex effect and these correlations were stronger in female than male CSZ. Conclusions: Odor discrimination deficits, which has not been consistently evaluated in schizophrenia studies, showed the strongest differentiation between patients with schizophrenia and controls. This is the first study to report relationship between odor deficits and DNMT and GABAergic mRNAs in human subjects. However, the negative correlations of odor scores with lymphocyte mRNA levels may not necessarily reflect neuronal processes. Keywords: Odor Deficits, Schizophrenia, DNMT, DNMT Disclosure: Nothing to disclose. P579. Neurovascular Water Exchange and Negative Symptoms in Schizophrenia Eric Goldwaser, Danny Wang, Bhim Adhikari, Joshua Chiappelli, Xingfeng Shao, Jiaao Yu, Tong Lu, Shuo Chen, Wyatt Marshall, Alexa Yuen, Mark Kvarta, Yizhou Ma, Xiaoming Du, Osamah Saeedi, Heather Bruce, Patrick Donnelly, Hugh O’Neill, Alan Shuldiner, Braxton Mitchell, Peter Kochunov, Elliot Hong Weill Cornell Medicine, New York, New York, United States Background: Negative symptoms limit real-world functioning and treatment response in patients with schizophrenia spectrum disorder (SSD). Mounting evidence supports cerebrovascular contributions to negative symptoms but with unknown mechanism. The blood-brain barrier (BBB) is at the nexus of neural-vascular exchanges, tasked with regulating cerebral homeostasis. BBB abnormalities in SSD, if any, likely occur at subclinical levels and imaging measures used to assess large molecule BBB leakage in major neurological events may not be sensitive enough to directly examine clinical evidence of BBB abnormalities in SSD. Methods: We tested the hypothesis that neurovascular water exchange (Kw) measured by non-invasive diffusion-prepared arterial spin label MRI is impaired in SSD and associated with negative symptoms. Peripheral vascular endothelial health was examined by brachial artery flow-mediated dilation (n = 44 healthy controls, n = 37 SSD) to assess whether centrally measured Kw is related to endothelial functions. The study design was cross-sectional and both sexes were included in the studies. Results: Whole-brain average Kw was significantly reduced in SSD (p = 0.005), particularly in the right parietal lobe, including the right supramarginal gyrus (p = 0.002) and right postcentral gyrus (p = 0.008). Reduced right superior corona radiata (p = 0.001) and right angular gyrus Kw (p = 0.006) was significantly associated with negative symptoms. Peripheral endothelial function was also significantly reduced in SSD (p = 0.0001). Kw in 94% of brain regions in controls positively associated with peripheral endothelial function, although the trend was not observed in SSD patients, where the correlation was inversed in 52% of the brain regions. Conclusions. Conclusions: This study provides initial evidence of negative symptoms in schizophrenia linked to subclinical neurovascular water exchange abnormality in specific brain regions. Keywords: Blood-Brain-Barrier, Neurovascular, Schizophrenia Spectrum Disorders, Endothelial Function, Negative Symptoms Disclosure: Nothing to disclose. P580. Adolescent Development of Cortical Circuits for Predictive Coding is Sex-Dependent and Relates to Maturation of Prefrontal Cortex Jordan Ross, Anna Rader, Connor Gallimore, Jordan Hamm* Georgia State University, Atlanta, Georgia, United States Background: Although not diagnostic, difficulties in the processing of sensory information in the context of past and present stimuli are common in a number of neuropsychiatric disorders, serving to ultimately undermine how affected individuals perceive and interpret their world. For example, this has been quantified in people with schizophrenia (SZ) as altered sensory cortical responses in the EEG to unexpected stimuli during “oddball” sequences— i.e. reduced “mismatch negativity” (MMN). Understanding the essential cell and circuit mechanisms generating healthy and impaired MMN remains a key goal in neuropsychiatry given the strong translational potential of this biomarker. Our past work in mice has shown that, during a visual oddball paradigm, long-range projections from prefrontal brain regions, such as the anterior cingulate area (ACa) in mice, may modulate activity in primary visual cortex (V1) via tonic input at oscillating at ≈10 Hz. We found that serves to enhance the activity of V1 VIP-positive interneurons while suppressing SST-positive interneurons. This SST-suppression effectively disinhibits ensembles of pyramidal neurons to release prediction-error-like “deviance detection” responses (DD) to unexpected stimuli (i.e. the MMN-like response). Because many neuropsychiatric diseases onset during adolescence in a sex-dependent manner, and because past work has shown that human EEG indices of predictive coding (MMN, P300) mature across adolescence, we sought to understand i) how such MMN-like responses develop in adolescence at the cellular level, and ii) how this development corresponds to the development of top-down inputs from prefrontal cortex. Methods: For part 1, awake mice (male (n = 8) and female (n = 10)) underwent two-photon calcium imaging at p28 pre-adolescence, p42 mid-adolescence, and p84 adult while viewing a visual oddball and a control sequence. Sex and developmental effects on DD was assessed with age by sex by stimulus type (deviant vs control) on cell-level responses. For part 2, local field potentials (LFP) were recorded in prefrontal (PFC; area ACa) and visual cortex (V1) in male and female mice aged p28 and p84 (n = 14 mice) to assess interregional synchrony (phase-locking factor- PLF). A one-way ANOVA was carried out on PLF to assess developmental effects of PLF. Results: Results show a sex by age interaction on deviance detection (F(2,2756)=21.11). Males did not develop DD until adulthood (p84) while females exhibited DD at all ages. Closer analysis shows that, regardless of sex, the DD present in pre-adult ages (p28, p42) was strongly predicted by simple feature selectivity in the local (<300µm3) network (i.e. the average orientation selectivity of neurons; r = 0.66, p < 0.01) while this relationship was not present in adulthood (r = 0.30). This suggests a more complex, network-driven predictive coding emerges during adolescence. Our LFP recordings in PFC and V1 were consistent with this interpretation, showing that prefrontal-visual synchrony increases strength from p28 to p84 (f(1,12)=9.51, p < 0.01). Conclusions: Altogether this suggests that difficulties in sensory-context processing may involve long-range cortical circuits which develop in late adolescence, corresponding to the onset of multiple neuropsychiatric diseases. Juvenile females may exhibit a form of DD which is more independent of prefrontal input, buttressed by stronger local feature selectivity in sensory regions, suggesting a potential protective factor explaining lower rates of adolescent onset psychotic disorders. Keywords: Biomarker, Adolescence, Mismatch Negativity, Two-Photon Calcium Imaging, LFP Disclosure: Nothing to disclose. P581. Comprehensive and Simultaneous 3-D Imaging of Interneuron Subtypes in CA1 Depicts Deficits in Interneuron Activity Resulting in Microcircuit Imbalance in a Mouse Model for the 22q11.2 Deletion Syndrome Stephanie Herrlinger, Bovey Rao, Anna Tuttman, Bert Vancura, Tristan Geiller, Joseph Gogos, Attila Losonczy Zuckerman Institute, Columbia University, New York, New York, United States Background: Individuals with the 22q11.2 deletion syndrome (22q11.2DS), one of the strongest genetic risk factors for schizophrenia, demonstrate cognitive impairments, including episodic memory (EM) dysfunction. Our group previously showed that EM is impaired in a mouse model for the 22q11.2DS (Df(16)A + /-). Place cells, cellular representations of EM, are under strong inhibitory control by heterogeneous subtypes of GABAergic interneurons, which have been implicated in the pathophysiology of schizophrenia. In this study, we examined the contribution of pyramidal cells and hippocampal interneuron subtypes to local microcircuit dysfunction in CA1. Methods: 2-photon imaging of CA1 pyramidal neuron population dynamics were performed in vivo to characterize plasticity during novel context exposure in a virtual environment in Df(16)A + / − and WT mice (n = 2 Wt, n = 2 Df16). Wild-type and Df(16)A + /− mice performed goal-oriented learning, random foraging and reversal learning tasks on a cued belt while undergoing large-scale, unbiased 3D GCaMP-Ca2+ imaging of in vivo CA1 interneuron dynamics (n = 6 Wt, n = 5 Df16). Molecular identification of major interneuron subtypes was performed post-hoc utilizing immunohistochemistry. Interneuron subtype activity was assessed through Pearson cross-correlations with velocity and through peristimulus time histograms around behavioral indicators. Results: In Df(16)A + / − mice we observe a significant decrease in CA1PC somatic bursting rate during context switch compared with WTs, suggesting that plasticity is suppressed in vivo (n = 2832 WT, n = 1732 Df(16)A + / − cells, p < 0.001). Interneurons exhibit subtype-specific alterations in activity during locomotion, and a significant overall decrease in place preference (n = 8 Wt, n = 7 Df(16)A + / − , F = 2.111, p = 0.045). Conclusions: Results examining CA1 principal neuron dynamics and plasticity collected in vivo and in vitro suggest that inhibitory circuits are either over-compensating in vivo or are intrinsically deficient themselves. We identify subtype-specific alterations in interneuron dynamics contributing to microcircuit dysregulation, ultimately resulting in a less stable microcircuit in CA1 during learning. Keywords: Cognitive Impairment Associated With Schizophrenia, Interneurons, 22q11.2 Deletion Syndrome, Memory and Learning Disclosure: Nothing to disclose. P582. Perineuronal Nets Degradation in the Ventral Hippocampus of Adult Rats Partially Recreates an Adolescent-Like Phenotype of Stress Susceptibility Débora Colodete, Anthony Grace, Francisco Guimarães, Felipe Gomes University of Sao Paulo, Ribeirao Preto, Brazil Background: The onset of some psychiatric disorders, such as schizophrenia, often occurs in late adolescence and/or early adulthood, which is a period of greater vulnerability to socio-environmental factors. Evidence suggests that stress during this period may affect brain structures that are important in regulating stress response, such as the ventral hippocampus (vHipp), leading to a dopamine system overdrive, which has been associated with psychotic symptoms. Parvalbumin (PV) interneurons in the vHipp are proposed as a site of vulnerability to adolescent stress. Here, we assessed the long-lasting effects of exposure to stress during adolescence or adulthood on behavior, the activity of dopamine neurons in the ventral tegmental area (VTA), and the maturation of PV interneurons and their associated perineuronal nets (PNNs) in the vHipp. We also tested whether the removal of PNNs in the vHipp in adult rats, which is proposed to reset PV interneurons to a juvenile-like state, recreates an adolescent-like phenotype of stress susceptibility. Methods: Male Sprague-Dawley rats were exposed to a combination of stressors, consisting of daily footshock (1.0 mA, 2 s, randomized every 60 ± 20 s) for 10 days (adolescence: PND 31-40; adulthood: PND 61-70), and three 1-hour restraint stress sessions (days 1, 2 and 10), right after the footshock session. Between 2-3 weeks after the end of the stress, the animals were submitted to different behavioral tests to evaluate anxiety [elevated plus-maze (EPM) and light-dark box], sociability (social interaction test), cognitive function [novel object recognition (NOR) test], anhedonia (splash-test), and hyperresponsivity to psychostimulants (locomotor response to the NMDA receptor antagonist MK-801). The impact of stress on in vivo electrophysiological activity of dopamine neurons in the VTA and the number of PV + and PNN + cells in the vHipp were also studied. Only males were used in this study since we have previously found that female rats were resistant to present behavioral and electrophysiological changes after exposure to the same stress protocol. In a second set of experiments, adult animals received intra-vHipp bilateral infusion of chondroitinase ABC (0.05U/μL, 700 nL), which degrades PNNs, or penicillinase (control – an enzyme inert in mammals). One week later, animals were submitted to stress and tested 2-3 weeks post-stress. Results: Adolescent stress exposure (n = 12 naïve and 12 stressed) produced in adult rats anxiety responses in the light-dark box (decreased the time in the light compartment, t(22)=4.4; p = 0.002), decreased social interaction (t(22)=3.1; p = 0.005), impaired cognitive function in the NOR test (t(22)=4.6; p < 0.0001), and increased locomotor response to MK-801 (t(22)=2.5; p = 0.02). Furthermore, adolescent stress increased the number of spontaneously active VTA dopamine neurons (t(10)=4.3; p = 0.001), along with a decrease in the number of PV + (t(10)=2.5; p = 0.03), PNN + (t(10)=7.6; p < 0.0001), PV + /PNN + cells (t(10)=4.9; p = 0.0006) in the vHipp. Correlation matrix analysis indicated that increased VTA dopamine system activity was highly correlated with anxiety behaviors, impairments in social interaction and cognitive function induced by adolescent stress. In contrast, adult stress (n = 12 naïve and 12 stressed) did not induce long-lasting changes in behavior, electrophysiology and the number of PV + , PNN + , PV + /PNN + cells in the vHipp. However, preliminary data indicate that the infusion of ChABC into the vHipp (n = 8-6/group) causes the adult stress to produce cognitive deficits in the NOR test and increase VTA DA neuron population activity (p < 0.05 vs. penicillinase-naïve), similar to that induced by the adolescent stress. The degradation of PNNs in naïve animals did not produce any change. Conclusions: Our findings indicate that adolescent stress induced a schizophrenia-like hyperdopaminergic state which was highly correlated with long-term anxiety-like behaviors, cognitive and social interaction impairments, suggesting that adolescence may be a period of higher vulnerability for the development of schizophrenia in adulthood. In addition, degradation of perineuronal nets in the vHipp of adult rats partially recreates an adolescent-like phenotype of stress susceptibility, further suggesting that PNNs play an important role in protecting PV + interneurons from the deleterious effects of stress. Financial support: Sao Paulo Research Foundation (18/17597-3; 21/03391-7). Keywords: Stress, Parvalbumin Interneurons/Perineuronal Nets, Ventral Hippocampus Disclosure: Nothing to disclose. P583. Cholinergic Modulation of Hallucination-Like Perception Katharina Schmack, Adam Kepecs Francis Crick Institute, London, United Kingdom Background: Psychosis involves excessive dopamine release in the striatum. Cholinergic interneurons are crucial for controlling striatal dopamine release and are reduced in numbers in psychotic disorders. However, the role of striatal acetylcholine in psychosis and its interplay with dopamine are not well understood, mainly because psychosis is challenging to study in animal models. Methods: Here, we employed a recently established task to measure hallucination-like perception in mice. Using optogenetics and dual-colour photometry, we tested how the activity of striatal cholinergic neurons relates to hallucination-like perception and to dopamine release. Results: We found that optogenetic inhibition of striatal cholinergic transmission significantly increased hallucination-like perception (F(1,3)=21.0, p = 0.02). Preliminary results indicated that activity of cholinergic neurons peaked before hallucination-like perception and was inversely related to dopamine release. Conclusions: These results suggest that reduced striatal cholinergic transmission might give rise to psychotic experiences via increasing dopamine release. If our findings are confirmed, novel antipsychotic treatment strategies could target striatal cholinergic interneurons. Keywords: Hallucinations, Psychotic Disorders, Acetylcholine, Striatum, Mouse Model Disclosure: Nothing to disclose. P584. What is Odd about the Oddball: A Time-Frequency Deconstruction of Auditory P300 Deficits in Schizophrenia Michael Avissar, Blair Vail, Heloise De Baun, Javier Lopez-Calderon, Pejman Sehatpour, Gaurav Patel, Daniel Javitt Columbia University, New York, New York, United States Background: The auditory P300 (P3) event-related potential (ERP) is among the best established biomarkers of cognitive dysfunction in schizophrenia (Sz). Sz-like deficits in P3 generation may be induced by ketamine, potentially implicating N-methyl-D-aspartate receptor (NMDAR)-medicated neurotransmission as well as by psychedelics such as psilocybin and LSD. However, the neural mechanisms underlying impaired P3 generation in Sz remain poorly understood at both local- and distributed- circuit levels. Here, we used a combined temporal dynamic and resting-state functional connectivity (rsFC) approach to evaluate both distributed and local circuit mechanisms underlying impaired P3 generation in Sz. Time-frequency deconstruction of the P300 allowed interrogation of distinct underlying processes reflected in alpha (thalamocortical input), theta (cortico-cortical interactions), and delta (neural entrainment) frequency bands. Methods: Twenty healthy controls (HCs) and twenty outpatients with Sz were enrolled in the study. Tone-matching thresholds were measured to assess baseline auditory pitch discrimination function. Participants completed an adaptive auditory oddball task during EEG acquisition that normalized difficulty across individuals. Both ERP and time-frequency analyses (TFAs) were conducted. Hierarchical mixed-model regressions with confirmatory mediation modeling of responses to standard and target stimuli were performed to elucidate relationships between TFA measures and P3 amplitude. In addition, participants underwent resting-state fMRI scans to measure functional connectivity (FC) between surface-based resting-state networks defined in the Gordon et al. 2016 atlas, which parcellates the cortical surface based on FC data. Mixed model regressions with FC to the auditory network and group as fixed factors and EEG delta entrainment as a dependent measure were performed. Results: Tone-matching thresholds were nearly identical between HCs and Sz patients (p = 1.0) suggesting the two groups demonstrated intact pitch discrimination. Mean mismatch negativity (MMN) amplitudes also did not differ between groups (p = 0.93), validating the normalization of oddball task difficulty. As expected, patients demonstrated deficits in P3 amplitudes compared to HC (p < 0.01). P3 generation was mediated by a combination of delta entrainment to standards (p < 0.01) and by early alpha activity to targets (p < 0.01). There were deficits in both delta entrainment (p < 0.001) and early alpha activity (p < 0.01) in patients. Delta entrainment strength was associated with FC of visual (p < 0.01) and sensorimotor hand (p < 0.001) networks to the auditory network. Furthermore, differential associations between FC to the auditory network and delta entrainment were observed between HCs and patients to sensorimotor hand (p < 0.001), default (p < 0.01), and cingulo-opercular (p < 0.05) networks in group by FC interactions. Conclusions: P3 generation may be conceptualized primarily as a prediction error involving the delta-oscillatory system. In Sz, deficits are observed in both the initial alpha-frequency cortical response to targets and to stimulus-induced delta entrainment to standards, which, in turn, predict impaired delta responses to targets. All components of this response are impaired in Sz, suggesting impaired input to auditory cortex. Delta entrainment represents a key component of “active sensing” during active auditory processing. Both ERP and resting-state fMRI findings suggest that individuals with Sz may need to use active top-down mechanisms to compensate for impaired function of low-level auditory systems. Keywords: Schizophrenia (SCZ), Neuronal Oscillations, Resting State Functional Connectivity, P300, EEG/ERP Electrophysiology Disclosure: Nothing to disclose. P585. Steady State and Harmonic Responses: Cross-Species Similarities and Differential Responses to the NMDA Antagonist, Memantine Juan Molina, Muhammad Raza, Makoto Miyakoshi, Yash Joshi, Peter Clayson, Neal Swerdlow, Gregory Light, Sivarao Digavalli University of California, San Diego, San Diego, California, United States Background: Translational biomarkers are critical for psychiatric drug discovery, to prioritize lead molecules, signal entry of the drug into the brain, and to demonstrate a functional change to help dose selection. Biomarkers that index circuit level processes may bridge cellular changes with functional and behavioral outcomes. Our investigative group has studied electroencephalographic (EEG) measures of the Auditory Steady State Response (ASSR) to a 40 Hz click train as a potential cross-species biomarker for treatment development for schizophrenia (SZ). When spectrally complex click trains elicit ASSR, in addition to entrainment at the driving frequency, time-locked EEG harmonics produce a Steady State Harmonic Response (SSHR); ASSR and SSHR exhibit distinct characteristics and support distinct cognitive functions and thus may have different predictive properties as biomarkers. While the ASSR at 40 Hz is viewed as a potential biomarker of NMDA receptor functioning, the SSHR has not been evaluated as a target engagement measure. Here we report on the ASSR 40 Hz response and its 80 Hz SSHR in rodents and humans, and their sensitivity to acute pharmacologic challenge with the non-competitive NMDA antagonist, memantine (MEM). Methods: Ten Sprague-Dawley rats (age 6-8 weeks) were implanted with epidural temporal (recording) and bilateral cerebellum (ground / reference) electrodes. After at ~ 2-weeks, rats were tested 90 min after MEM (0, 0.3, 1 and 3 mg/kg sc) in a repeated measures within-subject crossover design, with > 3 d between tests. EEG was recorded in individual Plexiglas cylinders equipped with a video camera and a house speaker. Data were acquired as 5-s sequential sweeps; click train used 5 mV monophasic, 1-ms square waves, 40/1 s, ~ 65 dB. Evoked ASSR and SSHR responses were band-pass filtered (35–45 and 75-85 Hz) and averaged from ~ 75 consecutive trials. Evoked Power (EP) and inter-trial coherence (ITC) were calculated using the 0.2-1.0 s data. Eighty-two human subjects, including 44 patients with schizophrenia (SZ) and 38 healthy subjects (M:F = 48:34; age = 33.4 ± 9.7 (mean±s.d)) were included. EEG was continuously recorded via established methods starting 385 min after MEM (0 or 20 mg, po) in a repeated measures within-subject crossover design, with 7 d between tests. The ASSR paradigm utilized 1 ms, 85 dB clicks presented in 500 ms trains at a frequency of 40 Hz. A total of 250 click trains were played through insert earphones with an inter-train interval of 0.5 s. For EP, the averaged epochs across the click trains were transformed into power spectrum by means of fast Fourier transform (FFT) using a bin width of 2 Hz. ASSR and SSHR ITC were calculated from wavelet coefficients obtained from continuous wavelet transformation; ITC was estimated by extracting and averaging across the 35–45 Hz and 75-85 frequency layers. Results: In rats, 40 Hz driving stimuli evoked both an ASSR at 40 Hz and an SSHR at 80 Hz. For ASSR, ANOVA of EP showed significant effects of frequency (p < 0.0001; 40 Hz > 80 Hz) and treatment (p = 0.03); post-hoc tests showed significant EP augmentation by the 3 mg/kg MEM dose (p = 0.001). ANOVA of ITC showed a significant frequency x treatment interaction (p = 0.015); post-hoc testing showed significant augmentation by the 3 mg/kg MEM dose (p < 0.05). For the 80 HZ SSHR, no significant effects of MEM were detected on either EP or ITC. In humans, 40 Hz driving stimuli evoked both an ASSR at 40 Hz and an SSHR at 80 Hz. For ASSR, ANOVAs of EP and ITC showed significant main effects of frequency (p’s < 0.0001; 40 Hz > 80 Hz); for EP at 40 Hz ASSR, there was a significant main effect of diagnosis and a significant diagnosis x treatment interaction (p’s < 0.05); post-hoc testing revealed that the interaction was driven by MEM-induced enhancement of EP in SZ patients. Similar trends were observed for ITC at 40 Hz ASSR. For the 80 Hz SSHR, no significant effects of MEM were detected on either EP or ITC (both ns). Conclusions: Across species, auditory stimulation by complex stimuli evokes EEG entrainment at both the stimulus driving frequency (ASSR) and its harmonics (SSHR). These two responses may be regulated by distinct neural substrates and may in turn regulate different cognitive processes. Compared to the 40 Hz ASSR, relatively less is known about the harmonic response to 40 Hz stimulation. Here, we report that, in rodents and humans, the 40 Hz ASSR exhibits greater power and inter-trial coherence than its 80 Hz second harmonic. Furthermore, across species, the 40 Hz ASSR power and coherence are enhanced by the non-competitive NMDA antagonist, MEM, while the SSHR is insensitive to MEM at the doses tested. In parallel studies, Digavalli et al. (in preparation) found that the 40 Hz SSHR to a 20 Hz driving frequency was disrupted by the NMDA antagonist, MK801, while the 20 Hz ASSR response was unaffected. The present results suggest that ASSR and SSHR: 1. can be studied across species; 2. produce different levels of evoked power and coherence (here, at 40 Hz, ASSR » SSHR), and these differences are maintained across species; 3. exhibit differential drug sensitivity, with MEM increasing 40 Hz ASSR but not its SSHR, and this difference is maintained across species. In total, ASSR and SSHR appear to be robust, non-redundant EEG signals suitable for cross-species analyses and potential biomarker development. Keywords: Cross-Species Translation, Biomarker Development, Schizophrenia Therapeutics Disclosure: Nothing to disclose. P586. State-Dependent Hippocampal Hyper Synchronous Activity in a Reverse Translational Psychosis Model Mouse Jun Yamamoto, Daniel Scott, Carol Tamminga University of Texas Southwestern Medical Center, Dallas, Texas, United States Background: Hyperactivity in the hippocampal (HPC) region was first identified as one of the prominent biomarkers in psychotic individuals, initially using PET brain imaging in early 2000’s, drawing significant interest in its underlying mechanisms. Moreover, the hyperactivation of the HPC has been identified as an informative human biomarker of schizophrenia especially in young patients. Recent studies of human schizophrenia post-mortem brain tissue have shown decreased expression of NMDAR subunit GluN1 in the dentate gyrus (DG) with evidence of hyperactivity downstream in the hippocampus downstream Cornu Ammonis (CA) subregions. There is however little insight into the emergence of such hyperactivities in the HPC subregions and the time course of their development. Therefore, our group has reverse translated this feature into a selective DG-inhibited mouse model and conducted immunohistochemistry, behavior tests and long-term in vivo electrophysiology studies to determine the dynamics of the hippocampal hyperactivity in both adolescent and adult conditions. Methods: 4 weeks old (for adolescent condition) and 8 weeks old (for adult condition) male C57BL/6 J mice were purchased from Jackson Labs. To specifically manipulate DG in a temporally controlled manner, we expressed an inhibitory DREADD (Designer Receptors Exclusively Activated by Designer Drugs, pAAV-CaMKIIa-hM4Di-mCherry) in the granule cells of the entire DG at 5 and 9 weeks of age, and started inhibiting the DG with Compound 21 (C21) for 21 days in adolescent (6 week) and adult (10 week) C57BL/6 J mice. For control, pAAV-CaMKIIa-eGFP was used. At 5th or 9th week of age, a 32 channel silicon linear array electrode was chronically implanted in the dorsal HPC to monitor the DG - CA1 axis. The daily 1 hour recording session started a few days before the start of the C21 administration to obtain baseline data and continued up to 2 months including the three weeks of C21 exposure between 6 to 9 weeks of age for the adolescent and 10 to 13 weeks of age for the adult conditions. Following removal of C21, behavior testing was conducted with social memory (SM) test and standard contextual and cued fear conditioning (CFC). One week after cessation of C21 exposure, brains were taken and assessed for cFos-positive cell density within the hippocampal subfields. For the in vivo electrophysiology, both wide-band local field potentials (LFPs) and multi-unit spike activity (MUA) were recorded at 32 kHz sampling rate. The LFPs were then band-pass filtered into sub-bands for all 32 recording points. The multi-unit spiking data was temporally aligned with LFPs and spike timing modulation in relation to the LFP amplitude were analyzed. Results: When the DG was suppressed by the DREADD system during adolescent period, the cFos-positive (+) cell density was significantly increased in both CA3 and CA1 subregions (N = 7/group, P < 0.05), but not in DG itself. However, when the DG was inhibited during adulthood, cFos+ cells decreased in DG as well as in CA3 (N = 6/group, P < 0.001, P < 0.01 respectively), but not in CA1. We interpret that the significant increase in cFos expression, which indicates the hyperactivity in both CA3 and CA1 during adolescent period, mimics the HPC hyperactivity that was reported earlier in human ScZ. Using this system, we then subjected the mice to the SM test and the CFC test. In both behavior testing paradigms, only the adolescent DREADD mice group showed memory phenotypes (N = 13, SM: P < 0.001, CFC: P < 0.05), but not in the adult DREADD mouse group (N = 18), suggesting that there is a risk time window to exhibit memory impairments with the abnormal level of hyperexcitation in the HPC. Next, we analyzed the LFP data (N = 5 adolescent pairs and N = 3 adults). While we monitored the activities on a day-to-day basis, short-lived large irregular potentials that are accompanied by a large population of MUA (dubbed Hyper-Synchronous Events: HSEs) emerged at around day 7 at extremely low occurrence (once every 5 minutes) while animals were in either quiet awake or slow-wave sleep states, but not during awake state. Subsequently, the HSEs became more and more obvious as the days went by, and by day 14, HSE became extremely frequent (once every 10-15 sec). Among 5 adolescent pairs, we observed epileptiform/seizure-like events developed in two DG-DREADD mice during quiet wake and/or slow-wave sleep periods. During high HSE occurring periods, the spontaneous homecage behavior deteriorated in such a way that their nest in the homecage became disorganized. Moreover, the reduced-level of HSEs (once every minute or so) persisted even after the C21 treatment, i.e. the DG inhibition period, was over. This HSE development was observed neither in the control adolescent mice group nor in the adult mice and was specific to the adolescent DG-DREADD mice group suggesting that the HSE-like abnormal electrical discharges might also be occurring in the human ScZ HPC. Conclusions: Adolescence age in mice appears to be a critical period in which decreased DG activity can induce hippocampal hyperactivity including epileptiform-like neural activity in the HPC, an outcome not observed in adult mice. This hippocampal hyperactivity originates as a transient increase in activity in CA3, and persists in CA1, defined by the presence of HSEs. Moreover, a psychosis-like behavioral phenotype tracks with CA3 activity. This suggests a sensitive period of development in which the brain is sensitive to alterations in hippocampal activity and can result in psychosis-like behavioral outcomes. Keywords: Psychosis, Hyperactivity, Hippocampus, In Vivo Electrophysiology Disclosure: Nothing to disclose. P587. Associations Between Structural Stigma and Allostatic Load: Results From the National Health and Nutrition Examination Survey Robert-Paul Juster, Caroline Rutherford, Katherine Keyes, Mark Hatzenbuehler University of Montreal, Montreal, Canada Background: Structural forms of stigma and discrimination are associated with a host of adverse outcomes across numerous stigmatized groups. Yet, the biological mechanisms underlying this relationship remain inadequately understood. To address this gap, the current study assessed the relationship between indicators of structural stigma surrounding lesbian, gay, and bisexual (LGB) individuals (i.e., state-level policies) and allostatic load indices representing physiological dysregulations. Methods: Pooled data from the continuous 2001-2014 National Health and Nutritional Examination Survey was analyzed (N = 21,774). Ten state-level LGB-related policies (e.g., employment non-discrimination protections, same-sex marriage rights) were used to index structural stigma. A sex-specific allostatic load index representing immune, metabolic, and cardiovascular biomarkers was calculated. Multi-level regressions adjusting for covariates were used. Results: Sexual minority men living in states with more policies protecting sexual minorities experienced significantly lower allostatic load (β = −.45, p = 0.02) than those living in states with fewer protective policies. Conclusions: This is the first study to assess structural stigma at the state level in association with allostatic load among members of a stigmatized group. Our novel findings provide a mechanistic understanding of how the social environment can ‘get under the skin and skull’ for sexual minority Americans, with potential implications for other marginalized groups. This work also has social policy implications by demonstrating direct impacts of structural stigma on indices of physiological dysregulation. Keywords: Allostatic Load, Sexual Orientation, Structural Stigma Disclosure: Nothing to disclose. P588. Effect of Lemborexant on Sleep Onset and Maintenance in Patients With Comorbid Insomnia Disorder and Mild Obstructive Sleep Apnea Margaret Moline, Jocelyn Y. Cheng, Dinesh Kumar, Alan D. Lowe, Barbara Ramos Eisai Inc., Nutley, New Jersey, United States Background: Insomnia and obstructive sleep apnea (OSA) are common sleep disorders that are frequently comorbid, a clinical entity referred to as comorbid insomnia and sleep apnea (COMISA). Studies of hypnotics in patients with OSA often focus on the impact of the drug on respiratory variables such as the apnea-hypopnea index (AHI), since commonly prescribed hypnotic drugs may exacerbate pre-existing respiratory dysfunction. Additional concerns about the selection of a hypnotic for concomitant therapy for OSA include residual sedation in a somnolent patient population and the known risks of hypnotics for the treatment of insomnia. Lemborexant (LEM), a dual orexin receptor antagonist, is approved in multiple countries for the treatment of insomnia disorder. Respiratory safety studies in patients with untreated mild-severe OSA indicated no negative impact on either the AHI or peripheral oxygen saturation following single or multiple doses of LEM 10 mg (LEM10), the higher approved dose (Cheng et al., J Sleep Res 2020). A post-hoc analysis was conducted to evaluate the effects of LEM, which has demonstrated respiratory safety in OSA, for the treatment of insomnia in patients with mild OSA. Methods: Study E2006-G000-304 (Study 304; NCT02783729) was a 1-month, randomized, double-blind, placebo (PBO)-controlled and active-comparator (zolpidem tartrate extended release 6.25 mg [ZOL]) study of LEM 5 mg (LEM5) and LEM10 (1006 randomized). Subjects (male and female) age ≥55 y who met criteria for insomnia disorder per DSM-5 and with verified sleep maintenance problems were enrolled; subjects were not required to have difficulty with sleep onset. A screening polysomnogram (PSG) with respiratory montage was used to rule out moderate to severe OSA. Subjects whose AHI was <15 events/h of sleep (mild OSA) were eligible, assuming other criteria were met. Sleep onset (latency to persistent sleep [LPS]); sleep maintenance (sleep efficiency [SE] = total sleep time / time in bed); wake after sleep onset (WASO); and WASO in the second half of the night (WASO2H) were assessed at Days 1/2 and 29/30 using PSG and averaged across consecutive nights. Pairs of PSG during the PBO run-in served as the baseline. Change from baseline (CFB) was analyzed using mixed-effect model repeated measurement analysis. CFB means are reported (rounded to the 10th decimal) as least squares geometric mean ratio: visit/baseline for LPS and least squares means for SE, WASO, and WASO2H. These post-hoc analyses focused on the subpopulation of patients with both insomnia disorder and mild OSA. Results: The Full Analysis Set (FAS) included 1006 subjects, of whom 410 (40.8%) had mild OSA. In the mild OSA subpopulation, median age was 65 y, 83.9% were female, and median body mass index was 27.56 (29.8% had BMI > 30); AHI (mean [SD]) on screening PSG was 9.33 (2.9) events/h. CFB for SE (%) was larger and statistically significant (increases = improvements) for both LEM5 and LEM10 (herein LEM5-LEM10) (13.1-15.8) vs PBO (3.4, P < 0.0001) and ZOL (11.2, P < 0.05) on Days 1/2; similar results were observed on Days 29/30 (LEM5-LEM10 12.8-14.0; PBO 4.4, P < 0.0001; ZOL 8.7, P < 0.0001). CFB for LPS, WASO, and WASO2H was larger and statistically significant (decreases = improvement) for LEM5-LEM10 vs PBO on Days 1/2 (LPS [ratio]: LEM5-LEM10 0.58-0.68 and PBO 0.89, P < 0.005; WASO [min] LEM5 LEM10 –48.8 to –57.6 and PBO –11.3, P < 0.0001; WASO2H: LEM5-LEM10 –28.6 to –36.1 and PBO –4.7, P < 0.0001) and Days 29/30 (LPS [ratio]: LEM5-LEM10 0.47-0.58 and PBO 0.80, P < 0.005; WASO [min]: LEM5-LEM10 –44.4 to –45.2 and PBO –16.0, P < 0.0001; WASO2H [min]: LEM5-LEM10 – 27.0 to –28.7 and PBO –9.2, P < 0.0001). LEM10 produced larger CFB vs ZOL for LPS, WASO, and WASO2H at Days 1/2 (ZOL LPS: 0.72, P < 0.01; WASO: –41.9, P < 0.001; WASO2H: –22.2, P < 0.0001) and Days 29/30 (ZOL LPS: 0.83, P < 0.0001; WASO: –33.1, P < 0.01; WASO2H: –18.7, P < 0.005), and LEM5 (P < 0.02 for all assessments) on days 29/30. LEM was well tolerated, with a safety profile in the subpopulation consistent with its known safety profile. Conclusions: These results demonstrated the effectiveness of LEM compared with both PBO and ZOL in an older patient population with untreated COMISA, characterized by sleep maintenance complaints meeting insomnia disorder criteria and mild OSA. These data and those from the respiratory safety studies suggest LEM may be an appropriate choice for the treatment of insomnia in patients with mild OSA. Keywords: Comorbid Insomnia Obstructive and Sleep Apnea (COMISA), Obstructive Sleep Apnea, Apnea-Hypopnea Index, Lemborexant, Dual Orexin Receptor Antagonist (DORA) Disclosure: Eisai Inc.: Employee (Self). P589. The Acute and Chronic Effects of Zolpidem and Lemborexant on Sleep Oscillation Expression in Older Adults With Insomnia Negin Sattari Barabadi, Abhishek Dave, Hamid Niknazar, Ivy Y. Chen, Ariel B. Neikrug, Ruth M. Benca, Bryce A. Mander University of California Irvine, Lake Forest, California, United States Background: Insomnia symptoms increase in prevalence with age and are among the most common sleep disturbances reported in older adults. Two biological systems targeted to treat insomnia include the GABAergic and orexigenic systems. It remains unknown how use of medications that target these systems to treat insomnia differentially impact the expression of sleep oscillations supportive of the physical and mental health functions of sleep, particularly among older adults with insomnia. Here, we compare the acute and chronic effects of zolpidem and lemborexant, in comparison to placebo, on quantitative sleep electroencephalography (EEG) among older adults with insomnia. Methods: One hundred sixteen older adults (71.01 ± 4.81 years, 78.67% female) with insomnia (Insomnia Severity Index 18.73 ± 3.13) completed a randomized, phase III clinical trial (E2006-G000-304; NCT02783729) comparing the effects of placebo (n = 29), zolpidem tartrate extended release (6.25 mg, n = 26; ZOL), and lemborexant (5 mg, n = 29 [LEM5]; 10 mg, n = 32 [LEM10]) on polysomnography(PSG)-recorded sleep across baseline (before), acute (1-2 days) and chronic (29-30 days) treatment. Spectral analysis was performed using the multitaper method on artifact-free EEG data during non-rapid eye movement (NREM) sleep epochs. Changes in absolute spectral power within canonical frequency bands (i.e., slow-oscillation [0.5-1 Hz], delta [1-4.5 Hz], theta [4.5-7.5 Hz], alpha [7.5-11 Hz], slow-sigma [11-13 Hz], fast-sigma [13-16 Hz], beta [16-28 Hz] and gamma [28-35 Hz]) from baseline were compared between groups at averaged frontal electrodes (i.e., F3, F4) across acute and chronic treatment phases using repeated measures ANOVA (RMANOVA) models with Fisher’s Least Significant Difference post hoc testing (fixed factors: visit, frequency; between factor: drug intervention). In addition, changes from baseline in objective measures of sleep quality [SE: sleep efficiency, SOL: sleep onset latency, WASO: wake after sleep onset] were calculated. Further, the calculated sleep measures at chronic were used in a multivariate ANOVA to test for difference in change in sleep quality measures from baseline after chronic use of medications. Results: Examining the effect of treatment on objective sleep quality, a MANOVA analysis model predicting objective sleep quality was significant (p = 0.02), with a significant intervention effect on WASO (p = 03) and SOL (p = 0.04) being detected. Post hoc comparisons revealed that chronic treatment impacted objective sleep quality differentially by treatment group. Compared to PBO, sleep quality improved significantly more following both LEM5 [SE p = 0.05, WASO p = 0.01] and LEM10 [SE p = 0.05, SOL p = 0.03, WASO p = 0.04] treatment. In addition, following chronic treatment, sleep quality, compared to ZOL, improved more for LEM5 [WASO p = 0.04, SOL p = 0.05] and LEM10 [SOL p = 0.02]. Examining the effect of treatment on absolute power, a 3-way RMANOVA revealed 1) a significant main effect of frequency [F(7,784)=8.05, p < 0.001], 2) a significant visit×frequency interaction [F(7,784)=2.68, p = 0.009], 3) a significant frequency×intervention interaction [F(21,784)=4.99, p < 0.001], and 4) a significant visit×frequency×intervention interaction [F(21,784)=2.67, p < 0.001]. Post hoc testing revealed a greater acute suppression in power for ZOL compared to the PBO [delta p < 0.001, theta p < 0.001, alpha p < 0.001], LEM5 [delta p = 0.00, theta p < 0.001, alpha p < 0.001], and LEM10 [delta p = 0.01, theta p < 0.001, alpha p = 0.001] treatment groups. Post hoc testing also revealed chronic suppression in power for ZOL compared to the PBO [delta p < 0.001, theta p < 0.001] and LEM10 [slow oscillation p = 0.04, delta p < 0.001, theta p = 0.004] groups. However, chronic ZOL treatment was also associated with greater increases from baseline in power compared to LEM5 [slow-sigma p < 0.001, fast-sigma p = 0.006, beta p = 0.06-trend] and LEM10 [slow-sigma p = 0.008] groups. Further, following chronic treatment, LEM5, compared to PBO, showed a suppression of delta (p = 0.02), theta (p = 0.04), alpha (p = 0.04), and slow sigma (p = 0.05). Conclusions: In older adults reporting moderate to severe insomnia symptoms, chronic treatment with lemborexant showed greater improvement in objective sleep quality relative to zolpidem and placebo. In addition, chronic treatment with lemborexant and zolpidem had distinct effects on sleep oscillation expression, with zolpidem suppressing low frequency activity and increasing high frequency activity, lemborexant 5 mg suppressing both low and high frequency activity, and lemborexant 10 mg showing no significant effect. Future studies should examine the functional consequences of these differential effects on sleep oscillation expression in insomnia patients. Keywords: Insomnia Disorder, Sleep Oscillations, Older Adults Disclosure: Nothing to disclose. P590. Sleep Disturbance Associated With Suicide Risk for Children in the Adolescent Brain Cognitive Development Study Rebekah Huber, Erin McGlade, Perry Renshaw, Deborah Yurgelun-Todd University of Utah School of Medicine, Huntsman Mental Health Institute, Salt Lake City, Utah, United States Background: Suicide risk in children is a growing concern. During the past decade, the pediatric suicide rate has nearly tripled (Curtin and Heron, 2019) and a recent meta-analysis estimated the prevalence of suicidal ideation in children younger than 12 years of age is 7.5% (Geoffroy, et al., 2022). There is a critical unmet need to identify risk factors associated with suicidal thoughts and behaviors (STB) during childhood to design developmentally appropriate preventative strategies. Sleep disturbances including insomnia, hypersomnia, and nightmares have been linked to risk for suicide in studies of adults and adolescents (Harris et al., 2020; Goldstein, Bridge, and Brent, 2008) yet, few studies have examined sleep disturbance and associated risk for suicide in children. The purpose of this study was to examine the relationship between sleep disturbance and STB in children. Methods: This study utilized baseline data (data release 4.0) from 11,869 participants in the Adolescent Brain Cognitive Development℠ Study (ABCD Study®), a longitudinal study that follows nine- and ten-year-old children through late adolescence to examine factors that influence developmental trajectories. Youth STB was assessed by the Kiddie Schedule for Affective Disorder and Schizophrenia (KSADS) suicide module completed separately by the parent and the youth. Recent child STB included any suicidal thoughts or attempts reported by the parent or child during the previous two weeks. Parents completed the Sleep Disturbance Scale for Children (SDSC) which assessed youth sleep disturbance in the previous 6 months including disorders of initiating and maintaining sleep, sleep-disordered breathing disorders, disorders of arousal, sleep-wake transition disorders, disorders of excessive somnolence and sleep hyperhidrosis. Generalized additive mixed models were used to examine the relationship between STB and sleep disturbance which modeled family nested within site as random effects controlling for age, sex, parent education, household income, and current symptoms of depression. Results: At baseline, participants had a mean age of 9.9 and the sample included 6188 (52%) males and 5681 (48%) females. 330 youth had recent STB and 11,324 youth did not have recent STB. 63% of children with recent STB slept less than 9 to 11 hours compared to 52% of children without recent STB. Children with recent STB had higher scores on total sleep disturbance (p = 0.01; Cohen’s d = 0.13), disorders of initiating and maintaining sleep (p < 0.001; Cohen’s d = 0.22), and disorders of excessive somnolence (p = 0.019; Cohen’s d = 0.12) compared to youth without recent STB. Forty-nine percent of children with recent STB had clinically significant sleep disturbance scores compared to 26% of children without recent STB had clinically significant sleep disturbance scores. There were no significant associations between recent STB and sleep breathing disorders, disorder of arousal, sleep-wake transition disorders. Conclusions: Overall, our study revealed that in this large sample of youth between 9 and 10 years old, children with recent STB had greater sleep disturbance, particularly difficulty falling and staying asleep and excessive somnolence. These findings have important implications, as insufficient sleep and poor sleep patterns have become a common issue for children and adolescents worldwide (Gariepy, et al., 2020). Sleep disturbance is associated with decreased attention and inhibition and may be related to difficulty controlling thoughts of suicide and regulating emotions thereby increasing risk for suicide. The current study is cross sectional and cannot address whether sleep plays a causal role in STB; however, studies with adolescents have shown sleep disruption predicted future STB. Disturbed sleep is modifiable and sleep interventions may inform suicide prevention efforts. Future research should examine the longitudinal relationship between sleep disturbance and risk for suicide. Keywords: Suicidal Behavior, Suicidal Ideation, Sleep Disturbances Disclosure: Nothing to disclose. P591. Acute Disruption of Affect and Daytime Alertness in Premenopausal Women Undergoing Experimental Sleep Fragmentation and Estradiol Withdrawal to Mimic Menopause Hadine Joffe, Leilah Grant, Margo Nathan, Jessica Harder, Primavera Spagnolo, Sybil Crawford, Aviva Cohn, Aleta Wiley, Tianyu Luo, Ellexa Menezes, Anna Joseph, Ancella Roy, Shadab Rahman Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts, United States Background: Animal studies show the adverse impact of sleep perturbation and estradiol withdrawal on both affective state and arousal and regulatory systems in females. However, few experimental studies translate these findings to humans, for whom middle-of-the-night sleep interruption and reproductive-hormone changes commonly co-occur with hot flashes (HF) during the menopause transition. Given the potential to intervene on HF-related sleep interruption as a modifiable target in order to improve the highly prevalent reduction in psychological well-being and daytime alertness among midlife women, we examined the acute effects of sleep fragmentation and estradiol withdrawal on these neuropsychological symptoms, and the moderating role of HF, using an experimental paradigm mimicking menopause. Methods: We studied 27 premenopausal healthy female volunteers (age 28.7 ± 5.6 years) in two 5-night inpatient studies in 1) the mid-to-late follicular phase (estrogenized) and 2) following hypoestrogenism induced by a gonadotropin-releasing hormone agonist that provoked HF (detected objectively using skin conductance methodologies) in 56% of study subjects. Each inpatient study had 2 nights of unfragmented sleep followed by 3 nights of experimentally induced sleep fragmentation to achieve approximately one hour of wake after sleep onset without reducing total sleep time. The following were assessed daily during the daytime of the unfragmented and fragmented sleep conditions: 1) affect on the Positive and Negative Affect Schedule (PANAS) to capture positive (PosAff) and negative affect (NegAff) (range 10-50 each), and, in a subset (n = 16), 2) daytime arousal and regulatory state through objective measurement of sustained attention using the Psychomotor Vigilance Task (PVT) mean reaction time (RT) in milliseconds and attentional failures (number of RT > 500 ms), as well as subjective measurement of sleepiness (Karolinska Sleepiness Scale, range 1-9). Acute effects of sleep condition (after one night of sleep fragmentation) and of estradiol withdrawal on PosAff, NegAff, PVT-RT, number of PVT-attentional failures, and sleepiness were examined using Generalized Linear Mixed Models testing interactions with HF. Results: One night of sleep fragmentation acutely and adversely disrupted affective state (PosAff and NegAff), PVT-RT, and sleepiness (all p ≤ 0.039) independent of estradiol state and HF. Estradiol withdrawal also had an adverse impact on PosAff, PVT-RT, and PVT-attentional failures (all p ≤ 0.001), but not on NegAff or sleepiness. The effect size for most outcomes was small to medium (Cohen’s d range 0.18–0.56). While HF did not independently influence these outcomes, HF were a robust effect modifier of the adverse impact of sleep fragmentation on both affective and arousal states (both 2-way interactions p ≤ 0.030), such that those with both HF and sleep fragmentation had the lowest PosAff and the most attentional lapses. In 3-way interactions, HF were a robust effect modifier of the impact of estradiol-dependent effects of sleep fragmentation: those with HF appeared to be most sensitive to the effects of sleep fragmentation on PVT-attentional lapses, whereas their subjective perception of sleepiness was attenuated after estradiol was withdrawn and HF emerged (both interactions p ≤ 0.033). Conclusions: Experimental studies in humans demonstrate acute adverse effects of both menopause-pattern middle-of-the-night sleep interruption and estradiol withdrawal on psychological well-being and daytime alertness. Having HF influence the deleterious impact of sleep fragmentation on these outcomes, notably with worse affect and more objectively measured attentional lapses despite acclimation to the perception of sleepiness after a period of exposure to HF and hypo-estrogenism. These findings highlight the potential therapeutic benefit of consolidating sleep, especially in combination with HF suppression, as a strategy to improve both affective state and sustained attention during the daytime in women undergoing the menopause transition. Keywords: Women’s Mental Health, Sleep Disturbance, Menopause, Estradiol, Hot Flashes Disclosures: National Institutes of Health, Merck, Pfizer: Grant (Self), Bayer, Eisai, Hello Therapeutics: Consultant (Self), Merck: Other Financial or Material Support (Spouse), Arsenal Biosciences: Employee (Spouse). P592. Effects of Electronic Noise Masking Ear Buds on Objective and Subjective Sleep Parameters in Health Care Providers Andrew Novick, Heinrich Haller, Susan Moore, Rachel Johnson, Mary D. Sammel, Katherine Green, C. Neill Epperson University of Colorado, Denver, Aurora, Colorado, United States Background: The importance of sleep amongst health care workers is undeniable, as good health and alertness are essential for the delivery of care to patients. The COVID-19 pandemic has had detrimental effects on sleep quality among health care workers, prompting the need to explore new interventions. The present study evaluated the effect of Bose Noise-Masking Sleepbuds(TM) in health care workers with reported sleep difficulties using both objective and subjective measures. Methods: Seventy-seven (n = 46 females, n = 31 males) hospital-based health care workers, aged 20-50, completed this pre-post efficacy study. Participants had to self-identify as light or moderate sleeps with self-reported difficulty falling or staying asleep, and score at least an 8 (“subthreshold insomnia”) on the Insomnia Severity Index. Objective sleep architecture data was obtained using the DREEM headband in-home sleep monitoring device, which records EEG signals, heart rate, movement and sound. Subjective measurements included perceptions of sleep onset latency, wake after sleep onset, total sleep time and scores on the Insomnia Severity Index. Participants first completed a 3-night baseline assessment wearing just the DREEM headband, followed by a 4-night adjustment period of wearing only the Sleepbuds(TM), and then a final 3-night intervention assessment wearing both the Sleepbuds(TM) and the Dreem headband. Primary outcomes were analyzed for statistical significance using mixed-effect linear regression and random intercept for each participant to model repeated measures over time. Results: Use of the Sleepbuds(TM) was associated with a significant decrease in perceived sleep onset latency compared to baseline, with a 6 minute decrease from the baseline mean to intervention mean (21% faster, 95% CI: 8%-35%, p = 0.003, Cohen’s d = −0.38). There was also a significant decrease in Insomnia Severity Index scores from baseline (p < 0.001) with a mean change score of -7.7 (4.4) points. There were no significant changes in objective sleep measures, including objective sleep onset latency, wake after sleep onset, number of awakenings or % time in various sleep stages (all p’s > 0.05). Conclusions: Compared to baseline assessments, the use of noise masking Sleepbuds(TM) in health care workers improved subjective sleep quality as indicated by a decrease in perceived sleep onset latency and decreases in Insomnia Severity Index scores. Lack of changes in objective sleep measures are likely due to the healthy sample population with lack of overt sleep pathology. Overall, the results suggest that noise masking ear bud technology holds promise as a non-pharmacological intervention to improve sleep in health care workers. Keywords: Technology, Sleep, Insomnia Disclosure: Nothing to disclose. P593. Conversion of Suvorexant to Lemborexant and Study Under Environmental Conditions Kazumaro Okino, Hirohisa Suzuki, Hiroi Tomioka, Akira Iwanami, Atsuko Inamoto Mental Care Center, Showa University Northern Yokohama Hospital, Tokyo, Japan Background: Insomnia negatively affects physical and mental health and also increases the risk of traffic and workplace accidents. Approximately 20% of Japanese adults have chronic insomnia, and the rate is currently increasing. Patients are more likely to experience insomnia during hospitalization due to environmental changes, noises such as alarms, and changes in sleep habits. Suvorexant was the first approved dual orexin receptor antagonist (DORA). However, it was reported to be more effective in treating difficulties in maintaining sleep rather than in initiating sleep and was found to be effective in only 70%–75% of patients. A new DORA, lemborexant, is reported to have a numerically larger effect size on sleep measures compared with placebo and suvorexant. This study aimed to examine the effects of a change in medication from suvorexant to lemborexant among patients with insomnia. The differences in sleep improvement effects between inpatient and elderly care facility environments are also examined. Methods: Medical records of patients from three medical centers who received suvorexant in January 2021, including inpatients at the Mental Care Center of Showa University Northern Yokohama Hospital and patients at two clinics, were retrospectively studied. The patients visiting the two clinics resided in a long-term care facility. Patients with chronic insomnia persisting for ≥3 months and who had been taking suvorexant for ≥3 months were selected. Participants were divided into two groups: the “modified” and “nonmodified” groups. When a drug change from suvorexant to lemborexant was required, informed consent was obtained from the patients and their families. The modified group was further divided into inpatient and facility groups. Four subtypes of insomnia (difficulty initiating sleep, difficulty maintaining sleep, early-morning awakening, and nonrestorative sleep) were investigated. Logistic regression was used to investigate improvements in both groups after 12 weeks. The study design was approved by the Showa University Yokohama City Northern Hospital Ethics Committee. Results: Of the 77 participants, 43 and 34 patients were included in the modified and nonmodified drug groups, respectively. In the comparison of sleep disorders between the two groups, we found significant improvement after 12 weeks in the modified drug group in terms of difficulty initiating sleep compared with the nonmodified drug group (odds ratio = 0.036, p = 0.008, confidence interval = 0.003–0.415). However, no significant differences were found between the two groups in terms of difficulty maintaining sleep, early-morning awakening, and nonrestorative sleep. There were only six and four cases of side effects in the modified and nonmodified drug groups, respectively. Of the 43 patients in the modified drug group, 12 were in the inpatient group and 31 were in the facility group. There were no significant differences between the two groups in any insomnia disorder subtype. Conclusions: Orexin recepter 2 (OX2R) has a stronger effect on maintaining arousal and circadian rhythm than orexin recepter 1 (OX1R). Similar to suvorexant, lemborexant shows an antagonistic inhibitory effect on both OX1R and OX2R, and lemborexant has a higher selectivity for OX2R than suvorexant. Therefore, compared with suvorexant, lemborexant increases non–rapid eye movement sleep and promotes sleep efficiency, resulting in shortened sleep latency and increased sleep maintenance effect. In this survey, the modified drug group showed significant improvements in difficulty initiating sleep compared with the nonmodified drug group. In addition, because suvorexant 15/20 mg is considered to be equivalent to lemborexant 5 mg, the average dose of lemborexant after 3 months was 6.750 (±2.750) mg. Lemborexant administration at ≥5 mg, as opposed to suvorexant at 20 mg, could have led to the significant improvement in the modified drug group. In this study, only six and four cases of side effects were observed in the modified and nonmodified drug groups, respectively. Moreover, no serious side effects were observed, and no significant difference was observed between the two groups. Lemborexant 10 mg reportedly has a higher risk of somnolence than suvorexant 15/20 mg. Therefore, the dosage of lemborexant can be increased up to 10 mg in cases in which the effect is insufficient at 5 mg; however, because the risk of side effects increases at high doses, caution is required. Sleep disorders can be treated by alleviating difficulties in initiating sleep by switching from suvorexant to lemborexant. In inpatient settings, where sleep disorders are more likely to occur, the effects were similar to those observed in facilities. In addition, it was confirmed that the change in drugs caused no serious side effects and that it was highly safe and tolerated. Therefore, the results of this study suggest that conversion to lemborexant is a promising and safe option for improving insomnia disorders in patients using suvorexant for difficulty initiating sleep. Keywords: Insomnia Disorder, Lemborexant, Sleep Disorders, Dual Orexin Receptor Antagonist Disclosure: Nothing to disclose. P594. A Novel Orally-Available Selective Orexin 2 Receptor Agonist, E2086 Enhances Wake-Promotion and Treats Narcolepsy-Like Symptoms in Narcolepsy Model Mice Ken Hatanaka, Hiroyuki Suzuki, Fumiko Michikawa, Reiko Koba, Yoshihide Osada, Yoshiaki Furuya, Yukio Ishikawa, Margaret Moline Eisai, Co., Ltd., Tsukuba-Shi, Japan Background: Orexin neurons in the hypothalamus are critical regulators of sleep/wakefulness states, and their loss is associated with narcolepsy type 1 (NT1). In patients with NT1, characterized by reduced orexin levels in cerebrospinal fluid (CSF), excessive daytime sleepiness and cataplexy are observed, both of which are diagnostic for NT1. Orexins act through two classes of G-protein coupled receptors, the orexin-1 receptor (OX1R) and the orexin-2 receptor (OX2R). Although both OX1Rs and OX2Rs are related to sleep-wake regulation, OX2Rs contribute more to sleep-wake regulation. Greater awake/non-rapid eye movement (NREM) sleep episode fragmentation and reduced duration of wakefulness in hypnograms have been seen for OX2R-knockout and double-receptor knockout mice compared with wild-type (WT) and OX1R-knockout mice. Therefore, an OX2R-selective agonist is expected to act as a wake-promoting drug. E2086 has demonstrated strong wake-promoting and cataplexy reduction in nonclinical studies. Here we report the in vitro and in vivo profiles of this novel, orally available OX2R-selective agonist. Methods: Four nonclinical studies were conducted. (1) Lenti-X™ 293 cell lines that constitutively and stably express either human, mouse, or rat OX2R or OX1R were used to assess OX2R or OX1R-agonistic activity via calcium influx assay. (2) To confirm selectivity of E2086 on OX2R, in vitro binding assays on 86 off-targets were conducted at 1 and 10 uM. (3 and 4) To evaluate E2086-mediated wake-promoting effects in (3) WT mice and in (4) orexin-neuron deficient mice (orexin/ataxin-3 hemizygous mice), EEG/EMG recordings were conducted with E2086 or vehicle treatment during the active phase. Results: (1) E2086 (0.03 – 10,000 nmol/L) activated human OX2R (EC50: 2.3 nmol/L) in the calcium influx assay without considerable species differences or activity on the human OX1R (EC50: 4700 nmol/L). (2) E2086 (1 and 10 uM) had no significant affinity (>50% inhibition in binding assay) on 86 receptors, transporters, and ion channels associated with important physiologic functions. (3, 4) Single dose oral administration of E2086 (0.3, 1, and 3 mg/kg) promoted wakefulness in both WT mice and orexin-deficient transgenic mice. Additionally, in orexin-deficient mice, latency of direct transitions from wake to REM sleep, which is a murine analog of human cataplexy, was also prolonged. After 14 days of repeated dosing in orexin-deficient mice, E2086 (3 mg/kg) significantly prolonged wakefulness duration and prevented cataplexy-equivalent episodes without tolerance throughout the treatment period. Conclusions: These results suggest that E2086 is a selective and potent OX2R agonist, with wake-promoting and anti-cataplexy effects. Thus, these results support the potential for E2086 to improve wakefulness and other orexin deficiency-related symptoms like cataplexy in patients with orexin network hypofunction such as narcolepsy Type 1 and suggest that E2086 may also improve wakefulness in patients with excessive daytime sleepiness across a range of orexin levels. Keywords: Orexin, Narcolepsy, Wake-Promoting Disclosure: Eisai, Co., Ltd.: Employee (Self). P595. Inhibition of Kynurenic Acid Synthesis Protects Learning and Memory Despite Prolonged Sleep Deprivation in Rats Ana Pocivavsek, Snezana Milosavljevic, Courtney Wright, Maria Piroli, Homayoun Valafar University of South Carolina School of Medicine, Columbia, South Carolina, United States Background: Sleep is highly conserved physiological process critical for learning and memory consolidation. Kynurenic acid (KYNA), a metabolite of the kynurenine pathway (KP) of tryptophan catabolism, has been shown to play a major role in sleep regulation, learning, and memory formation. KYNA is predominately synthesized by kynurenine aminotransferase II (KAT II) from its biological precursor kynurenine. KYNA released from astrocytes acts as an endogenous antagonist of N-methyl-D-aspartate and alpha7-nicotinic acetylcholine receptors, thereby hypothesized to modulate glutamatergic and cholinergic circuits involved in the regulation of sleep and cognition. Of clinical significance, elevated KYNA levels are found in brains of individuals with schizophrenia and bipolar disorder, who frequently experience sleep disturbances and cognitive impairments. We presently sought to determine if pharmacological inhibition of the KYNA synthesizing enzyme, kynurenine aminotransferase II (KAT II), may serve as a potential avenue to overcome sleep disturbances. Methods: To obtain further insight into an interplay between brain KYNA, sleep, and cognitive function, adult male Wistar rats (N = 4 – 12 per group) were sleep deprived (SD) during the latter half of the light phase, Zeitgeber time (ZT) 6 to ZT 12 for 5 consecutive days, using our novel sleep restriction chamber. During this period, we assessed spatial and reversal learning in the Barnes maze during 4 acquisition trials and 1 reversal learning trial conducted at ZT 6. Vehicle or PF-04859989 (30 mg/kg, s.c.), brain-penetrable KAT II inhibitor that reduces brain KYNA, were administered at ZT 6 immediately after each trial, and prior to the onset of SD (ZT6 - ZT12). Upon completion of behavioral testing on day 5, tissues were collected from rats at ZT 8. A separate cohort of animals was implanted with telemetric devices to acquire polysomnographic recordings that combine electroencephalography (EEG) and electromyography (EMG) to evaluate efficacy of our automated sleep restriction chamber across 5 consecutive days and sleep recovery with vehicle or PF-04859989 (30 mg/kg, s.c.) treatment. Results: Automated sleep restriction chamber effectively eliminates rapid eye movement (REM) sleep in rats during the SD period (P < 0.05). In the Barnes maze, vehicle-treated controls had a significantly reduced distance traveled between acquisition days 1 and 4, demonstrating learning in the behavioral paradigm (P < 0.05), while learning did not occur in vehicle-treated SD subjects. Importantly, PF-04859989-treated SD subjects also had reduced distance traveled across days 1 and 4 (P < 0.05). Further, reducing KYNA with PF-04859989 attenuated reversal learning deficits in SD animals (latency: *P < 0.01; distance traveled: P < 0.05; errors: P < 0.01). Prolonged SD significantly increased both plasma tryptophan and KYNA (P < 0.01), while KAT II inhibition prevented these increases, yet elevated plasma kynurenine (*P < 0.01) in SD rats. Conclusions: Taken together, we demonstrated for the first time that KAT II inhibition may attenuate learning deficits during extended sleep loss in rats. The present and future complementary experiments provide mechanistic value to understanding the role of KYNA in modulating sleep behavior and demonstrate that KAT II inhibition may serve as a potential therapeutic avenue for improving neurocognitive dysfunction associated with prolonged sleep loss. Keywords: Sleep Deprivation, Schizophrenia and Cognition, Alpha7 Nicotinic Acetylcholine Receptor, Electroencephalography, REM Sleep Disclosure: Nothing to disclose. P596. Sleep Oscillations in Insomnia and Aging Ruth Benca, Negin Sattari Barabadi, Abhishek Dave, Ivy Chen, Meredith Rumble, Brady Riedner, Bryce Mander Wake Forest, Atrium Health Wake Forest Baptist, Winston Salem, North Carolina, United States Background: Sleep EEG oscillations that subserve sleep functions such as learning, memory, and removal of toxins are affected by aging as well as by sleep disorders. The studies reported here identify relationships between changes in sleep oscillations and insomnia-related sleep state misperception, sleep-dependent learning, and Alzheimer’s disease (AD) biomarkers in older adults at risk for AD. Methods: Study 1: Sleep oscillations related to sleep state misperception were determined in subjects with insomnia and normal controls (n = 21) who underwent sleep recordings with high density (256 channel) EEG (hdEEG) for nights where they were awakened repeatedly during sleep and asked whether they had been awake or asleep. Study 2: hdEEG sleep and sleep dependent learning were assessed in cognitively intact older adults enriched for AD risk (n = 58) and correlated with insomnia, as well as inflammatory and AD biomarkers in CSF fluid. Study 3: PSG was recorded from older adults with insomnia disorder (n = 106) who participated in a randomized phase III clinical trial (E2006-G000-304; NCT02783729) examining the acute (1-2 days) and chronic (29-30 days) impact of zolpidem extended release (ER) and lemborexant relative to placebo on subjective and objective sleep expression. Results: Study 1: Subjects reported being awake despite the presence of EEG sleep prior to the awakening ~10% of the time, mostly during non-rapid eye movement (NREM) sleep stages N2 and N3; this was associated with increased alpha power (in the 10.5-12.5 Hz range) in posterior and mid-central brain regions. Study 2: Similarly, in older adults at risk for AD, insomnia was associated with increased alpha (7.5-11.5 Hz) and theta (4.5-7.5 Hz) power, albeit in more frontal regions. Study 3: Greater decreases in subjective wake after sleep onset (sWASO) were associated with greater increases in fast sigma activity (13-16 Hz) and slow oscillation power (0.5-1 Hz) following chronic zolpidem ER and lemborexant treatment. In addition, in chronic treatment with zolpidem ER, greater decreases in Alpha activity (7.5-11 Hz) were associated with greater decreases in subjective sleep onset latency (sSOL). Conclusions: Insomnia, particularly characterized by sleep state misperception, may be due in part to abnormal, local intrusion of waking EEG activity (alpha and/or theta) during sleep in both young and older adults, including those with increased risk of AD. Subjective insomnia measures are associated with sleep oscillation expression. Keywords: Sleep Oscillations, Insomnia Disorder, Preclinical Alzheimer’s Disease, Sleep-Dependent Learning Disclosures: Eisai: Grant (Self), Idorsia, Merck, Jazz: Consultant (Self), Genomind, Sage: Advisory Board (Self). P597. Effects of Endogenous Orexin and Dynorphin Corelease on Ventral Tegmental Dopamine Neuronal Activity Aida Mohammadkhani, Min Qiao, Stephanie Borgland* Hotchkiss Brain Institute, University of Calgary, Calgary, Canada Background: Dopamine neurons in the ventral tegmental area (VTA) respond to motivationally relevant cues and are key targets of addictive drugs. Orexins (ox; also known as hypocretin) and dynorphin (dyn) are co-expressed lateral hypothalamic (LH) neuropeptides that project to VTA. While LHox promotes drug-seeking behavior, dynorphin inhibits drug-seeking behavior. Furthermore, these peptides have opposing effects on the firing activity of VTA dopamine neurons. Previous work in our lab implicated that exogenously application of ox and dyn, modulate different VTA dopaminergic projections. However, it is unknown if dynorphin inhibition of these circuits in opposition to LHox is driven by the LHox/dyn input, rather than other sources. This study sought to determine the effects of endogenous LHox/dyn release on VTA dopamine neuronal activity. Methods: We expressed a cre-dependent channel rhodopsin2 selectively in LHox/dyn neurons of orexin-cre mice and photostimulated terminals in the VTA while recording VTA neuronal firing using patch clamp electrophysiology. VTA dopamine neurons were labeled with biocytin during recordings and posthoc imaged for tyrosine hydroxylase expression. Projection targets were identified using retrobeads injected into the nucleus accumbens lateral (lNAcSh) or medial shell (mNAcSh) as well as the basolateral amygdala (BLA). In some experiments, morphine tolerance was induced by delivering two daily injections of escalating morphine doses (5-100 mg/kg) over 5 days. Control mice were injected with saline. Results: We showed a diverse response of LHox/dyn photostimulation on dopamine neuronal firing rate. A 30-Hz stimulation, increased firing in 7/19 neurons and decreased firing in approximately 9/19 of lateral VTA dopamine neurons, an effect persisted in the presence of synaptic transmission blockers. SB334687, an ox1 receptor inhibitor or NorBNI, a kappa receptor inhibitor reversed the potentiation or inhibition of firing, respectively. Morphine tolerance reduced the number of neurons that had inhibitory response to optical stimulation of LHox/dyn terminals. Of lNAcSh projecting dopamine neurons, optical stimulation of LHox/dyn neurons increased firing in 6/10 and decreased firing in 4/10 neurons, whereas mNAcSh projecting neurons had 4/11 neurons increase firing and 7/11 neurons decrease firing. BLA projecting dopamine neurons were mostly inhibited (8/11) by optical stimulation of LHox/dyn terminals. Conclusions: Our findings provide evidence that LHox/dyn co-release tune the output of the VTA by simultaneously inhibiting and activating different VTA projection neurons. Morphine tolerance may lead to a shift in the balance of excitatory and inhibitory effects of LH ox/dyn co-release. Keywords: Dopamine, Orexin, Dynorphin, Opioid Tolerance, Slice Electrophysiology Disclosure: Nothing to disclose. P598. Cell-Type-Specific Epigenetic Priming of Gene Expression in Nucleus Accumbens by Cocaine Philipp Mews, Yentl Van der Zee, Hope Kronman, Ashik Gurung, Aarthi Ramakrishnan, Caleb Brown, Rita Futamura, Molly Estill, Abner Reyes, Simone Sidoli, Eric Nestler Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Cocaine use disorder (CUD) is an intractable syndrome and rising overdose deaths represent an enormous public health crisis. The urgent need for better mechanistic insight into this chronic relapsing brain disorder is driven by sharp increases in cocaine use and the lack of effective treatments. Chronic cocaine exposure induces persistent changes in gene regulation in the brain’s motivation and reward circuitry, coupled to neuroplasticity within the brain and vulnerability to relapse. Susceptibility to relapse is believed to involve stable changes in chromatin in the nucleus accumbens (NAc), a brain region that controls motivated behaviors, that alter transcription during long-term drug withdrawal. However, the molecular events that underlie maladaptive gene activity in cocaine addiction and other substance use disorders remain incompletely understood. A fundamental challenge is determining which neuronal subtypes are responsible: the NAc is composed primarily of two opposing types of medium spiny neurons (MSNs), the D1 and D2 dopamine receptor-expressing subtypes, which exhibit dramatic differences in activity and effects on drug reward. In these distinct subtypes, we examined how chronic cocaine modifies chromatin structure and characterized immediate versus persistent changes in gene regulation. Methods: The NAc primarily comprises (>90%) two functionally distinct subtypes of MSNs, making the cell-type-specific identification of epigenetic changes critical. In recent years, the assay for transposase-accessible chromatin using sequencing (ATAC-seq) has become a fundamental tool of epigenomic research. It is employed to assess chromatin structure genome-wide to detect “open” chromatin regions indicative of active gene transcription or priming. Here, we defined chromatin accessibility in D1 and D2 MSNs using fluorescence-activated nuclei sorting (FANS) coupled to ATAC-seq and RNA-seq. Combined with unbiased histone modification profiling by mass spectrometry and ChIP-seq, we distinguished acute versus long-term alterations in chromatin and gene expression. Specifically, we characterized persistent alterations in circuit-specific chromatin composition with prolonged withdrawal (30d) after chronic exposure to cocaine (10d cocaine i.p.). Results: We discovered that chronic cocaine persistently alters chromatin structure in D1 MSNs, involving dramatic depletion of the histone variant H2A.Z – a recently identified memory suppressor – at key neuronal genes related to synaptic plasticity. Genome accessibility is prominently increased at these genes even after prolonged withdrawal, linked to aberrant gene expression upon drug relapse. The histone chaperone ANP32E promotes the removal of H2A.Z, and we demonstrate that D1 circuit-selective ANP32E knockdown prevents cocaine-induced H2A.Z depletion and effectively blocks cocaine-conditioned place preference. In contrast, the D2-specific knockdown of ANP32E enhances cocaine-related reward learning in this animal model. Conclusions: Here, we describe a novel epigenetic mechanism whereby chronic cocaine exposure causes lasting chromatin and downstream transcriptional modifications in the NAc. We link prolonged withdrawal from cocaine to the depletion of the histone variant H2A.Z, coupled with increased genome accessibility and latent priming of gene transcription, in D1 MSNs that relate to aberrant gene expression upon drug relapse. The histone chaperone ANP32E removes H2A.Z from chromatin, and we demonstrate that D1 MSN-selective Anp32e knockdown prevents cocaine-induced H2A.Z depletion and blocks cocaine’s rewarding actions. By contrast, very different effects of cocaine exposure, withdrawal, and relapse were found for D2-MSNs. These findings establish histone variant exchange as an important mechanism and clinical target engaged by drugs of abuse to corrupt brain function and behavior. Keywords: Withdrawal, Drug Relapse, Epigenetics, Gene Priming Disclosure: EpiVario, Inc: Founder (Self). P599. Effects of Long-Term Intermittent Access to Alcohol on Glutamatergic Basal Forebrain Neurons During Aversion-Resistant Drinking Ankit Sood, Runbo Gao, Angel Tran, Margot Lortie, Jocelyn Richard University of Minnesota, Minneapolis, Minnesota, United States Background: Compulsive alcohol use is a major contributor to alcohol use disorder’s intractable and persistent nature. Prior research has shown that neuroadaptations in corticostriatal projections to nucleus accumbens are critical for the development of compulsive-like alcohol use behaviors, including “aversion-resistant” drinking, in which animals continue to consume alcohol despite its adulteration with bitter quinine. Yet it remains unclear how alcohol-induced changes in nucleus accumbens can impact sensitivity to aversive outcomes during consumption and seeking of alcohol. The nucleus accumbens projects to glutamatergic neurons in the ventral pallidum, lateral preoptic area, and lateral hypothalamus, which are implicated in aversive processing and constraint of reward-seeking behaviors. Our goal here is to examine the effects of long-term intermittent access to alcohol and aversion-resistant drinking on cell-type specific markers and activity in the ventral pallidum, lateral preoptic area, and lateral hypothalamus from rats tested for aversion-resistant alcohol consumption. Methods: Adult Long Evans rats (n = 68, n = 33 females, 35 males), were assigned to long-term (LTA, n = 38) and short-term (STA, n = 24) alcohol access groups, or to a no ethanol exposure group (n = 6). For 14 weeks, LTA rats had access to a bottle of 15% ethanol, for 24-hour periods every other day, whereas STA rats had access to an additional water bottle, but not ethanol. Rats then underwent testing for consumption of 15% ethanol with 0, 45, and 90 mg/L quinine. Brains were extracted immediately after a final 30-minute test with access to ethanol, ethanol plus quinine, or no solution. Brains were then processed via in situ hybridization with RNAscope for mRNA markers of neural activity (Fos), as well as markers for glutamatergic (Slc17a6 [[Vglut2]) and GABAergic neurons (Gad1). Results: Our preliminary results indicate that, in rats that have undergone a final test with quinine-adulterated alcohol, long-term intermittent access to alcohol reduces the percentage of vGlut2 positive cells that are positive for Fos mRNA. This suggests that alcohol exposure blunts the activation of glutamate neurons during aversion-resistant drinking. Additionally, we found that long-term access increased the percentage of vGlut2 neurons that are also Gad1 positive, suggesting that alcohol exposure may alter the downstream effects of these neurons in areas like the lateral habenula. Female rats also had a higher proportion of vGlut2 neurons that were Gad1 positive. Conclusions: Our results indicate that intermittent access to alcohol can alter the expression of cell-type specific mRNA markers and neural activity in the glutamatergic basal forebrain. Specifically, long-term access increased the colocalization of markers for glutamate and GABA neurons, and reduced Fos expression in glutamate neurons. Given that glutamatergic basal forebrain neurons have been implicated in aversive processing, increased colocalization could alter the processing of both rewards and punishment during conflict. Reduced activity and increased GABA release by neurons that normally respond to aversive events could be two mechanisms by which alcohol exposure alters sensitivity to negative outcomes. Keywords: Alcohol, Aversion, Glutamate GABA, Compulsive Drug Intake, Ventral Pallidum Disclosure: Nothing to disclose. P600. Endocannabinoids Influence Representations of Interval Timing in the Nucleus Accumbens Natalie Zlebnik, Joseph Cheer University of California, Riverside, Riverside, California, United States Background: Cannabinoids disrupt timing by interfering with dedicated brain timing circuits. The ability to perceive and respond to environmental information in the time domain is critical for adaptive survival, and striatal circuits play a central role in timing behavior. Our previous work demonstrated that phasic dopamine release in the nucleus accumbens (NAc) encodes interval timing and that CB1 receptor activation accelerates the perception of time and shifts temporally-engendered patterns of phasic dopamine release. Methods: Using in vivo fiber photometry and neuronal ensemble recordings, we examined how endocannabinoid signaling orchestrates timing-mediated NAc network dynamics in male mice. Results: We found that interval timing was encoded by bidirectional ramping activity of NAc ensembles and sustained levels of phasic dopamine release. Increasing levels of the endocannabinoid 2-AG via the MAGL inhibitor JZL184 (18 mg/kg, ip) resulted in an acceleration of time estimation and attenuation of interval encoding in a CB1 receptor-dependent manner. Conclusions: These results reveal a significant role for endocannabinoids in ventral striatal network dynamics associated with interval timing and may have important implications for the use of pharmacotherapies targeting the endocannabinoid system and for the recreational use of plant-based and synthetic cannabinoids. Keywords: Endocannabinoids, Timing, Dopamine, In Vivo Electrophysiology Disclosure: Nothing to disclose. P601. The Interaction of Biological Sex, Early-Life Stress, and Mesolimbic Dopamine Function on Vulnerability to Heroin Self-Administration Brianna George, Sara Jones Wake Forest School of Medicine, Winston-Salem, North Carolina, United States Background: The scale and severity of the opioid epidemic in the United States cannot be overstated. With the recent restriction on prescription opioids, there has been a shift to greater abuse of illegal drugs such as heroin. To combat the opioid crisis effectively, we need a deeper understanding of the factors that drive vulnerability to develop opioid use disorder (OUD). Chronic psychosocial stress has been linked to increased risk for a host of negative outcomes, including depression, anxiety, and substance use disorders (SUDs). Exposure to these stressors during adolescence drives an even higher risk for future psychiatric disorders that may engender or worsen SUDs in adulthood. Our group and others have shown that chronic adolescent social isolation (aSI) stress in male rodents leads to persistently increased drug responsiveness and negative affective behaviors when compared to adolescent group-housed (aGH) control. The robust impact of aSI on drug and alcohol self-administration (SA), coupled with strong evidence that aSI-induced alterations in the dopamine system potently regulate vulnerability to drug-seeking, lead us to predict these effects would apply to opioids as well. Here, we will assess the impact of aSI on heroin self-administration. Methods: Male (n = 112) and female (n = 40) Long-Evans rats were housed in groups (4/cage) or isolation (1/cage) from postnatal day (PND) 28-70 or 21-70, respectively. Following the housing paradigm, all rats were assayed for anxiety-like behavior and response to a novel environment, implanted with jugular catheters, and individually housed in operant chambers that serve as both the home cage and SA chamber. Following recovery, rats were given access to a lever and trained to self-administer heroin (FR1, 0.025 mg/kg/infusion). After acquiring heroin SA, rats were tested for dose-responsivity (FR1), motivation for heroin seeking using the progressive ratio (PR), and escalation of intake using a long access paradigm (unlimited infusions, 6 hr/session, FR1). Next, the combination of cue- and stress-induced reinstatement responses using the pharmacological stressor, yohimbine (1.25 mg/kg, IP), was evaluated following the extinction of responding. A group of aSI and aGH were given sham surgeries to serve as heroin-naïve controls. To measure dopamine alterations after chronic heroin exposure (or LgA), we utilized ex vivo fast-scan cyclic voltammetry (FSCV) to measure dopamine release and uptake kinetics and terminal receptor functioning in brain slices containing the nucleus accumbens (NAc). RNAscope in situ hybridization (ISH) was used to quantify the expression of D2 mRNA and D3R mRNA. Lastly, negative affect elicited by heroin withdrawal was assessed by recording ultrasonic vocalizations (USVs) before the last LgA session. Results: In these studies, we found that male and female aSI rats have increased anxiety-like behavior and locomotor response to a novel environment. Our SA results revealed that aSI rats have increased rates of heroin SA acquisition, escalation of heroin responding on LgA, responding during extinction sessions, and cue- and stress-induced reinstatement responding. However, female aSI and aGH rats displayed increased responses during LgA compared to male aSI and aGH rats. In addition, we found that aSI has increased responding to various doses of heroin on a PR schedule of reinforcement. Using FSCV, we found that heroin-naïve aSI rats have increased electrically stimulated dopamine release and uptake rats, indicating increased DA system functioning. Following LgA, stimulated dopamine release was reduced in both aSI and aGH rats, however; dopamine uptake rates were only reduced in heroin-exposed aSI rats, compared to their respective heroin naïve counterparts, suggesting a greater downregulation of the dopamine system in heroin-exposed aSI rats. In addition, we found that heroin aSI rats had increased activity at D3, but not D2, autoreceptors, which may play a role in the profound decrease in NAc dopamine terminal function in aSI rats after heroin. This finding was supported by the RNAscope ISH showing increased expression of D3 mRNA in the NAc of aSI rats. Further, analysis of USVs revealed increased 22 kHz calls in heroin aSI rats compared to heroin naïve aSI and heroin-exposed aGH rats. Consistent with our results demonstrating downregulated dopamine functioning in the NAc, this data suggests that heroin-exposed aSI rats experienced greater negative affect during withdrawal from heroin, which may, in part, drive the increased heroin seeking exhibited during SA. Conclusions: Our results demonstrate that exposure to chronic psychosocial stress during adolescence results in robust behavioral and neurobiological adaptions that lead to increased vulnerability to opioid seeking. In addition, the intersection of adolescent stress, biological sex, and dopamine functioning heroin vulnerability may be linked to greater heroin vulnerability. Keywords: Heroin Self-Administration, Dopamine (D2, D3) Receptors, Dopamine, Early Life Stress (ELS), Nucleus Accumbens Disclosure: Nothing to disclose. P602. Transcriptional Correlates of Drug-Associated Memories in the Nucleus Accumbens Freddyson Martinez-Rivera, Leanne Holt, Solange Tofani, Romain Durand-de Cuttoli, Angelica Minier-Toribio, Szu-ying Yeh, Molly Estill, Rita Futamura, Giselle Rojas, Damian Mason, Hossein Aleyasin, Scott Russo, Li Shen, Eric Nestler Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Background: Substance use disorders exemplify a maladaptive imbalance wherein drug seeking persists despite negative consequences or drug unavailability. This maladaptation correlates with neurobiological alterations that hijack reward seeking and consequently withdrawal, extinction, and relapse processes. Extinction, a form of learning in which drug-seeking responses are attenuated by repeated cue exposure in the absence of the drug, represents a valuable tool to suppress drug-associated memories at the behavioral and molecular levels. While there is increasing evidence linking addiction phases to faulty epigenetic and transcriptional modifications in brain reward regions such as the nucleus accumbens (NAc), there is a pressing need to characterize these molecular events in a phase, subregion, and cell-specific manner. Methods: Methods: Here, we used cocaine self-administration (SA) in rats combined with RNA-sequencing (RNAseq) of NAc subregions (core/shell) to transcriptionally profile the impact of extinction learning on counteracting drug memories in different contexts. Results: Results: As expected, we first observed that rats receiving extinction training in the original SA context (cues/no drug) significantly reduced active lever pressing when compared with rats receiving force abstinence in either their home cages or in the SA context (no cues/no drug). Further analysis revealed that rats undergoing withdrawal in the original drug context increased drug seeking and incubation. As for the animals receiving extinction in a different context (ABA), a heterogonous distribution of rats showing either extinction or renewal was observed. Consistent with these behavioral features, RNAseq data reveal specific transcriptional patterns that correlate with these distinct phenotypes and the influence of extinction training on modulating the transcriptional burden upon withdrawal. Subsequent motif and pathway analyses will identify hub genes that encode these phenotypes to manipulate these critical targets, virally. Complementary to these datasets, with the goal of subsequent cell-specific characterizations, we are using RNAscope, electrophysiology, chemogenetic and fiber photometry approaches on transgenic rats expressing Cre-recombinase selectively in D1 or D2 NAc medium spiny neurons. Conclusions: Conclusion: Together, these approaches will provide unprecedented evidence of how extinction, withdrawal, and renewal reprogram the transcriptome of the NAc to identify novel avenues to prevent drug relapse. Keywords: Addiction Phenotypes, Extinction, Transcriptomics, Cocaine Disclosure: Nothing to disclose. P603. Cocaine Taking and Craving Drives Differential Gene Expression in Midbrain Dopamine Neurons Alexander Margetts, Nikolai Rogalinski, Tate Pollock, Samara Vilca, Luis Tuesta Center for Therapeutic Innovation, University of Miami, Miami, Florida, United States Background: Repeated cocaine use induces long-lasting gene expression changes throughout the brain reward system. Of particular interest, understanding the gene expression changes that occur in midbrain dopamine (mDA) neurons during cocaine taking and craving could provide insights into regulation of the negative affective states associated with drug craving and relapse. To explore this question, we combined a cell subtype-specific nuclear tagging approach with a mouse model of cocaine intravenous self-administration (IVSA), and bioinformatics analyses to identify mDA-specific changes in gene expression during cocaine taking and cocaine craving. Methods: Male Dat-Cre heterozygous mice (8-12 weeks) received bilateral intra-VTA injections of a Cre-inducible virus expressing a nuclear membrane-bound HA tag (KASH-HA) to selectively label mDA nuclei, and were then implanted with a jugular catheter for cocaine IVSA studies. To this end, mice completed a food training protocol under a fixed ratio 5, time-out 20 (FR5TO20) schedule of reinforcement, in which 5 active lever presses (FR5) resulted in delivery of a food pellet and presentation of a 20-second cue light, indicating a time-out (TO20) period where lever pressing was recorded but had no effect. Mice were allowed to establish food training over consecutive daily 1 hr session for 7 days, at which point the reinforcer changed from food pellets to cocaine infusions [0.3 mg/kg/infusion] for 5 consecutive days, and then 1.0 mg/kg/infusion for 10 consecutive days. Following cocaine IVSA, mice underwent 21 consecutive days of forced abstinence (craving) in their home cages, during which mice did not have access to the drug. VTA samples were collected and enucleated following either food training (N = 5), cocaine maintenance (N = 7) or cocaine craving (N = 7). To obtain a pure mDA nuclear population, the suspended nuclei were immunostained for HA and sorted via fluorescence assisted nuclear sorting (FANS). RNA was extracted from both mDA and non-mDA populations, and nuclear RNA-sequencing libraries (pooled N = 2-3 samples per group) were prepared and sequenced. Differential gene expression analysis was then conducted using DESeq2, followed by Gene Ontology (GO) pathway analyses to evaluate the biological context for gene expression changes. Results: RNA-sequencing of mDA nuclei showed an enrichment in dopamine identity genes, Th, Dat, Vmat2, Ddc, as well as depletion of glial genes when compared to non-labeled nuclear samples, ensuring the purity of our sequenced samples (\|Log2FC \| >1.3 and padj<0.1). Three-way comparison between food training (N = 5), cocaine maintenance (N = 7), and cocaine craving (N = 7) showed that cocaine craving mice had the highest variation in differentially expressed genes, especially in direct comparison with food-trained controls (p < 0.05). Further, gene ontology pathway analyses revealed a pattern in which extracellular matrix (ECM) genes were upregulated during cocaine maintenance and then downregulated during cocaine craving (p < 0.05, q < 0.1), with ECM genes such as Bgn, Col1a1, Fn1, and Hspg2 showing near complete depletion (normalized gene count<1) during cocaine craving. Conclusions: While preliminary and only performed in males, upregulation of ECM genes during cocaine taking and subsequent downregulation during cocaine craving supports the role of the ECM in synaptic remodeling and plasticity. More broadly, these IVSA stage-specific and cell-specific gene expression changes suggest that mDA neurons differentially regulate their transcriptional profiles to adapt to environmental changes (i.e. presence or absence of a drug). As the nuclear capture method, we developed and employed retrieves both nuclear mRNA and chromatin, follow-up studies (chromatin profiling) will determine the transcriptional regulators underlying these changes. Keywords: Cocaine Self-Administration, Cocaine Craving, Dopamine Neuron, Cell-Specific, Transcriptome Disclosure: Nothing to disclose. P604. Distinct Ensembles Support Early Vs Late Stages of Cocaine Memory Encoding Marine Salery, Arthur Godino, Yu Qing Xu, Leanne M. Holt, John F. Fullard, Panos Roussos, Eric J. Nestler Icahn School of Medicine At Mount Sinai, NEW YORK, New York, United States Background: Learned associations between the rewarding effects of drugs and the context in which they are experienced are decisive for precipitated drug-seeking and relapse in addiction. These associative memories are stored in sparse and highly discriminative populations of concomitantly activated neurons defining drug-recruited ensembles. In this study, we explore the dynamics and molecular mechanisms of both the recruitment of these ensembles upon initial drug exposure and their contribution to the encoding, strengthening and ultimately expression of drug-associated memories. Additionally, we explore the intrinsic and acquired cellular properties favoring the allocation of specific cells to these ensembles and/or predicting their further reactivation. Methods: Capitalizing on the activity-dependent labeling in Arc-CreERT2 mice (Denny et al., 2014), we captured and permanently tagged (fluorophores, channel-rhodopsin) cocaine-activated ensembles in the nucleus accumbens for further characterization, optogenetics, and nuclei sorting. Cocaine-context associative memories have been assessed in a cocaine-conditioned place preference paradigm. Ensemble reactivation has been measured as the overlap between ensemble-specific fluorophore tag and the recent Arc activation. Tagged nuclei were isolated with Fluorescence Activated Cell Sorting to analyze their molecular signature. Results: We identified distinct subsets of neurons activated at both early and late stages of drug exposure and show that the reactivation of an initial ensemble correlates with behavioral sensitization. Similarly, re-exposure to a cocaine-paired context in a conditioned place preference (CPP) paradigm triggered cocaine ensembles’ reactivation. Using optogenetics-mediated artificial reactivation, we found that populations recruited at early versus late stages of drug exposure had opposite roles in CPP expression. In addition, these two distinct ensembles exhibited significantly different levels of endogenous reactivation following re-exposure to either a cocaine challenge or a cocaine-paired context. Single nucleus RNA Sequencing was then performed on FACS-isolated tagged neurons, and we successfully isolated a cluster of reactivated cells within the initially activated ensemble. Conclusions: By tracking ensemble reactivation at different stages of cocaine exposure, this work provides new insights into the fate of neurons recruited at early phases of cocaine-related memory formation. Together, this ensemble-specific approach represents a pivotal step in identifying highly specific cellular processes involved in the encoding of pathological memories associated with addiction. Keywords: Neuronal Ensembles, Arc, Reward Learning, Nucleus Accumbens, Addiction Disclosure: Nothing to disclose. P605. Loss of Microglial MyD88 Induces Aversion Resistant Drinking in Adolescent Male Mice Jerome Moulden II, Neil Rogers, Julia Dziabis, Dang Nguyen, Staci Bilbo Duke University, Durham, North Carolina, United States Background: Binge drinking and heavy alcohol use are a massive public health burden and have increased in the United States in the last few years. There is limited drug development and treatment therapy to effectively reduce alcohol use disorder (AUD) and its societal impact. Further elucidation of biological mechanisms involved in AUD could enhance prevention, treatment, and disease progression of AUD. Recently, neuroimmune responses have been described as potential therapeutic targets for AUD. Studies suggest that alcohol signaling through innate immune toll-like receptors (TLRs) on microglia, the brain’s primary immune cells, to stimulate the release of immune signaling molecules may be critical. Microglia also use neuroimmune molecules to interact closely with other cells in the brain, such as neurons and their synapses, providing a mechanism through which alcohol impacts neuroplasticity and behavior. MyD88 is an essential co-adaptor protein for nearly every TLR, and therefore is important for broad inflammatory signaling. Methods: Our lab previously developed and characterized a mouse line that allows for a microglia-specific MyD88 deletion (Cx3cr1-CreBT-MyD88f/f), thereby blunting inflammatory TLR signaling. MyD88 was removed from microglia using Bacterial Artificial Chromosome (BAC) Transgenic mice with Cre recombinase under the Cx3cr1 promotor (Cx3cr1-CreBT) crossed with MyD88f/f mice. To study the role of TLR signaling in drinking during brain development, we employed a drinking in the dark (DID) binge-drinking paradigm in adolescent male and female mice + /- microglial-specific MyD88 deletion. Mice were weaned from mothers between P26-P28 and group housed for 1-3 days before transfer to single housing. Water bottles were replaced with 30% ethanol for 2 hours for 3 days and 4 hours on the fourth day for two weeks. Tasting aversion quinine was added to the bottles of some groups at week four. Following adolescent DID, adult mice were sacrificed, and cerebellum and brain regions associated with addiction were punched and protein processed for analysis. (Two-way ANOVA used for data analysis) Results: MyD88 signaling alters alcohol consumption in adolescent mice in a sex dependent manner. At weeks 1 and 2 on binge drinking day, Cre+ males consumed more alcohol than females (Week 1, F(1,17)=6.340 P = 0.200; Week 2, F(1,21)=9.173 P = 0.0064. Surprisingly, loss of microglial Myd88 induced aversion resistant drinking in adolescent males (F(1,21)=0.3574 P = 0.3574). Preliminary data shows that alcohol consumption and MyD88 signaling alters expression of proteins in the cerebellum and various brain regions involved in addiction, decision making and memory. Following DID, microglia maker P2RY12 protein expression is decreased in the cerebellum of both Cre- and Cre+ males compared to females. Loss of microglial Myd88 increases protein expression of cerebellar parvalbumin in both male and female mice. Alcohol consumption reduces Iba-1 expression in the cerebellum. However, Iba-1 is elevated in the hippocampus of DID Cre+ males and females. Lastly, we observed lower protein expression of TMEM119 in the prefrontal cortex, nucleus accumbens and hippocampus of Cre- females compared to males following adolescent DID. (Alcohol consumption Cre- female n = 5, Cre+ female n = 5, Cre- male n = 6, Cre+ male n = 9); (Western Blot n = 1 per group). Conclusions: These data show an immune inflammatory TLR signaling component involved in excessive alcohol consumption. Ongoing studies will further examine the role of alcohol induced microglia activation, PVIs and PNNs in AUD. Keywords: Adolescent Binge Drinking, Microglia, Sex-Specific, Myd88 Disclosure: Nothing to disclose. P606. Distinctions in the Prefrontal Cortex Protein Profile of Rats Exhibiting Incubated Cocaine- Versus Sucrose-Seeking: Implications for Pharmacotherapy Fernando Cano, Laura Huerta Sanchez, Taylor Li, Hoa Doan, Gabriella Shab, Mathangi Sankaran, Tod Kippin, Karen Szumlinski* University of California, Santa Barbara, Santa Barbara, California, United States Background: Craving is a cardinal feature of both drug and food addiction that is elicited by re-exposure to reward-associated cues/contexts. Insidiously, the intensity of both cue-elicited drug and food craving incubates during protracted withdrawal and their parallel behavioral trajectories have led to the hypothesis that common time-dependent neuroadaptations may drive both phenomena. Over the past several years, our laboratory has sought to determine the functional relevance of protein correlates within the ventromedial prefrontal cortex (PFC) of incubated cocaine-craving induced by a history of long-access (6 h/day) cocaine self-administration procedures in rats and identified increased PI3K/Akt/mTOR signaling within the prelimbic (PL) subregion as necessary for the expression of incubated cocaine craving, while reduced mGlu1 and mGlu5 subtypes of metabotropic glutamate receptors impair the consolidation of extinction learning and an imbalance in Homer1 vs. Homer2 expression facilitates the reinstatement of cocaine-seeking following extinction. Whether or not the incubation of cocaine-craving induced by shorter-access cocaine-self-administration procedures or the incubation of food-craving is associated with comparable, behaviorally relevant, changes in protein expression is not known. Such knowledge would identify common and unique molecular adaptations important for the appropriate pharmacological targeting in cocaine use and over-eating disorders. Methods: At the outset of our studies, we hypothesized that if the incubation of cocaine- and food-craving involved similar mechanisms, then our results from a model of incubated sucrose-craving should parallel those observed in cocaine-incubated rats, regardless of their cocaine self-administration history. In our study of incubated cocaine-seeking, rats were trained to self-administer intravenous cocaine (0.25 mg/0.1 ml/infusion) under operant-conditioning procedures during which each cocaine infusion was paired with a 20-sec tone-light stimulus. Two different short-access self-administration procedures were probed; one cocaine group was trained for 2 h/day for 10 days (2-Hour), while the other cocaine group was trained for 6 h on Day 1, and 2 h/day for the remaining 9 days (Mixed). In the first study of incubated sucrose-craving, male and female rats were trained to self-administered 45 mg banana-flavored sucrose pellets for 6 h/day for 10 days, with each pellet delivery paired with the tone-light stimulus. On either withdrawal day (WD) 1 (sucrose) or 3 (cocaine) and on WD30, rats were tested for cue-elicited craving in the absence of the sucrose/cocaine reinforcer. In a second sucrose incubation study, rats were orally infused with either 1.0 mg/kg of the mTOR inhibitor Everolimus or vehicle prior to the test for craving to determine if this pretreatment, which effectively blocks incubated cocaine-seeking, would also reduce incubated sucrose-seeking. Following cue testing, PFC subregions were dissected out for immunoblotting in all studies. Results: Relative to cocaine-trained rats on WD3, the Mixed cocaine self-administration procedures elicited more robust incubation of cocaine-craving on WD30 than the 2-Hour procedure [Group X Withdrawal: F(2,63)=4.053, p = 0.023] and the incubated craving of the Mixed group was associated with a time-dependent increase in PL Homer2a/b [interaction: F(2,63)=4.19, p = 0.02] and phospho-CaMKII expression in the PL [interaction: F(2,59)=6.243, p = 0.004], although both cocaine groups exhibited elevated phospho-Akt within this subregion [interaction: F(2,64)=8.12, p = 0.001]. No incubation-related changes in either mGlu receptor or any of the AMPA or NMDA receptor subunits examined were detected in either subregion (p’s > 0.10). Relative to sucrose-trained rats on WD1, we observed a robust incubation of sucrose-craving in both male and female rats on WD30 [Withdrawal effect: F(1,59)=14.72, p < 0.0001]. However, opposite cocaine incubation, sucrose incubation reflected lower phospho-Akt expression within the PL, but this effect was selective for females [Sex X Withdrawal: F(1,51)=5.456, p = 0.024]. No other protein changes were detected in the PL of sucrose-incubated rats. In the IL, only male sucrose-incubated rats exhibited increased mGlu5, phospho-Akt and phospho-mTOR expression [for all proteins, F(1,55)>6.22, p’s<0.01]. However, pretreatment with 1.0 mg/kg Everolimus did not block the sucrose-incubated state in either male or female rats [Group effect: F(2,42)=9.118, p = 0.001, SNK post-hoc tests]. Conclusions: Our cocaine data to date indicate that while the PFC protein profile of cocaine-incubated rats varies as a function of their cocaine history, elevated indices of Akt/PI3K/mTOR signaling within the PL is a common molecular adaptation that may be key to driving the cocaine-incubated state. Our sucrose data indicate a clear sex difference in the protein profile of sucrose-incubated rats, particularly with respect to Akt/mTOR activation. However, we failed to detect any effect of oral pretreatment with Everolimus on incubated sucrose-seeking in rats of either sex, indicating that unlike incubated cocaine-craving, incubated sucrose-craving does not require mTOR activity. Taken together, our body of work argues that the incubation of cocaine- and sucrose-seeking reflect distinct molecular changes within PFC subregions of relevance to not only the appropriate targeting of pharmacotherapies to mitigate cue-induced craving for sweet food versus drug, but also their potential to induce off-target effects. Keywords: Incubation of Cocaine Craving, Incubation of Sucrose Craving, mTOR, Medial Prefrontal Cortex, mGluR5 Receptors Disclosure: Nothing to disclose. P607. Exploring a Role for GalR1-MOR Heteromers in Modulating the Cellular Effects of Opioids in the VTA Stephanie Foster, Jung Hoon Shin, Ewa Galaj, Alyssa Petko, Falyn Thomas, Carlos Paladini, Sergi Ferre, Veronica Alvarez, David Weinshenker Emory University School of Medicine, Atlanta, Georgia, United States Background: Opioids exert their rewarding effects by binding to mu opioid receptors (MORs) on GABAergic neurons and inhibiting GABA release onto ventral tegmental area (VTA) dopamine (DA) neurons. The endogenous neuropeptide galanin is thought to oppose the dopaminergic effects of opioids by signaling through galanin receptor 1 (GalR1) – MOR heteromers. While these heteromers represent an intriguing target for therapeutic intervention, their distribution in the brain and cellular effects on DA neurons remain unknown. Because visualizing G-protein complexes in vivo is technically challenging, we characterized GalR1 and MOR RNA co-expression in the mouse brain as an indicator of which brain regions and neuronal subtypes might be capable of assembling the heteromer. We examined GalR1 and MOR RNA expression and co-expression levels in regions that exert GABAergic control over VTA DA neurons: rostromedial tegmental nucleus (RMTg), nucleus accumbens (NAc), and the VTA itself. We also used slice electrophysiology to determine whether galanin alters morphine-induced disinhibition of VTA DA neurons through a GABAergic mechanism. Methods: RNAscope fluorescent in situ hybridization was performed on brain tissue from C57BL/6 J mice (males and females) using probes for GalR1, MOR, and GAD1 as a cell type-specific marker for GABAergic neurons. Confocal images were acquired, and a nuclear-based analysis of GalR1 and MOR expression among GAD1 + and GAD1- neurons was performed using Cell Profiler software. Whole-cell patch clamp studies were conducted using mouse VTA sections. GABAergic inputs to the VTA were electrically stimulated, and the resulting inhibitory postsynaptic current (IPSC) was recorded from VTA DA cells. IPSCs were recorded at baseline and following bath application of galanin, morphine, or both. Results: We found that the pattern of GalR1 RNA expression was similar across the three brain regions; ~25% of GABAergic cells co-expressed GalR1 and MOR mRNA, compared with only ~5% of non-GABAergic cells. Electrophysiology studies revealed that, as expected, morphine attenuated GABAergic IPSCs onto VTA DA neurons. Subsequent application of galanin had no effect, but pre-incubation of the slices with galanin prevented the effects of morphine. Conclusions: Our findings indicate that a significant fraction of GABAergic neurons that provide input to VTA DA neurons co-express GalR1 and MOR in mice, and are thus capable of containing GalR1-MOR heteromers. By contrast, most non-GABAergic cells within these brain regions do not co-express these receptors. Our electrophysiological results indicate that galanin opposes the GABA-mediated effects of opioids on VTA DA neurons under certain conditions. Future studies will examine how GALR1-MOR heteromer disruption impacts galanin’s ability to exert these effects. Keywords: Opioids, Galanin, Ventral Tegmental Area (VTA) Disclosure: Nothing to disclose. P608. Adenosine 2a Receptor Agonism Decreases Nicotine Seeking and Associated Accumbens Microglial Activation Emma Bondy, Shailesh Khatri, Erin Maher, Percell Kendrick, Terry Hinds, Jr., Wang-Hsin Lee, Cassandra Gipson University of Kentucky, Lexington, Kentucky, United States Background: Neuroimmune and glutamatergic mechanisms within the nucleus accumbens core (NAcore) are involved in nicotine-motivated behaviors. We found profound NAcore microglia activation and increased expression of the glutamate NMDA receptor subtype, GluN2B, by chronic nicotine self-administration (SA). Microglia are brain immune cells, and play a critical role in regulating neuronal activity and synaptic plasticity. Agonism of adenosine 2a receptors (A2aRs), located on microglia to regulate their proliferation and survival, is neuroprotective in disease models. Prior studies show a beneficial effect of modulating A2aRs on nicotine withdrawal. Thus, NAcore adenosine signaling may play a critical role in driving nicotine seeking behavior and associated microglial activation. Methods: Here we examined the ability of A2aR agonism to decrease cue reinstated nicotine seeking and associated NAcore microglial activation (N = 11-13/group). Rats underwent nicotine (0.06 mg/kg/infusion) or saline SA, extinction, and reinstatement, receiving either chronic systemic treatment with an A2a agonist (CGS21680; 0.4 mg/kg, IP), antagonist (SCH58261, 0.4 mg/kg, IP), or vehicle (saline, IP). Microglial morphology (via 3DMorph) or mRNA (via RT-qPCR) was then quantified from the NAcore. Linear mixed effects modeling (LME) was used for analysis. Results: Chronic systemic treatment with the A2a agonist significantly decreased cue-induced nicotine seeking as compared to both vehicle and antagonist treatment (p < 0.05). Antagonist treatment also significantly increased reinstatement-induced microglial activation compared to saline SA, as shown by 5 measures of cell morphology (p’s < 0.05 for each measure) and no differences were found between agonist-treated nicotine and saline SA rats. Agonist treatment decreased expression of both the A2aR coding gene, Adora2a, and the GluN2B coding gene, Grin2b, compared to vehicle and antagonist. Conclusions: These results suggest that rebalancing of A2a and GluN2B receptor expression may be involved in decreasing nicotine seeking. Together, our results indicate that A2aR agonism may be protective against NAcore microglial activation induced by nicotine seeking, and may thus be a viable therapeutic treatment strategy for smoking cessation. Keywords: Activated Microglia, Adenosine A2A Receptor, Nicotine Addiction, Cue Reinstatement, Nucleus Accumbens Core Disclosure: Nothing to disclose. P609. Molecularly-Defined Cell Types Within the Septal Complex and Their Putative Role in Opioid Dependence and Withdrawal Rhiana Simon, Madelyn Hjort, Pranav Senthilkumar, Madison Martin, Gabrielle Cooper, Garret Stuber University of Washington School of Medicine, Seattle, Washington, United States Background: The lateral septum (LS) is a limbic brain area that has been recognized as both a site of reinforcement and as a regulator of fear expression. Its connectivity with key brain structures in reward learning and in anxiety underlies the LS’ involvement in diverse behaviors. While there is evidence for the LS’ role in addiction-related behaviors, it is unclear how chronic drug use impacts genetically-defined cell types within the LS, and how disrupting these neurons may contribute to drug withdrawal, a highly aversive experience that facilitates drug seeking. Methods: We used single-cell RNA-sequencing (scRNAseq) to identify LS cell types that are involved in opioid dependence and withdrawal. To this end, we employed a morphine binge paradigm, in which both male and female C57/bl6 mice received a 7-day escalating dose of morphine (MOR, 10-70 mg/kg, 6 mice). Control mice received saline (SAL, 6 mice). Upon completion of the MOR schedule, some MOR mice received naloxone to precipitate opioid withdrawal (NAL, 1 mg/kg, 6 mice). Two additional groups of mice receiving only one dose of morphine (one-MOR, 6 mice) and solely a dose of naloxone (NAL-only, 6 mice). Septal tissue was then harvested and underwent single cell capture and sequencing via the 10X Genomics platform and Illumina HiSeq 4400. Analysis was carried out using Seurat v3.0. Next, we used hybridization chain reaction (HCR), a highly multiplexed in situ method, to assess the topography of each molecularly-defined cell type within mouse septal tissue (8 mice) and the induction of immediate early genes (IEGs) following NAL (3 groups, 4 mice each). We then assessed how Nts+ neuronal activity in vivo changes over MOR and NAL treatment using 2-photon deep brain imaging. To selectively image from Nts+ neurons in the LS, we injected male and female NTS-cre mice with a cre-dependent AAV delivering GCamp6s, a genetically-encoded calcium indicator. We also implanted a GRIN lens in the LS. 4 weeks following lens implantation, mice underwent the binge and withdrawal paradigm described above, where 2 mice received SAL and 2 mice received MOR. Both groups received NAL. During MOR and NAL administration, both spontaneous and evoked activity were recorded. Mice received random presentations of 10% sucrose (positive valence) and 20 psi air puffs (negative valence). Results: Our scRNAseq experiment yielded ~50,000 total cells across our 5 conditions, and over half of these cells were putative neurons (~26,000), determined by the expression of Stmn2 and Thy1. We identified 14 bioinformatically-defined cell clusters, 12 of which were GABAergic. 2 clusters were GLUergic. Among these neurons were both canonical LS cell types (e.g. Nts + , Sst + , Crhr2+), as well as novel, undescribed populations within the septum (e.g. Met + , Pax6+). Overall, chronic MOR admin profoundly alters gene expression across all LS neurons, enhancing genes involved in GLUergic signaling and depleting protein synthesis pathways. We trained a classifier to determine which cell cluster was the most transcriptionally perturbed in each condition. In doing this, we identified Nts+ neurons to be highly disrupted by MOR and NAL, whereas Met+ neurons to be the most altered by NAL and not chronic MOR. We selected genetic markers representative of 12 cell clusters and ran HCR. In situ mapping of selected cell types indicated that although there is genetic heterogeneity within the septum, septal cells co-express genes in a gradient-like fashion. For example, Nts is co-expressed with up to 65% of Col15a1 + , 49% of Met + , 41% of Onecut2 + , 38% of Sst+ cells. Despite extensive overlap, LS Nts+ neurons form a distinct layer across the A-P and D-V axes. We repeated HCR with mice treated with SAL, MOR and NAL to measure the expression of IEGs altered by MOR and NAL. Here, we discovered that although NAL induces Fos in Nts+ neurons (K-S test, D = 0.356, p < 0.0001, median diff of 16 copies), Fos is more highly induced in cells co-expressing Nts and Met (K-S test, D = 0.564, p < 0.0001, median diff of 25 copies). For deep-brain imaging experiments, we targeted the intermediate portion of the LS, where this co-expression occurs. 2-photon imaging of LS Nts+ neurons during MOR admin and NAL precipitated withdrawal revealed that NAL increases the number of Nts+ activity peaks during the first minute of NAL exposure (Two-way ANOVA, F(1,122) = 3.910, p < 0.05). Conclusions: Our scRNAseq results revealed that although chronic MOR transcriptionally alters all LS cell types, Nts+ neurons are among the most genetically perturbed by both chronic MOR and NAL, indicating that these neurons may play some role in addiction-related behaviors. In situ mapping of cell types identified in scRNAseq showed that there are gradient-like overlaps among each population. Further analysis suggested that cells co-expressing Nts and Met, a novel genetic marker, are the most predictive of LS activation during withdrawal. Finally, we showed using 2-photon imaging that Nts neurons are in fact activated by NAL in vivo, and we currently have ongoing work attempting to determine whether and how MOR and NAL disrupt evoked Nts+ activity patterns. Together, these results highlight a novel role for LS Nts+ neurons, an underappreciated cell type in limbic brain circuitry. Keywords: Opioid Abuse, Single-cell RNA Sequencing, Lateral Septum, 2-photon Techniques, Opioid Withdrawal Disclosure: Nothing to disclose. P610. Carbonic Anhydrase 4 Disruption Prevents Synaptic and Behavioral Adaptations Induced by Cocaine Withdrawal Subhash Gupta, Ali Ghobbeh, Rebecca Hebl-Taugher, Rong Fan, Jason Hardie, Ryan LaLumiere, John Wemmie University of Iowa, Iowa City, Iowa, United States Background: Cocaine use followed by withdrawal induces synaptic changes in nucleus accumbens (NAc), which are thought to underlie subsequent drug-seeking behaviors and relapse. Previous studies suggest cocaine-induced synaptic changes depend on acid-sensing ion channels (ASICs). Methods: Here we investigated potential involvement of carbonic anhydrase 4 (CA4), an extracellular pH-buffering enzyme. We examined effects of CA4 in mice on ASIC-mediated synaptic transmission in medium spiny neurons (MSNs) in NAc, as well as on cocaine-induced synaptic changes and behavior. Results: We found CA4 is expressed in the NAc and present in synaptosomes. Disrupting CA4 either globally, or locally, increased ASIC-mediated synaptic currents in NAc MSNs and protected against cocaine withdrawal-induced changes in synapses as well as cocaine-seeking behavior. Conclusions: These findings raise the possibility that CA4 might be a novel therapeutic target for addiction and relapse. Keywords: Alcohol and Substance Use Disorders, Synaptic Plasticity, Drug Seeking Disclosure: Nothing to disclose. P611. Synapse-Selective Association of Nucleus Accumbens Astrocytes Affects Synaptic Plasticity at Baseline and After Heroin Use Anna Kruyer, Brittany Kuhn, Daniela Neuhofer, Peter Kalivas Medical University of South Carolina, Charleston, South Carolina, United States Background: Astrocytes regulate excitatory activity at striatal synapses via gliotransmission, glutamate uptake, and spatial buffering of glutamate spillover at nearby spines and extrasynaptic receptors. Astrocyte insulation of synapses in the nucleus accumbens core (NAcore) is dynamic during extinction of addictive drug use and during drug seeking, and a high degree of synaptic insulation by NAcore astroglia serves to suppress cued drug seeking. Methods: To determine how astrocyte morphology impacts excitatory synaptic activity in the striatum, we used confocal microscopy to quantify astrocyte adjacency to the two main synapse types in the NAcore, D1 and D2 receptor-expressing synapses on medium spiny neurons (D1- and D2-synapses). We next used whole cell patch clamp electrophysiology to determine the impact of synaptic insulation by astroglia on D1- and D2-synapses in the NAcore. We compared tissue treated with an ezrin-targeted morpholino oligo, which induces astroglia retraction from synapses, with control oligo-treated tissue, to determine how synapses responded to electrical stimulation in the presence or absence of astroglial insulation. Results: We found that striatal astrocytes exhibit a bias in their synaptic association, with increased adjacency to D2-, vs. D1-synapses at baseline. After extinction of heroin seeking, this pattern is reversed, and astrocytes increase their insulation of D1-synapses, while simultaneously retracting from D2-synapses. By comparing traces from control vs. ezrin oligo-treated tissue, we found that astrocyte retraction from both D1- and D2-synapses reduced synaptic depression elicited by high frequency stimulation. Notably, high frequency stimulation-induced synaptic depression was more likely at D2- vs. D1-synapses. We also found that synaptic release probability was increased onto D1-, but not D2-synapses, in the absence of astrocytes insulation. Conclusions: Based on these data, we predict that astrocyte-synapse adjacency serves to dampen synaptic activity in the striatum, perhaps via different mechanisms at D1- and D2-synapses. Given the differences in astrocyte-synapse association at baseline and after drug withdrawal, we predict that astrocyte-synapse adjacency predicts the likelihood of synaptic plasticity at different striatal subcircuits relevant to drug seeking behavior. Keywords: Excitatory Synapses, Astrocyte-Neuron Interaction, Heroin Self-Administration, Short-Term Synaptic Depression Disclosure: Nothing to disclose. P612. Sex-Specific Effects of Oxycodone Withdrawal and Abstinence on Brain Region-Specific Transcriptomes in Heterogeneous Stock Rats Xiangning Xue, Lisa Maturin, Benjamin Williams, Sophia Miracle, Stephanie Puig, Julie Michaud, Joseph Seggio, Abraham A. Palmer, Olivier George, George Tseng, Ryan Logan Boston University School of Medicine, Boston, Massachusetts, United States Background: The prevalence of opioid use disorder and opioid dependence have increased dramatically over the recent decade. Despite current treatments, more than 90% of people being treated for opioid use disorder or dependence, eventually relapse. To improve treatment and to discover new, more effective therapeutics, a greater understanding of the molecular mechanisms contributing to dependence, and withdrawal and craving during abstinence is necessary. The individual differences in the vulnerability to opioid dependence and severity of withdrawal is due to a myriad of factors, including genetic diversity. Further understanding of the molecular mechanisms that contribute to this vulnerability within the context of individual differences will provide new insights into potential genetic factors that underlie the molecular and behavioral plasticity accompanying chronic opioid use. Powerful tools for investigating the genetics underlying opioid use include the population of Heterogeneous Stock (HS) rats. HS rats are derived from an eight-way cross of inbred and outbred rat strains to generate the most genetically diverse rat population. The recombination of these genomes results an expansion of phenotypic heterogeneity driven by enhanced genetic diversity. Leveraging the Oxycodone Biobank of HS rats at University of California San Diego, we investigated the transcriptional alterations associated with opioid use, withdrawal, and abstinence in male and female HS rats in several brain regions related to opioid use disorder and dependence. Methods: HS male and female rats underwent oxycodone self-administration followed by acute withdrawal and abstinence. Brain tissue was collected following oxycodone intoxication, acute withdrawal, and abstinence, from separate cohorts of HS male and female rats. An additional cohort of untreated, naïve HS rats were also used for comparison. Subregions of the nucleus accumbens (NAc), including the “core” and “shell” areas, along with the prefrontal cortex (PFC) and suprachiasmatic nucleus (SCN) were micropunched from frozen brain tissue sections then processed for RNA extraction. RNA-sequencing was conducted on NAc shell and core, PFC, and SCN from male and female HS rats (n = 6 group/sex/region, total n, 172 samples). Comparisons between treatment groups and sex were conducted using DESeq2 (Bioconductor) with the full model including interaction terms (treatment x sex). Differentially expressed (DE) transcripts were at log fold-change of plus/minus 0.26 and p < 0.01 thresholds. For DE transcripts, various enrichment analyses included pathways and gene set enrichment. Rank-rank hypergeometric ordering (RRHO) analyses compared changes in transcriptional patterns between oxycodone intoxication, withdrawal, and abstinence within and between sexes and brain regions. Results: Within each brain region, analyses investigated the interaction between sex and opioid treatment condition (i.e., intoxication, withdrawal, and abstinence) to identify DE transcripts that were altered in male and female HS rats. In DLPFC, there were 267 DE transcripts altered by sex and opioid treatment condition, along with 156 and 182 DE transcripts significantly altered in NAc shell and core, respectively. In SCN, 143 DE transcripts were identified. In the DLPFC and SCN, the majority of these transcripts were significantly changed by sex during abstinence (109 transcripts, DLPFC; 99 transcripts, SCN). Enrichment analyses showed pathways related to circadian rhythms and protein among others during oxycodone abstinence in DLPFC specifically in male HS rats. In NAc core, pathways were enriched for circadian rhythms and protein processing, including acetylation during both intoxication and abstinence, specifically in female HS rats. During withdrawal, pathways were mostly associated with growth factor signaling, mRNA splicing, and glial cell activation. Pathways enriched in the SCN were associated with corticosteroid signaling and extracellular matrix organization, also with sex-specific effects specific to oxycodone withdrawal and abstinence. Conclusions: Our findings begin to identify the sex-specific transcriptional alterations in brain circuitry relevant to opioid use disorder and opioid dependence. Together, our data also suggests associations between circadian rhythm pathways, extracellular matrix signaling, and others in oxycodone withdrawal and abstinence consistent with recent findings in human brain. Keywords: Oxycodone, Transcriptome Biology, Heterogeneous Stock Rats, Behavioral Genetics Disclosure: Nothing to disclose. P613. Simultaneous and Sequential Cocaine + Alcohol PSU Alters Neurocircuitry of Cocaine Seeking Javier Mesa, Lori Knackstedt University of Florida, Gainesville, Florida, United States Background: While many medications reduce the reinstatement of cocaine-seeking in animals, these agents show little clinical efficacy at preventing relapse in humans. This is possibly due to the fact that an estimated 60-90% of cocaine users also use alcohol, but animal models of polysubstance use (PSU) are seldom used. Here we developed a rat model of simultaneous cocaine+alcohol self-administration for the investigation of the neurobiology of drug seeking in a PSU condition. Methods: Male and female rats underwent IVSA of cocaine alone (n = 21) or a cocaine+alcohol solution (n = 21) for 2 hr/day for 5 consecutive days, then 6 hr/day, 2 days/week, for 5 weeks. Two doses of cocaine were tested (0.25 and 0.5 mg/kg/infusion) with and without alcohol (12.5 or 25 mg/kg/infusion). At the conclusion of IVSA, rats were tested on a progressive ratio schedule for 2-3 days, followed by tests for cued cocaine-seeking (relapse) at 1 and 30 days of abstinence. Results: For the low dose of cocaine, the addition of alcohol to the intravenous solution had no effect on cocaine intake during self-administration, breakpoint for self-administration, or cued relapse after 30 days of abstinence. For the high dose of cocaine, the addition of alcohol to the intravenous solution resulted in increased cocaine intake during self-administration (p = 0.0201), increased breakpoint for self-administration (p = 0.0026), and increased cued relapse after 1 day of abstinence (p = 0.0359). Conclusions: Simultaneous cocaine+alcohol self-administration increases cocaine intake and the motivation to seek drug and drug-associated cues in a dose-dependent manner. Ongoing work is examining the effects of alcohol on the neurocircuitry of cued cocaine-seeking. Sequential cocaine and alcohol uniquely alter patterns of neuronal excitation in the infralimbic cortex and ventral tegmental area relative to monosubstance cocaine use. Keywords: Polysubstance Abuse, Cocaine Seeking, Alcohol Use Disorder and Drug Addiction Disclosure: Nothing to disclose. P614. Determining Parabrachial Nucleus Physiology and Function During Naloxone-Precipitated Opioid Withdrawal Barbara Juarez, Derek Ban, Abi Elerding, Mary Loveless, Larry Zweifel University of Washington, Seattle, Washington, United States Background: The drive to relieve aversive somatic and affective symptoms that accompany opioid withdrawal is a contributor to opioid relapse and the progression of opioid use disorder (OUD). Understanding the neurophysiological regulation of neural circuits involved in opioid withdrawal will give us an opportunity to identify novel mechanisms to dampen these aversive experiences of withdrawal and possibly put a break on the cycle of OUDs. Calcitonin gene-related peptide (CGRP, gene name: Calca) of the parabrachial nucleus (PBN) have been demonstrated to be critical in relaying aversive spinovisceral information to brain substrates for appropriate behavioral response to threat or harm. Here, we seek to understand whether PBN CGRP neurons are involved in opioid withdrawal and the neurophysiological mechanisms that may be altered throughout the opioid exposure cycle. Methods: We performed patch-clamp ex vivo electrophysiology and in situ hybridization to profile the baseline intrinsic properties and potential neurophysiological regulators of PBN CGRP neurons in non-dependent mice. We next performed ex vivo single cell calcium imaging of PBN CGRP neurons to determine how this cell population responds to pharmacological activation of mu-opioid receptors. In order to determine how the PBN is involved across the opioid exposure cycle, we used a regimen of twice-daily i.p. injections of increasing concentrations of morphine across 5 days (20 mg/kg, 40 mg/kg, 60 mg/kg, 80 mg/kg, 100 mg/kg) to induce opioid-dependence mice. To precipitate opioid withdrawal, a 100 ug/kg dose of naloxone was administered (i.p.) 5 hours following the morphine injection of 100 mg/kg of morphine. Control mice were administered saline instead of morphine in the same frequency. Following this treatment, one cohort of mice was used to perform in situ hybridization for Fos, an immediate early gene and a marker of increased neural activity, in PBN tissue. In a separate cohort of mice, we performed in vivo fiber photometry of calcium dynamics in mice undergoing morphine treatment and naloxone precipitated withdrawal. Results: We were able to profile the spontaneous activity, intrinsic excitability and presence of M-Current and Hyperpolarization-activated current in PBN CGRP neurons. We were also able to determine the responsivity profiles of PBN CGRP neurons following pharmacological activation of mu-opioid receptor in ex vivo PBN slices. We found increased Fos expression on PBN CGRP neurons in morphine-dependent mice following naloxone-precipitated withdrawal (p < 0.05). Finally, using fiber photometry of calcium dynamics, we found that morphine reduces PBN CGRP neuron population activity (p < 0.05) and naloxone-precipitated withdrawal increase activity (p < 0.05). Conclusions: Based on the Fos and fiber photometry results in morphine dependent mice, PBN CGRP neurons are regulated by naloxone-precipitated opioid withdrawal. Future studies will determine the causal role these neurons may play in mediating opioid withdrawal symptoms. Keywords: Opioid Withdrawal, Parabrachial, Neurophysiology Disclosure: Nothing to disclose. P615. Midbrain Mechanisms in Fentanyl Use and Relapse Logan Fox, Taylor Seifert, Mahashweta Basu, Seth Ament, Megan Fox* Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States Background: Rates of opioid use and overdose death continue to skyrocket in the United States. Synthetic opioids such as fentanyl contribute to >70% opioid-related deaths, but synthetic opioid use remains broadly understudied. Like other substances with abuse potential, opioid exposure disrupts connections between the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Both NAc and VTA undergo molecular and physiological changes in response to opioid use, and altered NAc and VTA activity are heavily implicated in drug relapse. Numerous studies have established the importance of the dopaminergic VTA to NAc projection, however the GABAergic projection from NAc back to VTA is understudied in opioid use. Methods: All experiments were conducted in 8–10-week-old mice using sex as a biological variable (defined gonadally). Mice were trained to self-administer fentanyl (1.5 µg/kg/infusion IV, 5 days FR-1, 5 days FR-2) in operant chambers equipped with nose-pokes. To assess NAc->VTA projections in fentanyl relapse, mice received retrograde AAV-Cre in the VTA and Cre-dependent DREADDs or mCherry in the NAc after fentanyl self-administration (n = 5-7/sex/virus). Two-weeks later, mice were tested for fentanyl-seeking under extinction conditions with 1 mg/kg CNO. To assess molecular adaptations in the VTA, mice received transynaptic AAV-Cre in the NAc prior to fentanyl or saline self-administration training (n = 8-12/sex/condition). VTA nuclei were isolated from half the mice (pooled 4 mice/sex/condition) for single nuclei RNAseq with the 10X Genomics platform. Brains from the remaining mice were flash frozen and 16 µm sections containing the VTA were processed with RNAscope and imaged on a confocal microscope. Results: We found female mice self-administered more fentanyl compared with male mice (Day x Sex F(1,31)=3.5, p = 0.007), and female mCherry controls exhibited greater fentanyl-seeking behavior compared with male controls (Sex F(1,31), p < 0.001). Increased Gq signaling in NAc->VTA neurons abolished fentanyl seeking in both sexes (Virus F(2,31) =29.6, p < 0.001; female: p < 0.0001; male, p = 0.002; Dunnet’s post-hoc). Increased Gi signaling in NAc->VTA neurons showed a trend towards decreased fentanyl seeking in female mice (p = 0.059). We detected transsynaptic Cre from NAc in 2% of all VTA nuclei, with the most enrichment in cluster 3 (36% of Cre+ nuclei). Cluster 3 nuclei had more expression of dopamine neuron markers—Ddc, Th, Slc6a3, Slc18a2, Drd2 – in addition to enrichment for multiple GABA receptor subunits (Gabrb2,Gabrb1, Gabbr2, Gabrb3, Gabrg3) and vesicular glutamate transporter 3 (Slc17a8). We identified numerous differentially expressed genes in the VTA between fentanyl vs saline self-administering mice: 56 upregulated and 64 downregulated. We also identified sex-specific changes in gene expression: Female--13 down, 9 up; Male—277 down, 135 up. Gene Ontology analysis revealed enrichment in neuronal morphology GO terms (e.g. actin cytoskeleton reorganization, axon extension) neurotransmission terms (e.g. neurotransmitter secretion, synaptic vesicle exocytosis), and potassium channel terms. Conclusions: First, our data in mice replicate findings in rats where females self-administer more opioids compared with males. Second, the DREADDs experiments indicate that activity in the NAc->VTA projection is important for fentanyl seeking behavior after abstinence. Third, NAc likely targets both dopaminergic and GABAergic VTA neurons. Finally, fentanyl self-administration alters expression of genes important for neuronal structure and physiology in the VTA. On-going work aims to validate and expand the snSeq findings with RNAscope, and manipulate expression of molecular candidates. Keywords: Fentanyl, Self-Administration, Single-Nucleus RNA Sequencing, DREADDs Disclosure: Nothing to disclose. P616. Single Cell Sequencing Reveals Cell Type Specific Transcriptional Responses to Oxycodone and Buprenorphine by iPSC-Derived Brain Organoids From Patients With Opioid Use Disorder Ming-Fen Ho, Cheng Zhang, Irene Moon, Xiujuan Zhu, Joanna Biernacka, Brandon Coombes, Quyen Ngo, Cedric Skillon, Michelle Skime, Paul Croarkin, Tyler Oesterle, Victor Karpyak, Hu Li, Richard Weinshilboum Mayo Clinic, Rochester, Minnesota, United States Background: Oxycodone is one of the most prescribed opioid medications in the United States, whereas buprenorphine is currently the most prescribed medication for opioid use disorder (OUD) pharmacotherapy. This study was designed to identify gene expression profiles associated with drug treatment with oxycodone or buprenorphine drug treatment in induced pluripotent stem cell (iPSC)-derived brain organoids from patients with OUD. Our data showed cell type specific transcriptional responses to oxycodone and buprenorphine in iPSC-derived forebrain organoids. We then performed functional genomic studies of genes, transcription factors, and pathways identified during this series of experiments. Our findings enhance the understanding of drug mechanism(s) of action and the underlying pathophysiology responsible for opioid addiction. These results may provide novel mechanistic insight into drug action at single-cell resolution. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) using iPSC-derived forebrain organoids from three male subjects with OUD. The forebrain organoids were treated with vehicle, oxycodone or buprenorphine for seven days. The concentrations of oxycodone (50 ng/mL) and buprenorphine (2 ng/mL) used to perform these experiments were selected to fall within the range of blood drug concentrations in patients taking standard clinical doses of these two drugs. Functional validation was performed using iPSC-derived forebrain organoids and neural cells. Results: We hypothesized that oxycodone and buprenorphine might regulate somewhat similar biological pathways in iPSC-derived brain organoids from patients with OUD. We began by performing bulk RNA-seq using iPSC-derived brain organoids to test that hypothesis and observed that oxycodone and buprenorphine displayed surprisingly distinct gene expression profiles. Specifically, we identified 279 and 3333 genes for which expression was significantly altered (FDR < 0.05) after exposure to oxycodone or buprenorphine treatment, respectively, as compared to vehicle treatment. The most common and most highly affected biological pathways in the presence of oxycodone differed from buprenorphine. However, bulk RNA-seq cannot provide detailed insight into the molecular mechanism of drug action, i.e., specific cell type alternation. As a result, we took a step further by performing single-cell transcriptomics using the same samples. We analyzed 25787 nuclei using snRNA-seq after quality control filtering. The single-cell sequencing data revealed 16 transcriptionally distinct clusters containing treated and untreated organoids from three subjects. As a first step, we set out to determine the cell-type specific and drug-specific gene expression profiles in iPSC-derived forebrain organoids. Differential gene expression analysis was performed for each cluster to determine the effect of the drugs on iPSC-derived forebrain organoids in a cell-type specific fashion. Our results showed that oxycodone and buprenorphine displayed distinct gene expression profiles. Specifically, oxycodone affected transcriptional response primarily in neurons, whereas buprenorphine significantly influenced transcription regulation in glial cells. Pathway analysis showed that oxycodone induced the type I interferon signaling pathway in neural cells, whereas the mTOR signaling pathway was the most commonly affected pathway in response to buprenorphine treatment. We pursued functional genomic studies to confirm that IFNγ concentrations were induced by oxycodone in iPSC-derived forebrain neurons and forebrain organoids. However, buprenorphine had no effect on IFNγ concentrations. We next constructed a protein-protein interaction network using the differentially expressed genes in the interferon signaling pathways, and found that STAT1 appears to interact with several genes in the interferon signaling pathway, which could be activated by oxycodone but not buprenorphine. Strikingly, we also found that, at baseline, STAT1 expression in iPSC-derived forebrain neurons was significantly higher in patients with OUD as compared with unaffected control subjects. Even more striking, STAT1 expression was significantly induced by oxycodone only in patients with OUD but not in unaffected control subjects. We subsequently cultured iPSC-derived neurons and exposed them to fludarabine, a STAT1 inhibitor that causes a specific deletion of STAT1 but not other STATs. Of importance, a series of genes involved in the interferon signaling pathway significantly altered their mRNA expression in response to fludarabine treatment in a dose-dependent fashion. These results suggest that upregulation of STAT1 might be associated with OUD, and that STAT1 might play a role in transcriptional regulation in type I interferon signaling in response to oxycodone. Conclusions: Our results revealed cell type specific transcriptional responses to oxycodone and buprenorphine by iPSC-derived brain organoids from patients with OUD. We demonstrated that elevation of STAT1 expression associated with OUD might have a role in transcriptional regulation in response to oxycodone but not buprenorphine. Our results imply that different opioids do not share the same biological effects or mechanisms. This series of studies identified a novel layer of molecular regulation associated with OUD that might potentially open new avenues for future drug development to treat and/or prevent OUD. Keywords: Opioid Addiction, Oxycodone, Buprenorphine, Single-cell RNA Sequencing, Brain Organoids Disclosure: Nothing to disclose. P617. Insulin-Like Growth Factor 1 and Its Receptor in Prefrontal Cortex Regulates Heroin Addiction-Induced Maladaptation Zi-Jun Wang, Shuwen Yue, Yunwanbin Wang, Archana Singh, Guohui Li University of Kansas, Lawrence, New York, United States Background: Opioid use disorder (OUD) is a chronic relapsing psychiatric disorder with an enormous socioeconomic burden. Relapse is one of the most difficult challenges during recovery. None of the current treatment is effective to prevent relapse. Therefore, it is urgent to further understand the neurobiology of OUD. Clinical studies show that the neuronal responses to stimuli in the prefrontal cortex (PFC) from individuals with OUD are disrupted. Consistently, preclinical data also report opioid-induced synaptic dysfunction in the PFC. Given the critical role of PFC in regulating opioid-related behaviors, it is vital to investigate the molecular mechanisms underlying opioid-induced PFC dysfunction and its role in shaping opioid-induced behavioral plasticity. Increasing studies have shown that insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R) regulate synaptic transmission, but the involvement of IGF1/IGF1R in opioid addiction-induced synaptic dysfunction remains unknown. Here we used a mouse heroin self-administration (SA) model to investigate the role of IGF1/IGF1R on heroin-induced maladaptation in the PFC. Methods: Current study used both male and female C57BL/6 J mice. All the procedures are approved by the Institutional Animal Care and Use Committee. All animals were maintained according to the National Institutes of Health guidelines in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities. At age of P60, mice were trained to self-administer water on a fixed ratio schedule. After water training, animals underwent jugular surgery. After recovery, mice underwent 10 days of heroin SA (50 ug/kg/infusion, 3 h/session), followed by 14 days of forced abstinence. Then animals were sacrificed for testing IGF1 level in the PFC. Another set of animals received IGF1 infusion in the PFC during the last 5 days of abstinence, then were subjected to 1 hour of cue- and context-induced drug seeking test. To study the IGF1-dependent synaptic and molecular mechanisms that underlying heroin-induced behavioral plasticity, a different set of animals that received IGF1 infusion during abstinence were sacrificed without cue re-exposure for electrophysiology recording and RNA-sequencing. Results: We found that IGF1 in PFC was significantly decreased after prolonged abstinence from heroin SA (n = 6/group, t10 = 3.3, P < 0.01, t-test). Moreover, intra-PFC IGF1 administration attenuated (n = 7-8/group, t13 = 2.8, P < 0.05, t-test) while IGF1R selective knockdown in PFC pyramidal neurons potentiated (n = 8-10/group, t16 = 2.2, P < 0.05, t-test) heroin-seeking behavior. Furthermore, we used whole-cell patch-clamp method to detect changes in synaptic function. Our data showed that intra-PFC IGF1 administration restored heroin abstinence-induced decrease in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor- and N-methyl-D-aspartate (NMDA) receptor-mediated evoked excitatory postsynaptic currents (n = 8-20cells/3-5 mice/group, AMPAR: Ftreatment(2, 29)=7.3, p < 0.01; NMDAR: Ftreatment(2, 37)=6.1, p < 0.01; Two-way rmANOVA). In addition, IGF1 also recovered the elevated AMPA/NMDA ratio in response to heroin-associated cues in mice underwent heroin abstinence (F2, 38 = 20.7, p < 0.001, Two-way ANOVA). To explore the underlying molecular mechanisms, we used translating ribosome purification method coupled with RNA-sequencing. We found that Igf1 induces genome-wide restoration of genes involved in neural signaling in PFC pyramidal neurons from mice that underwent heroin abstinence. Conclusions: These data indicate that IGF1/IGF1R system in the PFC play a key role in regulating heroin-induced behavioral and synaptic plasticity, which will provide a novel therapeutic target for the development of OUD treatment strategies. Keywords: IGF-1, PFC, Heroin Self-Administration, Synaptic Function, RNA-seq Disclosure: Nothing to disclose. P618. Impact of THC Vape on the Proteomic Profile of Extracellular Vesicles in the Brain Valeria Lallai, TuKiet T. Lam, Kenneth Williams, Angus C. Nairn, Christie D. Fowler University of California - Irvine, Irvine, California, United States Background: With legalization of cannabis in the US, there is an urgent need to more clearly understand the drug’s effects on central signaling mechanisms. Extracellular (EVs) vesicles have been identified as intercellular signaling mediators, which contain a variety of cargo, including proteins, enzymes, and RNA transcripts. The focus of these studies was to examine whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV cargo in the brain. Methods: In vitro studies were first conducted to determine whether THC can act on primary epithelial cells derived from the dorsal third ventricle of rats. Next, to examine the impact of THC in vivo, male and female rats were exposed to aerosolized THC or vehicle in vapor chambers. The first cohort of rats (n = 12/group/sex) received a single session of exposure, and the second cohort (n = 12/group/sex) received 14 consecutive daily sessions of exposure. CSF was collected from the cisterna magna, and EVs were extracted with SBI SmartSEC and then processed for label free quantitative proteomics analyses via high resolution tandem mass spectrometry. Quantitative LFQ mass spectral data were analyzed using Progensis QI Proteomics software. Results: Cannabinoid receptor (CB1R) expression was localized in the choroid plexus, and THC upregulated the expression of c-fos, CB1R mRNA, and mir-204, a transcript localized in EVs. In the THC vape exposed rats, multiple EV proteins were identified as being differentially expressed following either acute or chronic exposure. Interestingly, opposing effects were found with some proteins (such as APOE), in which acute exposure decreased, but chronic exposure increased, expression. These findings are paralleled by our prior data examining RNA transcripts packaged in CSF EVs. Conclusions: Our findings reveal that cannabinoids can modulate intercellular signaling mechanisms in the brain, with differential effects following single or chronic exposure. Our data further support the contention that THC can act on CB1 receptors in the choroid plexus to mediate EV signaling, which may then integrate into different brain regions to modulate cellular function. Supported by NIH DA051831 (C. Fowler) and Yale/NIDA Neuroproteomics Center (Pilot to V. Lallai; Parent Grant NIH DA018343; SIG grant OD023651-01A1). Keywords: THC, Extracellular Vesicles, Proteomics Disclosure: Nothing to disclose. P619. Gene Expression Within Reward-Specific Ensembles Carl Litif, Lucio Vaccaro, Jason Gigley, Nicolas Blouin, Ana-Clara Bobadilla University of Wyoming, Laramie, Wyoming, United States Background: Reward-based positive reinforcement is an evolutionary strategy shared across species. However, in drug addiction, reward seeking becomes maladaptive and endangers survival. While drug and natural rewards such as sucrose involve overlapping brain nuclei, we have recently shown that drugs of abuse and natural rewards are linked to different neuronal ensembles, defined as a discrete number of neurons synchronously activated. Mounting evidence indicates that drug exposure strongly and uniquely impacts gene expression transcriptome-wide. However, aside from having identified a small number of plasticity-related genes in neuronal ensembles linked to methamphetamine seeking in rodents, little is known about how drug exposure alters mRNA expression profiles, specifically in drug-related ensembles. To address this gap in knowledge, we aim to investigate gene expression within reward-specific ensembles and how it differs in neurons activated while seeking different types of reward. We hypothesize that a discrete number of key genes, likely activity-dependent ones, are differentially expressed within each reward-specific ensemble and in cells responding to all types of reward. Methods: Using inducible cFosiCreERxAi14 reporter mice, we demonstrated we can fluorescently tag different reward-specific neuron ensembles in the nucleus accumbens core (NAcore), a hub of reward processing. To investigate gene expression in these different ensembles, we used this same approach to induce fluorescent tagging of 3 different groups: the cocaine-, sucrose- or overlapping ensembles. We then isolated the NAcore neurons from mouse brains, sorted the tagged ensembles using the FACSMelodyTM cell sorter, extracted RNA and performed RNA sequencing to compare gene expression within the 3 ensembles to the untagged, non-ensembles cells. Differences in gene expression profiles amongst the 4 cell groups were compared and used to identify cell types and to create cell clusters based on transcriptional profiles. Results: The first step of the proposed experiments was to establish a protocol for tissue collection that would allow us to sort the cells tagged in different ensembles, i.e., small percentages of 1% or less of the NAcore cells, using FACS technology without damaging the mRNA and hamper sequencing. The presentation will include an in-depth description of the protocol with the key takeaways and practical advice to increase reproducibility and yield of good quality mRNA. We then identified genes differentially regulated within each neuronal ensemble tagged, the cocaine-, sucrose-, and overlapping ensembles, compared to non-ensemble cells. Conclusions: Establishing differential gene expression profiles exclusively linked to drug-seeking raises the possibility to therapeutically targeting maladaptive drug seeking without affecting essential and biologically adaptive seeking of natural rewards. Keywords: Cocaine, c-Fos-Expressing Ensembles, Reward Neural Network Disclosure: Nothing to disclose. P620. Delta-9-THC in Marijuana and the Adolescent Amygdala Bertha Madras, Susan George, Meena Sivasubramanian, Jack Bergman, Sarah Withey McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Compared with adult onset, adolescent-onset cannabis use confers a higher risk for developing neuropsychiatric symptoms and disorders, some of which emerge during adulthood. The risks may be amplified currently as more youth are using cannabis daily and/or consuming higher potency cannabis (high THC: ∆9-tetrahydrocannabinol). These unprecedented trends present major gaps in our knowledge of age-dependent molecular adaptations engendered by exposure to frequent high THC exposure and of strategies to mitigate cannabis-induced maladaptive changes. Methods: In adolescent nonhuman primates, we measured the effects of daily long-term THC (1 mg/kg ∆9-tetrahydrocannabinol; 4 months; n = 4) or THC + CBD (1 mg/kg: 3 mg/kg; n = 4) or vehicle (n = 4) on gene (RNA-Seq) or protein expression of amygdala, whether CBD (cannabidiol) antagonized THC effects, and if findings were associated with sleep disturbances. Results: RNA-Seq analysis of left amygdala showed that THC dysregulated canonical pathways associated with sleep disorders (ranking first statistically), with dysregulated genes including TRPC7, IL1R, RBFOX1, and genes encoding hormone receptors. Other dysregulated canonical pathways were associated with cannabis use disorder, anxiety, emesis, dopamine neurodevelopment and psychotic disorders. THC + CBD produced overlapping but differently regulated genes than THC alone. An inflammatory marker (GFAP) was up-regulated in amygdala of THC-treated nonhuman primates which correlated with sleep fragmentation in the same subjects. Coadministration of THC with cannabidiol attenuated sleep fragmentation and GFAP up-regulation. Conclusions: Maladaptive changes in amygdala elicited by THC may contribute to a range of cannabis-induced adverse behavioral and physiological consequences, including sleep disturbances that are associated with early onset, heavy marijuana use by adolescents. As CBD mitigated some effects of THC, CBD content of cannabis and THC:CBD ratios conceivably can modulate specific negative outcomes of high frequency, high potency cannabis. Keywords: Marijuana, Adolescent, Sleep Disturbances, High Potency THC, Neurodevelopmental Disorders, Cannabidiol, CBDV, Translational Models, Clinical Trials Disclosure: Kennedys Law Firm: Consultant (Self). P621. Cocaine Use During Oxycodone Withdrawal Reduces Somatic Signs of Withdrawal and Exacerbates Oxycodone-Induced Glutamate Dyshomeostasis Shailesh Khatri, Emma Bondy, Erin Maher, Mei Hong, Hanaa Ulangkaya, William Stoops, Cassandra Gipson University of Kentucky, Lexington, Kentucky, United States Background: Opioid use disorder (OUD) is a leading public health crisis in the United States, and commonly co-occurs with cocaine use. Mechanistically, glutamate signaling within cortico-striatal circuitry underlies use of both opioids and cocaine, such that self-administration (SA) of these substances alters synaptic plasticity measured as changes in AMPA-to-NMDA current ratios (A/N) within the nucleus accumbens core (NAcore). Specifically, cocaine potentiates A/N, whereas opioids induce long term depression (LTD) of NAcore medium spiny neurons (MSNs). The current study determined whether cocaine use during oxycodone withdrawal (1) reduces somatic withdrawal signs, and (2) reverses oxycodone-induced LTD as measured by A/N. Methods: Long Evans male and female rats (N = 34) underwent oxycodone (0.03 mg/kg/infusion) or food and cocaine (0.25 mg/kg/infusion) SA in an A-B-A-B design. Rats underwent a minimum of 10 oxycodone SA (“A”) sessions prior to the first cocaine SA (“B”) phase, and following the second phase of oxycodone SA (“A”), rats underwent one cocaine SA session (“B”) at the 24-hr oxycodone or food withdrawal timepoint. Somatic signs of withdrawal were measured at 0, 22, and 24 hrs post-oxycodone or food SA. NAcore A/N was then measured via electrophysiological recordings. Results: Cocaine consumption significantly increased following oxycodone withdrawal, (p < 0.01, comparing cocaine consumption prior to and following 24 h oxycodone withdrawal), but not following food SA (p > 0.05). Further, cocaine SA significantly reduced somatic signs of oxycodone withdrawal (p < 0.05), but did not significantly alter somatic signs in the food control group (p > 0.05). Compared to food SA, we found that rats in withdrawal from oxycodone SA showed significantly lower A/N (p < 0.01). Further, A/N was significantly lower following cocaine SA during oxycodone withdrawal as compared to cocaine SA following food “withdrawal” (p < 0.01). Finally, compared to cocaine SA in a food control group, cocaine SA during oxycodone withdrawal decreased A/N. Conclusions: Cocaine intake during oxycodone SA reverses oxycodone withdrawal, which on its own renders synapses in a de-potentiated state. Together, these data suggest that oxycodone use induces persistent changes in glutamatergic signaling, which may exacerbate motivation for cocaine co-use due to alleviation of withdrawal symptomatology. Keywords: Co-Abuse, Opioid Withdrawal, Cocaine and Opioid Use Disorders, Glutamatergic Transmission, Glutamate Transporter (EAAT3) Disclosure: Nothing to disclose. P622. Sex Differences in Cortico-Striatal-Limbic Responses to Stress and Alcohol Cues Predict Future Heavy Drinking Outcomes Milena Radoman, Nia Fogelman, Cheryl Lacadie, Dongju Seo, Rajita Sinha Yale University School of Medicine, New Haven, Connecticut, United States Background: Growing research suggests that chronic alcohol use disrupts neural stress and reward pathways, and such dysfunction increases stress- and alcohol cue- related craving and underlies high alcohol relapse risk in alcohol use disorder (AUD). Previous research has shown that specific disruptions in the ventromedial prefrontal cortex (vmPFC) and striatal responses to stress and alcohol cues are associated with future heavy drinking days (HDD). While sex differences in neural stress and cue responses have been reported, their association with alcohol use outcomes has not been studied thus far. Therefore, the current study examined sex differences in the prospective association between neural stress and alcohol cue responses and future alcohol use outcomes during an 8-week behavioral AUD treatment. Methods: Seventy-two treatment-entering men and women with AUD (ages 18-60, 43 males and 29 females) underwent functional Magnetic Resonance Imaging (fMRI) scanning during which they participated in a well-validated cue provocation task exposing them to standardized and matched visual cues for stress (S), alcohol (A) and neutral control (N) cues over six successive runs in a randomized block design per condition. Whole brain, voxel-based second-level fMRI analyses were conducted using linear mixed effects models (3dLME, AFNI) covarying for age and number of days of abstinence at treatment initiation assessing Condition (S, A, N) x Drinking Outcome (i.e., %HDD), and Condition x %HDD x Sex as predictors and Participant as a Random effect. Subjective alcohol craving was repeatedly assessed in each block per condition. Results: Across all participants, S and A significantly increased alcohol craving with no sex differences (p’s < 0.01). In fMRI, significant Condition x %HDD interaction showed that greater right amygdala, caudate, putamen, hippocampus, fusiform gyrus, left vmPFC, dorsomedial thalamus, dorsolateral prefrontal cortex (dlPFC) as well as bilateral anterior insula (AIC), orbitofrontal cortex (OFC) and dorsal anterior cingulate (dACC) activation was associated with subsequent higher %HDD during treatment. Post-hoc analyses revealed that increased activation in the cortico-striatal regions (e.g., vmPFC, caudate, putamen) during A-N as well as limbic regions (e.g., amygdala, hippocampus) during S-N predicted higher %HDD. Greater putamen activation was associated with higher provoked alcohol craving in the entire sample. Notably, a significant three-way interaction indicated that decreased responses of bilateral bed nucleus of stria terminalis (BNST) and hypothalamus to S-N and increased responses of right OFC, caudate and bilateral vmPFC and dACC to A-N were associated with greater future %HDD in female relative to male AUD participants (all fMRI analyses, at p < 0.001 threshold, and additional whole brain cluster correction at α < 0.05). Conclusions: Taken together, these findings suggest specific altered neural responses within a distributed cortico-striatal-limbic circuit during both stress and alcohol cues that predicts subsequent higher %HDD in both sexes, with additional limbic mid-brain alterations being specific to women. Identification of these shared and unique neural markers of poor alcohol use outcomes in men and women with AUD are noteworthy, given that women are markedly more susceptible than men to alcohol-related harms, and timely considering worrisome recent statistics showing significant increases in problem drinking in women, who are catching up to men in levels of alcohol consumption and AUD risk. Our study has important implications for understanding sex-specific differences in neurobiology underlying response to stress and reward cues, vulnerability to relapse as well as development of novel, more effective AUD treatments tailored to each sex and improved prognostic predictions. Keywords: Alcohol Use Disorder, Cue Reactivity, fMRI, Sex Differences Disclosure: Nothing to disclose. P624. Body Mass Index as a Predictor of Extended-Release Naltrexone Treatment Retention in Individuals With Opioid Use Disorder Xinyi Li, Daniel Langleben, Kevin Lynch, Gene-Jack Wang, Corinde E. Wiers, Zhenhao Shi University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Despite the proven efficacy of injectable extended-release naltrexone (XR-NTX) for opioid use disorder (OUD), its clinical use is constrained by poor retention in treatment. Growing evidence suggests that food and opioids interact at the brain reward circuits modulating each others’ rewarding properties. However, implications of OUD patients’ nutritional status on the treatment outcomes are unknown. In this secondary analysis, we used body mass index (BMI) to explore the role of nutritional status on XR-NTX treatment retention in individuals with OUD. Methods: 61 OUD individuals who participated in XR-NTX treatment trials and had baseline BMI available, were included in this secondary analysis. Participants were offered up to three monthly injections of 380 mg XR-NTX. Treatment completion was defined as having received all three injections, whereas treatment noncompleters prematurely withdrew from study after recieving 1 or 2 doses. The association between pre-treatment BMI and probability of treatment completion was examined with univariate logistic regression. Moreover, a multivariate analysis was performed with BMI, opioid craving at baseline and age as factors and bootstrapped with 5000 samples. Results: A total of 41 individuals completed the 3-months’ study treatment protocol, whereas 20 dropped out prematurely. Univariate binary logistic regression [χ 2(1)=6.4, p = 0.01; Nagelkerke R2 = 0.14] demonstrated significant positive association between BMI and likelihood of treatment completion (OR = 1.2; 95% CI = 1.03- 1.4). BMI remains a significant predictor of treatment completion in the expanded multivariate logistic regression that controlled for covariates [χ2(3)=11.51, p = 0.009; Nagelkerke R2 = 0.25]. While opioid craving at baseline was not associated with BMI, both BMI and opioid craving demonstrated significant association with treatment completion in the multivariate analysis. Conclusions: In OUD individuals undergoing XR-NTX treatment, increased BMI was associated with beneficial effects in retaining individuals on treatment. Further research is needed to understand the causality underlying the observed interrelationship between BMI and opioid antagonist treatment outcomes, and examine the implications of our study findings on long-term abstinence and relapse. Keywords: Opioid Antagonist Treatment, Nutrition, Opioid Use Disorder, Treatment Outcome Disclosure: Nothing to disclose. P625. A Practice-Quit Model for Screening Medications for Alcohol Use Disorder Lara Ray, Steven Nieto, Erica Grodin, Wave-Ananda Baskerville, Diana Ho, Riley Russell, Artha Gillis, Karen Miotto University of California - Los Angeles, Los Angeles, California, United States Background: While developing novel medications to treat alcohol use disorder (AUD) remains a high priority research area, there are major opportunities to refine the process of screening novel compounds. The premise of this study is that screening novel AUD medications can be more efficient and clinically meaningful if early efficacy (phase 2) studies combine the internal validity of laboratory testing with the external validity of clinical trials. To that end, we developed and tested a novel laboratory model in which participants with AUD engage in a practice quit attempt lasting 1-week. In order to leverage the existing literature, we conducted a comparison of a placebo arm versus two established medications for AUD, namely naltrexone and varenicline. We hypothesize that both active medications would result in a (a) higher percentage of days abstinent (b) lower number of drinks per drinking day during the practice quit attempt, and (c) dampened cue-induced craving for alcohol, as compared to placebo. Methods: Participants were non-treatment seeking individuals with current AUD (moderate-to-severe) who completed a randomized, double-blind, placebo-controlled, crossover study of naltrexone (50 mg), varenicline (2 mg bid), or matched placebo. Participants took the study medication for one week prior to starting the 1-week practice quit attempt. During the quit attempt, participants completed daily interviews with the research staff. All participants completed an alcohol cue-exposure paradigm pre and post-randomization. Analysis of Variance (ANOVAs) followed by planned comparisons across the three medication conditions were conducted to examine the drinking outcomes. Additional analyses are underway to examine medication effects on cue-reactivity. This clinical trials is registered in clinicaltrias.gov, NCT04249882. Results: A total of 53 individuals with current AUD were randomized to one of three conditions: placebo (n = 18), naltrexone (n = 15) and varenicline (n = 17). Initial analyses, without covariates, found no overall differences across the three conditions on total number of drinks consumed during the practice week. Observed Means were 3.39 drinks for placebo, 7.79 drinks for naltrexone and 5.41 drinks for varenicline, [F(2,49)=1.26, p = 0.29]. Additionally, there was no medication main effect on the likelihood of remaining abstinent for the duration of the practice quit. The observed complete abstinence rates were 55.56% on placebo, 53.33% on naltrexone, and 41.18% on varenicline, [Chi square=0.82, p = 0.66]. Analyses are underway to consider multiple drinking outcomes as well as to test the effects of medication on cue-reactivity. Conclusions: These initial results do not support the sensitivity of the practice quit paradigm to detect medication effects for AUD. Ongoing analyses will consider other drinking outcomes, include relevant covariates, and test cue-reactivity measured before and after medication treatment. Together, these results and further analyses will develop the practice quit model of medication screening in conjunction with two established pharmacotherapies (naltrexone and varenicline) and leveraging an established screening tool (alcohol cue-reactivity in the laboratory). Keywords: Alcohol Use Disorder - Treatment, Clinical Trial, Clinical Trials Methodology Disclosure: Nothing to disclose. P626. Pharmacotherapy Effects on Alcohol Cue-Reactivity in the Human Laboratory: A Systematic Review Elizabeth Burnette, Lindsay Meredith, Steven Nieto, Han Du, Suzanna Donato, Howard Becker, Molly Magill, Lara Ray University of California - Los Angeles, Los Angeles, California, United States Background: Alcohol Use Disorder (AUD) is a prevalent but severely under-treated condition. The need for the development of novel, effective pharmacotherapies is critical. Testing novel compounds in humans using experimental psychopharmacology paradigms is a method proposed to detect initial efficacy for AUD treatment. One such human laboratory method is cue-reactivity (CR), which assesses a medication’s effect on individuals’ behavioral and biological responses to alcohol cues (e.g. craving for alcohol, heart rate, salivation, and blood pressure) in a controlled context, consisting of exposure to images of or in vivo alcoholic beverages. This systematic review aims to describe and evaluate the literature on alcohol cue-reactivity in the laboratory as a screening tool for AUD pharmacotherapies. Methods: Published reviews on AUD pharmacotherapies were used to identify 40 medications that have been screened using human laboratory paradigms. Searches were conducted in January 2022 with each of the 40 medications in combination with applicable search terms such as “alcohol cue-exposure”, “cue-reactivity”, “alcohol craving”, as well as “cues” and “craving” MeSH terms. Inclusion criteria for the alcohol cue-reactivity studies were: (1) the administration of a pharmacological agent approved or being developed for the treatment of AUD, (2) placebo-controlled, (3) cue exposure in the laboratory, (4) self-reported cue-induced craving, and (5) reported in the English language, or translated to English. PubMed searches yielded 358 studies that were screened on title and abstract, of which 59 were retrieved for full-text screening. Thirty-six eligible studies were included in the final analysis following full-text screening. All studies were independently screened and coded by two reviewers. Results: The 36 studies included in the final analysis comprised 19 medications, including two combination pharmacotherapy studies. Seventy-five percent of studies were conducted in populations with AUD, while 25% included heavy-drinking individuals. Studies were primarily mixed biological sex (males and females), on average 71% male; 8% of studies enrolled males only. At baseline, participants consumed an average of 190.89 drinks per month, with a range from 39.03 to 412.72 (information available in 72% of studies). Twenty-two percent of studies reported a within-subjects crossover design, while the remaining 78% used a between-subjects design. For cue-reactivity paradigms, 72% of studies exposed participants to in vivo cues, 25% to visual cues, 6% to auditory scripts, and 3% to odor cues. Thirty-six percent of studies also reported at least one biological outcome in addition to clinical assessments of craving. Conclusions: Alcohol cue-reactivity paradigms are often used to detect an early efficacy signal in medication development for AUD. However, there is a need to standardize experimental procedures. This systematic review identified wide variability with regards to many key methodological considerations, including sample characteristics and quantity of alcohol use, type of cue presentation, crossover versus between-subject designs, and collection of biological outcomes. These results can inform efforts to standardize and leverage cue-reactivity in the human laboratory as a screening tool for novel AUD pharmacotherapies. Further, there is little quantitative data demonstrating that a medication signal in the alcohol cue-reactivity paradigm predicts medication efficacy. Future directions aim to test the predictive utility of cue-reactivity results toward randomized clinical trial outcomes. The results from this work may help streamline the process of screening novel compounds for AUD. Keywords: Alcohol Use Disorder - Treatment, Cue-Reactivity, Human Laboratory, Alcohol Disclosure: Nothing to disclose. P627. Baseline C-Reactive Protein Levels are Predictive of Treatment Response to a Neuroimmune Modulator in Individuals With an Alcohol Use Disorder: A Preliminary Study Erica Grodin, Lindsay Meredith, Elizabeth Burnette, Karen Miotto, Michael Irwin, Lara Ray UCLA, Sherman Oaks, California, United States Background: Inflammation has been implicated in the development and maintenance of alcohol use disorder (AUD). Ibudilast is a novel neuroimmune modulator that has shown promise for the treatment of AUD. AUD is a highly heterogeneous disorder, subtypes of which present distinct characteristics and may require distinct treatment strategies. High inflammation, as indicated by high circulating levels of c-reactive protein (CRP), represents a possible subtype of AUD which may identify individuals who are likely to be more responsive to anti-inflammatory AUD medications like ibudilast. The current study evaluated CRP as a predictor of treatment response to ibudilast; hypothesizing that ibudilast would be more effective at reducing drinking and alcohol cue-reactivity in individuals with higher CRP levels. Methods: This is a secondary analysis of a clinical trial of ibudilast for AUD, which found that ibudilast reduced heavy drinking in individuals with AUD (ClinicalTrials.gov identifier: NCT03489850). Fifty-one individuals were randomized to receive ibudilast (n = 24 [16 M/8 F]) or placebo (n = 27 [18 M/9 F]) for two weeks. Participants provided blood samples at baseline to assess CRP levels, completed daily assessments of alcohol use, and an fMRI alcohol cue-reactivity task at study mid-point. General linear models were used to evaluate the effects of medication, CRP levels, and their interaction on drinks per drinking day and alcohol cue-reactivity. Tukey post-hoc tests were used to conduct pairwise comparisons to identify group differences. Results: There was a significant interaction between medication and baseline inflammation (F(1,44) = 3.80, p = 0.03, η_p^2 = 0.11).Tukey post hoc tests showed that individuals in the ibudilast high CRP group had significantly fewer drinks per drinking day compared to individuals in the ibudilast low CRP group (p = 0.007). There was a significant interaction between medication and baseline inflammation on alcohol cue-elicited brain activation in a large cluster extending from the left inferior frontal gyrus through the anterior cingulate cortex to the right caudate and right putamen (Z = 4.55, p < 0.001). Follow-up analyses identified several group differences, driven by attenuated alcohol cue-reactivity in the ibudilast high CRP group relative to the ibudilast low CRP group and placebo high CRP group (Z’s > 3.33, p’s < 0.04). Conclusions: In this proof-of-concept study, baseline CRP levels significantly moderated the effect of ibudilast treatment on alcohol consumption during the two-week trial, suggesting that individuals with elevated CRP levels showed a beneficial treatment response compared with placebo. Neuroimaging results showed that baseline CRP levels were associated with neural alcohol cue-reactivity in mesocorticolimibic regions associated with incentive salience and motivation attributed to alcohol-related stimuli. This study serves as an initial investigation into predictors of clinical response to ibudilast treatment and suggests that a baseline proinflammatory profile may enhance clinical efficacy. Keywords: Alcohol Use Disorder - Treatment, C-Reactive Protein, Ibudilast, Inflammation, Functional MRI (fMRI) Disclosure: Nothing to disclose. P628. Mifepristone as a Pharmacological Intervention for Stress-Induced Alcohol Craving: A Translational Randomized Trial Carolina Haass-Koffler, Rajita Sinha, Robert Swift, Lorenzo Leggio Brown University, Providence, Rhode Island, United States Background: Preclinical and clinical work suggests that mifepristone, a glucocorticoids receptor antagonist, may represent a treatment for alcohol use disorder (AUD). We investigated the safety, tolerability, neuroendocrine variation, alcohol craving and consumption after oral administration of mifepristone in a human laboratory comprised of administration of yohimbine, a cue-reactivity procedure and alcohol self-administration. Methods: This was an outpatient, cross-over, randomized, double-blind, placebo-controlled, human laboratory study with non-treatment-seeking individuals with AUD (N = 32). The design was within-subject, with two counterbalanced stages with mifepristone and/or placebo administered for a week. Outcomes were assessed using Generalized Estimating Equations and mediation and moderation analyses assessed mechanisms of action and precision medicine targets Results: Safety outcomes were excellent and no statistically-significant difference between the mifepristone, compared to placebo, in the hemodynamic response, alcohol subjective effects and pharmacokinetics parameters. Mifepristone significantly reduced alcohol craving, urge and salivary output and increased cortisol level. Cortisol was not a mediator of the relationship between condition and alcohol craving indicators. Moderation analysis testing the interaction between family history density of AUD (FHDA) and mifepristone, suggested that reduced craving and urges were present in individuals with low, but not high FHDA. Conclusions: The study confirms the safety and tolerability of mifepristone when co-administered with yohimbine and alcohol. Mifepristone reduced the cue-induced alcohol craving; however, this effect was not related to mifepristone-induced increase in cortisol. Moderation of FHDA suggested the importance of evaluating AUD subtypes in order to inform a personalized medicine approach in AUD. Keywords: Alcohol Use Disorder - Treatment, Neuroendocrine Responses, Cortisol Response to Stress, Glucocorticoid Antagonists Disclosure: Nothing to disclose. P629. Substituted Benzothiazole Analogues as Dopamine D4 Receptor Selective Ligands to Treat Substance Use Disorder Comfort Boateng, Ashley Nilson, Franziska Jakobs, R. Benjamin Free, David Sibley, Rana Rais, Barbara Slusher, Kent Stewart, Thomas Keck High Point University, High Point, North Carolina, United States Background: The dopamine D4 receptor (D4R), a G protein-coupled receptor, is predominantly expressed in the prefrontal cortex in which it plays an important role in cognition, attention, and decision making. Studies have indicated that the D4R is a promising therapeutic target for the treatment of neuropsychiatric conditions such as substance use disorders (SUD). D4R ligands have been shown to alter cognition and behavior in animal models of drug addiction. A better understanding of D4R-mediated signaling is essential to treating D4R-associated disorders, including SUD. Despite its clinical importance, there are no currently approved medications that selectively target the D4R for cocaine use disorder. The present study focuses on the design of D4R ligands based on a benzo[d]thiazole scaffold template as the initial parent compound. Methods: D4R ligands were designed and synthesized following computational modeling predicting favorable interactions. Compounds were purified and analytically characterized followed by CHN combustion elemental analysis. In vitro binding affinities were determined via [3H]N-methylspiperone radioligand binding using membranes prepared from HEK293 cells expressing dopamine D2-like receptors (D2R, D3R, or D4R). The ligands were also studied in β-arrestin recruitment and cAMP inhibition assays for their effects on D2R-like function. We calculated in silico brain penetration using central nervous system multiparameter optimization of chemical features (CNS MPO) and performed Caco-2 membrane permeability tests of selected D4R compounds. We further performed in vitro and in vivo pharmacokinetic analyses with selected compounds. Results: We produced a library of eighteen compounds with varied substitutions on the pyrimidinylpiperidinyl (PP) ring and/or the benzothiazole moieties. Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cAMP inhibition assays, and computational modeling. Metabolic stability in rat and human liver microsomes, followed by in vivo pharmacokinetics analyses were performed on a subset of ligands. We identified several compounds with nanomolar D4R binding affinity and excellent D2-like subtype selectivity (>91-fold vs. D2R and D3R). Several compounds were selective D4R antagonists in functional β-arrestin recruitment and cAMP inhibition assays. Based on the analysis profiles, lead compounds were selected for in vitro metabolic stability in rats and human liver microsomes of which some displayed acceptable stability profiles. One of the lead candidates was selected for in vivo pharmacokinetics assessment in rats where it displayed excellent brain penetration with AUCbrain/plasma>3. Conclusions: These novel ligands display high binding affinity and subtype selectivity for D4R with advantageous pharmacokinetic profiles. Further development of these compounds may provide insights to targeted drug discovery leading to a better understanding of the role of D4Rs in neuropsychiatric disorders such as SUD. Keywords: Dopamine D4 receptor, Antagonist Ligands, benzo[d]thiazole, Substance Abuse Disorder Disclosure: Nothing to disclose. P630. A Peri-Ceorulear Neuropeptidergic Pathway for Modulating OFC-Mediated Reward Seeking Kasey Girven, Katherine Motovilov, Elena Judd, Azra Suko, Luis de Lecea, Richard Palmiter, Larry Zweifel, Michael Bruchas University of Washington, Seattle, Washington, United States Background: Research demonstrates that both acute and chronic stress can reduce as well as potentiate an animal’s drive for seeking reward, depending on the modality. In humans, anxiety disorders are comorbid with depression and substance use disorders. Previous work indicates that a relatively unknown neuropeptide called neuropeptide S (NPS) acts to reduce anxiety-like behavior as well as drives reward-seeking through activation of its cognate Gq-coupled protein receptor NPSR1. We generated both NPS-Cre and NPSR1-Cre driver mouse lines for accessing and examining the neuromodulatory circuit components of NPS/NPSR1-mediated behaviors. We isolated a population of NPS-containing cells that reside partly within and surrounding the locus coeruleus (LC), known as the periLC. The LC’s and peri-LC are known to be involved in stress and cue-processing throughout the brain. They also project to the cortex where there is dense expression of NPSR1, particularly in the orbitofrontal cortex (OFC). Previous work has shown that NPS administered through intracerebroventricular infusions promotes cue-induced reinstatement of reward-seeking behavior, while intracranial infusion of NPSR1 antagonist to the lateral hypothalamus blocks drug-seeking. In addition to the observed involvement of NPS and NPSR1 in drug-seeking, intracranial infusion of NPS into the amygdala reduces conditioned fear responding and increases exploration. These prior studies indicate that NPS and its receptor, NPSR1 may regulate reward-seeking behavior and anxiety-like states and suggest this neuronal pathway may be sensitive to stress. In our preliminary studies, we found that the periLC sends excitatory monosynaptic projections onto OFC NPSR1-expressing neurons. Thus, the OFC is a strong candidate for NPS-regulation of avoidance and reward-like behavior. Here I tested the hypothesis that periLC-NPS neurons act to modulate reward-seeking behavior through positive gain control in OFC-NPSR1 neurons. Methods: Whole cell patch clamp electrophysiology: NPSR1-Cre reporter mice (n = 8 [4 female/4 male]) expressing AAV-CaMKIIA-ChR2 in the periLC were recorded in NPSR1-positive cells from the OFC. OFC-NPSR1 neurons that receive input from the periLC were identified using ChR2-assisted circuit mapping. In vivo Fiber Photometry: Both NPSR1-Cre (n = 16 [8 female/8 male]) and NPS-Cre (n = 16 [8 female/8 male]) mice received unilateral infusions of AAVDJ-DIO-GCaMP6s-eYFP to either the OFC or periLC, respectively. In addition, all mice received unilateral fiber photometry implants to the OFC on the same side as the viral injection. This cohort underwent classical conditioning, a fixed ratio 1 (FR1) task, and a social interaction task. A separate cohort (n = 16 [8 female/8 male]) then underwent fentanyl’ self administration in a FR1 task. In vivo 2-Photon Imaging: NPSR1-Cre mice (n = 8 [4 female/4 male]) received unilateral infusion of AAVDJ-DIO-GCaMP6s-eYFP to the OFC with a 1 mm diameter GRIN lens positioned above. After recovery, mice underwent a head-fixed classical conditioning task where they were exposed to multiple cues that predicted delivery of water (neutral), 10% sucrose (appetitive), or 10% quinine solution (aversive) through individual lick spouts. All experiments were carried out in accordance with the NIH Guide and approval of the University of Washington IACUC. Results: We determined that the periLC sends excitatory projections to the orbitofrontal cortex (OFC) that connect directly with NPSR1-expressing neurons (n = 12/35). Bath application of NPS caused a significant increase in the peak amplitude of the optically evoked excitatory postsynaptic current (paired t-test, p = 0.0131). We also found that OFC-NPSR1 neuron activity was strongly associated with delivery of a conditioned stimulus that predicted delivery of a sucrose pellet (paired t-test, p = 0.0044). We also found during and FR1 task where mice could nosepoke for fentanyl access, NPSR1 activity was enhanced during drug cue delivery (paired t-test, p = 0.0475). In addition, the OFC-NPSR1 neuron activity dipped during consumption of fentanyl reward (paired t-test, p = 0.0001) showing an interesting pattern of activity across cue and drug delivery. Finally, in our preliminary 2p experiments, we are observing that there is mixed heterogeneity in the responsivity of OFC-NPSR1 neurons during delivery of stimuli with varying modalities. Conclusions: A better understanding of potential targets that are important to anxiety and substance use disorder are necessary for the development of more effective therapeutic strategies. Examining the mechanisms of less understood neuromodulators, such as NPS in driving reward-seeking behavior and anxiety-like phenotypes addresses this behavioral connection. Future work aims to combine Crispr with fiber photometry to gauge NPS transmission’s role in the calcium dynamics observed in the OFC-NPSR1 neurons. These experiments serve as the groundwork for developing our understanding of the basic neurobiology of the NPS system in reward. This work was supported by NIH grants F32DA0055480 (Kasey Girven), DA007278, and MH112355. Keywords: Neuropeptide S, Locus Coeruelus, Orbitofrontal Cortex, 2-Photon Techniques, Electrophysiology Disclosure: Nothing to disclose. P631. Neural Basis of Reward Pursuit and Threat Avoidance Blair Vail, Seth Koenig, Benjamin Hayden, Seng Bum Michael Yoo, David Darrow, Alexander Herman* The University of Minnesota, Minneapolis, Minnesota, United States Background: Background: Reward pursuit and threat avoidance comprise fundamental building blocks of behavior that are impacted in a range of neuropsychiatric disorders. The orbitofrontal cortex (OFC), amygdala, and anterior cingulate cortex (ACC) have all been implicated in networks differentiating reward and threat, but these are rarely assessed in naturalistic tasks or using invasive recording in humans. The objective of our study was to determine whether activity in these areas differs between dynamic states of reward pursuit and threat avoidance in humans. Methods: Electrophysiology data were recorded from electrodes implanted in the OFC, amygdala, and ACC of epilepsy patients while they performed a naturalistic pursuit/avoidance task based on the “Pacman” video game. In one condition they had to pursue “prey”, and in another they had to both pursue “prey” and avoid “predators”. For each condition, wavelet convolution was performed to obtain induced power at frequencies from 2 to 150 Hz over a one second window from 500 ms before the appearance of predators and prey to 1000 ms after this event. We then subjected each time-frequency (TF) point in this window to a linear mixed effects model with induced power as a function of predator/prey condition, and with subjects and channels as random effects. We corrected for multiple comparisons within areas using permutation testing and between areas with a Bonferroni correction. Clusters of adjacent TF points with a corrected p value of < 0.05 were then treated as significant differences between predator and prey conditions. Results:. In OFC (5 subjects, 41 channels), theta power (4-8 Hz) was elevated in the predator condition compared to the prey condition at approximately 365-625 ms after the appearance of prey/predators, and again at 650-1000 ms; delta power (2-4 Hz) was elevated at 565-1000 ms, and beta (12-30 Hz) and gamma power (30-150 Hz) were reduced at 775-885 ms. In the amygdala (5 subjects, 32 channels), delta and beta power were elevated in the predator condition at approximately 625-895 ms and 685-800 ms, respectively. In ACC (4 subjects, 18 channels), alpha (8-12 Hz) and beta power were reduced in the predator condition at 200-270 ms, as were beta and gamma power at 330-450 ms, and gamma power again at 770-815 ms. Theta and delta power in ACC were increased in the predator condition at 585-810 ms and 845-1000 ms, respectively. Conclusions: The multiple clusters of significantly different activity between the prey and predator conditions in the Pacman task suggest that the OFC, amygdala, and ACC all participate in differentiating between reward pursuit and threat avoidance under dynamic, naturalistic conditions. The naturalistic task and human intracranial data give these results an unprecedented level of ecological validity. Keywords: Intracranial EEG, Approach/Avoidance, Prefrontal Cortex Disclosure: Nothing to disclose. P632. Divergent Relationships Between Excitatory and Inhibitory Responses in the Nucleus Accumbens and Sign Tracking / Goal Tracking Behavior Kyle Duffer, Sara Morrison* University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: The ability of reward-associated cues to produce approach and/or interaction varies widely among individuals. For example, if a cue (e.g., extension of a lever) predicts a reward in a different location (e.g., a sugar pellet delivered to a food cup), some rats will preferentially approach and interact with the lever – a behavior known as sign tracking (ST) – and others will approach the site of reward delivery, a behavior known as goal tracking (GT). A propensity towards ST has been linked to susceptibility to drug-taking, relapse, and related behaviors, and it has been shown that sign trackers and goal trackers have distinct patterns of NAc dopamine release during the acquisition of reward-seeking behavior. Moreover, we and others have shown that ST is relatively resistant to extinction and reward devaluation. We have previously shown (Gillis and Morrison, 2019) that cue-evoked excitations in the NAc encode the vigor of both ST and GT behavior. At the same time, among sign tracker individuals (but not goal tracker individuals), reward-related excitations showed a sharp decrease over the course of training, which may reflect a decreasing reward prediction error encoded by phasic dopamine release. However, a substantial subset of NAc neurons respond to reward-predictive cues with inhibitions that, similar to excitations, encode the vigor of approach during instrumental tasks (Morrison et al., 2017). Therefore, we set out to compare the patterns of excitatory and inhibitory cue- and reward-evoked responses during the acquisition of ST and GT in sign tracker and goal tracker individuals. In addition, we wished to test the hypothesis that cue-evoked responses, whether excitatory or inhibitory, would be relatively insensitive to reward devaluation in sign tracker individuals. Methods: Using custom-constructed microelectrode arrays, we recorded the activity of individual neurons in the NAc core throughout the acquisition and expression of ST and/or GT behavior. We also recorded neural responses during extinction sessions following either reward devaluation or sham devaluation. Subjects were male and female Long-Evans rats. Results: Similar to excitatory cue-evoked responses, we found that both the proportion of inhibited cells and the magnitude of inhibitory responses increase over the course of learning; this is true for both sign tracker and goal tracker individuals. Unlike cue-excited neurons, reward responses in cue-inhibited neurons, which are predominantly (but not exclusively) inhibitory, do not diverge between sign tracker and goal tracker individuals. During extinction sessions without reward devaluation, some cue-evoked excitations appear to “extinguish” their response, but others do not; we found that this is also the case for cue-evoked inhibitions. Following reward devaluation, on the other hand, the proportion of cue-excited neurons drastically decreased while the proportion of cue-inhibited neurons and non-responsive neurons increased. This difference was apparent even among the first few cue presentations following reward devaluation. Finally, we were surprised to find that some cells retained robust excitatory or inhibitory responses to omitted reward during extinction sessions, even following reward devaluation. Conclusions: Inhibitory responses in the NAc, like excitations, evolve during learning; but our findings suggest that, unlike excitations, their activity does not directly reflect a dopaminergic reward prediction error signal. This is consistent with a previous report that cue-evoked inhibitions, unlike excitations, are not sensitive to dopamine receptor antagonists (du Hoffman and Nicola, 2014). Interestingly, some inhibitory responses can be “unmasked” by dopamine receptor blockade; our results suggest that a similar phenomenon may occur when approach behaviors are suppressed following extinction or reward devaluation. Finally, the preservation in some neurons of responses to cues and/or at the time of omitted reward might provide a substrate for the maintenance of cue-reward associations and recovery/relapse after extinction. Keywords: Nucleus Accumbens, Single-Unit Electrophysiology in Vivo, Sign-Tracking, Reward Devaluation, Extinction Disclosure: Nothing to disclose. P633. Brain Mechanisms of Hypersensitivity to Distress in Substance Use Disorder Kristen Platt, Francesca Filbey The University of Texas at Dallas, Dallas, Texas, United States Background: Negative valence systems, such as stress, depression and anxiety, are known to increase one’s risk for the development of substance use disorder (SUD) and vulnerability for SUD relapse; hence, the high comorbidity between SUD and mood disorders. Based on the scientific premise that the lateral habenula (LHb) encodes negative motivational stimuli, thereby, playing a critical role in regulating mood, reward and valence-based learning, we tested a model that hypersensitivity within the brain’s lateral habenula (LHb) is related to negative valence and substance use disorder (SUD). We hypothesized that increased functional connectivity between the LHb and areas reported during motivational processing (Filbey et al., 2013) will be associated with greater stress and negative mood symptoms as well as negative motivation. Furthermore, we tested that severity of substance use mediates the relationship between LHb connectivity and negative valence and motivation. Methods: We used fMRI resting state data from 69 adults with self-reported substance use (age: M = 27, SD = 9; males= 45). Negative valence was measured using the Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), the Perceived Stress Scale (PSS), and the Early Life Stress (ELS). The Monetary Incentive Delay (MID) task was used to measure negative motivation. LHb whole brain resting state functional connectivity (rsFC) was evaluated using left and right LHb seeds in CONN Toolbox. Regression analyses were conducted that included connectivity z-scores [right and left LHb seed connectivity with a priori ROIs - right frontal pole (rFP), right cingulate gyrus (rCg), right anterior cingulate gyrus (rACG), right fusiform gyrus (rFFG), right postcentral gyrus (rPoCG), right putamen (rPU), left cerebellum (lCBM), left parahippocampus (lPHP), and left amygdala (lAMG)], BDI, BAI, PSS, and ELS as well as substance use. Mediation analyses were then conducted to determine whether LHb connectivity mediates the relationship between negative valence and symptoms of SUD from the Structural Clinical Interview for DSM-5 (SCID). Results: The regression analyses found multiple significant correlations between LHb rsFC and measures of negative valence. BAI was significantly positively correlated with right LHb-right rFP rsFC (r2 = 0.06, p = 0.05). PSS scores showed a significant positive correlation with left LHb-rFP rsFC (r2 = 0.09, p = 0.05), as well left LHb-rFFG rsFC (r2 = 0.10, p = 0.05). ELS scores were significantly positively correlated with right LHb-lCBM rsFC (r2 = 0.09, p = 0.04) and left LHb-lCBM rsFC (r2 = 0.08, p = 0.05). BDI scores were not correlated with LHb rsFC. LHb rsFC was also positively correlated with negative motivation such that number of successful punishment trials during the MID task was significantly positively correlated with right LHb-lCBM (r2 = 0.06, p = 0.05), right LHb-left LHb (r2 = 0.14, p = 0.01), left LHb-rFP (r2 = 0.07, p = 0.04), and left LHb-lAMG rsFC(r2 = 0.08, p = 0.03). Mediation analyses of the above significant correlations found significant mediating effect of symptoms of SUD. Alcohol use disorder mediated the relationship between (1) PSS and left LHb-rFP rsFC (r2 = 0.24, F(3,47)= 5.04, p = 0.004) and (2) PSS and left LHb-rFFG rsFC (r2 = 0.25, F(3,53)= 5.27, p = 0.003). Symptoms of hallucinogen use disorder mediated the relationship between ELS scores and right LHb-lCBM rsFC (r2 = 0.15, F(3,58)= 3.32, p = 0.03), and between ELS scores and right LHb-lCBM rsFC (r2 = 0.141, F(3,58)= 3.17, p = 0.03). Symptoms of other SUD mediated the relationship between PSS and left LHb-rFP rsFC (r2 = 0.14, F(3,53)= 2.75, p = 0.05), and PSS and left LHb-rFFG rsFC (r2 = 0.14, F(3,53)= 2.9, p = 0.04). SCID scores for alcohol use disorder (r2 = 0.21, F(3,53)= 4.62, p = 0.01), cannabis use disorder (r2 = 0.18, F(3,59)=4.17, p = 0.01), opioid use disorder (r2 = 0.17, F(3,58)= 4, p = 0.01), cocaine use disorder (r2 = 0.16, F(3,58)= 3.77, p = 0.02), hallucinogen use disorder (r2 = 0.16, F(3,58)= 4.17, p = 0.01), and other SUD (r2 = 0.1, F(3, 58)= 3.82, p = 0.01) mediated the relationship between negative motivation via response during punishment trials and left LHb-right LHb rsFC. Other SUD scores further mediated the relationship between left LHb and the rFP and response during negative motivation (r2 = 0.12, F(3,58)= 2.83, p = 0.05). Conclusions: These results provide evidence for the relationship between LHb intrinsic network connectivity with brain motivational networks, negative valence, and SUDs. Our findings further suggest that SUDs exacerbate the disruptions in negative valence systems and LHb connectivity. Future studies should examine the direction of these effects. Keywords: Alcohol and Substance Use Disorders, Negative Valence System, Resting State Functional Connectivity, Lateral Habenula Disclosure: Nothing to disclose. P634. Dopamine Release in the Nucleus Accumbens Core Controls Behavior in Response to Aversive Stimuli Stephanie Cajigas, Jennifer Zachry, Patrick Melugin, Jennifer Tat, Shannon Kelly, Munir Kutlu, Erin Calipari Vanderbilt University, Nashville, Tennessee, United States Background: Understanding how behavior is controlled by environmental stimuli is critical to understanding how and why behavior occurs, as well as how this is dysregulated in disease. The addiction field has focused on understanding how drugs drive future behavior (i.e. positive reinforcement), how repeated drug use results in deficits in these processes, and how associations between drugs and environmental stimuli (classical conditioning, conditioned reinforcement) underlie craving and relapse. These behavioral processes, as well as the interactions between them, are critical drivers of substance use disorders, making the understanding of neural circuits that underlie these behaviors a major goal of the field. Therefore, by manipulating the environment of subjects (i.e. mice) we can begin to understand what and how decisions are made and the deficits that arise with disease. Methods: Here we utilized fear conditioning and negative reinforcement paradigms to investigate dopamine release patterns during passive and active behavioral responses while keeping stimulus valence constant (i.e. negatively valenced). The optical sensor dLight in conjunction with in-vivo fiber photometry in the NAc core was used to monitor real-time dopamine responses during each behavioral paradigm. We timestamp each component of the behavior task (cues, shocks, and conditioned responses). This allows us to obtain precise temporal information about when behavior occurred along with a custom pipeline for robust and reproducible analysis. Sample sizes range from 2-5 mice depending on the experiment, and included both males and females. Results: In the first experiment, we modulated the duration of a discriminative cue (the cue that signaled that responses would be reinforced) in a negative reinforcement task and recorded dopamine responses in the NAc. When the cue was shorter, animals learned more slowly, and the dopamine signal to this cue was decreased. Once animals learned to respond in the task the dopamine response increased to the cue. We first hypothesized that this was due to learning about the escapable nature of the shock. Thus, we compared a fear conditioning task – where the shocks were inescapable – with animals that had learned negative reinforcement. We did find that dopamine was reduced following a fear cue (where the shock presentation was inescapable), and was increased to cues signaling negative reinforcement (where the shock was escapable). However, introducing novel and neutral stimuli into the fear conditioning context caused an increase in dopamine in response to the fear cue – even though the shock remained inescapable. Thus, the directionality of the dopamine response changed in the absence of a change in the contingencies or predictions. Additionally, the temporal dynamics of the dopamine response changed in the presence of novelty where dopamine levels remained elevated for more than 30 seconds. Conclusions: Dopamine signaling is complex and more than a valence or prediction error detector as the direction of its response to aversive cues differed based on auxiliary information present in the environment (even when that information did not signal changes in predictions or changes in value). Not only does this dopamine signal appear to track novelty in a temporally defined window, but how long dopamine remains elevated is also modulated by the novelty of introduced stimuli. Critically, these cue-induced elevations in dopamine are well-known to modulate the speed of behavior acquisition which holds important implications for dopamine-based disease states such as addiction and how dopamine may be playing a role in observed deficits in cognition. Keywords: Addiction, Dopamine, Nucleus Accumbens, Negative Reinforcement, Fiber Photometry Disclosure: Nothing to disclose. P635. Associations Between the Endocannabinoid System and Drug Craving in Chronic Cocaine Users Sara Kroll, Ann-Kathrin Kexel, Carola Boost, Franziska Pahlisch, Cathrin Rohleder, Franz-Markus Leweke, Boris Quednow Psychiatric University Hospital Zurich, University of Zurich, Zurich, Switzerland Background: Stress has been proposed as a crucial risk factor for developing drug addiction and relapse, consequently maintaining the vicious circle of substance use disorder. Recent evidence from preclinical and clinical studies suggests that the endocannabinoid system (ECS), directly or indirectly activated by the endocannabinoids anandamide (AEA), 2-arachidonoyglycerol (2-AG), palmitoyl- ethanolamide (PEA), and oleoylethanolamide (OEA) binding at the type-1 cannabinoid (CB1) receptors, plays a key modulatory role in stress vulnerability and resilience. Accordingly, activation of the ECS has been suggested to be directly linked to stress- induced substance craving and reward in animals and humans due to its stress-buffering effects. Although recent preclinical studies indicate the role of the ECS in substance use disorder, human translational studies have been limited so far. Therefore, the present study aimed to assess potential alterations of the ECS in chronic cocaine users. Methods: We used plasma samples of 195 participants (women and men) from Zurich Cocaine Cognition Study (ZuCo2St). We compared plasma concentrations of the endocannabinoids: 2-AG, AEA, OEA, and PEA between cocaine-naïve control group (n = 92) and chronic cocaine users (n = 103; 69 recreational cocaine users [RCU] and 34 dependent cocaine users [DCU]). Cocaine dependence was diagnosed following the DSM-IV criteria, with DCU fulfilling and RCU not meeting these criteria. Further inclusion criteria for the chronic cocaine users were cocaine use of at least 1 g per month, cocaine as primary used illegal substance, and a current abstinence duration <6 months. Exclusion criteria for the user groups were past or current use of opioids, a polytoxic substance use pattern, and an axis-I DSM-IV adult psychiatric disorder with the exception of cocaine, cannabis, and alcohol use disorder as well as history of affective disorders and attention deficit hyperactivity disorder (ADHD). Exclusion criteria for the control group were acute or history of any axis-I DSM-IV psychiatric disorder. Endocannabinoid plasma analysis was performed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyses of covariance (ANCOVAs) were used, with Group as the fixed factor, to control for well-known confounding variables sex, age, and acute cannabis use (THC and CBD plasma levels). For our secondary and exploratory analyses, we used stepwise linear regression analyses, forward and backward method, within chronic cocaine users to investigate the association between endocannabinoid plasma concentrations and specific subjective and objective cocaine use variables. For significant findings, we further checked if the results are driven by the cocaine user GROUP (DCU or RCU) by adding GROUP as an additional regressor into the linear regression analysis in a second step (model 2). The statistical comparisons were carried out with a significance level of p < 0.05 (two-tailed). Results: ANCOVAs showed a significant group effect for 2-AG (F(2,188)=3.4, p = 0.035, eta2 = 0.04) but no group differences were found for AEA, PEA, and OEA (p’s > 0.176). More precisely, post-hoc pairwise comparisons identified the highest 2-AG plasma concentrations specifically in the DCU group compared to controls (F(1,20)=6.6, p = 0.012, eta2 = 0.05) and on a trend-level to RCU (F(1,97)=3.7, p = 0.059, eta2 = 0.04), whereas the control and RCU group did not differ in 2-AG plasma levels (F(1,155)=1.3, p = 0.715). Within chronic cocaine users (RCU and DCU), 2-AG was a significant regressor for cocaine craving (F(1, 101)=7.7, p = 0.006, R2 = 0.07). Model 2 of the linear regression analysis including the cocaine user GROUP did not significantly explain more variance of the dependent variable (F(1, 100)=1.6, p = 0.216, R2 = 0.09) indicating model 1 including only 2-AG as a predictor for cocaine craving as the best fit. Conclusions: Elevated 2-AG baseline levels in the DCU group might indicate higher reward response to cocaine in this group, which may lead to increased vulnerability of cocaine dependence. Moreover, our results suggest that elevated baseline 2-AG plasma levels might have stress-buffering effects in terms of cocaine craving, which was observed overall chronic cocaine users and not specific to cocaine dependence. Therefore, the ECS might be a promising pharmaco-therapeutic target for novel treatments of cocaine use disorder to prevent stress-induced drug relapse and improve abstinence in the context of cocaine craving. Interestingly, our current preliminary findings of endocannabinoid plasma concentrations in non-medical prescription opioid users yielded elevated AEA, OEA, and PEA baseline levels in chronic opioid users (n = 23) compared to healthy controls (n = 29), but no differences in 2-AG levels. These results indicate distinct alterations of the ECS in cocaine and opioid users suggesting substance specific targets of the ECS in a pharmaco-therapeutic context. Our preliminary endocannabinoid findings in opioid users will be discussed in more detail at the ACNP meeting. Keywords: Endocannabinoids, Cocaine Addiction, Addiction, Craving Disclosure: Nothing to disclose. P636. Astrocyte Activities in the External Globus Pallidus Regulate Action-Selection Strategies in Reward-Seeking Behaviors Sa-Ik Hong, Shinwoo Kang, Seungwoo Kang, Minryung Song, Minsu Yang, Matthew Baker, Jeyeon Lee, Sang Wan Lee, Doo-Sup Choi Mayo Clinic College of Medicine and Science, Rochester, Minnesota, United States Background: In the striatopallidal circuits, the external globus pallidus (GPe) has been considered an integrative hub for behavioral flexibility in reward-related behaviors because it coordinates the top-down neurotransmission from the two distinctive dorsal striatum regions, the dorsomedial and dorsolateral striatum (DMS and DLS). These regions are the central neural substrates for goal-directed and habitual reward-seeking behaviors, respectively. Indeed, the GPe redistributes the dorsal striatal inhibitory GABAergic tone and signals through the fast-spiking prototypical and slow-spiking arkypallidal neurons in the GPe to the downstream brain region. Interestingly, recent studies demonstrate that the GPe contains abundant astrocytes, which are distinguishable to the neighboring brain regions, and its astrocytes directly modulate the neuronal activities in the GPe, leading to the consequent behavioral changes. However, the role of GPe astrocytes in goal-directed and habitual action strategies is poorly understood. Methods: We utilized the two operant tasks upon effort- or time-based reward (10▯▯l of 20% sucrose solution) delivery to establish the dominance of goal-directed behavior or habit. Then, we employed in vivo calcium imaging to probe the temporal dynamics of GPe astrocytes during goal-directed and habitual learnings. Subsequently, using a support vector machine, we tested whether the type of operant tasks is predictable with GPe astrocytic calcium dynamics. Lastly, we determined whether enhancing the calcium signaling in GPe astrocytes and attentional stimulus increase behavioral flexibility. Results: Using in vivo calcium imaging with fiber-photometry, we found significantly silenced GPe astrocytic activity during habitual learning compared to goal-directed learning. Moreover, the support vector machine (SVM) analysis predicted whether mice perform goal-directed or habitual behaviors. Interestingly, chemogenetic activation of the astrocytes dampened GPe neuronal firing and increased goal-directed reward-seeking behavior. Similarly, attentional stimuli shifted the habitual to goal-directed behaviors, indicating increased behavioral flexibility. Conclusions: Our study demonstrated that GPe astrocytic dynamics are reduced during habit learning. Chemogenetic and attentional stimulation can prevent the dominance of habitual reward-seeking, at least partly through the activation of the Gq pathway in GPe astrocytes. This novel finding may help to treat maladaptive habit-related disorders such as addiction and obsessive-compulsive disorders. Keywords: Astrocyte, GPe, Habit, Goal-Directed, Reward Disclosure: Peptron Inc: Advisory Board (Self). P637. Lifespan Development of Thalamic Nuclei and Differential Sensitivity to Alcohol Use Anna Huang, Qiaochu Jiang, Kaidi Kang, Alexandra Moussa-Tooks, Baxter Rogers, Simon Vandekar, Neil Woodward Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: The thalamus is a heterogeneous structure composed of multiple nuclei with critical roles in sensorimotor and cognitive function, and guiding cortical development. Abnormal thalamic morphology has been implicated in the pathophysiology of multiple neurodevelopmental and aging disorders (e.g. Schizophrenia and Alzheimer’s Disease). Substance use is highly comorbid and has been identified as a risk and maintenance factor, and emerging preclinical and clinical studies also indicate the thalamus may be especially sensitive to the deleterious effects of substance use, particularly in Alcohol Use Disorder (AUD). To inform the etiology of thalamic dysfunction in psychiatric and neurological disorders, and clarify the effects of alcohol use and thalamic nuclei, the current study 1) characterized age effects on thalamic nuclei volumes across the lifespan, 2) quantified associations between thalamic nuclei and cognitive functions and 3) determined whether thalamic nuclei were differentially affected in AUD. Methods: The cross-sectional Human Connectome Project Lifespan dataset (n = 1952, age range 5-100, 1071 Female) was used to establish age effects on thalamic nuclei volumes. Linear and curvilinear models of age were compared for nine nuclei of the thalamus spanning higher-order and sensorimotor nuclei, controlling for sex, sibling relationship, and total intracranial volume. The association between thalamic nuclei volumes and cognitive function as measured by the NIH Toolbox were also examined. The Enhanced Nathan Kline Institute – Rockland Sample (NKI-RS), a large-scale, cross-sectional, community ascertained cohort was used to determine the effects of AUD on thalamic structure (AUD group: n = 45, mean age = 44.9, 27 Female, Healthy Controls: n = 45, mean age = 44.4, 31 Female). Results: Thalamic nuclei volumes showed curvilinear effects of age characterized by three patterns. The first pattern, present in mediodorsal and centromedian nuclei showed decreasing volumes in youth that stabilized in young adulthood and further decreased in older adults. The second pattern, observed in the pulvinar and medial geniculate nuclei showed relatively steady decrease from youth to older adults. The final pattern observed in the ventral posterolateral, ventrolateral, ventral anterior and lateral geniculate nuclei showed stable volumes in youth and young adults followed by a prominent decrease in older adults. Mediodorsal and pulvinar nuclei volumes showed a positive association with executive function scores (F = 9.365, p < 0.01 and F = 13.570, p < 0.001 respectively) and this effect did not show any interactions with age. AUD was associated with smaller thalamus volumes compared to healthy controls (F = 5.065, p < 0.05, ηp2 = 0.057), due to smaller right pulvinar volumes (F = 8.152, p < 0.01, ηp2 = 0.088). Conclusions: Our finding that thalamic nuclei have 1) different effects of age on volumes, 2) different associations with executive functions and 3) differential effects in AUD all highlight the importance of examining individual thalamic nuclei when studying thalamus abnormalities in psychopathology. The most prominent differences in age effects on thalamic nuclei volumes were observed in youth and young adults, suggesting that it is especially important to examine individual thalamic nuclei in neurodevelopmental disorders. Keywords: Thalamus, Brain Development, Ageing, Subcortical Volume, Alcohol Use Disorder Disclosure: Nothing to disclose. P638. Social Navigation in Individuals With Cocaine Use Disorder and Childhood Trauma Matthew Schafer, Philip Kamilar-Britt, Vyoma Sahani, Yasmin L. Hurd, Daniela Schiller, Keren Bachi Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Navigating social interactions depends on social mapping which represents the spatial-like organization and use of abstract social information, similar to that of physical space. It is hypothesized that interaction between the hippocampus and prefrontal cortex may guide social navigation similar to their role in physical navigation with the hippocampus building maps and the prefrontal cortex directing map-based decision-making. Cocaine use disorder (CUD) and childhood trauma both show concurrent social dysfunction and neural dysfunction in these regions with prefrontal dysfunction characteristic of CUD and hippocampal dysfunction common in childhood trauma. Here, we use a naturalistic social interaction task to test whether social relationships are tracked differently in navigation-related regions in the prefrontal cortex of individuals with CUD relative to healthy controls (HC), and differently in the hippocampus as a function of childhood trauma. Methods: Demographically matched groups of CUD (n = 30) and HC (n = 33) completed the social interaction game during functional magnetic resonance imaging (fMRI). In the task, participants interact with fictional characters to accomplish social goals (e.g., find a job). Unbeknownst to participants, their decisions with each character were modeled as a series of locations through a social affiliation by power space – the “social navigation” of relationships. To probe the neural correlates of this navigation-like behavior, subject-level fMRI activity was regressed onto the angles from the point-of-view of the participant to the characters’ locations (the characters’ dynamic power to affiliation ratio, relative to the participant). We used regions-of-interest analyses to test two predictions. 1) We predicted that tracking of social locations would be reduced in prefrontal cortex in people with CUD - specifically in right inferior frontal gyrus, a region involved in processing navigational demands (Javadi et al., 2017). To test this, we compared the groups with a t-contrasts in a flexible factorial model, as well as with group classification (a Decision Tree with leave one out cross-validation and permutation-based significance testing). 2) We predicted that individuals with higher childhood trauma would have a reduced social location effect in left hippocampus, given the region’s sensitivity to stress and its role in tracking the social angles in this task (Tavares et al., 2015). To test this, we modeled the interaction between childhood trauma (Childhood Trauma Questionnaire score) and group (CUD v. HC) in the same factorial model and used t-contrasts. Results: As predicted, voxels in the right inferior frontal gyrus showed a smaller parametric modulation effect in CUD than HC (peak voxel FWE-corrected P < 0.05). Group classification was well above chance using this region (mean accuracy=66%, P < 0.001) but below chance using a control region (motor cortex). Also as predicted, childhood trauma negatively correlated with the activity of a left hippocampus region previously shown to track abstract social locations. Regardless of CUD status, there was negative hippocampus effect (peak voxel FWE-corrected P < 0.01). Conclusions: Our findings suggest that both CUD-related prefrontal dysfunction and childhood trauma-related hippocampal dysfunction extend to the neural tracking of social relationships. Childhood trauma had similar reductions in left hippocampal effects in both CUD and HC participants, suggesting that the effect of trauma is separable from the effects of cocaine addiction in representing social relationships. CUD and childhood trauma may thus impact the representation of social navigation in different but potentially complementary ways. Further research is needed to fully characterize these effects – such as the interaction between these regions in both CUD and childhood trauma, as well as implications for real-world social behavior. Keywords: Social Cognition, Cocaine Use Disorder, Functional MRI (fMRI), Childhood Trauma, Cognitive Neuroscience Disclosure: Nothing to disclose. P639. Metabotropic Glutamate Receptor Subtype 5 and Memory in Methamphetamine Use Disorder Megan McClintick, Robert Kessler, Tarannom Mahmoudie, Daicia Allen, Olivia Jarrett, Shane Marohnic, Dara Ghahremani, Jean Baptiste Pochon, Judah Farahi, Edwin Partiai, Larissa Mooney, Andrew Dean, Mark Mandelkern, Edythe London University of California, Los Angeles, Los Angeles, California, United States Background: The group-I metabotropic glutamate receptor subtype 5 (mGlu5) is implicated in methamphetamine (MA) intake and preference. Mice selectively bred for high methamphetamine oral consumption exhibit higher mGlu5 levels in the nucleus accumbens than those bred for low MA drinking (Szumlinski et al., 2017). Expression of mGlu5 receptors in the nucleus accumbens correlates with MA-conditioned place preference, and repeated daily doses of MA increase mGlu5 receptor function, indexed by agonist-stimulated glutamate release in the nucleus accumbens of mice (Szumlinski et al., 2017). In humans, mGlu5 receptor availability is lower in the striatum and orbitofrontal cortex (OFC) in individuals with cocaine dependence as compared to healthy control subjects (Martinez et al., 2014). Associations between cognitive function and mGlu5 have also been observed. A positive association between hippocampal mGlu5 binding and episodic memory was observed in a PET study comparing adults with Alzheimer’s Disease and controls (Mecca et al., 2020). mGlu5 positive allosteric modulators also alleviated MA-induced memory deficits in rats (Reichel et al., 2011). Associations between mGlu5 receptors, cognitive function, and MA use have not been studied in human subjects. Methods: Twelve individuals with severe Methamphetamine Use Disorder (MUD; 11 men and 1 woman) and fourteen individuals with no history of drug use (6 men and 8 women) participated in this study in which mglu5 glutamate receptor volumes of distribution (VT) were measured in brain with the radiotracer [18 F]FPEB. Participants with MUD were in outpatient (n = 2) or residential (n = 10) treatment programs and were abstinent from substances of abuse for fewer than 70 days (mean 41 days). All participants completed a drug use questionnaire that assessed lifetime and recent drug use. Those with MUD self-reported their craving for methamphetamine on the Methamphetamine Craving Questionnaire – Brief (MCQ-Brief). Verbal memory was assessed on the Rey Auditory Verbal Learning Test (RAVLT; Rey, 1964), with the variable of interest being immediate memory (age-adjusted Z-score trials A1-A5 total score). To measure mglu5 volumes of distribution (VT), [18 F]FPEB was administered intravenously (5 mCi bolus/infusion [kbol 205 min]). PET data were acquired on a Siemens Biograph mCT, combined into three images containing data averaged across 10 minutes and corrected for motion with FSL MCFLIRT. The images were co-registered with FSL FLIRT to T1-weighted structural MRI scans. Volumes of interest (VOIs) were derived from MPRAGE images using FSL FIRST (left and right hippocampus) and manually placed masks (ventral striatum, lateral OFC, medial OFC). Time-activity curves provided average tissue (brain) activity in each VOI. Venous blood samples collected at 10-minute intervals during PET data acquisition were used to calculate average decay-corrected plasma activity from 109 to 139 min after the start of the radiotracer, when radioactivity in the brain was at a steady state. VT was calculated as the ratio of activity in brain to plasma activity of the free tracer. Group differences in mGlu5 VT in bilateral ventral striatum, lateral OFC and medial OFC VT and immediate memory were tested with unpaired t tests. Associations of MA use and craving with VT in the ventral striatum, lateral OFC, and medial OFC were tested with correlational analyses controlling for cigarettes smoked per day and age. The association of immediate memory and right and left hippocampal VT were tested with correlational analyses, controlling for cigarettes smoked per day. Significant sex differences were not observed in RAVLT scores, self-report measures, or VT; thus sex was not included as a covariate. Results: Individuals with MUD exhibited lower ventral striatal (t = 2.5, p = 0.02), lateral OFC (t = 2.3, p = 0.04), and medial OFC (t = 2.2, p = 0.04) mGlu5 VT than control subjects. MUD VT in these regions was 17-19% lower than control values. Self-reported craving, duration of abstinence, and frequency of methamphetamine use before entering treatment (days per month) did not correlate with VT, though lifetime exposure (years of use X grams per day) approached significant association (p = 0.07) with ventral striatal VT. Immediate memory scores were significantly lower in MUD versus control groups (t = 2.1, p = 0.04), and correlated with left hippocampal (r = 0.60, p = 0.0019) and right hippocampal (r = 0.60, p = 0.0019) mGlu5 VT. Secondary analyses by group found an association between hippocampal VT and immediate memory only in the controls (left hippocampus r = 0.75, p = 0.0049; right hippocampus r = 0.70, p = 0.012). Conclusions: Severe MUD is associated with lower ventral striatal and OFC mGlu5 VT than controls, similar to the difference between participants with Cocaine Dependence and controls observed using a different mGlu5 receptor radiotracer (Martinez et al., 2014). A deficit in mGlu5 expression may impair cognitive function during early abstinence from chronic methamphetamine use, and thereby could interfere with cognitive-based treatments for MUD. Keywords: Metabotropic Glutamate Receptor 5 (mglu5), Methamphetamine Use Disorder, Verbal Memory Disclosure: Nothing to disclose. P640. Ventral Striatal Activation During Anticipation of Smoking Rewards—But Not Monetary Rewards—Predicts Relapse During a Quit Attempt: Exploratory Analyses From a Randomized Clinical Trial Maggie Sweitzer, Jason Oliver, Angela Pisoni, Ryann Giummo, Stacey Daughters, Scott Kollins, F. Joseph McClernon Duke University Medical Center, Durham, North Carolina, United States Background: Cigarette smoking remains a leading cause of death, and the majority of quit attempts end in relapse. Growing evidence supports the role of a “hijacked” reward system, in which chronic smoking is associated with both hypersensitivity to smoking and related cues and hyposensitivity to alternative reinforcers. In our own previous work we found that withdrawal from smoking was associated with an increase in ventral striatal (VS) activation to smoking reward, and a parallel decrease in activation to monetary reward, suggesting a clear dissociation and supporting the hijacking model. However, a recent meta-analysis found that decreased activation to both smoking cues and non-smoking reward cues within the striatum among smokers was associated with greater severity of nicotine dependence, suggesting an overall deficit in reward-related functioning. Direct comparisons between smoking and non-smoking reward processing are challenging due to fundamentally different fMRI task paradigms typically used for each reward type (smoking cue reactivity versus monetary incentive delay tasks). As such, it is unclear whether heightened sensitivity to smoking reward, deficits in sensitivity to non-drug rewards, or a global deficit in reward processing may be most critical to maintaining smoking behavior. In the present analysis, we leveraged existing data from a small randomized clinical trial with a baseline fMRI scan to examine VS activation during anticipation of both smoking and monetary rewards as a predictor of relapse during a quit attempt. Methods: Thirty-five daily smokers completed an fMRI scan and were then randomized to one of two treatment conditions. During the fMRI scans, participants completed a previously validated rewarded guessing task designed to evaluate striatal BOLD response during anticipation of both smoking and monetary rewards (both delivered immediately after the scan). In order to mimic the early stages of the quit attempt, participants abstained from smoking for 24 hours prior to the scan, but were provided with a 21 or 14 mg nicotine patch. The primary purpose of the study was to examine the effects of a combined behavioral and pharmacological intervention on reward-related brain function and quit outcomes. Participants in both conditions were provided with very low nicotine content Spectrum cigarettes and nicotine patches for 4 weeks prior to their quit date. They also completed 8 behavioral treatment sessions (4 pre-quit and 4 post-quit) consisting of either behavioral activation or a health education control condition. Following the quit date, participants were provided with a standard 8-week course of nicotine replacement therapy and were followed for 10 weeks to assess quit outcomes. Present analyses examined signal change extracted from within right and left VS regions of interest for the money > neutral anticipation and smoking > neutral anticipation contrasts. Extracted values were entered in survival analyses to predict time to relapse, controlling for age, sex, treatment group, and baseline nicotine dependence. Results: A total of 29 participants had useable fMRI data. Of these, 11 (38%) relapsed during the quit attempt, defined as smoking at least 1 cigarette for 7 consecutive days. An additional 4 participants (14%) withdrew prior to completion and were presumed to have relapsed. Neither money > neutral anticipation nor smoking > neutral anticipation for right or left VS was correlated with baseline smoking characteristics, including cigarettes smoked per day or nicotine dependence. However, smoking > neutral anticipation in the right VS was positively correlated with craving at the time of the scan (r = 0.43, p < 0.05) and with the number of cigarette puffs smoked immediately after the scan (out of a possible 12 puffs earned; r = 0.40 for the right VS and r = 0.43 for the left VS, both p’s < 0.05). In survival analyses, greater right VS activation for smoking > neutral anticipation was associated with greater likelihood of relapse (Odds ratio = 63.4, p < 0.05). This association remained significant when analyses were restricted to those with complete quit outcome data, and when both contrasts (money > neutral and smoking > neutral) for right VS activation were included in the same model. Conclusions: We found that a heightened incentive value of smoking reward reflected in right VS activation—rather than a deficit in non-drug reward processing—was predictive of smoking behavior both immediately after the scan and weeks later during a quit attempt. These findings partially support a reward hijacking model, but indicate a less critical role for non-drug reward processing than seen in our prior work. Given nicotine’s reward-enhancing effects, it is possible that the use of the nicotine patch at the time of the scan and during the quit attempt served to normalize non-drug reward deficits that could otherwise have been present during withdrawal. As such, these preliminary findings could provide a better window into neural processes contributing to relapse when a quit attempt is aided by nicotine replacement therapy. Future work should further characterize reward-related changes in a larger sample and examine potential avenues for attenuating hypersensitivity to smoking reward. Keywords: Reward Anticipation, Nicotine Addiction, Smoking Cessation Disclosure: Nothing to disclose. P641. Speech and Viewing Choice as Naturalistic Markers of Drug-Biased Salience Attribution in Individuals With Heroin Use Disorder Ahmet Ceceli, Sarah King, Greg Kronberg, Natalie McClain, Devarshi Vasa, Nelly Alia-Klein, Rita Goldstein Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Drug addiction is characterized by excessive salience attribution to drug at the expense of nondrug cues. Previously, in a verbal fluency task, individuals with cocaine addiction generated more addiction-related words than healthy controls (HC), illustrating a drug-biased neuropsychological speech-based function. Similarly, in a picture-based choice task that served as an estimate of simulated drug seeking, compared to HC, individuals with cocaine addiction made more choices to view drug images, as predictive of actual drug use outside the lab 6 months later. We tested whether individuals with heroin use disorder (iHUD) exhibited a similar drug bias in speech fluency and behavioral choice. In an entirely novel computational natural language processing (NLP) approach, we further tracked drug bias in salience attribution using the participants’ recounting of a drug-themed movie, assessing correlations with the other measures. Methods: Thirty-two inpatient, medication-stabilized iHUD (age = 40.8 ± 9.0 years, 7 women) and 21 age and sex-matched HC (age = 39.8 ± 10.8 years, 8 women) participated in the study. In the fluency task, participants were instructed to generate as many drug words as possible in 1 min. In the choice task, participants made deterministic selections between two fully-visible heroin, pleasant, unpleasant, or neutral pictures presented side by side. In the movie task, subjects viewed the first 17 minutes of the heroin-themed movie, Trainspotting, during fMRI; their verbal recounting of the movie was recorded immediately after the scan. We transcribed recordings and a priori segmented the movie into drug and nondrug scenes. Independent raters analyzed the proportion of drug vs. nondrug scenes recalled and speech duration. NLP of the speech samples extracted the number of words used, identified frequency of drug-related words based on a word bank derived from all drug words generated in the fluency task, and frequency of first- and third-person pronouns to approximate participants’ attribution of personal relevance. Results: Similar to the patterns in individuals with cocaine addiction, iHUD generated significantly more drug-related words compared to the HC in the fluency task (p = 0.020; no group difference in nondrug words: p = 0.100). In the choice task, consistent with previous results, iHUD made more frequent choices to view drug pictures compared to the HC (p = 0.007). After watching the heroin-related movie, iHUD recalled proportionally more drug compared to nondrug scenes (p = 0.045), with a similar pattern for drug vs. non-drug scene related speech duration (p = 0.024). NLP measures indicated comparable use of number of words between the groups (p = 0.365), but higher drug word frequency (p = 0.022), and higher first-person pronoun use in iHUD compared to HC (p = 0.004), suggesting a more drug-biased and personal recounting of the movie in iHUD. Importantly, the more frequent use of drug words as indicated by NLP, the more the drug vs. pleasant picture choices in iHUD (p = 0.004), suggesting a link between naturalistic measures of drug-biased functioning as potentially predictive of risk for future drug use outside the lab. Conclusions: We demonstrate pronounced drug bias in fluency and choice measures in iHUD, as previously documented in cocaine addiction. Using machine learning, we show that the semantic content of speech samples from matched iHUD and HC indicates a more personal and drug-biased recounting of the movie in the former, despite comparable speech volume. Importantly, these verbal/semantic and choice features were interrelated, potentially of use for identifying at-risk individuals for early prevention efforts. The neural substrates of this drug biased, multi-dimensional behavioral profile remain to be explored. Keywords: Heroin, Computational Neuroscience, Natural Language Processing (NLP), Decision Making, Incentive Salience Disclosure: Nothing to disclose. P642. Leveraging Daily Diary Methods in Alcohol Use Disorder: Influence of Real-World Cue Exposure on Drinking Lindsay Meredith, Carrie Lee, Craig Enders, Erica Grodin, Lara Ray UCLA, Los Angeles, California, United States Background: Alcohol cue reactivity paradigms are frequently used in laboratory settings to capture the effect of alcohol stimuli on measures of craving, mood, physiological response, and future drinking behavior. Yet, a paucity of research has assessed the impact of exposure to alcohol cues in the natural environment, such as through ecological momentary assessment (EMA) or daily diary methods, in clinical samples of alcohol use disorder (AUD). As such, the current study sought to assess, across a two-week period, the daily impact of alcohol-relevant cues in the natural environment on quantity of alcohol intake among non-treatment seeking adults with AUD. Our primary hypothesis for this exploratory analysis is that greater daily exposure to alcohol-relevant cues in the natural environment would predict same-day increases in the number of drinks consumed. We further tested whether one’s degree of exposure to alcohol-relevant cues during the two-week period would predict an increase in average number of drinks consumed. Methods: This study utilized data collected during a two-week randomized clinical trial (NCT03489850) of ibudilast that enrolled 52 non-treatment seeking participants with AUD. During the trial, participants completed three in-person visits and morning daily diary assessments (DDAs). For the 14 days of the trial, participants were asked to complete DDAs each morning to report on their previous day’s experiences in terms of alcohol intake, craving, and exposure to alcohol-relevant cues. Of primary interest, participants reported on their exposure to any of eight alcohol cues in their natural environment. If participants reported alcohol intake, only daily cue exposure before drinking initiation was included, to capture the effect of cues on subsequent alcohol intake. Participants who completed at least one DDA during the study (n = 50: females = 17, males = 33) were included in these analyses. A daily total cue exposure variable was created (range 0 – 8) and served as the main predictor. Important level-2 covariates included in the model were biological sex, AUD severity (DSM-5 AUD symptom count), and medication condition. As this data comes from a micro-longitudinal trial with repeated measures, a multilevel model with random intercepts and random effect of daily cue exposure was conducted to account for clustering of observations within participants. Model-based multiple imputation was used to treat missing data prior to fitting the regression model, as this procedure readily accommodates mixtures of numerical and categorical missing values and reduces potential bias from random effects. In Blimp 3, we created 50 imputed data sets from 50 MCMC chains that included two additional auxiliary variables: baseline heavy drinking days and daily urge. We then used MPlus 8.6 to fit the multilevel model with an unstructured covariance matrix and pool the resulting parameters estimates and standard errors. Results: Across all participants, 653 DDAs were completed (92.6% completion rate) with participants missing between 0 to 4 days of diaries and reporting an average of 7.92 drinking days and 3.52 drinks per day during the two-week period. For the cue exposure predictor variable, 53 observations were missing and for the outcome variable, number of drinks, 54 observations were missing. The analysis results from model-based Multiple Imputation show us that, after accounting for relevant covariates, one’s daily degree of alcohol cue exposure was significantly associated with same-day number of drinks (p = 0.008). The slope coefficient for cue exposure, β1 = 0.27 (SE 0.10) tells us that we would expect a relative 0.27-point increase in same-day number of drinks for each additional cue seen. However, average cue exposure over the course of the two-week period, β2 = 0.20 (SE 0.26) was not significantly associated with number of drinks consumed (p > 0.05). In terms of covariates, only AUD symptom severity, but not biological sex nor medication condition, was significantly associated with average number of drinks (p = 0.004), such that greater AUD severity at baseline predicted greater drinking over the two-week period. Conclusions: This investigation furthers the field’s understanding on the impact of naturalistic alcohol cues on drinking in adult clinical samples of AUD. Results showed that daily fluctuations in one’s exposure to a range of alcohol cues, such as a bar, alcohol advertisements, other people drinking alcohol, and places alcohol is typically consumed, predicted relative changes in daily drinking behaviors. These findings are in line with those from laboratory cue-reactivity paradigms and speak to the potential impact of real-world exposure to alcohol cues on relapse and heavy drinking. Additional planned analyses with this dataset include examining moderators of cue-reactivity in the natural environment, to potentially identify behavioral phenotypes of AUD that display enhanced incentive sensitization. Future research should extend these findings to treatment-seeking and higher severity samples of AUD and explore sex differences by utilizing ecologically valid reports. Keywords: Alcohol Use Disorder, Cue Reactivity, Naturalistic Drug Cues, Statistical Methods Disclosure: Nothing to disclose. P643. Neural and Hormonal Factors Underlying Sex Differences in Poor Inhibitory Control and IV Alcohol Self-Administration Jessica Weafer, Michael Wesley, Justin Verlinden University of Kentucky, Lexington, Kentucky, United States Background: Poor inhibitory control is a well-established risk factor for AUD, and recent evidence suggests this is especially true for women. Female drinkers display poorer inhibitory control than male drinkers, and poor inhibitory control is a stronger prospective predictor of escalation of binge drinking in women. Further, we have preliminary data showing less brain activity during response inhibition among heavy drinking women compared to men, particularly in the early follicular phase of the menstrual cycle, when sex hormones (estradiol and progesterone) are low. However, little is known regarding the degree to which neural and hormonal factors underlying sex differences in inhibitory control influence current drinking behavior. Methods: Female and male heavy drinkers, matched on demographic and alcohol consumption measures, performed the stop signal task to assess inhibitory control while undergoing fMRI. Women were tested at three phases of the menstrual cycle: early follicular phase (low estradiol and progesterone), late follicular phase (high estradiol, low progesterone), and mid-luteal (moderate estradiol, high progesterone), and men were tested three times at matched intervals. Blood samples were taken to assess serum hormone levels at each session. Immediately following the fMRI scan, participants completed a 60-minute free-access IV alcohol self-administration period. Results: Data collection is currently ongoing and to date 9 women and 5 men have completed the study. Analyses of estradiol and progesterone levels confirmed that we successfully achieved the targeted menstrual cycle phases. Preliminary analyses show less brain activity in right frontal regions implicated in response inhibition (i.e., right supplementary motor area and inferior frontal gyrus/insula) in women compared to men across sessions. Further, women showed less brain activity during the early follicular phase (low hormones) compared to the late follicular and mid-luteal phases (high hormones). Finally, activity in the IFG was negatively related to IV alcohol administration in women, such that women who had less brain activity during inhibition self-administered greater amounts of alcohol. Conclusions: These findings replicate our previous findings showing that female drinkers display less neural inhibitory function than males, and that women display less neural inhibitory function when sex hormones are low. Importantly, these results extend previous findings to show an inverse relationship between less neural inhibitory function in women and increased alcohol self-administration. Taken together, these preliminary findings suggest that the early follicular phase, when hormones are low, could be a time of pronounced risk for excessive alcohol consumption in women due to impairments in neural mechanisms of inhibitory control. Keywords: Alcohol, Women, Hormones, fMRI, Inhibitory Control Disclosure: Nothing to disclose. P644. A Neural Circuit Selective for Fast Drug Reward in Humans Peter Manza, Dardo Tomasi, Ehsan Shokri-Kojori, Michele-Vera Yonga, Evan Dennis, Allison Johnson, Leah Vines, Diana Sotelo, Rui Zhang, Kai Yuan, Gene-Jack Wang, Nora D. Volkow National Institutes of Health, Bethesda, Maryland, United States Background: The faster an addictive drug enters the brain, the greater its rewarding effects. This can have powerful implications. When a drug like methylphenidate (MP) is taken orally, resulting in slow brain delivery and slow increases in the neurotransmitter dopamine, it is therapeutically beneficial for attention deficit-hyperactivity disorder. However, when MP is administered intravenously (IV) it enters the brain quickly and like cocaine is highly rewarding. Yet we lack basic knowledge about what underlies this phenomenon in the human brain: which circuits are sensitive to the speed at which a drug enters the brain? This information would be invaluable for development of new therapies to combat addiction. Methods: We used simultaneous PET-fMRI imaging to triangulate dynamic changes in brain dopamine signaling, functional brain activity, and the self-reported experience of ‘high’ as healthy adults received doses of MP at different speeds. Twenty individuals received MP in slow (oral 60 mg) and fast (IV 0.25 mg/kg) doses on different sessions in a double-blind, counterbalanced, placebo-controlled study. We first developed a novel PET method to assess dynamic dopamine increases, which we validated in microdialysis recordings of extracellular dopamine in five rats given IV MP, and in two monkeys given oral MP. Results: Applying this model to our human data, we found that the speed of dopamine increases was higher for IV than oral MP (p < 0.0001, paired t-test), as hypothesized. We then tested where brain activity significantly correlated with speed of dopamine increases to oral and IV MP, using multiple regression. This analysis yielded two distinct circuits. The first involved the orbitofrontal cortex, significantly decreasing its activity with both slow (oral) and fast (IV) dopamine increases. However, it’s activity did not significantly correlate with ‘high’ ratings. A second circuit involved the dorsal anterior cingulate cortex (ACC) and its connections with the striatum, which was activated and responded only to fast (IV) MP. Remarkably, ACC activity significantly correlated with IV dopamine increases in all 20 subjects, and it was the only brain region whose activity significantly correlated with ‘high’ ratings (p < 0.05 FDR-corrected). Conclusions: These data provide first evidence in humans for a network selective for fast drug-induced dopamine increases. This work identifies the ACC as a key region for the subjective perception of drug reward. Keywords: Substance Use Disorder, Dopamine, Pharmacokinetics, positron Emission Tomography (PET), fMRI Disclosure: Nothing to disclose. P645. Behavioral and Brain Reactivity to Uncertain Stress Prospectively Predicts Binge Drinking in Youth Stephanie Gorka, Milena Radoman, Jagan Jimmy, Stacey Culp The Ohio State University, Dublin, Ohio, United States Background: Alcohol abuse is associated with tremendous burden and there is an urgent need to understand who is vulnerable, and why/how, to facilitate accurate detection and prevention. Prior studies indicate that one potential source of risk for problematic alcohol use is exaggerated reactivity to uncertain threat (U-threat) – or threat that is unpredictable in timing, duration, or intensity. Our lab and others have shown that compared with controls, individuals with alcohol use disorder (AUD) reliably demonstrate greater behavioral reactivity (i.e., startle eyeblink potentiation) to U-threat, but not predictable threat (P-threat). Individuals with AUD also exhibit heightened neural reactivity to U-threat, reflected in increased anterior insula (aINS) and dorsal anterior cingulate cortex (dACC) reactivity and connectivity. Magnitude of startle and brain U-threat reactivity correlates with AUD symptom severity and coping-oriented motives for alcohol use. Taken together, robust cross-sectional evidence indicates that AUD is characterized by exaggerated behavioral-brain reactivity to U-threat. It is theorized that this brain-based individual difference factor emerges early in life and contributes to the onset and escalation of alcohol problems. No study to date, however, has tested this theory using a within-subjects longitudinal design. It is therefore unclear to what extent increased reactivity to U-threat reflects a risk phenotype that can be targeted in early detection and prevention strategies. Methods: The current study was a multi-session laboratory paradigm with a 1-year tracking period. Given that onset and escalation of alcohol problems typically occurs in late adolescence and early adulthood, male and female participants were required to be 17-19 years old at enrollment. Participants were young adults from the community (n = 95; M age = 18.4 ± 0.7; 67.4% Caucasian) with minimal initial alcohol exposure (consuming > 1 but <100 lifetime alcoholic beverages). Individuals were selected to be at-risk for onset of alcohol abuse by virtue of affiliating with risky peers and self-reporting easy access to alcohol. Participants completed two laboratory sessions, approximately 7 days apart. Both laboratory sessions included administration of a well-validated No-Predictable-Unpredictable (NPU) threat-of-shock task designed to probe reactivity during anticipation of U-threat and P-threat – once during collection of startle eyeblink potentiation as a behavioral index of aversive reactivity and once during functional magnetic resonance imaging (fMRI). Standardized residual scores were calculated to quantify startle eyeblink potentiation during U-threat relative to no-threat and P-threat relative to no-threat. Extracted parameter estimates from anatomical bilateral aINS and dACC masks were used to capture brain reactivity during U-threat > No-threat and P-threat > No-threat. At baseline and 1-year later, participants reported their drinking behavior using a 90-day time-line follow-back procedure. Binging drinking at the 12-month follow-up was the primary dependent variable. To test our hypotheses, we fit a series of multilevel hurdle models which allowed us to model the binary outcome of whether binge drinking occurred (yes or no) and the continuous outcome of number of binge drinking episodes. Results: We used the zero-inflated submodels to estimate the probability of binge drinking versus not binge drinking at the 12-month follow-up. These submodels revealed that greater baseline startle reactivity to U-threat (b = −0.64, p = 0.009), greater bilateral aINS reactivity to U-threat (b = −0.67, p = 0.030), and greater dACC reactivity to U-threat (b = −0.69, p = 0.035) were associated with increased probability of binge drinking one-year later. There were no associations between behavioral or brain reactivity to P-threat and probability of binge drinking. We used the conditional submodels to estimate the positive count process, or frequency of binge drinking at the 12-month follow-up. No significant associations emerged between any brain or behavioral threat measure and binge drinking in these conditional submodels. All results were adjusted for biological sex and baseline drinking behavior. Conclusions: Results indicate that behavioral-brain reactivity to U-threat prospectively predicts binge drinking one-year later in a cohort of at-risk youth. These results demonstrate that exaggerated reactivity to U-threat is not just an AUD disease marker; it is a brain-based individual difference factor that connotes risk for problem drinking. These findings also add to a growing literature implicating aINS and dACC (together labeled the ‘salience network’) dysfunction in the pathophysiology of alcohol abuse. Taken together, exaggerated reactivity to U-threat is a relatively novel objective AUD prevention target, anchored in the brain’s salience network. Keywords: Alcohol Abuse, Anxiety and Stress, Salience Network, Acoustic Startle Response Disclosure: Nothing to disclose. P646. Methamphetamine Reduces Striatal Neural Activation to Monetary Reward Natania Crane, Hanna Molla, Katherine Fesperman, Harriet de Wit University of Illinois at Chicago, Chicago, Illinois, United States Background: Stimulant drugs like methamphetamine (MA) activate brain reward circuitry, which has been linked to subjective drug reward and the development of drug abuse. However, it is not clear how drugs like MA alter immediate neural responses to a non-drug reward. In this study, we examined if an acute dose of MA altered neural response to receipt of a monetary reward. We hypothesized that MA (vs. placebo) would increase mesolimbic neural activation to reward. Methods: In a within-subject, randomized, double-blind, placebo-controlled design, 41 healthy adults completed the Doors monetary reward task during fMRI approximately 70 minutes after ingestion of placebo or 20 mg MA. We examined effects of the drug on neural response to reward receipt using a priori anatomical striatal regions of interest (nucleus accumbens (NAcc), caudate, putamen). We also ran exploratory whole-brain analysis to examine drug effects on neural response to receipt of monetary reward. Results: MA resulted in less NAcc BOLD activation during Win>Loss compared to placebo (p = 0.007). The drug did not significantly alter caudate or putamen BOLD activation during Win>Loss (p-values > 0.05). Exploratory whole brain analysis revealed that MA decreased BOLD activation during Win>Loss in the striatum, including the NAcc, caudate, and putamen, compared to placebo (p < 0.05, corrected). Conclusions: Our findings suggest that stimulant drugs like MA that are known to activate brain reward circuitry may acutely decrease mesolimbic neural response to the receipt of non-drug, monetary rewards. It is possible that MA administration masked the expected neural response to reward by increasing basal dopaminergic tone. Keywords: fMRI, Mesolimbic Reward Circuitry, Methamphetamine, Healthy Individuals Disclosure: Nothing to disclose. P647. Anterior Insula to Nucleus Accumbens Brain Tract Structure but Not Functional Connectivity Predicts Relapse to Alcohol Use Claudia Padula, Daniel M. McCalley, Lea-Tereza Tenekedjieva, Kelly MacNiven, Melanie Comejo-Coffigny, Brian Knutson, Leanne M. Williams VA Palo Alto Health Care System, Palo Alto, California, United States Background: Great strides have been made recently in identifying the biopsychosocial factors that contribute to and are associated with alcohol use disorders (AUD). Recent findings indicate that structural and functional connectivity between the anterior insula (AIns) and the nucleus accumbens (NAcc) may play a critical role in our understanding of addiction. Specifically, white matter integrity is reduced among individuals with AUD relative to healthy controls, while functional connectivity is elevated while viewing alcohol cues. Yet little is known about whether these metrics within the AIns-NAcc circuit can predict treatment outcomes. Therefore, we sought to determine whether structural and functional qualities of tracts projecting from the AIns to the NAcc were associated with relapse to alcohol use six months after treatment. Lastly, we tested whether the relationship between structural connectivity and relapse was mediated by functional connectivity. Methods: A total of N = 74 Veterans (18 women) in AUD treatment were scanned with 3 T functional MRI during an alcohol cue-reactivity task, diffusion tensor imaging, and were followed for 6 months post-treatment. DTI data were processed using mrDiffusion and fMRI data were analyzed using CONN. DTI measures included right and left functional anisotropy (FA), radial diffusivity (RD), medial diffusivity (MD) and axial diffusivity (AD). Functional connectivity was defined as correlated BOLD signal between the AIns and NAcc while viewing alcohol images and neutral images. Binary logistic regression models with backward conditional parameter estimates were used to determine structural and functional predictors of relapse, with bootstrapped validation, followed by a mediation analysis. Results: Following treatment, 73% of the sample had relapsed by six months. Structural connectivity metrics between the AIns and NAcc were significantly predictive of relapse. Specifically lower right FA (β = −.713, p = 0.032) and higher left MD (β = 1.176, p = 0.029) suggested that lower white matter integrity in the AIns-NAcc tract predicted relapse in this sample. Classification accuracy of a model including these two variables was 83.8% and bootstrap results were significant (right FA CI: -1.448 to -.099, p = 0.015; left MD CI: .285 to 2.577, p = 0.029). Functional connectivity metrics during the alcohol cue-reactivity task did not predict relapse (p > 0.05), thus the mediation test was not performed. Gender was included as a covariate in all models and results remain unchanged. Conclusions: These findings highlight a structural target for predicting relapse to alcohol use following standard treatment. In addition, functional connectivity during an alcohol-cue reactivity task was not predictive of relapse in this sample, nor was functional connectivity associated with structural metrics. There has been recent interest in transcranial magnetic stimulation (TMS) to deeper cortical targets, as well as preliminary evidence that repetitive TMS may increase white matter integrity (specifically FA). Thus, future studies targeting the AIns for AUD treatment may be warranted. Keywords: Alcohol Use Disorder, Addiction Circuitry, Structural and Functional Connectivity, Relapse Biomarkers Disclosure: Nothing to disclose. P648. Hydrocortisone Alters the Formation of Alcohol-Related Episodic Memories Bailey Harris, Brynn Sherman, Nicholas Turk-Browne, Rajita Sinha, Elizabeth Goldfarb Yale University, New Haven, Connecticut, United States Background: Memory plays a crucial role in alcohol use disorder (AUD), including by promoting relapse in contexts where patients used to drink. We recently showed that, relative to social drinkers, patients with AUD remember alcohol-related episodes differently, with preferentially enhanced memories for alcohol-paired contexts (Goldfarb, Fogelman and Sinha 2020 Neuropsychopharm). One possible mechanism driving this bias is related to the stress response: acute stress also enhances memory for emotionally salient contexts. The memory-altering effects of stress are often driven by glucocorticoids, which are dysregulated in AUD and acutely elevated in response to alcohol. However, the impact of glucocorticoids on the formation of alcohol-related memories, and the consequences of this exposure for later drinking behavior, are unknown. Here we used a pharmacological-fMRI approach to investigate the cognitive and neural mechanisms by which glucocorticoids influence memory formation for alcohol-related episodes. Methods: We used a double-blind, placebo-controlled, cross-over design. Participants (N = 27 social drinkers) each performed two rounds of encoding and delayed retrieval (24 h after encoding). In one round, they received 20 mg of hydrocortisone ~1 hour prior to encoding; in the other round, they received a visually identical placebo tablet. fMRI data was acquired during encoding. During encoding, participants formed associations between 80 photographs of objects and neutral scenes and rated their emotional responses to each object/scene pair. Of these 80 objects, 50% were neutral (handheld household objects) and 50% were alcoholic beverages, previously validated to be comparable in perceptual features. During retrieval, participants were asked to remember different components of these episodes, including item-level (object recognition) and context (recognition of scene associated with object) representations. Immediately following these retrieval tests, participants had an opportunity to freely consume alcohol in the laboratory as part of a previously validated alcohol taste test. Results: We found that glucocorticoid administration prior to encoding amplified the perceived emotional salience of object/scene pairs (b = 0.08 [.04], p = 0.03). This change was adaptive for later memory. Specifically, following hydrocortisone, higher emotional salience was associated with enhanced memory for items and contexts. However, this association was flipped following placebo, with higher emotional salience impairing both forms of memory (arousal x drug; item: F1,71 = 11.34, p = 0.001; context: F1,71 = 10.2, p = 0.002). We also identified key associations between glucocorticoid effects and risky drinking behavior. First, we found that glucocorticoids induced a memory bias similar to one that we previously observed among patients with AUD. Following placebo, participants had worse memory for salient alcohol-paired contexts (replicating our past findings in social drinkers; b = 0.09 [.03], p = 0.02). However, these same individuals showed enhanced context memory for these salient alcohol/scene pairs following hydrocortisone (b = 0.09 [.03], p = 0.009), a similar bias to that shown previously by participants with AUD. Second, we found that individuals who engaged in riskier drinking behavior (higher AUDIT scores) achieved higher breath alcohol levels in the laboratory, demonstrating the ecological validity of the alcohol taste test (F1,25 = 11.53, p = 0.002). Critically, these risky drinking individuals also consumed more alcohol after retrieving memories that had been encoded under hydrocortisone (AUDIT x drug: F1,72 = 9.36, p = 0.003), suggesting that glucocorticoid-induced effects at encoding had consequences for later drinking behavior. Preliminary analyses of brain responses during encoding indicate that glucocorticoids modulated memory-related processes in the hippocampus (altering connectivity between subfields) and amygdala (influencing representational similarity). Specifically, hydrocortisone increased intrahippocampal connectivity, and changed the relationship between connectivity and subsequent memory performance. Hydrocortisone also promoted pattern separation in the amygdala for alcohol-related episodes, which was in turn associated with less “gisty” alcohol-related context memories. Conclusions: These results highlight the importance of glucocorticoids in the formation of biased alcohol-related memories. Consistent with past studies of emotional memory, we found impaired recall of contexts paired with emotionally salient objects following placebo. However, we found that hydrocortisone flipped this pattern, selectively amplifying context memory for these emotionally salient experiences. Intriguingly, elevated glucocorticoids at encoding promoted both a bias toward remembering alcohol experiences similarly to patients with AUD and, among riskier drinkers, led to more drinking following retrieval of those experiences. Together, these findings begin to elucidate a novel neurocognitive mechanism in which elevated glucocorticoids bias memory formation and promote later drinking. Keywords: Cortisol, Episodic Memory, Alcohol, Emotional Arousal, Task fMRI Disclosure: Nothing to disclose. P649. The Neural Processes Underlying Avoidance Learning Dysfunction in Problem Drinking Thang Le, Micaela Ciambrone, Takeyuki Oba, Chiang-Shan Li Yale University, New Haven, Connecticut, United States Background: Drinking as a pain-avoidance coping behavior plays a central role in the maintenance of problem drinking and escalation to alcohol use disorders. Specifically, individuals often seek alcohol to alleviate or avoid painful physical and emotional states. As drinking escalates, consumption is progressively driven by individuals’ heightened sensitivity to the painful consequences of alcohol intake cessation. Paradoxically, chronic alcohol use heightens pain reactivity which further motivates drinking as an avoidance coping strategy. Over time, this maladaptive behavior becomes increasingly less amenable to cognitive control, trapping drinkers in a spiraling cycle of drinking and distress. Yet, the underlying circuits of avoidance learning in problem drinking are poorly understood. Methods: We acquired fMRI and behavioral data that assessed avoidance learning in 30 problem drinkers and 32 social drinkers who performed a probabilistic learning go/no-go task. The task involved learning to associate visual cues with outcomes to avoid painful electric shocks and optimize monetary reward. We hypothesized that relative to social drinkers, problem drinkers would exhibit (1) poorer avoidance learning, (2) weakened prefrontal cortical activation to avoidance learning; and (3) greater pain circuit activity in responses to pain. Results: Our findings confirmed the hypotheses. Specifically, problem drinkers showed lower learning rates during pain avoidance conditions, coupled with reduced dorsolateral prefrontal cortical activity. Brain regions implicated pain reactivity including the insula, dorsal anterior cingulate cortex, amygdala, and periaqueductal gray showed hyperactivation during shock feedback and this activity was positively correlated with the drinking-to-cope measure. Conclusions: The current work sheds light on the relationships between problem drinking and the neural as well as cognitive processes underlying avoidance learning dysfunction. The characterization of these relationships offers further understanding of negative reinforcement drinking in humans, thus bridging the gap with preclinical research. Keywords: Alcohol Abuse, Pain, Approach/Avoidance, Cognitive Control, Brain Imaging, fMRI Disclosure: Nothing to disclose. P650. Mapping Social Behavior in Non-Treatment-Seeking Heavy Drinkers Irene Perini, Hanna Karlsson, Markus Heilig Center for Social and Affective Neuroscience, Linkoping, Sweden Background: Individuals with alcohol use disorder (AUD) present with social and environmental difficulties that severely impact their quality of life and mental well-being. Progressive impairment of social function is both a consequence and a cause of relapse, creating a vicious circle for people suffering from AUD. Therefore, understanding what factors shape social processing in AUD becomes critical. Using a simulated social media environment, we previously showed that female adolescents with nonsuicidal self-injury, a clinical group with high levels of social stress and at high risk of developing addiction, presented with a negative social bias. Compared to controls, individuals with nonsuicidal self-injury felt rejected significantly more often than controls, liked to see their face less, and were more sensitive to being rejected by other players. Importantly, this negative social bias was associated with engaging in self-harm, and with a distinct brain pattern during anticipation of social feedback. Here, using the same task, we examined social bias and social behavior in a group of young adults that engage in heavy versus light social drinking. Methods: After screening and upon inclusion, we tested N = 30 light social drinkers (N = 15 men drinking < 15 drinks/week and N = 15 women drinking < 10 drinks/week) and N = 30 heavy, non-treatment-seeking social drinkers (N = 15 females). We used a task developed by our group, which simulates an online interaction, where participants like and dislike photos of other putative players. Similarly, they anticipate and receive feedback from other players. During and after the game, participants were presented with questions aimed at addressing a potential social bias. In addition, to measure social behavior, the percentage of negative feedback towards other players was collected. The Alcohol Use Disorders Identification Test (AUDIT) and Drug Use Disorders Identification Test (DUDIT) were collected to measure alcohol and drug use severity. Personality traits and impulsivity were collected using the NEO-FFI questionnaire and the Baratt Impulsiveness Scale (BIS-11) respectively. We used a 2×2 ANOVA with group and sex as between factors for all measures. In addition, to identify potential predictors of social behavior, we ran stepwise regressions using percentage of negative social feedback as a dependent variable. Results: In the heavy (but not in the light) drinkers, AUDIT and conscientiousness scores were significant predictors of negative feedback towards others, explaining about 42% of variance (F(1, 28) = 9.78, p < 0.001, R2 = 0.42). No significant predictors were identified for the light drinkers (p > 0.5). There was no between-group difference in self-reports during and after the online game, indicating no social bias in the heavy drinkers (p > 0.05). And there was no significant between-group difference in percentage of negative feedback towards others (p > 0.6). AUDIT scores were higher in the heavy drinkers, indicating good group categorization (p < 0.001). In addition, the heavy drinkers had lower conscientiousness scores (p = 0.002) and marginally higher extraversion scores (p = 0.064). We identified some sex effects. Females liked to see their face significantly less than males (p = 0.003). In addition, females had higher neuroticism (p = 0.009), openness (p = 0.029), and agreeableness (p < 0.001) scores. All participants were free from psychiatric diagnoses. Conclusions: The negative social bias we initially observed in the clinical population of female adolescents with nonsuicidal self-injury was not evident in our sample of young adults that engage in heavy drinking. This suggests that social bias might emerge only after long-term exposure to harmful behaviors, that is, after drug use has had a clear impact on social function. However, social behavior seems to be influenced by heavy drinking already early stages, as severity of alcohol use predicted negative feedback towards others in the heavy drinking group. In addition, low conscientiousness predicted negative feedback towards others. Altogether, this study identified factors that might influence social processing in people engaging in harmful drinking and that are both dependent and independent on the onset of problematic alcohol use. Keywords: Alcohol Use, Social Processing, Social Behavior, Big Five Personality Factors, Sex Differences Disclosure: Nothing to disclose. P651. Sex Differences in Corticolimbic Microglia Levels in Individuals With Alcohol Use Disorder Compared to Healthy Controls Yasmin Zakiniaeiz, Karissa McCright, Ansel Hillmer, Hannah Shi, Nabeel Nabulsi, Yiyun Huang, David Matuskey, Gustavo Angarita-Africano, Sherry McKee, Kelly Cosgrove Yale University, New Haven, Connecticut, United States Background: Alcohol use is one of the leading causes of disability in the United States and female drinkers are more vulnerable than male drinkers to many of the consequences of alcohol use (NIAAA, 2017). Alcohol stimulates microglia, the resident immune cells of the brain, triggering signaling pathways that activate microglia to carry out repair functions. However, excessive activation releases substances such as inflammatory cytokines and reactive oxygen species that contribute to neuronal dysfunction and death. These pathways may contribute to alcohol-induced neurodegeneration. Neurodegeneration occurs over years of alcohol use and impairs neurocognition, further precipitating alcohol drinking and driving the addiction cycle. Women with alcohol use disorder (AUD) have greater neurocognitive impairments than men with AUD. Importantly, neurocognitive deficits predict poorer treatment outcomes. The goal of this study was to investigate sex differences in the neuroimmune system that may underlie sex differences in neurocognition in AUD. While our group and others (Hillmer, Mol Psychiatry, 2017; Kalk, Transl Psychiatry, 2017) previously reported that individuals with AUD have lower levels of TSPO, a microglia marker, than controls, few women were included. We hypothesized that women with AUD would show lower levels of microglia relative to sex-matched healthy controls in hippocampus, frontal cortex, and cerebellum and that lower microglia levels would be related to poorer cognitive performance. Methods: To date, twenty-four individuals with AUD (mean 13 drinks/day, 20 drinking years; 8 females) and 24 age-, sex-, smoking status- and rs6971 single-nucleotide polymorphism genotype-matched healthy control subjects have undergone positron emission tomography (PET) scans with [11 C]PBR28. [11 C]PBR28 measures levels of 18-kDa translocator protein (TSPO), a marker of microglia. PET scans were acquired for 120 min after bolus injection of 541 ± 177 mBq. [11 C]PBR28 on a High Resolution Research Tomograph with arterial blood sampling acquired throughout to measure the metabolite-corrected input function. The outcome measure, volume of distribution (VT), was estimated regionally with multilinear analysis (t=30 min) as a measure of TSPO availability. Univariate analysis of variance models (one per brain region of interest – hippocampus, frontal cortex, and cerebellum) were conducted with VT as the dependent variable, diagnostic group (AUD vs. healthy control) and sex (male vs. female) as between-subject factors, and genotype (‘high’ vs. ‘moderate’ affinity binders) as a fixed-factor. A priori post hoc analyses were conducted to compare VT values between women with AUD women and sex-matched controls. A subset (n = 35; n = 6 AUD women, n = 3 healthy control women) of subjects completed a cognitive battery. Performance on three cognitive tasks related to verbal learning and memory, executive function, and motor function was compared between the AUD and healthy control group using independent-samples t-tests. Exploratory analyses of relationships between VT and relevant cognitive task performance were conducted using linear regressions. Results: We found a main effect of diagnostic group such that individuals with AUD had significantly (or trending) lower levels of TSPO availability than their healthy control counterparts in the three brain regions of interest (0.073 ≤ p ≥ 0.017, effect sizes: 0.073 ≤ ηp2 ≥ 0.125). We found preliminary evidence of AUD-related sex differences in TSPO availability. Interactions of diagnostic group and sex were trending (p ≤ 0.010, ηp2 ≥ 0.051) for all three brain regions. A priori pairwise comparison between groups within each sex revealed that women with AUD had significantly lower VT in all three regions (hippocampus p = 0.034, ηp2 = 0.100; frontal cortex p = 0.030, ηp2 = 0.104; cerebellum p = 0.018; ηp2 = 0.123) compared to sex-matched controls. Preliminary analyses revealed that the AUD group performed worse than the healthy control group on the executive function (p = 0.003, Cohen’s d = 0.993), and trended in the same direction on the verbal learning and memory (p = 0.061) and motor function tasks (p = 0.108). Preliminary analyses revealed that VT values were not related to cognitive performance (p ≥ 0.125). Conclusions: Consistent with our prior work (Hillmer, 2017), TSPO availability was significantly (or trending) lower in individuals with AUD compared to healthy control subjects in hippocampus, frontal cortex, and cerebellum. Preliminary analyses revealed that this group difference may have been driven by differences between AUD women and their sex-matched controls. Consistent with our hypotheses, TSPO availability was lower in women with AUD than healthy control women in all three brain regions. While individuals with AUD performed worse on cognitive tasks than their healthy control counterparts, this was not related to TSPO availability. These data suggest that neuroimmune suppression may be more pronounced in women with AUD compared to sex-matched controls and that this could underlie reports of greater neurodegeneration in women. Future analyses will explore brain-behavior neurocognition relationships in an expanded sample with more women. Keywords: Alcohol Use Disorder, Positron Emission Tomography, Sex Differences, Microglia, Translocator Protein (TSPO) Disclosure: Nothing to disclose. P652. Resisting Tobacco Smoking is Linked to Faster Evidence Accumulation During Decision-Making Chungmin Han, Elena Molokotos, Adam Leventhal, Daniel G Dillon, Amy Janes National Institute of Drug Abuse, NIH, DHHS, Baltimore, Maryland, United States Background: Tobacco smoking remains a primary cause of mortality. Continued substance use despite such well-known consequences may reflect disrupted neurocognition, resulting in impaired decision-making. To test this hypothesis, we applied the Hierarchical Drift Diffusion Model (HDDM) to probabilistic reward task (PRT) data; the data were acquired from adult smokers who also completed a relapse analog task (RAT). PRT performance typically measures response bias, which is used to index reward sensitivity. The HDDM offers a richer set of measurements. It conceptualizes decisions in the PRT as a process of evidence accumulation towards response boundaries, and thus generates quantitative estimates of three key parameters: threshold (the distance between the response boundaries, indicating how much evidence must accumulate before a decision is made), starting point bias (whether the evidence accumulation process is shifted towards one response boundary vs. the other at the outset of each trial), and drift rate (the speed of evidence accumulation). A fourth parameter, non-decision time, captures the time needed to perceive stimuli and execute a motor response once a decision has been reached. By relating these four parameters to successful vs. unsuccessful abstinence on the RAT, we attempted to identify specific cognitive mechanisms (threshold, starting point bias, or drift rate) that might relate to the ability to forgo smoking. Drift rate was of key interest, as this parameter has shown sensitivity to psychopathology in several prior studies. Methods: 122 otherwise healthy nicotine dependent individuals performed the PRT following smoking as usual. During the PRT, a cartoon face was shown onto which a mouth was quickly flashed (mouth duration: 100 ms). Participants were asked to identify whether the mouth was short (11.5 mm) or long (13.0 mm). For each participant, correct responses to one mouth length (e.g., short, the “rich” stimulus) resulted in a monetary reward three times more frequently than correct responses to the other length (e.g., long, the “lean” stimulus). This asymmetric reinforcement rate was used to induce a response bias (RB), such that participants were more likely to respond “rich” than “lean”. The ability to discriminate between the two stimuli was also measured. Prior to a second, independent visit, participants abstained from smoking for a minimum of 16 hours. Abstinence was verified by a breath carbon monoxide (CO) level of <10 ppm. Participants then performed a RAT, where they could earn money by delaying smoking for the 50-minute session. At the beginning of the RAT, participants were given 8 cigarettes and told that every 5 minutes of abstinence would result in $0.20 reward. Most participants waited either 0-minutes (n = 36) or the full 50-minutes (n = 44) before smoking a cigarette, thus the PRT data was compared between these two groups. Results: No differences between the 0-min and 50-min groups were noted for age (0- min: 43.69 ± 10.59 yr; 50-min: 40.64 ± 10.88, p = 0.209), sex (Fisher’s exact = 0.228), or education (p = 0.250). Nor were there group differences in nicotine dependence level as defined by the Fagrestrom test of nicotine dependence (0-min: 5.83 ± 1.94; 50-min: 4.93 ± 2.33, p = 0.067) or average number of cigarettes per day (0-min: 17.79 ± 5.44; 50-min: 16.5 ± 7.52, p = 0.398). In the PRT, a response bias developed over the 3 blocks (p < 0.01) and “rich” responses were faster than “lean” responses (p < 0.001). There was no group difference in RB. However, the 50-min group had better discriminability than the 0-min group (p = 0.002). Moreover, the HDDM revealed that drift rate was higher in the 50-min group (1.21 ± 0.08) vs. the 0-min group (0.79 ± 0.09; q < 0.001). There was no group difference in threshold, non-decision time, or starting bias. Conclusions: Compared to adults who smoked immediately, those who abstained for the full 50-minutes showed better discriminability and higher drift rates in the PRT. This indicates that the speed of evidence accumulation—which is captured by drift rate and critical for discriminability—was higher in the abstainers. This is important because evidence accumulation is implicated in a wide range of decisions, not just in laboratory tasks like the PRT. Consequently, the data suggest that adults who fail to abstain from substance use may show decision-making deficits on a range of tasks due to slow evidence accumulation, although this remains to be confirmed. By contrast, no group difference was found for response bias. This suggests that individual differences in reward sensitivity were not a critical determinant of abstinence in this study. Keywords: Smoking, Reward Sensitivity, Computational Models of Decision-Making Disclosure: Nothing to disclose. P653. Electrophysiological Markers of Aberrant Cue-Specific Exploration in Heavy Drinkers Ethan Campbell, Garima Singh, Eric Claus, Katie Witkiewitz, Vincent Costa, James Cavanagh, Jeremy Hogeveen University of New Mexico, Albuquerque, New Mexico, United States Background: To optimize behavior in an uncertain world, it is often necessary to test novel and unfamiliar actions at the expense of the foregone value of familiar actions. Managing these competing demands is known as the ‘explore/exploit’ tradeoff in decision-making. We recently demonstrated that the neurocomputational mechanisms underlying explore/exploit decisions are conserved across human and nonhuman primate species (Hogeveen et al., 2022, Neuron), suggesting this represents a powerful translational paradigm for basic and translational science studies on patients with pathological decision-making. To wit—heavy drinking is associated with pathological alcohol-related decision-making, but little is known about the mechanisms that drive heavy drinkers to explore novel alcohol cues in the first place. Here, we merge behavior, computational modeling, and electroencephalography (EEG) to elucidate the electrophysiological correlates of aberrant alcohol-related exploration in heavy drinkers. Methods: We used computational model-based decomposition of explore/exploit decision-making during a 3-armed bandit reinforcement learning paradigm in heavy drinkers (N = 27; N = 24 of whom met criteria for a lifetime diagnosis of alcohol use disorder) and matched controls with a current alcohol use disorder identification test (AUDIT) score less than or equal to 3 (N = 27). Participants made speeded choices between three neutral images which were rewarded at a low (p = 0.2), medium (p = 0.5), or high (p = 0.8) probability. Occasionally, a novel stimulus insertion trial occurred: a novel image with a randomly assigned reward probability was inserted in lieu of one of the existing options. Half of the novel insertion trials contained an alcohol cue, whereas the other half of the insertion trials contained a non-alcohol beverage cue. In the immediate trials after each novel insertion, participants faced the difficult decision to either exploit the most valuable familiar option, or explore the novel option to learn its reward probability. An optimal decision policy was derived using a Partially-Observed Markov Decision Process model (POMDP), which derived separate estimates of each participants’ tendency to make decisions weighted by the relative future value of exploring novel options, versus their tendency to more heavily weight the immediate value of exploiting familiar options. Separate estimates were generated for the relative future valuation of alcohol cues (i.e., explore-alc-value), relative future valuation of non-alcohol cues (i.e., explore-nalc-value), immediate valuation of alcohol cues (i.e., exploit-alc-value), and immediate valuation of non-alcohol cues (i.e., exploit-nalc-value). Critically, continuous EEG data were collected across the scalp during the bandit task. We specifically focused on the P3a event-related potential over mid-frontal electrodes. The P3a is known to respond and habituate rapidly to novel stimuli, and is thought to reflect a top-down executive orienting response that might be essential for driving novelty-driven exploration on our task. Results: Observed explore/exploit decision-making behavior was modeled well by the POMDP (r = 0.74, 95% CI = 0.60-0.84). Participants tended to exploit the best alternative more often than the novel stimulus (p = 0.008), but in turn they also explored the novel stimulus more often than they chose the worst stimulus (p < 0.001). The POMDP explore-alc-value parameter demonstrated a two-way interaction between group and novel stimulus condition (p = 0.038). This was specifically driven by individuals in the heavy drinking group demonstrating greater weighting of the relative future value of exploring novel alcohol cues compared to control participants (p = 0.031). Groups did not differ in their weighting of the explore-nalc-value parameter, and the tendency to weight both the exploit-alc-value and exploit-nalc-value parameters did not differ between groups. This heighted motivation to explore novel alcohol stimuli based on the POMDP derived parameters was associated with a heightened P3a response to novel alcohol cues in the heavy drinking group. Explore-alc-value estimates were predicted by a two-way interaction between chosen stimulus type and P3a amplitude (p = 0.005), and paired comparisons showed a significantly larger relationship between exploration value and P3a amplitudes (p = 0.005), and separated by group, this cue-specific effect was found only in the heavy drinking group (p = 0.004). Conclusions: The current data assembles behavioral, electrophysiological, and computational evidence to examine the heightened tendency for heavy drinkers to explore novel alcohol stimuli. Our POMDP-derived latent value estimates suggest that individuals with clinically-significant drinking behaviors tend to overvalue the potential future reward value of exploring novel alcohol stimuli. Critically, evoked P3a responses to novel alcohol cues may represent a neurocomputational marker of heightened exploration tendencies in response to alcohol cues in heavy drinkers. Whereas the preponderance of existing studies of value-based decision-making in the alcohol field have focused on tendencies to learn to exploit alcohol cues based on prior reinforcement, this represents the first evidence for a potential biomarker that may help to explain why some individuals—but not others—engage in problem drinking behavior when the stimuli are novel and their reinforcement value is not known. Keywords: Value-Based Decision-Making, Computational Models of Decision-Making, Alcohol, Drinking Disorders, EEG Biomarkers Disclosure: Nothing to disclose. P654. Identification and External Validation of a Problem Cannabis Use Brain Network Sarah Lichenstein, Dustin Scheinost, Brian Kiluk, Kathleen Carroll, Marc Potenza, Godfrey Pearlson, Sarah Yip Yale University School of Medicine, New Haven, Connecticut, United States Background: Cannabis is the most commonly used illicit drug [1], and substantial evidence indicates that its use is associated with clinically-significant harms for a subset of users [2]. In the context of recent decriminalization/legalization of cannabis—in tandem with significant increases in potency [3]—further work is urgently needed to (i) identify who is most vulnerable to cannabis-related harms and (ii) elucidate neurobiological mechanisms of risk. Existing studies examining neural correlates of cannabis use in adolescence yield inconsistent results [4], and provide insufficient information to distinguish neural risk factors of problem-level vs. recreational use. Connectome-based predictive modeling (CPM) is a novel, data-driven, whole-brain, machine learning approach to identify neural networks related to specific behaviors of interest [5]. Therefore, the aims of the current study were to 1) apply CPM to identify a neuromarker of problem-level cannabis use in a non-clinical sample of adolescents/emerging adults, and 2) validate the identified network in an independent clinical sample of individuals entering treatment for cannabis use. Methods: Data were drawn from the NIAAA-funded Brain and Alcohol Research in College Students (BARCS) Study, a 2-year longitudinal study of alcohol and substance use behavior. The current analyses include N = 191 participants for whom usable baseline neuroimaging data were available. Problem cannabis use was defined as self-reported use ≥10 times/month [6] at any clinical follow-up. Reward task (Monetary Incentive Delay Task) fMRI data were used to compute functional connectivity matrices that were entered into connectome-based predictive models (CPMs). CPM identifies features that are positively and negatively associated with problem-level use and to fit a linear model that can be applied to novel data using internal cross validation. Notably, this method allows for the identification of neural features underlying successful models, facilitating a better understanding of the neurobiological mechanisms underlying risk for problem cannabis use. Clinical relevance of the identified network was assessed in an independent clinical sample of N = 33 individuals entering cannabis use treatment who were enrolled in randomized clinical trials of cognitive behavioral treatments for substance use. Results: CPM of reward task data successfully identified a network predictive of problem-level cannabis use in college-age adolescents/emerging adults (r = 0.20, p = 0.005). Consistent with other CPM work, identified networks were complex and included both cortical and subcortical connections. Despite this complexity, the spatial extent included only 1175 edges (618 positive, 557 severity), or less than 3.3% of possible connections. Network anatomy indicated that increased connectivity between the motor sensory network and medial frontal and frontoparietal networks, and decreased connectivity between the cerebellar network and medial frontal and frontoparietal networks was linked to problem-level cannabis use. Out-of-sample analyses supported the clinical relevance of the identified network: increased problem cannabis use network strength was positively associated with addiction severity at treatment entry (rho = 395, p = 0.023) and negatively associated with cannabis abstinence during treatment (r = −.393, p = 0.024) in an independent sample of individuals seeking treatment for cannabis use. Conclusions: These data for the first time identify a neural network predictive of problem-level cannabis use in adolescence/emerging adulthood. Furthermore, follow-up analyses in an independent clinical sample validate the clinical relevance of the identified problem cannabis use network. Elucidating neural mechanisms of risk for problem use is crucial to facilitate the development of targeted prevention/intervention approaches to mitigate cannabis-related problems for persons at risk. Keywords: Predictive Models, Marijuana, Problem-Level Use Disclosure: Nothing to disclose. P655. Where is the Main Target of the Most Commonly Used TDCS Electrode Montages for the Treatment of Neuropsychiatric Disorders? Ghazaleh Soleimani, Kelvin Lim, Hamed Ekhtiari University of Minnesota, Minneapolis, Minnesota, United States Background: Background: Transcranial electrical stimulation (tES) over the dorsolateral prefrontal cortex (DLPFC) has been widely used to modulate neurocognitive functions in both healthy and clinical populations. Most clinical trials place stimulating electrodes over F3/F4 location in EEG standard system based on the assumption that the active electrode will mainly stimulate the underlying brain region, DLPFC, as the main target. However, it has been shown that the maximal electric field (EF) during tES can fall outside the area under the active electrodes as commonly assumed. Here, we computationally assessed the spatial distribution and strength of the stimulation dosage during tES over DLPFC at both group and individual levels with the aim of determining the prefrontal regions that are targeted by DLPFC montages. We present our results for a subset of healthy subjects in the Human Connectome Project (HCP) database. To consider an independent clinical population, analyses were replicated with structural MRI of participants with methamphetamine use disorder (MUD) from our pre-registered trial data (NCT03382379). Here, we propose that the frontopolar cortex would receive the highest EF intensity in the DLPFC montages. Methods: Methods: To generate individualized head models, unprocessed T1 and T2-weighted MRIs from 80 (44 female) randomly selected healthy adults (age (year) between 22-35) were obtained from the freely available HCP database. For each model, two common montages for modulating DLPFC (asymmetric: F4/Fp1, symmetric: F4/F3) were simulated. We extracted the averaged EF strength in (1) the center of stimulating electrode (F4), and (2) the top 1% of voxels in individualized EF maps by using a whole brain 99th percentile threshold. Paired sample t-tests with FDR corrections were used to compare EFs between the center of the electrode and hotspots. Inter-individual variabilities were quantified with standard deviation (SD) in terms of (1) hotspot location, and (2) hotspot value. Atlas-based parcellation of head models was also used to determine averaged EFs in main subregions of PFC using the Brainnetome atlas. All steps were replicated with 66 participants with MUD as an independent clinical population (age (year) 18-60, all male). The overlap between hotspot locations in healthy subjects and MUDs was calculated using Euclidean distance. Results: Results: Results related to healthy subjects showed that, despite inter-individual variability in strength and location, hotspots were found in the medial frontopolar area; a region occupying the anterior portion of the brain’s frontal lobe (corresponding to Brodmann’s area 10) which is distinct from DLPFC (which is located on the lateral and dorsal part of the medial convexity of the frontal lobe) where the stimulating electrode was placed (comprises Brodmann’s areas 9, 46). EF hotspot was significantly higher than the EF “under” the stimulating electrode (F4) in both symmetric (hotspot: 0.41 ± 0.06, F4: 0.22 ± 0.04; mean ± SD in V/m) and asymmetric (hotspot: 0.38 ± 0.04, center of electrode: 0.2 ± 0.04) montages with large effect sizes (Hedges’g for: symmetric = 0.86, 95% CI (0.53,1.19), asymmetric = 0.76, 95% CI (0.43,1.09)). Inter-individual variabilities showed that, in symmetric montage, group-level location for hotspots in MNI space was [-1.21, 57.66, 18.67] with [6.43, 4.37, 8.08] as SD such that all hotspots were placed in a cube with a volume of 29 cm3 (X = 3.1, Y = 1.9, Z = 4.9). EF strength ranged from 0.31 to 0.68 V/m. In the asymmetric montage, group-level location for the hotspots in MNI space was [-2.62, 49.00, -4.54] with [7.05, 7.71, 8.94] as SD and all hotspots were placed in a cube with a volume of 46 cm3 (X = 3.3, Y = 3.4, Z = 4.1). EF strength ranged from 0.28 to 0.59 V/m across the population. Using atlas-based parcellation of head models, cumulative EF strength within the right frontopolar (A10m + A10l) was significantly higher than right DLPFC (A9/46 v + A9/46d) in both montages (P < 0.01 with Hedges’ g = 0.34). Results for the MUDs were in line with healthy subjects and EF strength in the medial frontopolar (symmetric: 0.31 ± 0.07, asymmetric: 0.32 ± 0.07) where EF hotspots were located was significantly (P < 0.001) higher than DLPFC (symmetric: 0.18 ± 0.05, asymmetric: 0.19 ± 0.05) underneath the stimulating electrode with large effect sizes (Hedges’g for symmetric = 1.6190 with 95% CI (1.19, 2.04) and asymmetric = 1.3599 with 95% CI (0.95, 1.77)). Averaged peak coordinates in symmetric [1.88, 60.34, 19.12], and asymmetric montages [-3.14, 56.9, 6.33] were also located within the frontopolar cortex in both montages. However, there were inter-group variations between healthy participants and MUDs (maximum 4.3 cm3 distance between the averaged location of hotspots). Conclusions: Conclusions: Here, we discussed that in common tES montages which are frequently used with the goal of modulating DLPFC, DLPFC was not maximally targeted. Considering inter-individual and inter-groups variability, our results highlighted the frontopolar area as the area receiving the highest electrical field in DLPFC montages. Optimizing targets in electrical stimulation at both group and individual levels is recommended for future trials with the hope of ultimately maximizing clinical benefits. Keywords: transcranial direct current stimulation (tDCS), bipolar electrode montage, dorsolateral prefrontal cortex (DLPFC), frontopolar cortex, individualized head models Disclosure: Nothing to disclose. P656. Chronic Cocaine Use is Associated With Impairment in Utility Prediction Error Signal in Dopaminoceptive Regions in Humans Anna Konova, Ahmet Ceceli, Guillermo Horga, Scott Moeller, Nelly Alia-Klein, Rita Goldstein Rutgers University - New Brunswick, Piscataway, New Jersey, United States Background: People with drug addiction struggle to adapt to changing reinforcement contingencies, a tendency often attributed to impaired computation of dopaminergic prediction error (PE) signals. PEs, the discrepancy between actual and expected reward value, are broadcast by dopamine neurons to downstream targets like striatum to guide value updating. Prominent theories of the acquisition of cocaine addiction posit PE alterations caused by dopamine oversupply, leading to increased expectation of reward that cannot match actual reward and thus continued drug pursuit. However, further preclinical work has shown chronic cocaine disrupts this system, causing globally degraded phasic dopamine firing and release and reduced PEs. Thus, a primary deficit in the computation of PE may explain how dopamine dysfunction drives addictive behavior as an inability to appropriately update the value of the drug, or the value of healthier alternatives to the drug. While preclinical models support this notion, it is unclear if PE deficits translate to humans. There are surprisingly few studies that have formally assessed mesolimbic PE encoding in people with addiction, and the data do not firmly support or refute PE differences from health. Thus, while alterations in dopamine receptors and transmission in cocaine addiction in humans are well documented, we do not know if these changes result in functional dysregulation of PE signals needed for value updating. The question is important because it is these teaching PE signals that have been proposed as a functional mechanism mediating and maintaining addictive behavior and that may provide a bridge for clinical translation. We aimed to address this gap using a detailed computational fMRI-based assessment of PEs in human cocaine addiction based on contemporary theories of dopamine function. Methods: Participants were n = 39 individuals with cocaine use disorders who had used cocaine for 18 years on average and n = 35 matched controls (74% males). We adapted for fMRI a decision-making task previously used to probe single cell responses to a risk-sensitive form of PE (“utility PE”) in nonhuman primates. Like sensory systems that encode stimulus intensity nonlinearly (typically as a saturating function), emerging data show phasic dopamine firing and release encode a nonlinear PE signal whose shape can be captured by rescaling reward size by individual preference for risk. Therefore, to obtain a robust estimate of individual risk preferences, participants completed the task three times: 3 weeks before an fMRI scan, on the day of the scan outside of the scanner, and during fMRI acquisition. Behavioral data were fit with an economic expected utility model to derive participant-specific risk preference parameters which were compared between groups with nonparametric tests. The multi-band fMRI data were preprocessed and quality checked using robust pipelines (fMRIPrep) and model-based fMRI analyses were used to isolate (nonlinear) utility PE signals based on individual risk preferences, focusing on unbiased regions of interest in bilateral ventral striatum. Competing neural models were tested via Bayesian model comparison: models that assumed objective (i.e., completely linear) encoding of reward vs. utility, as well as those that decomposed the PE signal into its expected reward and received reward terms. Results: Risk preference parameters had moderate to high test-retest reliability across sessions, with ‘1-k’ ICCs ranging from 0.69 [0.47, 0.83] (95% CI) in cocaine users to 0.77 [0.60, 0.88] in controls. They were invariant to current clinical state in the cocaine group, but cocaine users had higher risk tolerance in aggregate than controls (Z = 3.01, P = 0.003) especially those with earlier age of onset (rho = −0.36, P = 0.02), possibly reflective of longer-term vulnerability for substance use. Across the entire sample, utility PEs were robustly encoded in bilateral ventral striatum as observed whole-brain (FWE-corr P < 0.05) and in region of interest analyses (t = 4.12, P = 9.81×10-05). This striatal PE signal was better explained by the (nonlinear) utility PE model than the linear PE model (exceedance prob=1). Consistent with theoretical PE accounts, ventral striatum activity scaled positively with utility of received reward (P = 1.25×10-05) and negatively with its expected utility (P = 0.02). Focusing on this computationally defined utility PE signal, we found strong evidence for impairment in cocaine addiction: cocaine users had markedly reduced PE encoding in ventral striatum (t = −3.10, P = 0.003) and this was driven by reduced response to received reward (t = −3.02 P = 0.003), with no differences in expectancy encoding (t = 0.08, P = 0.94). Exploratory analyses showed impaired PEs across the mesolimbic circuit, notably orbitofrontal cortex. Conclusions: These findings provide unambiguous evidence for impaired PE signals in dopaminoceptive regions of human cocaine users, specifically utility PEs such as those encoded by dopamine cells. Our data underscore a relationship between human drug addiction and this core teaching signal that has so far eluded translational neuroscience despite its central role in addiction theories. An important future direction will be to evaluate the time course of PE deficits in relation to vulnerability and development of addiction. This would be a major advance in our understanding of how early dopamine disruption culminates in the chronic drug pursuit that characterizes addiction. Keywords: Reward Prediction Error, Risk, Cocaine Use Disorder, Dopamine, Functional MRI (fMRI) Disclosure: Nothing to disclose. P657. Explainable Machine Learning Analysis Reveals Sex and Gender Differences in the Phenotypic and Neurobiological Markers of Cannabis Use Disorder Gregory Niklason, Eric Rawls, Sisi Ma, Erich Kummerfeld, Andrea Maxwell, Leyla Brucar, Gunner Drossel, Anna Zilverstand University of Minnesota, Minneapolis, Minnesota, United States Background: Cannabis Use Disorder (CUD) has been linked to a complex set of risk factors, including neurobiological, individual-level (e.g., personality, cognitive), and environmental factors. These factors have, until now, often only been investigated in a fragmented way, with researchers focusing on a small number of factors in each study or focusing on a single domain of interest. Therefore, while many studies have revealed sex/gender differences for single risk factors, sex/gender differences in the relative importance of a complex set of risk factors has not been described. Methods: We evaluated the relative importance of a wide variety of risk factors associated with high cannabis use levels (lifetime uses) and cannabis dependence (semi-structured interview), as well as potential sex/gender differences in a well-described community sample [Human Connectome Project (HCP); N = 1204, aged 22-35; 54% female; 72% White, 13% African American, 15% Other]. The sample included 9% individuals with cannabis dependence (N = 109, 26% female). We included a wide array of self-report, diagnostic, and behavioral measures, as well as structural (FreeSurfer) and functional (both resting-state and all fMRI tasks) indices of brain function as potential risk factors, in total about ~2000 variables. We employed state-of-the-art machine learning methods [XGBoost (eXtreme Gradient Boosting)], a tree-based ensemble machine learning algorithm in combination with a feature ranking tool [SHapley’s Additive exPlanations (SHAP)] to assign relative importance (i.e., SHAP values) to each of the associated risk factors. Results: We confirmed that among a very large number of input factors (~2000), a small number of previously identified environmental (education level, social support), personality (openness), mental health (e.g., externalizing symptoms, childhood conduct disorder), neurocognitive (e.g., working memory), and brain factors (e.g., hippocampal volume) were consistently highly ranked (in the top 20 variables) for their contribution to the classification of cannabis use levels and diagnostic status. When comparing the average Area Under the receiver operating characteristic Curve (AUC) for different data modalities (phenotypic, FreeSurfer, resting-state fMRI, task fMRI), the phenotypic models had the highest AUC (0.74), with bimodal models slightly outperforming unimodal models (e.g., phenotypic + FreeSurfer: 0.80). Sex/gender was consistently ranked in the top 5 factors in all models that contained phenotypic data. Further inspection of sex/gender interactions revealed systematic sex/gender interaction effects. Risk factors with a larger contribution to the classification in men included personality (high openness), mental health (high externalizing, high childhood conduct disorder, high fear somaticism), neurocognitive (impulsive delay discounting, slow working memory performance) and associated brain factors (low hippocampal volume, increased postcentral thickness). Conversely, risk factors with a larger contribution to the classification in women included environmental factors (low education level, low instrumental support) and associated brain factors (smaller super temporal area). Conclusions: In summary, environmental factors contributed more strongly to CUD in women, whereas individual factors had a larger importance in men. Results confirm the utility of machine learning approaches in describing sex/gender differences and suggest the importance of understanding how these differences may inform the development of sex/gender-specific treatment approaches for addiction medicine. Keywords: Cannabis Use Disorder, Gender Differences, Addiction Phenotypes, Big Data Analysis, Machine Learning Classification Disclosure: Nothing to disclose. P658. Adolescent Decision-Making Trajectories are Altered in Environmental and Genetic Models of Addiction Susceptibility Stephanie Groman, Kaitlyn LaRocco, Mary Jack, Justin Hill, Peroushini Villiamma University of Minnesota, Minneapolis, Minnesota, United States Background: Adolescence is a critical neurodevelopmental period associated with robust biobehavioral changes. These age-related changes include improvements in decision-making functions that we have found to predict drug use in adulthood. Deviations in these neurodevelopmental mechanisms may the mechanism by which addiction susceptibility emerges in individuals. We hypothesized that if adolescent decision-making trajectories are critical for regulating drug-taking behaviors, that adolescent decision-making would be altered in established models of addiction susceptibility. Methods: To test this hypothesis, we assessed decision-making throughout adolescence and adulthood in rats that were 1) selectively bred to prefer or not prefer alcohol (e.g., P [N = 15] and NP [N = 15] rats; genetic model of addiction susceptibility) and in rats that were 2) either socially housed (N = 11) or isolated during adolescence (N = 10) or adulthood (N = 9; environmental model of addiction susceptibility). Rats were trained to acquire and reverse three-choice, spatial discrimination problems using a reversal-learning task and performance repeatedly assessed at P30, P60, P90, and P120. Trial-by-trial choice data was fitted with a reinforcement-learning model to obtain an estimate of reward-based decision-making. Results: We found that the performance of P rats in the PRL task was significantly poorer than that of NP rats at each of the ages examined. The reinforcement-learning model indicated that the difference in performance was due to an attenuation in reward-based decision-making functions in P rats. Age-related changes in PRL performance were also attenuated in isolated rats compared to socially housed rats and rats that were only isolated during adulthood. The impairment in isolated rats was also found to be due to disruptions in reward-based decision-making functions. Conclusions: Our data demonstrate that adolescent decision-making trajectories are altered in both the genetic and environmental model of addiction susceptibility. Collectively, these data suggest that decision-making disruptions that are predictive of drug use susceptibility emerge during adolescence and studies investigating adolescent neurodevelopment could provide critical insights into addiction susceptibility. Our ongoing studies are integrating proteomic and genomic approaches to investigate the neurodevelopmental mechanisms mediating decision-making trajectories and drug use susceptibility. Keywords: Reversal Learning, Reinforcement Learning, Juvenile Social Isolation, Alcohol-Preferring (P) Rats, Orbitofrontal Cortex (OFC) Disclosure: Nothing to disclose. P659. Prenatal THC Exposure Promotes Aberrant Reward Seeking, Exacerbated Dopaminergic Encoding of Reward-Predictive Cues and Transcriptomic Alterations in the Midbrain of Adult Rats Miguel Angel Lujan Perez, Reana Young-Morrison, Cali Calarco, Sonia Aroni, Kate Peters, Megan Fox, Basu Mahasweta, Seth Ament, Gautam Kumar, Miriam Melis, Mary K. Lobo, Joseph Cheer University of Maryland, School of Medicine, Baltimore, Maryland, United States Background: Marijuana is the most common illicit drug used by pregnant women and is associated with offspring’s attention and learning deficits from early childhood until later in life. Previous research has identified midbrain dopaminergic neuronal alterations related to in utero exposure to THC. However, the effect of prenatal cannabis exposure (PCE) on future impairments of motivational processing in adulthood has not been specifically addressed. Methods: In order to address this, we conducted a behavioral characterization of addition-like endophenotypes in PCE rats trained to obtain food (palatable pellets) and opioid (remifentanil) rewards. Results: In utero cannabis exposure increased total food and opioid consumption, breaking points in a progressive ratio task as well as the intrinsic motivational value of both rewards under an economical demand task. Inhibitory motor control measures reported that PCE rats present higher impulsivity. In vivo fiber photometry dopamine recordings unveiled increased dopaminergic encoding of reward-predictive cues in the NAc, which was accompanied by a facilitation of cue-induced reinstatement of remifentanil-seeking behavior. Furthermore, the addition of sex as a co-variate revealed that male rats were more vulnerable to the effects of prenatal cannabis exposure. To explore the origin of such maladaptations, we conducted transcriptomic analyses (RNA-seq) in VTA, NAc and mPFC brain samples of PCE adult rats. Weighted Gene Co-expression Network (WGCNA), Differential Gene Expression (DGE) and Gene Ontology (GO) analyses all identified single-gene and network-wide alterations of VTA mitochondrial function related to PCE, reporting similar sex-dependent effects than the behavioral experiments. Conclusions: Altogether, our results suggests that prenatal cannabis exposure is inducing, in a sex-dependent manner, an aberrant reward-seeking phenotype with excessive encoding of opioid and natural rewards that is accompanied by long-lasting, transcriptomic alterations in midbrain’s mitochondrial function. Keywords: Cannabis, Dopamine, Pregnancy, Opioid Abuse, Transcriptomics Disclosure: Nothing to disclose. P660. Brain Circuits for Maternal Sensitivity and Pain Involving Anterior Cingulate Cortex Functional Connectivity in Mothers Treated With Buprenorphine for Opioid Use Disorder James Swain, Shaun Ho Stony Brook University, South Setauket, New York, United States Background: Opioid-induced deficits in maternal behaviors are well-characterized in preclinical rodent models. The prevalence of opioid use disorder (OUD) among pregnant women has risen sharply on a background of epidemic OUD. Despite gold standard buprenorphine treatment (BT) for withdrawal, related public health issues of postpartum depression, polysubstance use, relapse, and developmental risks to the infant plague affected mothers. We know that an evolutionary conserved Maternal Behavior Neurocircuit (MBN) governs maternal caring and aggressive behaviors, critical to healthy child outcomes. In the MBN, the anterior cingulate gyrus (ACC) participates in mother-child emotional bonding and attachment. However, ACC is also part of a “pain matrix” that processes the affective dimension and is extremely sensitive to opioid modulations. We know that prescription opioids affect resting-state functional connectivity (rs-FC) between the dorsal ACC, rostral ACC, and other regions related to MBN in healthy participants without OUD. Preliminary evidence suggests that prescription opioids may produce physical and emotional ‘analgesic’ effects through disruption of specific ACC-insula and ACC-putamen connectivity. However, even if acute use of opioids can reduce functional connectivity between these MBN-related regions, the effects of BT in mothers with OUD, who have chronically used prescription opioids remain unknown. We conducted a pilot longitudinal study of mothers receiving BT for OUD to examine ACC rs-FC. Methods: In this exploratory study, we studied 32 mothers who completed fMRI scans at 1-month (T1) and 4-months postpartum (T2), including 7 mothers receiving BT for OUD (BT/OUD, years of age: M = 29.86, SD = 6.62) and 25 non-OUD mothers as a comparison group (CG, years of age: M = 27.62, SD = 8.46). The participants underwent a 6-minute resting-state fMRI scan at each time point. According to the literature, region-of-interest spheres (10 mm) in the DACC and RACC centered on MNI coordinates [0, 6, 40] and [0, 46, 2]. Thus, we restricted our analyses to insula and putamen, regions modulated by acute prescription opioid administration. Results: We examined the Time by Group interaction effects on the rs-FC between the two seeds (RACC and DACC), and the regions insula and putamen. We found that the time-by-group interaction effects were significant in the RACC-dependent rs-FC with the left insula (MNI: [-40, 22, 2], 48 voxels, Z = 4.62, p = 0.003, family-wise error corrected) and the DACC-dependent rs-FC with the right putamen (MNI: [24, 16, -8], 169 voxels, Z = 5.17, p < 0.001, family-wise error corrected). BT mothers, as compared to CG mothers, showed lesser left insula-RACC rs-FC but greater right putamen-DACC rs-FC at T1, with these between-group differences reversed at T2. Conclusions: Though preliminary, the current study pioneers the study of buprenorphine effects on mothers affected by OUD. Given that BT is currently the best practice for pregnant women suffering OUD, due to the potential adverse effects of any exogenous opioids on maternal behavior, it is important to examine whether buprenorphine, as a partial µ-opioid agonist and κ-opioid antagonist, exert beneficial or harmful effects on maternal brain and behavior during the postpartum. Previously, we have established that BT mothers differed from CG in periaqueductal grey-dependent rs-FC with the hypothalamus, amygdala, insular cortex, and other brain regions at T1 and many of these differences disappeared at T2, suggesting potential therapeutic effects of continuing buprenorphine treatment, i.e., normalizing the dysregulation of OUD. Similarly, in the current pilot study, we found time-by-treatment interaction effects on the DACC and RACC-dependent rs-FC. Our work may have identified a brain mechanism for the potential benefits of BT on reversing dysfunction of maternal brain and behavior: the initial group differences in rs-FC flipped by four months postpartum. This work anticipates studies to ascertain how BT affects maternal behaviors, mother-child bonding, and intersubjective function, involving rs-FC in the pain matrix and ACC, as targets for novel interventions. Keywords: Opioid Use Disorder, Maternal Brain, Affective Components of Pain, Resting State Functional Connectivity, Anterior Cingulate Cortex (ACC) Disclosure: Nothing to disclose. P661. Pathways of Risk for Opioid Misuse: Traumatic Stress and Neural Connectivity Jacquelyn Meyers State University of New York Downstate Medical Center, Brooklyn, New York, United States Background: In addition to impaired executive functioning, altered connectivity of large-scale brain networks (e.g., as measured by EEG functional connectivity) has been observed separately among individuals exposed to trauma and among those who misuse opioids. While there have been previous studies demonstrating alpha, beta, and theta band EEG coherence differences in OUD, they have had small and heterogeneous study populations (Ns: 15-36; mixed ages, genders, polysubstance abuse) and examined a limited number of frequency bands and electrodes), making the mixed findings difficult to interpret. Relatively larger studies (Ns~150) of EEG coherence differences in individuals exposed to trauma have found atypical left hemispheric alpha, beta, and theta coherence, and that decreases in alpha coherence mediated the effects of trauma on psychiatric impairment. It remains unknown what aspects of EEG coherence differ among those with OUD and how this varies as a function of trauma exposure. Further, the influence of sex and other common co-substance use/disorders (alcohol and cannabis) on EEG connectivity are unknown. Methods: Drawing on data from the Collaborative Study on the Genetics of Alcoholism (COGA), a large family sample enriched for substance use disorders (N: 16, 866; 53% female, 24% Black; 18% of whom ever used opioids; 6% of whom with a DSM-5 Opioid Use Disorder; 70% trauma exposed), we examined the association of OUD with inter- and intra-hemispheric EEG coherence measures (27 inter- and intra-hemispheric bipolar pairs across theta, alpha, and beta frequencies) in males and females. Assessments of opioid misuse and DSM-5 substance use disorders were ascertained using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) interviews. Assessments of traumatic exposures were also derived from the SSAGA, which includes information on 21 potentially traumatic events. Based on evidence that traumatic events cluster together, and our preliminary finding that sexual assaultive trauma has the greatest impact on neurodevelopment, in addition to an ‘all’ trauma composite variable (sum score ranging from 0-21), three non-mutually exclusive, composite variables were also examined, representing report of one or more physical assaultive traumas (sum score ranging from 0-7: stabbed, shot, mugged, threatened with a weapon, robbed, kidnapped, held captive), sexual assaultive traumas (sum score ranging from 0-2: rape or molestation), and non-assaultive traumas (sum score ranging from 0-5: life-threatening accident, disaster, witnessing someone seriously injured or killed, unexpectedly finding a dead body). EEG Recording protocols have been detailed previously. Bipolar electrode pairs were derived to reduce volume conduction effects, and the 27 coherence pairs used in this study were selected based on previous studies on EEG coherence. Conventional Fourier transform methods were used to calculate EEG interhemispheric and intrahemispheric coherence at the following frequency bands: theta (3-7 Hz), alpha (8-12 Hz), and beta (13-20 Hz). Utilizing Mplus v8.1, we examined the association of opioid use and OUD, four trauma exposure variables, and their interactions, with topological patterns of inter- and intra-hemispheric EEG connectivity. Initial models were conducted on all individuals and included the following covariates: sex, self-reported “race/ethnicity”, age, birth cohort, familial relatedness. Secondary models were conducted stratified by sex. Results: 3,086 (18.3%) participants responded affirmatively as having ever used an opioid not as prescribed, or as directed, by their doctor. 962 (5.7%) participants had a diagnosis of DSM-5 OUD. Among the 962 individuals with an OUD, 21.7% (N: 209) have an OUD only (without a co-morbid SUD), 78.3% also have an AUD, and 56.1% have a CUD. Opioid use, OUD, AUD and CUD were more common among males as compared with females, and rates of opioid use (but not OUD), and AUD were greater among White participants as compared with Black participants. 69.5% of the sample experienced at least one trauma. 31.6% of the sample experienced physical assaultive trauma (males: 43.6%, females: 20.2%), 13.4% sexual assaultive trauma (males: 4.3%, females: 21.5%), and 53.8% non-assaultive trauma (males: 59.0%, females: 48.8%). Rates of physically assaultive and non-assaultive trauma (but not sexually assaultive trauma,) were greater among Black participants (41.9%, 63.8%) as compared with Whites (26.2%, 49.0%). The most prominent effects (i.e., surviving Bonferroni test correction, p < 0.001) for OUD among those who are trauma exposed in the COGA data were observed with inter-hemispheric frontal alpha coherence among females. Conclusions: Given the findings of altered neural network connectivity in OUD most prominently in those who are trauma exposed, the important neurodevelopmental changes observed across the lifespan, and the recent success of clinical interventions using neuroplasticity-based approaches, EEG coherence research can inform the development of therapies involving neurofeedback, cognitive remediation, and brain stimulation methods which draw on modifying neural network connectivity. Keywords: Trauma Exposure, Opioid Addiction, EEG Electrophysiology Disclosure: Nothing to disclose. P663. Trends in Characteristics of Individuals Seeking Inpatient Treatment for Alcohol Use Disorder, 2005-2021: Sex Differences, Disorder Severity, and Psychiatric Comorbidity Melanie Schwandt, Vijay Ramchandani, Nancy Diazgranados, David Goldman National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: Nationally representative survey studies have identified long-term changes in alcohol use and misuse in the United States during the past 15-20 years. Among the trends highlighted in these studies are a more pronounced increase in alcohol use and binge drinking among women compared to men, and a narrowing of the difference between men and women in the prevalence of past-year alcohol use disorder (AUD). These findings are informally referred to as “closing the gap”. Irrespective of these findings, alcohol treatment utilization has steadily remained much lower among women compared to men, and several studies have shown this disparity is not accounted for by sex differences in severity of alcohol-related problems. The current study examined more than 15 years of data collected from individuals seeking inpatient treatment at the National Institute on Alcohol Abuse and Alcoholism (NIAAA) intramural clinical research program. The aims of this study were to 1) examine sex disparities over time among individuals seeking inpatient treatment for AUD, and 2) evaluate trends in severity of AUD, severity of withdrawal, alcohol consumption levels, and psychiatric comorbidity among treatment-seeking men and women. Methods: Data were drawn from 1,711 inpatient admissions (1182 M, 529 F) occurring across a 17-year period (2005-2021) at the NIH Clinical Center. All individuals were seeking treatment for AUD, diagnosed using the Structured Clinical Interview for DSM-IV (SCID IV) or DSM-5 (SCID 5), and were enrolled in NIAAA natural history protocols. Individuals were assessed for AUD severity (Alcohol Dependence Scale, ADS), withdrawal severity (Clinical Institute Withdrawal Assessment for Alcohol-Revised, CIWA-Ar), recent alcohol consumption (Timeline Follow-back, TLFB), psychiatric comorbidity (SCID IV or SCID 5), and anxiety and depression symptom ratings (Brief Scale for Anxiety, BSA; Montgomery-Asberg Depression Rating Scale, MADRS). Analyses included descriptive statistics, data visualization, and chi-square, Fisher’s exact, and t-tests to compare outcomes between men and women overall and by year. Results: Overall, 69.1% of treatment-seekers were men while 30.9% were women. This disparity remained more or less consistent across the years studied (all p-values <0.05 except for 2011). On average, women had higher AUD severity (p < 0.0001) and withdrawal severity (p < 0.0001), but this was not consistent across years. The number of heavy drinking days was not different between men and women seeking treatment for AUD, although men generally consumed more drinks per drinking day (p < 0.0001) and more drinks per week (p < 0.0001). A trend for increasing prevalence of comorbid mood disorders (current and lifetime) was observed for both men and women, with treatment-seeking women having a higher prevalence than men on average (p < 0.001 for both current and lifetime). Current and lifetime anxiety disorder prevalence was also higher among women compared to men (p < 0.0001 for both current and lifetime). Similarly, anxiety and depression symptom ratings were on average higher for women than men, but again this was not consistent across years. Differences between men and women in prevalence of comorbid substance use disorders were nominal. Conclusions: On average and by year, men outnumbered women 2 to 1 among individuals seeking inpatient treatment for AUD. This is generally consistent with national statistics on prevalence of AUD in the population as a whole, although this gap between men and women has narrowed in recent years. On average, women seeking inpatient treatment for AUD showed higher AUD severity and psychiatric comorbidity, despite overall alcohol consumption levels being lower than that of men. Studies suggest that while the number of barriers to any form of treatment for AUD does not differ between men and women, the perceived need for treatment may differ – for example, women may be more likely to believe that they can get better on their own. Furthermore, increased severity of illness and comorbid depression and anxiety are associated with a higher probability of alcohol treatment utilization. These observations suggest that among women with AUD, those with greater disorder severity and/or psychiatric comorbidity may be more likely to seek inpatient treatment. Further research is needed on the motivation for, and barriers to, inpatient alcohol treatment in men and women. Keywords: Alcohol Use Disorder - Treatment, Sex Differences, Psychiatric Comorbidity Disclosure: Nothing to disclose. P664. Lifetime Alcohol and Cannabis Use Among the U.S. Adolescents Across Age Groups: Differential Patterns by Sex and Race/Ethnicity Mehdi Farokhnia, Lorenzo Leggio, Renee Johnson National Institutes of Health, Baltimore, Maryland, United States Background: Alcohol and cannabis are among the most commonly used drugs in the U.S. and worldwide, leading to high morbidity, mortality, and economic/societal burden. Earlier age of onset of substance use, including alcohol and cannabis, is associated with poor long-term outcomes, such as increased likelihood of developing a substance use disorder in adulthood. The aim of this study was to examine the prevalence of alcohol and cannabis use among adolescents of different age groups, and to investigate differential patterns by sex and by race/ethnicity. Methods: Data from the 2019 National Survey on Drug Use and Health (NSDUH), a cross-sectional nationally-representative survey, were used and the analyses were limited to 12-17-year-olds who reported being non-Hispanic White, non-Hispanic Black, or Hispanic/Latino (n = 11,830). Deming regression was applied to find the line of best fit between age groups and percent of lifetime alcohol/cannabis use, and the slopes of the regression lines were compared between the groups. Results: In the full sample, lifetime prevalence of alcohol and cannabis use was 28.4% and 17.4%, respectively. At age 12, 6.4% of the respondents reported lifetime alcohol use and 1.3% reported lifetime cannabis use. At age 17, 53.2% of the respondents reported lifetime alcohol use and 35.9% reported lifetime cannabis use. Girls had a steeper increase for lifetime alcohol use than boys [F (1, 8) = 3.40, p = 0.09], while there was not a sex difference for cannabis. The increase in lifetime alcohol use across age was similar among non-Hispanic White and Hispanic/Latino respondents, whereas the rate of increase was significantly flatter among non-Hispanic Black respondents [F (2, 12) = 21.26, p = 0.0001]. For cannabis, non-Hispanic White and non-Hispanic Black respondents had similar increases in lifetime use by age, while Hispanic/Latino respondents had a comparatively higher rate of increase [F (2, 12) = 3.17, p = 0.07]. Conclusions: The prevalence of lifetime use of alcohol and cannabis increased substantially from age 12 to age 17 in the NSDUH survey. Increases in alcohol use were steeper for girls (vs. boys), and among White and Hispanic/Latino (vs. Black) respondents. There were no sex differences in increases in cannabis use, although Hispanic/Latino respondents had steeper increases than White and Black youth. The quick increase in the prevalence of substance use from 12-17 points to the need for early interventions in adolescents to prevent the development of substance use disorders. Furthermore, the sex and race/ethnicity differences here described highlight the need for evidence-based preventive interventions tailored for specific demographic groups. Keywords: Alcohol, Cannabis, Adolescence, Sex Differences, Racial/Ethnic Differences Disclosure: Nothing to disclose. P665. Initiation and Retention of Buprenorphine and Methadone in Pregnant Individuals With Opioid Use Disorder: An Analysis of Insurance Claims in the United States (2006-2016) Kevin Xu, Hendrée Jones, Davida Schiff, Caitlin Martin, Jeannie Kelly, Ebony Carter, Laura Bierut, Richard Grucza Washington University in St. Louis, St Louis, Missouri, United States Background: The USA has experienced escalating rates of overdoses and adverse birth outcomes among women of childbearing age with opioid use disorder (OUD). The initiation and retention rates for buprenorphine and methadone have been seldom characterized using national data in underserved populations, such as among people using public insurance and minoritized individuals. The objectives of the current study were to (1) calculate the initiation and retention rates for buprenorphine and methadone in reproductive-aged people with OUD using national data, and (2) characterize disparities in initiation and retention by insurance status and race. Methods: In this retrospective cohort study, we analyzed data from female-identifying persons, ages 16-45 years, in the MarketScan databases (2006-2016). OUD and pregnancy status were identified based on inpatient or outpatient claims for established ICD-9/10 diagnoses and procedure codes. The main outcomes were buprenorphine and methadone initiation and retention, determined using pharmacy claims. Adjusting for age and cooccurring psychiatric and substance use disorders, we used logistic regression to estimate buprenorphine and methadone initiation, and Cox regression to estimate buprenorphine and methadone retention, stratifying analyses by insurance status and race. Results: Our sample included 103,038 reproductive-aged women in the USA with OUD (mean age 30.7 years, 64.6% Medicaid, 84% White), of whom 2,982 (2.9%) were pregnant. 67,521(65.5%) were initiated on psychosocial treatment without MOUD, in comparison to 29,435 (28.6%) and 6,082 (5.9%) initiated on buprenorphine and methadone respectively. 52% of buprenorphine and 41% of methadone episodes were discontinued at 180 days. Medicaid status was associated with a significant decrease in buprenorphine initiation (OR = 0.26 [0.26-0.27]) in comparison to commercial insurance. In contrast, Medicaid status was associated with a nearly 5-fold increase in methadone initiation (OR = 4.92[4.47-5.43]). Decreased buprenorphine initiation was observed for treatment episodes among BIPOC individuals (OR = 0.72[0.65-0.80]) relative to White peers; in contrast, BIPOC individuals were more likely to be initiated on methadone (OR = 1.52[1.37-1.68]). Relative to commercial insurance, Medicaid status was associated with a nearly 20% increase in buprenorphine discontinuation (HR = 1.19[1.17-1.21]), mirrored by a nearly 20% decrease in methadone discontinuation (HR = 0.81[0.75-0.87]). We observed an increase in both buprenorphine (HR = 1.13[1.04-1.23]) and methadone (HR = 1.18[1.09-1.28]) discontinuation in association with treatment episodes among BIPOC people relative to White individuals. Conclusions: Most reproductive-aged women with OUD in the USA do not receive buprenorphine or methadone. Large insurance-based disparities were observed such that Medicaid enrollees are less likely to be initiated and retained on buprenorphine as commercial insurance enrollees, and more likely to be initiated and retained on methadone. BIPOC individuals were significantly less likely to be initiated and retained in buprenorphine treatment; even though BIPOC individuals were more likely to be initiated on methadone, methadone retention rates among BIPOC patients were lower than White counterparts. Keywords: Pharmacoepidemiology, Health Disparities, Opioid Agonist Treatment, Health Services, Women’s Mental Health Disclosure: Nothing to disclose. P666. Probing Disparities in Exposure to Smoking Contexts Using Computer Vision Jason Oliver, Matthew Engelhard, Baylee Stevens, Julia McQuoid, F. Joseph McClernon University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States Background: Substantive efforts by healthcare providers, scientists and policymakers have helped dramatically reduce cigarette smoking rates over the past 70 years. Yet, high rates of smoking and poor cessation outcomes persist among many minoritized and underserved groups (e.g., women, low SES, racial and sexual minority groups). Numerous potential explanations for tobacco use inequities have been posited, including targeted tobacco industry marketing, social minority stressors, differences in nicotine metabolism, treatment access and compliance, social norms, and the types of tobacco products (e.g., menthol flavor) most commonly used. However, less attention has been paid to the environments that these groups are exposed to in everyday life, how daily environments differ between groups, and the potential influence specific environments may have on smoking behavior. For example, two recent studies indicate the effects of SES on smoking behavior are mediated by increased exposure to environments where smoking is allowed. This topic has received substantially less attention to date as a potential cause for tobacco use inequities and existing research has been limited by exclusive reliance on self-report for assessing exposure to smoking and non-smoking contexts. In the present analysis, we used a novel machine learning algorithm developed and validated in our prior work (Engelhard et al., 2021) to examine differences in exposure to environments where smoking is allowed or the risk of smoking is high. Methods: Daily cigarettes smokers residing in Durham, NC (N = 48) attended a baseline visit that included a detailed assessment of demographic characteristics and smoking behavior. They then completed a 14-day ecological momentary assessment (EMA) protocol during which they were asked to photograph their current environment each time they smoked and following six random prompts throughout the day. Each prompt also assessed the time since last cigarette, time of day and current location (e.g., home, work). An out-of-sample convolutional neural network model based on MobileNetV2 was pretrained on the ImageNet database and then trained to predict: (1) The probability that smoking was permitted; and (2) The probability of actual smoking based on the photograph and other contextual information. Mixed effect models were then used to examine overall differences in algorithm-predicted probabilities as a function of nicotine dependence, gender identity, race, sexual orientation and SES, as well as whether these effects differed across smoking and non-smoking contexts. Results: Participants were diverse with respect nicotine dependence level (27% low, 42% moderate, 31% high), gender identity (65% women), race (48% non-white), sexual orientation (19% non-heterosexual) and SES (46% “do not” or “just” meet basic expenses). A total of 7,843 images were included in analyses. The only main effect of demographic characteristics was a relatively weak effect indicating higher algorithm-predicted smoking risk for men relative to women (F = 4.3, p = 0.046). However, interactions with context type (i.e. smoking versus non-smoking) revealed stark differences in both outcomes as a function of nicotine dependence (Permitted: F = 43.0, p < 0.001; Smoking: F = 46.2, p < 0.001), gender identity (Permitted: F = 35.0, p < 0.001; Smoking: F = 20.1, p < 0.001), race (Permitted: F = 68.8, p < 0.001; Smoking: F = 51.9, p < 0.001), and sexual orientation (Permitted: F = 89.7, p < 0.001; Smoking: F = 56.0, p < 0.001). In all cases, effects were driven by smaller differences in algorithm-predicted probability values between smoking and non-smoking contexts among participants with greater nicotine dependence or who were members of minoritized groups. The only effect of SES was relatively weak (F = 4.4, p = 0.036) and indicated higher algorithm-predicted probability that smoking was permitted in actual smoking contexts among low SES participants. Conclusions: Findings indicate that individuals with higher nicotine dependence and those who are members of minoritized and underserved groups are exposed to substantially different environments with regards to smoking risk and the permissiveness of smoking than individuals with lower dependence and belonging to more privileged groups. Surprisingly, these differences were not driven by higher predicted risk for smoking or smoking permissiveness across all environments. Instead, effects were driven by a reduced difference in algorithm-predicted risk between smoking and non-smoking environments. This lack of differentiation between smoking and non-smoking environments offers a potential new insight into smoking disparities. Interestingly, these effects were not accounted for by SES and indeed the effects of SES were quite limited when adjusting for other demographic characteristics. While speculative, clearer differentiation between contexts where smoking does and does not occur may have a protective effect and improve odds of successful cessation. Greater understanding of the specific environmental characteristics that drive these effects may be informative. Future work applying computer vision approaches may help yield answers to these questions and provide fresh lines of inquiry for identifying environmental drivers of tobacco-related inequities and informing treatment and policy interventions. Keywords: Smoking, Health Disparities, Machine Learning Disclosure: Nothing to disclose. P667. A Single-Cell Atlas of Gene Expression and Chromatin Accessibility Changes Associated With Cocaine Addiction in the Rat Amygdala Jessica Zhou, Giordano de Guglielmo, Aaron Ho, Marsida Kallupi, Hairi Li, Lieselot Carrette, Olivier George, Abraham Palmer, Graham McVicker, Francesca Telese* University of California - San Diego, La Jolla, California, United States Background: The amygdala plays a key role in the negative emotional states associated with drug withdrawal and leading to relapse. Neuroanatomical and functional observations have uncovered the role of discrete amygdala subregions in different aspects of these negative affective states. However, the underlying transcriptional regulatory programs driving the function of distinct amygdala cell types remain unknown. Methods: Here we generated an atlas of single nucleus gene expression and chromatin accessibility in the amygdala of male rats with low and high cocaine addiction-like behaviors that were subjected to prolonged abstinence from extended access to cocaine intravenous self-administration. We e utilize single-nuclei multiomic methods to measure gene expression and chromatin accessibility in the amygdala of rats with low or high addiction indexes. We perform validation experiments by combining pharmacological inhibition with electrophysiological recordings in tissue slices and cue-induced cocaine-seeking behavior in rats. Results: Between rats of different addiction indexes, there are thousands of cell type-specific differentially expressed genes that are enriched for molecular pathways, including energy metabolism and GABAergic synapses in excitatory and somatostatin neurons. We demonstrate that higher addiction severity is linked to excessive GABAergic inhibition and, using pharmacological inhibition, we find that addiction-like phenotypes are regulated by the metabolite methylglyoxal. By analyzing differences in chromatin accessibility, we predict upstream transcriptional regulators associated with addiction-like behavior and find discordant regulation of key transcription factors among distinct cell populations. Conclusions: Overall, we provide a comprehensive characterization of cell type-specific transcriptional changes in the amygdala and the regulatory mechanisms that shape them in the development of addictive behaviors. Keywords: Transcriptomics, Epigenomics, Cocaine Addiction, GABA-A Receptors Disclosure: Nothing to disclose. P668. Integration of snRNA Sequencing and Spatial Transcriptomics Data Identifies Brain Region and Cell-Type Specific Expression Changes in Mice After Chronic Alcohol Exposure Nihal Salem, Gayatri Tiwari, Olga Ponomareva, Lawrence Manzano, Amanda Roberts, Marisa Roberto, R. Dayne Mayfield* The University of Texas at Austin, Austin, Texas, United States Background: Alcohol use disorders (AUDs) affected 8.6% of men and 1.7% of women globally and attributed to 5.3% of all global deaths in 2016. Transcriptomic studies identified dysregulated signatures in post-mortem brain samples from alcohol-dependent individuals as well as animal models of alcohol consumption and dependence. Using AUDs brain transcriptomic signature to identify potential therapeutics showed promise in animal models to reverse the escalation of alcohol drinking. Identification of cell-type specific transcriptomic changes in chronic alcohol use will improve our understanding of mechanisms mediating escalation of alcohol use and consequently refine targetable mechanisms to which therapeutics can be developed. In this work we aim to identify cell-type brain region specific changes mediating escalation of alcohol use. We utilized chronic intermittent exposure treatment paradigm (CIE), an alcohol dependence model that produces escalated drinking in mice, and neurobiological and behavioral adaptations in mice that mimic those found in humans with AUD. Methods: C57Bl6/J mice were exposed to CIE paradigm. Briefly, animals were exposed to 16 hours of ethanol vapor/day (or air as control) for 4 days followed by 72 hours of forced abstinence then with 2 bottle choice for 2 hours; this paradigm was repeated for four cycles followed by animal sacrificing and brain harvesting. We performed single nuclei RNA sequencing (snRNA-seq; 10x Genomics) on micro-punches obtained from pre-frontal cortex (PFC) of CIE and control mice, to determine cell-type-specific alcohol-induced gene expression changes. To understand the spatial context of CIE transcriptomic changes, we generated 10x Genomics Visium Spatial transcriptomics data from coronal PFC sections (10 µm) from CIE mice and controls. We utilized an ‘anchor’-based integration workflow introduced in Seurat on snRNA-seq data obtained from PFC micro-punches (reference) and the spatial RNA sequencing data (query), this pipeline outputs, for each spatial capture spot, a prediction score for each of the snRNA-seq derived cell types. Results: We utilized transcriptomic data to identify cell types and subtypes of each cell. We identified differentially expressed (DE) genes and their enriched pathways in each subtype. Oligodendrocytes, astrocytes, and a subtype of inhibitory cells were the most susceptible to CIE. Integration pipelines identified the anatomical location of each subtype, we show regional specificity of each of the excitatory and inhibitory cell types identified in our snRNA seq data. Most susceptible inhibitory cell subtype was defined to specific layers across the cortical area of the section. We clustered the spatial loci based on their gene expression similarities, followed by differential expression in each cluster. We show that the clusters with highest number of differentially expressed genes are located within regions of myelin-dense fiber tracts, consistent with our results from snRNA sequencing showing the oligodendrocyte as a highly susceptible cell type, other highly affected spatial cluster is located in the olfactory tubercle within the ventral region of the section, this region is enriched in tyrosine hydroxylase genes, suggesting involvement of dopamine signaling pathways. To understand transcriptomic regulation mechanisms, we utilized the snRNA sequencing data and constructed gene co-expression networks in each cell type. Oligodendrocytes and inhibitory cells subtype C, two highly dysregulated cell types, contained gene co-expression modules with Cpa6 as a hub gene, most of gene members of those co-expression modules were dysregulated in CIE samples. Cpa6, a metallocarboxypeptidase is involved in the regulation of neuropeptides and previously linked to high alcohol consumption in human GWAS studies. Additionally, we identified Pde4b as a hub gene of highly dysregulated module, and as an upregulated gene in oligodendrocytes. Pde4b gene has been previously shown to be a potential therapeutic target to reduce binge drinking. In the microglia we identified Plxdc2, a microglial enriched gene previously shown to be upregulated by alcohol in rat microglial culture, to be upregulated in CIE samples and to be a hub gene of a dysregulated module involved with cell migration and motility. Our data provide new cell-type specific insight into the molecular mechanisms underlying AUD. Conclusions: Our data identifies (1) spatially defined cell types highly susceptible in chronic alcohol exposure models and dysregulated pathways in each cell type, (2) gene co-expression networks that link previously identified alcohol target to their highly dysregulated downstream network allowing for targeting those networks to reverse escalation of alcohol use. (3) glial cells involvement in chronic alcohol use Keywords: Single Cell RNA-Seq, Spatial Transcriptomics, Alcohol Use Disorder, Alcohol Dependence, Gene Expression Disclosure: Nothing to disclose. P669. Genetic Relationships Among Seven Models of Addiction-Related Traits in Heterogeneous Stock Rats Keita Ishiwari, Christopher King, Jordan Tripi, Apurva Chitre, Oksana Polesskaya, Alexander Lamparelli, Anthony George, Connor Martin, Hao Chen, David Dietz, Leah Solberg Woods, Abraham Palmer, Paul Meyer* University at Buffalo, Buffalo, New York, United States Background: Both environmental and genetic factors place certain individuals at greater risk for developing substance use disorder. Furthermore, this vulnerability is associated with non-drug traits such as impulsivity, sensation seeking, response to novelty, attentional control, social interaction, and cue-responsivity. Rat models of these intermediate phenotypes allow the combination of forward genetic approaches with detailed behavioral analysis to identify biological pathways associated with substance use disorder. Methods: To characterize the genomic regions associated with these behaviors, we performed a genome-wide association study (GWAS) using up to 1645 male and female heterogeneous stock (HS) rats tested in several behavioral paradigms, including locomotor response to novelty, choice reaction time, social reinforcement, visual (light) reinforcement, delay-discounting, Pavlovian conditioned approach (PavCA), and cocaine-conditioned cue preference. Further, genetic correlations with other GWAS projects, including nicotine self-administration, were also calculated. Results: Single nucleotide polymorphism (SNP) heritability for the key measures acquired for each of these traits ranged from 0.12 (goal-tracking during PavCA) to 0.30 (distance travelled during the locomotion test). Further, some traits were genetically correlated with nicotine self-administration, including measures of delay-discounting (0.86, p = 0.006) and average reaction time during the choice reaction time task (0.65, p = 0.009). Finally, many loci and candidate genes were identified. For example, four measures of reaction time reached genome-wide significant associations on chromosome 20 which contains Abcfl and also has a strong cis-acting expression quantitative trait locus (eQTL) that maps to this interval. Another region reaching significance for delay discounting on chromosome 15 includes the gene Otx2, a gene involved in the formation of dopaminergic neurons and already implicated in this trait. Conclusions: These studies demonstrate the utility of using HS rats for GWAS: many of the identified candidate genes have been implicated by human GWAS, and others are novel candidates that will identify new avenues of research and potentially new treatment approaches for human psychiatric diseases. Keywords: Substance Use Disorder, Genome-Wide Association Study, Cue-Reactivity, Impulsivity, Cocaine Sensitivity Disclosure: Nothing to disclose. P670. Using Whole Blood for Transcriptome-Based Drug Repurposing in a Model of Alcohol Dependence Laura Ferguson, Amanda Roberts, R. Dayne Mayfield, Robert Messing Dell Medical School UT Austin, Austin, Texas, United States Background: Alcohol Use Disorder (AUD) is a chronic, relapsing condition and a major public health problem with few pharmaceutical treatments available. Recent evidence in rodents suggests that brain gene expression profiles can discriminate between alcohol dependent and non-dependent subjects and predict pharmaceuticals that decrease drinking. However, this methodology has limited clinical potential because it is not possible to obtain brain specimens from patients. We hypothesized that whole blood could be a more accessible transcriptome for diagnostic and therapeutic applications. Methods: We used whole blood transcriptome profiles from mice exposed to chronic intermittent ethanol vapor (CIE) or air (N = 40 total; 10/sex/group) to define a blood CIE signature as the top DEGs based on p-value and fold change (the top 2% of each). To identify pharmaceutical candidates that might decrease ethanol consumption, we compared the blood CIE signature to those of pharmaceuticals in the NIH Connectivity Map database (CMap). Working under the hypothesis that medications with opposing effects on gene expression as the blood CIE signature will reduce voluntary alcohol consumption, we prioritized the compounds with negative connectivity scores (i.e., reversers). Additional prioritization included FDA-approval status, whether multiple drugs from the same category were predicted, and whether the same drug (or drug category) was predicted for both sexes. A proof-of-principal study was conducted with the top candidate compound to determine its effects on voluntary alcohol consumption in C57BL/6 J mice (N = 20 total; 5/sex/group) using the same CIE drinking protocol from which the transcriptome data were derived. Statistical significance was assessed using a three-way ANOVA with sex (male and female) and treatment (drug and vehicle) as between-subjects factors and time as a repeated measure. All experimental protocols in animal studies were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Results: The CMap analysis identified 39 reversers for females and 29 reversers for males (permutation p < 0.05). Of these, several cardiac glycosides were predicted to be reversers for both sexes. Digoxin is relatively safe and is the most prescribed cardiac glycoside for the treatment of heart failure, so it could be repurposed if proven to be effective for AUD. We administered digoxin (1 mg/kg; i.p.) or vehicle (5% DMSO in saline; i.p.) to male and female mice once daily beginning after the fourth CIE vapor session and continuing throughout the 5-day voluntary drinking test and one more CIE vapor and 5-day voluntary drinking test. The time x treatment interaction was highly significant, and digoxin decreased alcohol intake compared with vehicle in both sexes (by ~50% in males and ~70% in females). Conclusions: CMap analysis can predict pharmaceuticals that reverse the CIE transcriptional signature in blood. One predicted reverser, digoxin, decreased drinking in male and female C57BL/6 J mice. These results indicate that blood is an accessible tissue that can be used for transcriptome-based drug repurposing through the application of advanced computational approaches to identify therapeutics for AUD and potentially personalize AUD treatment. Keywords: Gene Expression, Systems Pharmacology, Alcoholism, Alcohol Use Disorder - Treatment Disclosure: Nothing to disclose. P671. Cell Adhesion Molecule 2 Deletion Reduces Impulsivity and Voluntary Cannabinoid Intake, and Impairs Physiological Response to Δ9-Tetrahydrocannabinol in Mice Hayley Thorpe, Malik Talhat, Sandra Sanchez-Roige, Abraham Palmer, Jibran Khokhar University of Guelph, Guelph, Canada Background: It is estimated that 3.9% of the global population uses cannabis annually and that approximately 9% of those who use cannabis within their lifetime develop cannabis use disorder. Consequently, there is a growing need to identify risk factors of cannabis use initiation and problematic use. Polymorphisms in Cell Adhesion Molecule 2 (CADM2), a gene associated with externalizing behaviours, were recently correlated with cannabis use initiation by genome-wide association studies. We hypothesized that these human findings would be recapitulated in a Cadm2 knockout (Cadm2-/-) mouse line, which was used to investigate the causal relationship of Cadm2 with voluntary cannabinoid intake, pharmacological response to Δ9-tetrahydrocannabinol (THC), and cognitive phenotypes associated with substance use. Methods: Adult male and female Cadm2 + /+, Cadm2 + /-, and Cadm2-/- mice (n = 11-18/genotype) were used for each experiment. To assess cannabinoid self-administration and preference, a two-edible choice preference test was employed with use of either pure THC or THC-dominant cannabis oil in cookie dough. Drug consumption and preference across escalating THC doses were calculated based on consumption of drug- and vehicle-containing dough. In a separate cohort, the role of Cadm2 genotype in pharmacological response to THC was determined using the cannabinoid tetrad assay (i.e., assessment of THC-induced hypolocomotion, hypothermia, catalepsy, and analgesia) after single or repeated intraperitoneal injection of moderate or high (3 or 10 mg/kg) THC doses. Finally, a range of executive functions were assessed in a drug-naïve group of animals using the 5-choice serial reaction time task (5CSRTT) under drug-free and THC challenge conditions. Parametric data were analyzed using one-way or repeated measures ANOVA where appropriate; nonparametric data were analyzed using Kruskal-Wallis H test or aligned rank transformed ANOVA where appropriate. Results: Significant differences in THC dough preference (F(2,32)=4.85, p < 0.05), cannabis oil dough preference (F(2,34)=4.65, p < 0.05), and cannabis oil dough consumption (F(2,36)=3.57, p < 0.05) were observed according to Cadm2 genotype. Cadm2-/- mice showed lower preference and consumption (p < 0.05) of drug dough compared to Cadm2 + /+ and Cadm2 + /- littermates. Cannabinoid tetrad assessment revealed an effect of genotype on baseline locomotor activity (F(2,76)=37.90, p < 0.001) and thermal pain tolerance (F(2,77)=4.64, p < 0.05), as well as THC-induced locomotor (moderate THC: F(2,34)=50.92, p < 0.001; high THC: F(2,35)=63.69, p < 0.001), body temperature (high THC: F(2,36)=13.52, p < 0.001), and analgesic (moderate THC: F(2,32)=6.23, p < 0.01) responses. Under drug-free conditions, Cadm2-/- mice were hyperlocomotive (p < 0.01) and exhibited greater thermal pain tolerance (p < 0.01) than littermates. Moderate (p < 0.01) and high (p < 0.05) THC doses exacerbated hyperlocomotion in Cadm2-/- mice, whereas characteristic hypolocomotion to high THC administration was observed in littermates (p < 0.05). In addition, acute injection of high-dose THC produced hypothermia in Cadm2 + /+ and Cadm2 + /- mice only (p < 0.001), and analgesic sensitization following repeated administration of moderate dose THC in Cadm2-/- mice (p < 0.01). Finally, the 5CSRTT revealed that Cadm2 expression affected performance accuracy (F(2,32)=3.58, p < 0.05) and premature responding (H(2)=16.10, p < 0.001) that was suggestive of attentional deficits (p < 0.05) and lower impulsivity (p < 0.01) by Cadm2 deletion. Acute administration of 2 mg/kg of THC did not affect impulsivity or accuracy in the 5CSRTT within any genotype. Conclusions: These data indicate a role for Cadm2 in cannabinoid preference and consumption, pharmacological response to THC, and executive functions relevant to substance use disorders. Notably, these results suggest that Cadm2 expression is a risk factor in voluntary cannabinoid intake, possibly by affecting pharmacological response to THC and promoting impulsivity. These findings provide support for human correlational studies that propose CADM2 polymorphisms affect externalizing phenotypes, including impulsivity and substance use. Keywords: Cannabis Use, Heritability of Substance Use Disorder, Drug Self-Administration, Executive Function, Translational Animal Models Disclosure: Nothing to disclose. P672. Metabolic-Epigenetic Exchange During Voluntary Alcohol Intake Gabor Egervari, Natalia Quijano-Carde, Connor Hogan, Cassidy Hemphill, Desi Alexander, Mariella De Biasi, Shelley Berger University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Alcohol metabolites contribute to brain histone acetylation by the direct deposition of alcohol-derived acetate on histones. This reaction is dependent on the metabolic enzyme acetyl-CoA synthetase 2 (ACSS2), which is nuclear and chromatin-bound in neurons. Mice lacking ACSS2 do not deposit acetate onto histones in the brain and show no conditioned place preference for ethanol reward. Here, we explore the role of this pathway during voluntary alcohol intake. Methods: We used the drinking-in-the-dark (DiD) paradigm to assess alcohol consumption in ACSS2 knock-out (KO) mice and wild-type littermates. We characterized histone acetylation (chromatin immunoprecipitation coupled with high throughput sequencing) and gene expression (RNA sequencing) to test the effect of alcohol-derived acetate during voluntary drinking. Results: We found that ACSS2 KO mice consume significantly less alcohol while undergoing DiD and, surprisingly, show significantly elevated blood alcohol concentrations. Histone acetylation and gene expression changes in the hippocampus, the nucleus accumbens and other brain regions point to impaired expression of genes that have previously been linked to alcohol and other substance use disorders. Conclusions: Direct incorporation of alcohol metabolites into histone acetylation has profound effects on gene expression in the brain, and potentially drives behaviors related to alcohol use disorder. Since inhibiting ACSS2 leads to decreased voluntary alcohol intake in mice, targeting this pathway could be a promising new therapeutic avenue. Keywords: Alcohol, Epigenetics, Drug Metabolism Disclosure: Nothing to disclose. P673. Behavioral and Brainstem Transcriptome Analysis of FVB/N Substrains in a Mouse Model for Neonatal Opioid Withdrawal Syndrome (NOWS) Kelly Wingfield, Kayla Richardson, Teodora Misic, Mia Rubman, Jacob Beierle, Emily Yao, Camron Bryant Boston University School of Medicine, Boston, Massachusetts, United States Background: Concomitant with the opioid epidemic, there has been a rise in pregnant women diagnosed with opioid use disorder and infants born with neonatal opioid withdrawal syndrome (NOWS). NOWS refers to a heritable set of symptoms following cessation of prenatal opioid exposure that comprise low body weight and hyperirritability. However, the genetic factors contributing to differences in withdrawal symptom severity remain poorly understood. We aim to use mouse models to identify genetic variants contributing to NOWS severity and translate these findings to humans. Methods: All mouse experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. First, we phenotyped genetically similar inbred substrains of the FVB/N strain (NJ, NCrl, NHsd, and NTac) to identify behavioral differences in NOWS model traits, including delayed growth, hyperalgesia, and hyperirritability. Male and female neonatal pups were injected twice daily from postnatal day 1 (P1) to P15 with morphine (10 mg/kg, s.c.; MOR) or saline (SAL). This early life exposure model represents the approximate third trimester in humans and is characterized by developmental milestones in the brain, including synaptogenesis and myelination. This exposure period is necessary and sufficient for inducing NOWS traits. Importantly, this model avoids dam exposure that could affect maternal care and permits control of MOR dosing across pups. We assessed several phenotypes during spontaneous MOR withdrawal (16 h) on P7 and P14, including ultrasonic vocalizations (USVs) followed by nociceptive testing (hotplate latency (52.5 C). On P8 and P15, we administered MOR or SAL, and recorded USVs to determine the effect of MOR treatment on USVs. In pups, USVs indicate emotional states and are emitted in response to thermal change, distress, and maternal separation. We explored the composition of USVs to determine if certain syllables were associated with opioid withdrawal, an aversive state. We also looked for divergence among FVB substrains in USV composition that would implicate genetic differences in USV model traits for NOWS. We used the machine learning software, DeepSqueak, to classify USV syllables, and developed a supervised classifier to distinguish USVs based on spectrotemporal characteristics. On P16 during withdrawal, brainstem tissue was collected for RNA-sequencing, which was conducted at the Microarray and Sequencing Resource Core Facility at Boston University using a 200 cycle P2 flow cell and Illumina NextSeq 2000 (100×100 bp paired-end reads). Results: All MOR pups vs. SAL pups displayed lower body weights and showed thermal hyperalgesia (reduced nociceptive latency) on the hot plate assay (SAL, n = NCrl: 11-17, NJ: 13, NHsd: 19-25, NTac: 10; MOR = NCrl: 11-15, NJ: 8-10, NHsd: 14-23, NTac: 8-9). During withdrawal on P14, MOR pups emitted more USVs, consistent with the excessive crying in NOWS infants (t-test, **p < 0.0001). Additionally, there was an increase in the percentage of complex 3 syllables in MOR (n = 40) vs. SAL (n = 51) pups (t-test, p < 0.0001). Interestingly, in the NJ substrain, female MOR pups (n = 5) emitted more USVs (Two-way ANOVA F(1,18)=10, p = 0.00540. Bonferroni correction *padjusted=0.0002) and a greater proportion of complex 3 syllables compared to male MOR pups (n = 5) (Two-way ANOVA (F(1,18)=2.9, p = 0.101. Bonferroni correction padjusted=0.00980). On P15, we administered a relief dose of MOR or SAL. MOR reduced the total number of USVs emitted (Two-way ANOVA F(1,134)=36.37, **p < 0.0001) and reduced the proportion of complex 3 syllables emitted by MOR pups toward a control-like level (Two-way ANOVA F(1,134)=36.4, p < 0.0001), suggesting that complex 3 syllables could be a unique marker associated with the opioid withdrawal state. RNA-seq analysis of NJ brainstem tissue (SAL,n = 4; MOR, n = 4) revealed downregulation of myelin basic protein, Mbp (log FC = −0.410, padjusted=0.00589), and proteolipid protein 1, Plp1 (log FC = −0.387, padjusted=0.0104), and upregulation of the µ-opioid receptor, Oprm1 (log2FC = 0.977, padjusted=0.0454), k-opioid receptor, Oprk1 (log2FC = 0.339, padjusted=0.00553), in addition to upregulation of the dopamine transporter, Slc6a3 (log2FC = 3.62, padjusted=0.0859), that was driven by the females. Conclusions: We expanded the third trimester-approximate model for NOWS to FVB/N substrains to show decreased weight gain and increased nociceptive sensitivity during withdrawal. MOR pups emitted more USVs than controls and a greater proportion of complex 3 syllables that were alleviated by a subsequent MOR maintenance dose. Based on the connection between USVs and emotional states in pups, our data suggest that complex 3 syllables could indicate negative emotional states associated with opioid withdrawal. Additionally, several addiction-relevant genes were upregulated in NJ MOR pups, while genes associated with myelination and development were downregulated. These results highlight sex and substrain differences in physiological and emotional withdrawal-associated phenotypes and provide insight into the biological mechanisms contributing to differences in NOWS severity. A future direction will involve quantitative genetic analysis and gene discovery underlying substrain variance in NOWS model traits in a reduced complexity cross. Keywords: Behavioral Pharmacology, Neonatal Opioid Withdrawal, Addiction Genetics, Genetic Mapping Disclosure: Nothing to disclose. P674. The Neuron -Navigator-1 (Nav1) Gene Regulates Self-Administration of Cocaine and Palatable Food in Mice Jared Bagley, Yalun Tan, Gary Peltz, James Jentsch Binghamton University, Binghamton, New York, United States Background: Intravenous self-administration (IVSA) of potentially addictive drugs in laboratory animals models the volitional initiation and progression of drug use in humans and provides opportunities to discover the neurogenetic influences on this behavior. Cocaine IVSA is a complexly determined trait, and a substantial proportion of individual differences in cocaine use in both humans and animals is determined by genetic variation, though the specific causal genes and alleles remain mostly unknown. Cocaine IVSA procedures can be used in genetically diverse laboratory animal populations to rigorously and rapidly identify relevant genetic influences. We have utilized the Hybrid Mouse Diversity Panel (HMDP) to characterize cocaine IVSA and perform haplotype-based computational genetic mapping for this trait. These efforts nominated Neuron-Navigator1 (Nav1) gene on chromosome 1 as a cocaine IVSA candidate gene. Nav1 is known to be expressed in post-mitotic neurons and to affect neuritogenesis; however, relatively little is known about the effects of altered Nav1 expression on the nervous system function or behavior, particularly potential effects on addiction-relevant functions or behavioral traits. In order to validate the role of this gene in cocaine IVSA, we developed a genetically engineered Nav1 knockout (KO) model to test in cocaine IVSA. Methods: CRISPR was utilized to induce a null allele at the Nav1 gene on the C57BL/6 J background and gene expression was measured to confirm that the mutation eliminates expression of functional Nav1. Mice were bred with male and female heterozygous mice to produce litters that include all 3 genotypes (homozygous wilde-type (wt), homozygous mutant (mutant) and heterozygous (het)). Some subjects were surgically catheterized, providing access to the jugular vein. Mice were tested in a between-subjects dose-response (0.1, 0.5 and 1.0 mg/kg) cocaine IVSA procedure (n = 14-15 per genotype/dose, sex approximately balanced across groups). Testing occurred in operant boxes under an FR1 schedule, in which a press of the active lever caused an infusion, presentation of a conditioned cue (flashing house light for 20 sec) and a 20 sec timeout. Sessions were terminated after 2 hours or when 65 infusions were earned. An additional cohort of naïve (no surgery) mice (n = 10-11 per genotype, sex approximately balanced across groups) were tested in a similar IVSA procedure with a palatable food as a reinforcer. Results: Cocaine IVSA testing across three doses revealed that mutant mice in the Nav1 KO line self-administered more cocaine [main effect of genotype F(2,113)=6.4, p = 0.002] relative to wild-type and heterozygous mice. We did not find evidence that this genotype effect varied across cocaine dose [genotype by dose interaction F(4,113)=0.9, p = 0.496]. Thus, this data suggests that KO of the Nav1 gene produces an upward shift of the dose-response curve. In testing the effects of genotype on operant responding for a food reinforcer, we found that homozygous mutant mice again earned more reinforcers [main effect of genotype F(2,26)=6.5, p = 0.005]. Conclusions: Our forward genetic approach for cocaine IVSA in the HMDP nominated Nav1 as a candidate gene that may moderate intake of cocaine, and the present data has validated this role of Nav1. We found that KO of Nav1 increased intake of cocaine and palatable food in an operant self-administration procedure. These data suggest that the effects of Nav1 generalize across drug and non-drug reinforcers. Previous study of this KO line revealed additional phenotypes associated with homozygous mutant mice including altered response to opioid reward and analgesia, lower than expected production of mutant mice, locomotor hyperactivity and a potential higher incidence of seizures. Future research will seek to determine the effects of Nav1 on key neurobiological mechanisms that may mediate Nav1 effects on reward-seeking behaviors and other associated phenotypes. Keywords: Addiction, Genetics, Cocaine, Self-Administration, Genomics Disclosure: Nothing to disclose. P675. Heritable Differences in Impulsive Behavior Associate With Altered Indices of Dopaminergic Transmission in the Orbitofrontal Cortex and Nucleus Accumbens in BXD Recombinant Inbred Mice Lauren Bailey, Anushree Karkhanis, James Jentsch Binghamton University, Binghamton, New York, United States Background: Impulsivity is a heritable phenotype in humans that is elevated in chronic drug and alcohol users, in part because it is a risk factor for the development of addiction. One hypothesis explaining the strong relationship between impulsivity and addiction is that genetic regulation of neuronal signaling within the orbitofrontal cortex (OFC), an area of the frontal cortex that exerts control over reward-guided behavior and plays a key role in addiction neurobiology, is involved. Methods: Here, we measured dopaminergic function within the OFC and its efferent target, the nucleus accumbens (NAc), in six recombinant BXD strains selected for a high or low impulsive behavioral phenotype in a reversal learning task. Adult mice underwent reversal learning or were euthanized and the OFC and NAc regions were collected. HPLC or rtPCR was conducted on tissue for quantification of dopamine, serotonin, and their metabolites or relative expression levels of Drd1 and Drd2, respectively. Results: Our measure of impulsive behavior and each of the measures of dopamine turnover and receptor expression were significantly heritable, with broad sense heritability estimates ranging from .076 to .180. HPLC analysis revealed increased dopamine turnover in the NAc of high impulsive strains (F[1,82]=5.620, p < 0.05, N = 86), lower DOPAC levels in the OFC of high impulsive strains (F[1,61]=5.011, p < 0.05, N = 65). rtPCR identified lower expression of Drd1 (F[1,86]=8.033, p < 0.01, N = 90) and Drd2 (F[1,86]=7.254, p < 0.01, N = 90) in the NAc of high impulsive strains. Conclusions: These results thus far would indicate heritable differences in the dopaminergic system of the NAc, and potentially OFC, of high impulsive mouse strains, most notably elevated dopamine metabolism and lower Drd1/Drd2 expression in the NAc. Data from this study facilitates understanding of dopaminergic dysfunction in the cortico-striato-thalamo-cortical pathway that may underlie impulsive behaviors and associated susceptibility for substance use and use disorder. Keywords: Impulsive Behavior, Dopamine, Nucleus Accumbens (NAA), Orbitofrontal Cortex (OFC) Disclosure: Nothing to disclose. P676. Cross-Ancestry Meta-Analysis of Tobacco Use Disorders Based on Electronic Health Record Data Uncovers Novel Loci and Reveals Associations With Numerous Health Outcomes Sandra Sanchez-Roige, Mariela Jennings, Sylvanus Toikumo, Hyunjoon Lee, Travis Mallard, Sevim Bianchi, Benjamin Pham, Natasia Courchesne-Krak, Maria Niarchou, Million Veteran Program, PsycheMERGE SUD Workgroup, Dana Hancock, Ke Xu, Jordan Smoller, Lea Davis, Amy Justice, Hank Kranzler, Rachel Kember University of California San Diego, San Diego, California, United States Background: Tobacco use disorders (TUD) are the most prevalent substance use disorder in the US, with a high proportion of smokers meeting criteria for dependence. These individuals often have difficulty quitting, experience withdrawal symptoms when they stop, and continue smoking despite negative mental, social, and medical consequences. Genetic factors influence smoking behaviors and strides have been made in understanding aspects of tobacco initiation and use via genome-wide association studies (GWAS). However, due to limited sample sizes, GWAS of TUD still account for only a small amount of the variance in these traits. In addition, nicotine-related GWAS have primarily focused on individuals of European ancestry. Methods: Here we leverage access to multiple biobanks (Vanderbilt University Medical Center, Mass General Brigham, Million Veterans Program, UK Biobank) to perform a cross-ancestral meta-analysis of TUD (derived via electronic health records, EHR) in 740,361 individuals of European, African American, and Latin American ancestries. Results: The cross-ancestral meta-analysis identified 31 independent loci, including the nicotinic acetylcholine receptor CHRNA3/A4 gene cluster, which has been consistently associated with smoking behaviors. Other promising candidate genes include PDE4B, previously associated with smoking initiation and problematic alcohol use, and PTPRF, recently implicated in opioid addiction and impulsive behaviors. TUD-EHR was also genetically correlated with traits derived from traditionally ascertained cohorts, including nicotine dependence and smoking cessation. Surprisingly, the genetic correlation between cigarettes per day and TUD-EHR, although significant and positive, was moderate in magnitude (rg < 0.5), suggesting that the genetic architecture of consumption and misuse is distinct. Lastly, we evaluated the use of TUD-EHR polygenic scores as genomic predictors of 1,335 psychiatric and medical traits, and revealed hundreds of associations, including HIV infection, heart disease, and pain. Conclusions: This work furthers our biological understanding of TUD and its shared genetic risk with other mental and physical traits, and establishes that EHR are useful sources of phenotypic information for studying the genetics of TUD. Keywords: Tobacco, GWAS, Substance Use Disorders, Electronic Health Record (EHR), Addiction Comorbidity Disclosure: Nothing to disclose. P677. Elevated DNA Damage in Specific Cell Types Associated With Opioid Use Disorder in Human Postmortem Brain Madelyn Ray, BaDoi Phan, Marianne Seney, Jill Glausier, Allison Tipton, Shelley Russek, David Lewis, Andreas Pfenning, Ryan Logan Boston University School of Medicine, Boston, Massachusetts, United States Background: In the United States, rates of opioid use disorder (OUD) and deaths from overdose are unprecedented. Despite the enormous public health impact of OUD, we have a limited understanding of the changes in the brain in patients with OUD. Few studies have directly examined the cellular and molecular changes in the human brain associated with OUD. A recent study from our group utilized bulk RNA sequencing in human postmortem brain in the dorsolateral prefrontal cortex and nucleus accumbens in OUD and control subjects. This study identified significant alterations in transcripts enriched for neuroinflammatory and extracellular matrix signaling in the brains of subjects with OUD. Additionally, cell type specific enrichment of microglia markers in demonstrates a potential primary role for microglia in OUD. Collectively, this study suggests that OUD may induce the upregulation of neuroinflammatory markers, particularly in microglia. However, a limitation to using bulk RNA sequencing is the lack of cellular resolution. To address this limitation, we are utlizing single nucleus RNA sequencing in human postmortem brain. Human neuroimaging studies investigating OUD have implicated functional changes in the dorsal striatum, an area integral in reward processing, habitual drug-seeking, craving, and relapse. The dorsal striatum is comprised of the caudate nucleus and putamen. In the current study, we conducted single nuclei RNA sequencing on human postmortem caudate nucleus and putamen in subjects diagnosed with OUD and comparison control subjects in order to identify cell-type specific transcriptional changes associated with opioid use disorder. This study is the first of its kind to identify key changes in transcriptional signatures associated with opioid use disorder at the single nucleus level in human postmortem dorsal striatum. Methods: In the current study, we conducted single nuclei RNA sequencing on postmortem brain tissue from male and female subjects diagnosed with opioid use disorder (N = 6; 3 females and 3 males; average age: 41.17) or comparison subjects (N = 6; 3 females and 3 males; average age: 44.67). For each subject, both the caudate and putamen were processed. Nuclei were isolated from 24 fresh frozen post-mortem human brain samples. Briefly, ~50 mg of tissue was dounce homogenized with 1,000 µl nuclei lysis buffer containing dapi using a glass douncer with 9 pestle strokes. Homogenate was filtered using a 40um cell filter. Samples were sorted on a BD FACS Aria II Flow Cytometer at the Boston University Flow Cytometry Core using Fluorescent Activated Cell Sorting (FACS) for dapi. Nuclei were sorted at a flow rate of 1.5-2.0 with gating criteria set to hierarchically select whole, single nuclei. For each sample, approximately 150,000 nuclei were collected into a tube containing 11.40 µl PBS with 0.04% BSA. Samples were evaluated for nuclei concentration and viability on a hemocytometer. 7,000 nuclei were targeted using the 10x single cell dual index 3’ gene expression protocol to prepare dual index next generation sequencing libraries. Finally, libraries are pooled and sequenced on the Next-seq 2000 (illumina) at the Boston University Single Cell Sequencing Core. Sequencing targeted 7,000 nuclei per sample and 50,000 reads per nuclei. Following completion of sequencing, data was pre-processed. Pre-processing included aligning the reads (STARsolo), ambient mRNA detection and correction, empty droplet and doublet filtering, and QC analysis. Next, data was labeled and verified using a dorsal striatum macaque dataset (He and Kleymen et al., 2021). Finally, full analysis was conducted using analyses for differential expression, DNA damage signatures, and differential abundance. Results: We identified transcriptional alterations associated with OUD in specific cell types of the striatum, including significant shifts in the expression of dopamine receptor subtypes in GABAergic medium spiny neurons. When examining differentially expressed genes (DEGs) by cell types comparing OUD and control subjects, DEGs were found in the following cell types: astrocytes, endothelial, microglia, oligodendrocytes, D1-matrix, D1-striosome, D1/D2 hybrid, D2-matrix, D2-striosome, interneuron. Next, we analyzed DNA damage signatures. DNA damage signature analyses revealed increased DNA damage specific to OUD subjects compared to control subjects (p = 0.002). When looking at cell type specific DNA damage, mural cells have a higher proportion of DNA damage (p = 0.025) in OUD subjects. Additionally, looking at average DNA damage scores across cell types revealed increased DNA damage in OUD subjects in microglia (p = 0.004), endothelial (p = 0.006), oligodendrocytes (p = 0.021), and interneurons (p = 0.023). Additional exploratory approaches are currently underway to reveal cell-specific alterations in gene expression and compare different cell types across striatal subregions and sex. Conclusions: Our results are the first to demonstrate significant alterations in gene expression across different cell types of the striatum in the human brain associated with OUD using single nuclei RNA-seq. Additionally, our results suggest that OUD is associated with higher rates of DNA damage, particularly in microglia subtypes. Uncovering molecular mechanisms of opioid use will propel the identification of new therapeutic targets and the development of successful treatment strategies for OUD. Keywords: Opioid Addiction, Human Genetics, Single-Nucleus RNA Sequencing, Postmortem Human Brain Tissue, Cell-Type Specific Transcription Disclosure: Nothing to disclose. P678. Sex-Specific Associations of Genetic Variation With Treatment Outcomes in Men and Women With Alcohol Use Disorder Victor Karpyak, Brandon Coombes, Man Choi (Ada) Ho, Antony Batzler, Josef Frank, Colin A. Hodgkinson, Michelle Skime, Ming-Fen Ho, Falk Kiefer, Marcella Rietschel, Richard Weinshilboum, Stephanie O’Malley, Karl Mann, Raymond Anton, David Goldman, Joanna Biernacka Mayo Clinic, Rochester, Minnesota, United States Background: Sex and gender-related differences are known to be associated with alcohol consumption and risk for development of alcohol use disorder (AUD). Studies also indicate potential male/female differences in tendency to enter treatment programs and treatment response, including time to return to heavy drinking as well as vulnerability to risk factors, including negative affect or social pressure (Erol and Karpyak, 2015). So far, no differences have been found in treatment response to acamprosate, naltrexone or brief intervention between men and women with alcohol dependence. We recently completed a genome-wide association study (GWAS) of AUD treatment outcomes and demonstrated that genetic variation may have a polygenic effect on AUD treatment response, and that some genetic variants may be associated with medication-specific effects (Biernacka et al., 2021). Here we explored potential sex differences in genetic markers associated with treatment response to acamprosate, naltrexone and placebo. Methods: We used genetic and clinical data from three studies of acamprosate and/or naltrexone treatment of AUD (COMBINE N = 498, PREDICT N = 266, and CITA N = 252) reported in our original GWAS of AUD treatment outcomes (Biernacka et al., 2021). This dataset includes a total of 1016 participants of European ancestry (24% female), including 484 patients treated with acamprosate, 200 with naltrexone, 101 with both medications and 231 with placebo. We now performed sex-stratified GWAS of time until relapse to any drinking and time until relapse to heavy drinking (i.e., more than 4 drinks/day for men and more than 3 drinks/day for women) based on 3-month treatment outcomes. For each outcome, we performed sex-stratified analyses within each study, and then meta-analyzed the results across studies using METAL. Results: Of the 1016 study participants in the analysis, 639 (61% of men and 68% of women) relapsed to light drinking and 564 (54% of men and 60% of women) relapsed to heavy drinking. In the GWAS of women, two SNPs (rs7801472 in AUTS2 and rs72949008 in RP11-146N18.1) were associated with time until relapse at a genome-wide significant level (p = 3.2e-8 and p = 4.2e-8, respectively). One SNP (RP11-73O6.3 rs3818792; p = 4.8e-8) was associated with time until relapse to heavy drinking in women. No variants were associated with time until relapse or time until heavy relapse in men at a genome-wide significant level. Conclusions: We observed two significant genetic associations with time until relapse in women undergoing AUD treatment that map to AUTS2 and RP11-146N18.1. While little is known about RP11-146N18.1, AUTS2 (activator of transcription and developmental regulator) is a gene known to be involved in brain development and regulation of neuronal gene expression. Mutations in this gene cause intellectual disability, microcephaly, and other phenotypes, and common variants in AUTS2 have also been found to be associated with alcohol consumption, alcohol dependence, heroin dependence as well as autism spectrum disorder, impulsivity, ADHD, schizophrenia, and bipolar disorder. We also found significant association between heavy relapse in women and rs3818792, which is in a genomic region that includes L3MBTL3 (L3MBTL histone methyl-lysine binding protein 3), associated with transcriptional repression, SAMD3 (sterile alpha motif domain containing 3), associated with age at depressive onset and RP11-73O6.3, which is antisense lncRNA of L3MBTL3 and SAMD3. None of these findings in women had corresponding significant associations with the treatment response in men. While these results are preliminary, if replicated, they may guide investigation of potential sex-specific differences in genetic contribution to known triggers for relapse (e.g., negative emotional states) or relapse prevention measures (e.g., medication types). If successful, this line of research may contribute to development of biomarker-based, sex-specific recommendations for men and women seeking treatment for AUD. Keywords: Alcohol Use Disorder - Treatment, Genetic Association Study, Sex-Specific Effects Disclosure: Nothing to disclose. P679. CHRNA5 Gene Polymorphism and Early Smoking Onset Effect on Adult Smoking and Alcohol Measures Shyamala Venkatesh, Bethany Stangl, Natalia Quijano Cardé, Mariella De Biasi, Paule Joseph, Khushbu Agarwal, Yupeng Wang, Vijay Ramchandani National Institute of Health, Bethesda, Maryland, United States Background: The onset of smoking at an early age increases the risk for later nicotine dependence and alcohol use disorder (AUD). A non-synonymous polymorphism (rs16969968) in the CHRNA5 gene has shown a significant interaction with smoking onset age in causing heavy smoking and nicotine dependence in adults. Furthermore, a recent pre-clinical study has identified a sex-dependent association between CHRNA5 mutation and adolescent alcohol or nicotine exposure on subsequent intake of the opposite drug in adulthood. The current study aimed to determine the effect of the rs16969968 polymorphism and age at onset of smoking on adult smoking and alcohol traits. Methods: This study included 29,423 participants from the UK Biobank study sample who were current smokers and alcohol drinkers. We divided the study participants into early and late smoking onset groups depending on their smoking onset age (early onset = ≤16 years, late onset = >16 years). Smoking and alcohol measures, including the number of cigarettes smoked per day (CPD) and Alcohol Use Disorders Identification Test (AUDIT) scores, were compared between onset age groups and by CHRNA5 (rs16969968) genotype, with age and gender included as covariates. Additionally, we conducted a complementary analysis in the NIAAA study sample that included 188 healthy adult non-AUD drinkers who were also smokers. In this sample, smoking and alcohol measures included Fagerström Test for Nicotine Dependence (FTND), pack-years, AUDIT, Timeline Followback (TLFB), and Lifetime Drinking History (LDH). These measures were compared between smoking onset groups and CHRNA5 genotype groups, with age, gender, and Ancestry Informative Markers scores (AIMs) included as covariates. In all analyses, the main effect of the rs16969968 polymorphism was tested using a dominant model (GG vs. AA/AG) and the effect of smoking onset age was analyzed in the full sample as well as in the GG and AA/AG genotype groups separately. Results: In the UK Biobank sample, we observed a significant main effect of the CHRNA5 genotype on CPD with higher mean scores in the AA/AG genotype group than the GG genotype group (p < 0.001). Individuals with early smoking onset showed an increased quantity of nicotine consumption compared to the late smoking onset group which was consistent across both genotype groups. There was a main effect of the CHRNA5 genotype on AUDIT-C (consumption) with higher mean scores in the GG genotype group than the AA/AG genotype group (p = 0.016). However, there was no significant interactive effect of the genotype and smoking onset age on smoking or alcohol measures. In the NIAAA sample, a main effect of the CHRNA5 genotype and the interactive effect of genotype and smoking onset age groups were not observed on smoking or alcohol measures. The early smoking onset group showed an increased number of years of smoking compared to the late smoking onset group in the full sample and in the GG genotype group but not in the AA/AG genotype group. The AA/AG genotype group showed an increased quantity of cigarette smoking per week and TLFB-total drinks in the early smoking onset group than the late smoking onset group; this effect was not observed in the full sample or in the GG genotype group. Conclusions: This study provides further evidence for the main effect of the CHRNA5 (AA/AG) genotype and early smoking onset on increased nicotine consumption. Individuals carrying either the GG or AA/AG genotype are at an elevated risk for increased nicotine consumption in their adulthood if they initiate smoking at an early age. Also, there is an increased risk for a greater quantity of alcohol consumption in smokers carrying the CHRNA5 GG genotype compared to individuals carrying the AA/AG genotype. The risk for increased alcohol consumption was also observed in individuals with CHRNA5 AA/AG genotype who initiated smoking at an early age. Keywords: CHRNA5, Smoking Onset Age, Adult Smoking and Alcohol Traits Disclosure: Nothing to disclose. P680. Stress and Alcohol Exposure Accelerate Epigenetic Aging Jeesun Jung, Daniel McCartney, Josephin Wagner, Andrew Bell, Lucas Mavromatis, Daniel Rosoff, Colin Hodgkinson, Alicia Smith, Rosie Walker, Archie Campbell, David Porteous, Andrew McIntosh, Steve Horvath, Riccardo Marioni, Kathryn Evans, David Goldman, Falk Lohoff* National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: Stress contributes to premature aging and susceptibility to alcohol use disorder (AUD) and AUD itself is a factor in premature aging; however, the interrelationships of stress, AUD and premature aging are poorly understood. Methods: We constructed a composite score of stress (CSS) from thirteen stress-related outcomes in a discovery cohort of 317 individuals with AUD and controls. We then developed a novel methylation score of stress (MS Stress) as a proxy of CSS comprising 211 CpGs selected by a penalized regression model. The effects of MS Stress on health outcomes and epigenetic aging were assessed in a sample of 615 AUD patients and controls using epigenetic clocks and DNAm telomere length (DNAmTL). Statistical analysis with an additive model using MS Stress and a methylation score for alcohol consumption (MS Alcohol) were conducted. Results were replicated in two independent cohorts (Generation Scotland GS n = 7028 and the Grady Trauma Project GTP n = 795). Results: CSS and MS Stress were strongly associated with heavy alcohol consumption, trauma experience, epigenetic age acceleration (EAA) and shortened DNAmTL in AUD. Together, MS Stress and MS Alcohol additively showed strong stepwise increases in EAA. Replication analyses showed robust association between MS Stress and EAA in the GS and GTP cohort. Conclusions: A methylation-derived score tracking stress exposure is associated with various stress-related phenotypes and EAA. Stress and alcohol have additive effects on aging, offering new insights into the pathophysiology of premature aging in AUD, and potentially, other aspects of gene dysregulation in this disorder. Keywords: Epigenetics, Aging, Alcohol, Stress, PTSD Disclosure: Nothing to disclose. P681. Novel Underlying Biology and Therapeutic Targets for Alcohol Use Disorder and Problem Drinking Identified Using Proteomics and Mendelian Randomization Daniel Rosoff, Josephin Wagner, Andrew Bell, Lucas Mavromatis, Jeesun Jung, Falk Lohoff National Insitutes of Health, Bethesda, Maryland, United States Background: Alcohol use disorder (AUD) is a common neuropsychiatric disorder that is a leading cause of morbidity and mortality worldwide; however, only a few pharmacological treatment options are currently available, highlighting the need for novel and safe drug development. While protein biomarkers with causal genetic evidence are promising novel drug target candidates for AUD, scans of brain proteins have not been performed. Methods: We integrated genome-wide association summary statistics (GWAS) for AUD and alcohol consumption behaviors, i.e., problem items from the Alcohol Use Disorders Identification Test (AUDIT-P), binge drinking, and total drinks per week (DPW), and applied cis-instrument Mendelian randomization (MR) to perform a proteome-wide MR with data from >1,700 brain proteins within the dorsolateral prefrontal cortex (dlPFC) to investigate their causal relevance for AUD and alcohol consumption behaviors. Results: We identified 34 unique brain protein-alcohol associations that emerged as causal mediators of AUD and alcohol consumption behaviors. Novel proteins not previously implicated in alcohol consumption behaviors included CAB39L, TESC (P-value=1.99×10-7 (AUD)), ERLIN1 (P-value=2.31×10-12 (AUDIT-P)), CPS1 (P-value=6.9×10-6 (binge drinking)), HDGF (SLC5A6 (P-value=1.53×10-7 (DPW)). CAB39L was consistently associated with increased drinking across alcohol phenotypes (with P-values ranging from 8.66×10-10 (AUD) to 1.69×10-114 (DPW)). We were able to replicate proteins using independent dlPFC protein and gene expression datasets. 11 of the proteins also showed evidence of a shared causal variant between the brain protein and respective alcohol consumption behavior, including CAB39L, HDGF, and SLC6A5 with DPW and CPS1 with binge drinking. Single-cell analysis showed enrichment predominantly in excitatory neurons within the dlPFC. Exploratory MR identified corresponding associations for 31 of the 34 alcohol-related proteins with 22 neuropsychiatric endpoints, highlighting pleiotropic associations with a range of disorders and endpoints such as cognition and smoking behaviors. TESC, SLC6A5, HDGF, and CPS1 were not associated with other neuropsychiatric endpoints, suggesting potentially specific roles in alcohol consumption. Finally, several of the proteins are considered actionable drug targets suggesting possible drug repurposing opportunities. Conclusions: Our findings highlight the power of integrating genetics, proteomics, and transcriptomics in elucidating causal biology underlying AUD and alcohol consumption behaviors, linking them with neuropsychiatric disorders, and identifying novel drug targets that may aid the development of new therapeutics aimed at reducing problematic drinking. Keywords: Mendelian Randomization, Proteomics, Alcohol Consumption, Alcohol and Substance Use Disorders, Single-cell RNA Sequencing Disclosure: Nothing to disclose. P682. Unbridled Amygdalae! Opioid Patients Carrying a Genetic Variant Linked to Over-Response of the Stress (Cortisol) System Show Heightened Resting Connectivity Between the Amygdala and the Ventromedial Prefrontal Cortex (vmPFC) Anna Rose Childress, Kanchana Jagannathan, Richard C Crist, Paul Regier, Teresa Franklin, Reagan Wetherill, Kimberly Young, Michael Gawrysiak, Kyle Kampman, Daniel Langleben, Charles O’Brien University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States Background: Honed by eons of survival-driven priorities, our brain leaps into action at the first whiff of danger – or reward. Within the brain, our amygdalae are critical first responders, well-studied for their rapid response to signals, e.g., for threat, and for rewarding drugs of abuse. In the healthy brain, these important functions are ‘bridled’, tightly regulated by other (inhibitory) brain regions, so that we are not in a chronic state of fear or reward motivation. As stress can undermine the inhibitory ‘bridle’, we recently tested and confirmed that cocaine and opioid patients carrying a genetic variant linked to over- response of the cortisol stress system (i.e., the minor G allele of rs3800373, for the FKBP5 gene) have heightened (‘unbridled’) responding to drug reward cues in motivational circuits (CPDD 2019; SfN 2019). This over-response to appetitive cues underscored an intriguing ‘cross-talk’ between aversive and appetitive motivational systems in the brain – and also begged a further question: Could the impact of the vulnerability allele be detected even when the brain is at rest, representing a brain that is tuned up and ‘over-ready’ to respond (to signals for threat or for drug reward)? Indeed, we found (SfN 2021) that cocaine patents who carried the vulnerability (‘unbridled’) allele had heightened functional connectivity between the amygdala and motivational circuitry nodes (VMPFC and insula). For the current presentation, we tested whether the ‘signature’ of heightened amygdala resting functional connectivity (for G allele carriers) could also be detected within a cohort of individuals with opioid use disorder (OUD). Replication in an additional cohort is important, given statistical concerns about studies (whether imaging, behavioral or clinical) that focus on a single nucleotide polymorphism (SNP), even if the examination is hypothesis-driven. This is because most psychiatric disorders and medical conditions are emphatically polygenic. Intriguingly, however, specific allelic variants of FKBP5, including the functional SNP studied here, have been shown to account for a significant outcome variability in complex behaviors, e.g., the vulnerability (high cortisol) allele is linked to healing, vs. progression to chronic musculoskeletal pain, in those with similar initial back injuries. This robust finding was replicated across two large pain cohorts (n = 948; n = 1607), stimulating the current studies in the appetitive (addiction) domain. Methods: As part of a large study of brain predictors of relapse to drug use, treatment-seeking opioid outpatients received an fMRI session (BOLD, 3 T) prior to their induction onto depot naltrexone. Resting state was collected at the outset of the session, prior to a variety of fMRI tasks. Resting state data (5 minutes) was analyzed within the SPM8 connectivity toolbox, generating voxel-wise functional connectivity maps for (R; L) amygdalae in carriers of the minor G (vulnerability) allele (TG, GG; n = 18) for rs3800373 of FKBP5, and in TT homozygotes (n = 13). The resulting correlation maps (r to Z transformed) were examined for each group, and then formally compared across the two genetic subgroups (group difference t maps thresholded 2 < t < 5 for display; FWE- corrected). Results: For both opioid allelic subgroups, there was robust positive “intra-limbic” connectivity between the amygdala (R; L) and multiple motivational nodes (e.g., the ventral tegmental area, ventral pallidum, ventral striatum/putamen, and insula) – and both allelic groups lacked inverse (‘bridling’) connectivity with the dorsomedial prefrontal cortex (PFC) found in studies of healthy controls – indeed, the connectivity was in the positive direction. In addition, carriers of the minor (vulnerability) allele for FKBP5 showed heightened resting connectivity between the amygdala (L and R amyg) and the ventromedial PFC, and the ventral striatum (L amyg), as compared to the TT homozygotes. Conclusions: Encouragingly, these new data in an opioid cohort do replicate the patterns of findings from the earlier cocaine sample: overall, both the opioid and cocaine patients exhibit the ‘unbridled’ amygdala relationships with the cortical regulatory regions – and both cohorts show that carriers of the vulnerability alle have heightened connectivity of the amygdala with the subcortical VMPFC, as compared to the TT homozygotes. These replicated results give confidence that the brain findings may be a marker of vulnerability across substance use disorders: carriers of the minor G (vulnerability) allele of rs380037 for the FKBP5 gene may be at additional risk for psychopathologies related to amygdala over-activity, whether in the domain of aversive (e.g., pain /anxiety /stress /PTSD) or appetitive (e.g., drug reward/relapse) motivation. Finally, though the current findings are in adult individuals struggling with addiction, accruing neurodevelopmental cohorts (e.g., the ABCD study) offer the unique opportunity to combine resting state functional connectivity with genetics as potential markers of vulnerability to the future development of affective or substance use disorders. If validated prospectively, these combined brain and genetic markers would encourage future (behavioral, pharmacologic, or neuromodulatory) interventions to boost or to restore regulation of the amygdala, especially for carriers of the vulnerability allele.FKBP5 Keywords: FKBP5, Resting-State fMRI, Cocaine and Opioid Use Disorders, Amygdala, Functional Connectivity Disclosure: Nothing to disclose. P683. An Increase in Epigenetic Age Acceleration Among People Who Use Substances Ke Xu, Xiaoyu Liang, Brooklyn Bradley, Rajita Sinha Yale University School of Medicine, New Haven, Connecticut, United States Background: Substance misuse can have a significant impact on overall health. People who use substances present greater risk of morbidity and mortality than non-users. Biological aging has been proposed as an indicator of the adverse effects of substance misuse on health and frailty. Recently developed epigenetic “clocks” employ cellular DNA methylation (DNAm) as a measure of the aging process. DNAm-based epigenetic clocks were shown to be more sensitive and precise measures of cellular age than other genomic measures (e.g., transcriptome, telomere length). In this study, we tested whether substance misuse (i.e., alcohol, cigarette smoking, and cannabis use) altered epigenetic clocks (i.e., HorvathAge, HannumAge, PhenoAge, GrimAge, and MonoAge). We further examined the mediating role of stress and BMI in substance misuse related epigenetic age acceleration Methods: All analyses were performed in a community-based sample from Greater New Haven, Connecticut (N = 509). All participants were determined healthy without major medical or psychiatric disorders. Substance use was based on self-reported data. Alcohol consumption was measured by using the 10-item Alcohol Use Diagnosis Identification Test (AUDIT). Heavy alcohol drinking was defined as an AUDIT score ≥8 for men and an AUDIT score ≥7 for women. The average AUDIT score was 5.69 among all participants in the cohort. Cigarette smoking and cannabis use were categorized as current, past, and never users as well as quantified number of uses in the past 30 days. DNA methylation in the blood methylome was profiled by using Illumina HumanMethylation Beadchip 450 K. Each clock of interest was estimated using the established algorithms for each sample. Pearson’s correlation between DNAm age and chronological age was performed in each group. Epigenetic age acceleration (EAA) was defined as the residuals of regressing DNAm age on chronological age. We calculated the mean difference in EAA between heavy users and non-heavy users, smokers and non-smokers, cannabis users and non-cannabis users. Mediation analysis was conducted to test whether stress and BMI mediated the EAA in each substance misuse Results: We found significant associations of each epigenetic clock with frequency of alcohol use per week (pHorvathAge = 0.03, pHannumAge=0.02, pPhenoAge=0.007, pGrimAge=0.002, pMonoAge=0.01), AUDIT-10 (pPhenoAge = 0.003 and pMonoAge=0.03), cigarette per day (pHorvathAge = 6.49E-05, pHannumAge=7.38E-05, pPhenoAge=2.27E-06 pGrimAge=2.18E-08, pMonoAge=0.0001 and cannabis use in the past 30 days (pHorvathAge = 0.0013, pHannumAge=0.02, pPhenoAge=0.002, and pMonoAge=0.012). For epigenetic age acceleration, AUDIT score was associated with HorvathAge (p = 0.02), HannumAge (p = 0.001), and GrimAge acceleration (p = 0.0006). Cigarette per day was associated with PhenoAge (p = 0.001) and GrimAge only (p = 5.73E-10). Cannabis use in the past 30 days was significantly associated with GrimAge acceleration (p = 0.002). These results suggest that each epigenetic clock employs slightly different functionalities to capture epigenetic age in each substance misuse type. Conclusions: Our results demonstrate that substance misuse increases epigenetic age acceleration in a healthy young population. We also show that stress partially mediates the substance misuse-related age acceleration. These findings indicate the importance of early prevention to potentially reserve adverse effects of substance misuse. Keywords: Epigenetic Age Acceleration, Substance Abuse, Lifetime Stress Disclosure: Nothing to disclose. P684. In Vivo Imaging of Brain Cortisol Regulation in Alcohol Use Disorder Terril Verplaetse, Shivani Bhatt, Henry Huang, Sherry McKee, Kelly Cosgrove Yale University School of Medicine, New Haven, Connecticut, United States Background: Stress dysregulation is associated with the maintenance of and relapse to alcohol use. Stress is also a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis, initiating the release of glucocorticoid hormones. Levels of glucocorticoids (e.g., cortisol, cortisone) present in the brain are dependent on the enzyme 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyzes the conversion of cortisone to cortisol and amplifies the action of glucocorticoids in the brain. 11β-HSD1 is located in brain regions critical in the negative feedback of glucocorticoids and in addiction, including the prefrontal-limbic circuit. Thus, high brain glucocorticoid levels, driven by 11β-HSD1 and induced by stress, may contribute to risky drinking. We used positron emission tomography (PET) imaging with the novel 11β-HSD1 specific radioligand [18F]AS2471907 to assess 11β-HSD1 expression in participants with alcohol use disorder (AUD) vs. healthy controls. Methods: We imaged ten individuals with moderate to severe AUD (n = 6 men, n = 4 women; mean age=38 years) and 12 healthy controls (n = 7 men, n = 5 women; mean age=29 years). Participants received 93.5 ± 15.6 MBq [18F]AS2471907as a bolus injection at high specific activity and were imaged for 150-180 minutes on the High-Resolution Research Tomograph (HRRT; 2-3 mm resolution). 11β-HSD1 availability was quantified by [18F]AS2471907 volume of distribution (VT; mL/cm3), the ratio at equilibrium of [18F]AS2471907in tissue to un-metabolized [18F]AS2471907in arterial plasma. A priori regions of interest included amygdala, anterior cingulate cortex (ACC), hippocampus, ventromedial PFC (vmPFC) and caudate, as these corticolimbic regions are involved in HPA axis regulation, stress pathophysiology, and addiction. Individuals were required to be overnight abstinent from drinking. Levels of 11β-HSD1 were correlated with past 30-day alcohol use on the Timeline Followback and self-reported childhood trauma (Childhood Trauma Questionnaire [CTQ]). Results: Individuals with AUD consumed 53.72 drinks/week and had 5.81 drinking days/week. Healthy controls consumed 2.75 drinks/week and had 1.30 drinking days/week. Preliminary data suggest that 11β-HSD1 levels are higher in amygdala, ACC, hippocampus, vmPFC, and caudate in individuals with moderate to severe AUD compared to healthy controls (p < 0.03). The AUD group demonstrated positive correlations between drinks per week and drinks per drinking episode with 11β-HSD1 levels in vmPFC (p = <0.01; r = 0.85 and r = 0.78, respectively) and caudate (p = 0.05; r = 0.67 [drinks per week only]). Childhood trauma was associated with higher 11β-HSD1 levels in ACC in individuals with AUD (p = 0.05; r = 0.66). Conclusions: This is the first in vivo examination of 11β-HSD1 levels in individuals with AUD. These preliminary findings suggest higher brain cortisol-producing 11β-HSD1 in AUD compared to healthy individuals, and a possible relationship between [18F]AS2471907 VT and self-reported alcohol use and childhood trauma. These results are consistent with work suggesting that childhood trauma may be related to increased stress and alcohol use in adulthood. Future studies will further investigate [18F]AS2471907as a marker of brain cortisol regulation in relation to stress-related drinking. Keywords: PET Imaging, Cortisol, Alcohol Use Disorder, Stress, Trauma Disclosure: Nothing to disclose. P685. Identification of ∆9-Tetrahydrocannabinol (THC) Impairment Using Resting-State Neuroimaging Nisan Ozana, Michael Pascale, Kevin Potter, Brian Kendzior, Gladys Pachas, A. Eden Evins, Jodi Gilman* Harvard Medical School, Boston, Massachusetts, United States Background: Intoxication from cannabis impairs cognitive performance due to the effects of Δ9-tetrahydrocannabinol (THC, the primary psychoactive compound in cannabis) on prefrontal cortex (PFC) function. There are currently no evidence-based methods to detect cannabis-impaired driving, and current field sobriety tests with gold-standard, drug recognition evaluations are resource-intensive and may be prone to bias. We tested whether a portable and inexpensive imaging method, functional near infrared spectroscopy (fNIRS), could be used to objectively detect impairment from cannabis at resting state. Methods: 169 participants with regular cannabis use were given a single dose of up to 80 mg of dronabinol, an FDA-approved synthetic THC ingredient in MARINOL® Capsules or identical placebo. Participants underwent two fNIRS sessions; one before dronabinol administration (“pre-THC”), and the other at approximately two hours after dronabinol administration (“post-THC”), which is the median peak of pharmacokinetic effects of dronabinol. During each session, 6 minutes of resting-state functional data were collected using a continuous-wave NIRS device, in which 8 Sources and 7 detectors were placed on the forehead, resulting in 20 channels covering PFC regions. fNIRS analysis was conducted using the CONN toolbox (https://web.conn-toolbox.org). Results: 76 participants had concordant clinical and algorithm ratings indicating that they were impaired/intoxicated following THC at (mean dose of 35.6 mg ± 11.5) and 57 participants had concordant ratings of not impaired (mean dose of 34.8 ± 16.1). The 80 impaired participants had greater subjective (DEQ) and physiologic (heart rate) compared with those who were not impaired post-THC. We found a significant change in resting-state connectivity from pre-THC to post-THC only among those participants who were impaired (group A, p < 0.05; FDR- corrected); those who were not impaired, but received THC, did not show a change in resting-state connectivity (group B). When comparing post-THC to post-placebo, only those who were impaired showed a significant decrease in resting state connectivity (group A, p < 0.05; FDR-corrected). Those who were not impaired (group B) did not show any significant differences between post-THC and post-placebo scans, even after receiving equivalent doses of THC. Conclusions: There is a growing public health need for an objective, reliable, unbiased method to detect impairment due to THC. This is not achievable with per se blood or body fluid THC or metabolite concentration cutoffs. Findings suggest PFC response can objectively determine who is impaired from THC intoxication, with potential applications in roadside settings. Future work is warranted to determine specificity to acute THC impairment. Keywords: Cannabis, Functional Impairment, Resting State, Functional Near-Infrared Spectroscopy Disclosure: Inventor: Patent (Self). P686. Cigarette Smoking Reduces a Marker for Neuroinflammation in People Living With HIV: A [18 F]FEPPA Positron Emission Tomography Study Arthur Brody, Anna Mischel, Andre Sanavi, Alvin Wong, Ji Hye Bahn, Brinda Rana, Carl Hoh, David Vera, Kishore Kotta, Arpi Minassian, Erin Morgan, Jeffrey Meyer, Neil Vasdev, Jared Young University of California, San Diego, San Diego, California, United States Background: Microglia become activated as part of neuroinflammation, which leads to increased expression of the translocator protein 18 kDa (TSPO). Positron emission tomography (PET) studies, using radiotracers that label TSPO, have recently been used to examine TSPO availability as a marker of neuroinflammation in a variety of conditions. In such PET studies, people living with HIV (PWH; HIV+) have been found to have higher levels of radiotracer binding than seronegative individuals (HIV-). In PET studies of cigarette smokers, people who smoke (Smok+) exhibit lower radiotracer binding than non-smokers (Smok-). Based on this prior research, we hypothesized that HIV + individuals (and HIV- controls) who are either cigarette smokers or nonsmokers would have the following order of the marker for neuroinflammation on PET scanning: HIV + /Smok- > HIV + /Smok += HIV-/Smok- > HIV-/Smok + . Methods: 53 otherwise healthy participants who were in one of four categories completed the study: HIV + /Smok- (n = 16), HIV + /Smok + (n = 11), HIV-/Smok- (n = 17), and HIV-/Smok + (n = 9). Participants underwent baseline assessments to confirm eligibility, genetic testing for a genotype known to affect TSPO affinity, PET/computed tomography (CT) scanning for 90-min following bolus injection of the radiotracer [18 F]FEPPA, and magnetic resonance imaging to assist in localization of regions on the PET/CT images. Smokers were scanned in the satiated state. The primary outcome measure was whole brain standardized uptake value (SUV). Results: An overall analysis of variance with whole brain SUV as the dependent variable, participant group as a factor of interest, and genotype as a nuisance covariate revealed a significant effect of participant group (F = 3.0;df=3,48;p = 0.04). In clarifying this overall result, smoking status had a significant main effect on whole brain SUV (F = 5.6;df=1,48;p = 0.02), while main effect of HIV status (F = 1.5,n.s.) and the interaction of HIV and smoking statuses (F = 1.2,n.s.) did not reach significance. Within the HIV + group, a main effect of smoking status was also found (F = 7.9;df=1,24;p = 0.01). Group whole brain SUV values (mean + /-SD) were in an order very similar to the hypothesized order cited above: HIV + /Smok- (1.27 + /-0.26), HIV-/Smok- (1.17 + /-0.21), HIV + /Smok + (1.11 + /-0.11), and HIV-/Smok + (1.10 + /-0.24). Conclusions: PET/CT findings using a marker for neuroinflammation in HIV + smokers were highly consistent with prior research examining the effects of HIV and cigarette smoking statuses separately. HIV + nonsmokers had the highest levels of the marker for neuroinflammation, while cigarette smokers (both HIV + and HIV-) had the lowest levels of the marker for neuroinflammation. Thus, cigarette smoking appears to have an anti-inflammatory effect on the elevated neuroinflammation levels found in PWH. Keywords: Human Brain Imaging, Tobacco Dependence, Human Immunodeficiency Virus, Positron Emission Tomography Disclosure: Nothing to disclose. P687. Acute White Matter Integrity Increases After the Moderate Dose of Alcohol Administration Hideaki Tani, Ryo Ochi, Mutsuki Sakuma, Fumihiko Ueno, Sakiko Tsugawa, Hiroyuki Uchida, Masaru Mimura, Shunji Oshima, Sachio Matsushita, Shinichiro Nakajima Keio University School of Medicine, Tokyo, Japan Background: Chronic heavy alcohol drinking adversely affects physical and mental health. On the other hand, a protective effect of light to moderate drinking has some biological plausibility. However, it is not well known the effects of modest alcohol intake on brain structure. In the present study, we aimed to examine the changes in white matter integrity after a modest dose of acute alcohol administration using diffusion tensor imaging (DTI) in healthy subjects. We hypothesized that the acute intake of modest alcohol would increase the white matter integrity. Methods: We recruited healthy volunteers aged 20-29 who carried ALDH21/2 genotype, understood the study design, and gave written informed consent. The participant received an intravenous alcohol infusion using the alcohol clamp method to keep a target blood alcohol concentration of 0.50 mg/mL. We conducted MRI scans and assessed self-reported clinical responses to alcohol administration (i.e., stimulus, physical, or sedative) using the visual analogue scale (VAS) at baseline (T0), 90 minutes after alcohol administration started (T1), and 90 minutes after alcohol administration ended (i.e., 180 minutes from baseline) (T2). MRI data were acquired on a 3 T MRI scanner (MR750; GE Discovery) equipped with a 12-channel head coil at Kurihama Medical and Addiction Center. DTI data were obtained via the use of a single-shot echo-planar sequence with diffusion gradients (b = 1000 s/mm2) applied in 30 noncollinear diffusion directions along with five diffusion-unweighted images with b = 0 s/mm2 (TE = 74.5 ms, TR = 16,000 ms, flip angle = 90, 128 × 128 matrix, 1.0×1.0×2.5 mm). After preprocessing using the FMRIB Software Library, we compared whole-brain fractional anisotropy (FA)/mean diffusivity (MD) values and FA/MD values for 48 regions of interest (ROI) extracted from the JHU-ICBM-DTI-81 white matter atlas across time using repeated-measure analysis of variance (ANOVA). Multiple comparisons for significant regions were performed using Bonferroni correction. Additional analyses were conducted to examine the correlation between baseline FA/MD value or FA/MD value changes and changes in behavioral VAS scores. Furthermore, we performed tract-based spatial statistics (TBSS) to compare white matter integrity between conditions (i.e., T0 vs T1, T0 vs T2, T1 vs T2). Results: Eighteen subjects participated in this study (mean age=25.1 ± 3.0 years, male=7 (38.9%)). Repeated measures ANOVA showed no significant differences in the whole FA (F(2,51)=0.26, p = 0.77) or FA values in any DTI ROIs among the three time points. TBSS analysis revealed no significant differences in FA between T1 and T0 or T2 and T1. However, there were significant increases in FA in the cerebral white matter voxel cluster (>1000 voxels) at T2 compared to T0 (p = 0.001, FWE-corrected), whereas no significant decreases were found. For MD values, repeated measures ANOVA indicated no significant differences in the whole MD (F(2,51)=0.03, p = 0.97) or MD values in any DTI ROIs among the three time points. In addition, TBSS analysis found no significant differences in MD between T1 and T0 or between T2 and T1. However, there were significant decreases in MD in the voxel clusters (>1000 voxels), including right anterior corona radiata, right inferior fronto-occipital fasciculus, corticospinal tract, left uncinate fasciculus, anterior thalamic radiation at T2 compared to T0 (p < 0.05, FWE-corrected) whereas no significant increases were found. The correlation analysis found that the whole FA change from T0 to T1 was negatively correlated with the change in the sedative score from T0 to T1 (r = −0.51, p = 0.03). This finding did not survive after the multiple comparison correction. The whole FA change between T0 and T2 was not associated with the sedative score change between T0 and T2. No significant correlation was found between the change in the whole FA values and the stimulus or physical score changes (p’s > 0.05). No significant association was found between whole MD changes and changes in behavioral score changes from T0 to T1 or from T0 to T2. No correlation was shown between baseline FA/MD value and the FA/MD value changes or behavioral score changes from T0 to T1 or T0 to T2. Conclusions: We found an acute increase in white matter integrity after the modest dose of single alcohol intake. Further research is needed to identify the effect of repeated administration of a modest amount of alcohol on brain structure in the long run. Keywords: Alcohol, Diffusion Tensor Imaging, Modest Dose, Acute Effect Disclosure: Nothing to disclose. P688. Glucocorticoid Receptors in the Orbitofrontal Cortex Mediate Cocaine-Induced Response Biases Michelle Sequeira, Kathryn Stachowicz, Shannon Gourley Emory University, Emory National Primate Center, Atlanta, Georgia, United States Background: Many addictive drugs, including cocaine, increase circulating stress hormones. Additionally, addictive drugs and corticosterone (CORT) weaken the ability of organisms to select actions in a flexible manner (causing a reliance on habitual behavior), and loss of dendritic spine densities on excitatory neurons in the ventrolateral orbitofrontal cortex (VLO) is common. We hypothesize that cocaine causes decision-making biases by increasing circulating stress hormone levels, activating low-affinity glucocorticoid receptors (GR), ultimately leading to dendritic spine loss in the VLO. Methods: Here we used pharmacological and site-selective gene silencing strategies to reduce Nr3c1, the gene for GR, in male and female mice. To quantify action selection strategies, mice were trained to generate two distinct responses for food, then required to update response strategies when one behavior was no longer reinforced. We used Thy1-driven YFP for the visualization of layer V excitatory neurons in the VLO. We then used Fos2A-iCreER (TRAP2) transgenic mice to express excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in neuronal ensembles in the OFC when mice must update the association between an action and its outcome. Results: Cocaine increased circulating CORT, and exogenous CORT exposure was sufficient to disrupt flexible behavior. Cocaine had the same effects, while inhibiting CORT synthesis blocked cocaine-induced response biases. Reducing Nr3c1 in the VLO also protects against cocaine-induced response biases, concurrent with dendritic spine modifications on excitatory deep-layer neurons. Activating neuronal ensembles involved during memory updating in cocaine-exposed mice rescued flexible behavior. Conclusions: Our findings indicate that cocaine-induced decision-making biases are driven by repeated activation of GRs in the VLO. Additionally, cocaine-induced spine loss may be rescued by GR reduction. Finally, we found that cocaine impedes the ability of OFC neurons to stably store action-outcome memories, thus destabilizing contingency learning. Keywords: Stress Hormones, Orbitofrontal Cortex, Cocaine, Rodents, Dendritic Spines Disclosure: Nothing to disclose. P689. Scarcity Early in Life Alters Both the Basolateral Amygdala Transcriptome and Addiction-Related Behaviors Amelia Cuarenta, Reza Karbalaei, James Flowers II, Alexandra Hehn, Molly Dupuis, Atiba Ingram, Claire Deckers, Sydney Roth, Sydney Famularo, Mathieu Wimmer, Debra Bangasser Temple University, Philadelphia, Pennsylvania, United States Background: Adversity is a risk factor for psychiatric disorders, however, stress that is not overwhelming can promote resilience. In our laboratory, we use the limited bedding and nesting (LBN) to model mild adversity. Our prior research discovered LBN reduces morphine self-administration in adult males and decreases impulsive choice and risk-taking behavior in the probability discounting (PD) task. These findings suggest that LBN induces neurobiological alterations that reduce some addiction-related behaviors. These behaviors rely on cues as a driver of behavior and performance. Exposure to cues previously paired with drug taking can induce craving and drug-seeking behavior following periods of abstinence. Thus, we are investigating whether LBN alters incubation of morphine craving, a cue-driven behavior. We extend this work to another drug, cocaine, to determine whether LBN causes similar changes to cocaine self-administration. Finally, we are beginning to explore the molecular changes induced by LBN in the basolateral amygdala (BLA), a region important for responses to stress and for the integration of cues. Methods: Long Evans rats were reared in LBN or control housing conditions from postnatal day (PND) 2 through 9. The LBN condition consisted of dams and pups placed in a limited resource environment where a metal grate prevented access to bedding and dams were given a single paper towel to use as nesting material. Control animals were reared in standard laboratory housing conditions with ample bedding, two cotton nestles, and one enrichment tube. On PND10 LBN rats were moved back to standard laboratory housing conditions. E1: Rats (n = 10-11/group) were placed in operant chambers and permitted to lever press on a fixed ratio 1(FR1) schedule for morphine infusions (0.75 mg/kg/infusion). Presses on the “active lever” resulted in one infusion accompanied by a 5-s light cue and a subsequent 20-s timeout period during which the house light was off and lever presses were recorded but had no corresponding drug infusion. Sessions began at the start of the animals’ dark cycle (8PM) and ended 12 hours later (8AM) resulting in 12 hours of access to the drug. This was performed on 10 consecutive days. Following the 10 days on FR1 morphine self-administering rats were tested for behavioral signs of drug seeking during early (day 1) and late (day 30) abstinence utilizing a within-subjects design. during drug-seeking tests, lever presses were reinforced by previously drug-paired cue presentations, but morphine was not available. E2: Rats had daily 6-hour access to cocaine self-administration (0.5 mg/kg/infusion) on an FR1 reinforcement schedule for 10 days. Presses on the “active lever” resulted in one infusion accompanied by a 5-s light cue and 20-s timeout period. Sessions began at the start of the animals’ dark cycle (9AM) and ended 6 hours late (3PM) for 6 hours of drug access. This was performed for 10 consecutive days. E3: RNA sequencing was conducted to delineate the effect LBN had on the transcriptional profile of the BLA in adult rats (n = 4-5 rats/group). BLA tissue from adult, behavioral naive rats were sequences on an Illumina HiSeq 4000. Fastqc version 0.11.8 was used to evaluate the quality of reads with adaptors and non paired reads removed using Trimmomatic version 0.39. The Rank-Rank Hypergeometric Overlap (RRHO) version 2 test evaluated the degree of overlap in gene signatures between sexes. Differentially expressed genes (DEGs) were identified using an adjusted P value of <0.1 and a 50% change in the expression as cutoffs to determine significance. Results: We investigated whether rats exposed to LBN had alterations in incubation of craving of morphine during early and late abstinence. At this dosage (0.75 mg/kg) there is no difference in morphine taking between LBN and control rats. Both LBN and control rats showed the incubation effect, pressing more after 30 days of abstinence than on day 1 (F (1,18)=25.94, p < 0.0001). However, there was no difference in lever pressing between LBN and control rats in males (F(1,18)=.325, p = 0.5757) or females (F(1,20)=.217, p = 0.646). Therefore, LBN does not reduce craving behavior elicited after prolonged abstinence suggesting that LBN does not elicit protective effects on the relapse model of craving associated with morphine-taking behavior. Our preliminary behavioral analysis demonstrates that LBN does not alter cocaine self-administration in male or female rats. LBN-induced sex-specific changes in transcription. RRHO analysis revealed distinct genes upregulated and downregulated in males and females due to LBN. There was minimal overlap between genes either upregulated or downregulated in males and females. A large proportion of genes were upregulated by LBN in males and downregulated in females. We narrowed our analysis to genes showing a significant difference between control and LBN and found 209 DEGs in females and 149 DEGs in males. These gene expression changes were predominantly sex specific as only 11 genes were altered by LBN in males and females. Heatmaps organized by fold change of LBN DEGs displayed different patterns of upregulated and downregulated genes in males and females. Conclusions: LBN reduces morphine drug taking in males, but there is no effect on cocaine drug-taking. Furthermore, LBN does not affect cue-driven incubation of morphine craving. LBN also induces sex-specific patterns of gene transcription within the BLA. Keywords: Basolateral Amygdala, Cocaine, Morphine, Transcriptome Disclosure: Nothing to disclose. P690. The Endogenous Ghrelin Antagonist LEAP-2 is Modulated by Alcohol Exposure and Pharmacological Manipulations of the Ghrelin System: Findings From Human Laboratory Studies Andras Leko, Mehdi Farokhnia, Lindsay Kryszak, Shelley N Jackson, Lisa Farinelli, Lorenzo Leggio National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland, United States Background: Investigating the neurobiological and pathophysiological mechanisms of excessive alcohol consumption is critical for developing new medications for AUD. One promising area of research is the gut-brain axis and its related endocrine pathways, which may play important roles not only in control of homeostatic and hedonic eating but also in reward processing and addiction. The stomach-derived ‘hunger hormone’ ghrelin is a key regulator of metabolism, food intake and reward. Consistent with preclinical research, human studies show that ghrelin levels decrease after acute alcohol administration and are positively associated with alcohol craving. Ghrelin administration increases cue-induced alcohol craving and alcohol self-administration, while preliminary human work suggests that the ghrelin receptor (GHS-R1a) inverse agonist PF-5190457 reduces alcohol cue-elicited craving. The liver expressed antimicrobial peptide-2 (LEAP-2) was recently discovered as an endogenous antagonist of the GHS-R1a. LEAP-2, produced predominantly in the liver, jejunum, and duodenum, is a key member of the ghrelin system and LEAP-2:acyl-ghrelin molar ratio is a marker of GHS-R1a function. In preclinical experiments, LEAP-2 administration inhibits ghrelin-induced food intake and growth hormone (GH) release. In humans, contrary results show LEAP-2 administration, decreases food intake and GH levels. Given the close link between alcohol consumption and the ghrelin system, our aim was to investigate LEAP-2 levels and the LEAP-2:acyl-ghrelin molar ratio, in relation to alcohol consumption, ghrelin administration and inverse agonism of GHS-R1a. Methods: We measured blood LEAP-2 and acyl-ghrelin concentrations via enzyme-linked immunosorbent assay (ELISA) in AUD patients enrolled in three placebo-controlled, human laboratory studies. Study 1 (n = 18; NCT01779024) was randomized, crossover, double-blind, placebo-controlled study, and included concomitant intravenous (IV) ghrelin administration and IV alcohol self-administration. Study 2 (n = 11; NCT02039349) was a single-blind, within-subject, placebo-controlled, Phase 1b human laboratory study, with oral PF-5190457 (50 mg BID and 100 mg BID) treatment and an oral alcohol challenge. We analyzed LEAP-2 levels with 100 mg BID treatment only in order to make the analyses consistent across Study 2 and 3. Study 3 (n = 32; NCT02707055) was a randomized, crossover, double-blind, placebo-controlled Phase 2a study, and included dosing with PF-5190457 (100 mg BID) without alcohol co-administration for up to 14 days. For statistical analysis, we used linear mixed modeling with covariates including baseline concentration, age, sex, body mass index, breath alcohol concentration (BrAC) and acyl-ghrelin level. Bonferroni correction was used to control for multiple comparisons during post hoc analysis. Results: In Study 1, we established that IV ghrelin administration increases LEAP-2 levels (p < 0.001). Under placebo conditions, IV alcohol self-administration does not change LEAP-2 levels significantly, but reduces acyl-ghrelin, resulting in a higher LEAP-2:acyl-ghrelin ratio (p < 0.05). Furthermore, acyl-ghrelin levels correlated positively (r = 0.228, p = 0.035) with LEAP-2, and sex was found to be a significant covariate (p = 0.016), with females having higher LEAP-2 levels. In Study 2, we found that LEAP-2 decreases 90 minutes after oral alcohol intake (p < 0.05), but ghrelin receptor inverse agonist, PF-5190457 diminishes the effect of alcohol. Consistent with Study 1 results, LEAP-2:acyl-ghrelin ratio increased after oral alcohol administration (p < 0.001), because acyl-ghrelin showed a more expressed decrease than LEAP-2. In Study 3, PF-5190457 decreases LEAP-2 levels (p < 0.001) and LEAP-2:acyl-ghrelin ratio (p < 0.05), when administered for a minimum of 6 days. Interestingly, PF-5190457 administration led to a pronounced decrease of LEAP-2 levels after the first day of dosing, however LEAP-2 showed a compensation via a gradual, modest increase from the minimal levels on the second day of treatment. Conclusions: In summary, we provide the first description that IV and acute oral alcohol administration results in a higher LEAP-2:acyl-ghrelin ratio, suggesting an inhibition of GHS-R1a. This is congruent with earlier results showing lower acyl-ghrelin levels after acute alcohol administration. IV ghrelin, co-administered with IV alcohol, increased LEAP-2 levels, while GHS-R1a inverse agonism reduced LEAP-2 and LEAP-2:acyl-ghrelin ratio, and inhibited alcohol’s effect on LEAP-2 levels. These results suggest a compensatory interplay between the endogenous ligand (agonist) and antagonist of the GHS-R1a, namely acyl-ghrelin and LEAP-2. Our findings are highlight the complex intersect between the ghrelin system and AUD toward the development of new potential therapeutical targets. Keywords: Ghrelin, Alcohol and Substance Use Disorders, Alcohol Consumption, IV Alcohol Disclosure: Nothing to disclose. P691. Depletion of the Gut Microbiome Influences Morphine Reward and Medial Prefrontal Cortex Gene Expression in an Age-Dependent Manner in Males and Females Rebecca Hofford, Ava Shipman, Violet Kimble, Drew Kiraly Wake Forest School of Medicine, Winston Salem, North Carolina, United States Background: Adolescence is a period of significant development and corresponds to a time of high rates of onset of mental health conditions. In addition to maturation of several crucial brain areas such as the medial prefrontal cortex (mPFC), there are major changes occurring in organ systems throughout the body – including major shifts in the composition of the gut microbiome. Data from our lab has demonstrated that knockdown of the microbiome influences cocaine and opioid reward as well as gene expression in the nucleus accumbens. Yet, it is unknown if perturbation of the microbiome might influence opioid reward and gene expression in an age-dependent manner, since both the microbiome and the brain are undergoing marked changes during this time. The current set of studies investigated the role of the microbiome on morphine reward and gene expression in the mPFC in adolescent and adult mice. Methods: To test whether microbiome depletion would influence morphine reward in an age-dependent manner, adolescent and adult male and female C57BL/6 J mice (n = 5-6 males, n = 7-8 females) were given a cocktail of broad-spectrum antibiotics (Abx) in their drinking water for 5 days before the start of behavior. This Abx treatment regimen is shorter than what has been utilized by the lab in the past and was chosen due to the short time that mice remain in adolescence. This was followed by a five-day morphine conditioned place preference (CPP) paradigm at 5.6 and 10 mg/kg morphine. Data was analyzed separately by age using two-way ANOVAs with morphine dose and Abx treatment as factors. Additional planned pairwise comparisons were conducted to assess the effects of Abx within sex and dose and were corrected for multiple comparisons. Experiment 2 used a separate group of mice to determine the effects of microbiome knockdown on gene expression in the mPFC. For this study, adolescent and adult male and female C57BL/6 J mice (n = 4-5) were placed on Abx for five days before receiving daily injections of 20 mg/kg morphine or saline (s.c.) for five days. This generated four treatment groups within each age and sex: H2O Sal (control), Abx Sal, H2O Mor, and Abx Mor. One hour after their last injection, mice were euthanized and their mPFC and cecal contents were removed and flash frozen. DNA was extracted from cecal contents using Qiagen DNA PowerSoil kit before bacterial 16 s sequencing on an Illumina MiSeq. The number of observed taxonomic units was used to assess microbiome diversity; this was analyzed using separate two-way ANOVAs with treatment group and age as factors. Principle component analysis using Unifrac dissimilarity was used to determine microbiome similarity across groups. RNA was isolated from whole mPFC tissue using Qiagen RNeasy kit and was sequenced using an Illumina HiSeq2500. Differentially expressed genes were identified using t-tests with an FDR-corrected p < 0.05 against each groups’ age- and sex-matched control (H2O Sal). Functional pathways were identified using G:profiler and Ingenuity Pathway Analysis. Results: Microbiome knockdown reduced morphine place preference in adolescents (Abx effect: F(1, 47) = 4.356, p = 0.0423, no other significant effects), but not adults (Abx effect: F(1, 51) = 0.3204, p = 0.5739, significant effect of dose F(1, 51) = 7.218, p = 0.0097). Planned comparisons indicated that adolescent females with a reduced microbiome demonstrated a place aversion at 10 mg/kg morphine (p = 0.034) but adult females were not affected by Abx (p = 0.2584). Adolescent males demonstrated reduced place preference at both doses (p = 0.0232), but adult males did not (p = 0.5626). While there were some interesting sex-specific effects, taken together adolescents showed a significant reduction in morphine place preference that was not demonstrated by adults, suggesting that adolescents are more sensitive to microbiome disruption than adults. For experiment 2, 16 s analysis confirmed that adolescent and adult male mice, but not female mice had unique microbiomes at baseline. However, males and females of both ages demonstrated shifts in the microbiome compositions after Abx, this was confirmed by reductions in microbiome diversity in both males and females (males: main effect of group: F(3, 31) = 45.39, p < 0.0001, interaction: F(3, 31) = 6.604, p = 0.0014, no age effect; females: main effect of group: F(3, 30) = 99.7, p < 0.001, interaction: F(3, 30) = 19.94, p < 0.0001, age: F(1, 30) = 83.40, p < 0.0001). Finally, in these same groups of mice, the combination of microbiome reduction and morphine treatment had the greatest effect on gene expression in the mPFC in both males and females at both ages. Abx Mor adolescents had a larger number of differentially regulated genes than their sex- and treatment-matched adults. Thus, like that observed in CPP, adolescents were more sensitive to the effects of microbiome depletion on gene expression in the mPFC. Pathway analysis identified more terms related to histone modification in male adolescents than adults, suggesting that alterations to chromatin structure could contribute to the age-specific effects on behavior and gene expression observed here. Conclusions: This series of studies demonstrated that adolescents are more sensitive to brief microbiome disruption than adults- observed effects were seen in both behavior and gene expression in the mPFC. Further work will demonstrate whether microbiome manipulations during the adolescent period can have long lasting effects on brain and behavior into adulthood. Keywords: Adolescence, Opioid, Microbiome Disclosure: Nothing to disclose. P692. Abstinence From Cocaine Self-Administration Drives Microglial Phagocytosis of Astrocytes Jonathan VanRyzin, Anze Testen, Tania Bellinger, Kathryn Reissner University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: Prolonged abstinence from drugs of abuse, such as cocaine, produces lasting physiological alterations in the brain, many of which prime the system for susceptibility to relapse. These alterations are not exclusive to neurons; two critical non-neuronal cell types- astrocytes and microglia- are also affected both by exposure to cocaine and duration of abstinence. We have reported that prolonged abstinence following long-access (6 h/day) cocaine self-administration leads to a pronounced reduction (~40%) in nucleus accumbens (NAc) astrocyte volume and synaptic colocalization, suggesting significant impairment in the ability of these pathological astrocytes to appropriately regulate synaptic function (Kim et al., 2022.04.06.487393). Separately, reports in the literature indicate that cocaine can directly trigger TLR4-dependent microglial activation. As professional phagocytes, microglia are appreciated for their ability to phagocytose dead or stressed cells and prune synapses in both homeostasis and disease. Thus, we hypothesized that cocaine induces aberrant microglial phagocytosis of astrocyte processes resulting in astrocyte dysfunction during prolonged abstinence. Methods: To test this hypothesis, we implanted indwelling jugular catheters and microinjected AAV-GfaABC1D-Lck-GFP virus (1 µL/hemisphere) into the NAc of adult male Sprague-Dawley rats for later quantification of astrocyte morphology. After 5 days of recovery, animals underwent 10 days of long-access (6 h/day) cocaine or saline self-administration, followed by either 1 or 45 days of home cage abstinence. We used confocal microscopy and three-dimensional modeling to determine the extent to which astrocyte membranes were internalized to microglia across abstinence (day 1 vs day 45). In a separate experiment, adult male Sprague-Dawley rats underwent 10 days of long-access (6 h/day) cocaine self-administration, followed by 30 days of home cage abstinence. Prior to self-administration, all rats received intra-NAc microinjection of AAV5-GfaABC1D-Lck-GFP virus (1 µL/hemisphere). Beginning on abstinence day 1, rats received intra-NAc infusion of neutrophil inhibitory factor (NIF) peptide (0.5 µL/hemisphere) or vehicle every 7 days to block microglial phagocytosis throughout abstinence (4 microinjections total). On abstinence day 30, all rats were tested for cue-primed drug seeking behavior, to determine whether blocking microglia phagocytosis would reduce drug seeking. Results: Microglia from cocaine self-administering rats contained significantly more GFP + inclusions compared to saline controls on abstinence day 45 (p < 0.001). This corresponded with an increased colocalization of both the lysosomal marker CD68 (p < 0.01) and separately the “eat-me” signal C3 (p < 0.01) in microglia from cocaine self-administering rats at this time point. Moreover, we found that NIF peptide-treatment reduced lever-pressing (p < 0.05) compared to vehicle-treated controls. Studies are currently underway to determine whether these measures of astrocyte engulfment are present during early abstinence (day 1) and the extent to which NIF peptide-treatment normalizes astrocyte volume and synaptic localization. Conclusions: These findings indicate that NAc microglia become highly phagocytic and engulf astrocyte processes following cocaine self-administration. Astrocytic pruning occurs throughout prolonged abstinence, which we predict is related to the previously observed astrocyte dysfunction. Drug seeking behavior is reduced by inhibiting phagocytosis during abstinence, which highlights the utility of targeting the neuroimmune system for potential therapeutic interventions. Keywords: Microglia, Astrocytes, Cocaine Self-Administration, Abstinence, Cocaine-Seeking Behavior Disclosure: Nothing to disclose. P693. Chronic Progesterone Treatment Reduces Nicotine Consumption and Accumbens Microglial Activation in a Sex-Specific Fashion Percell Kendrick, Emma Bondy, Erin Maher, Shailesh Khatri, Cassandra Gipson University of Kentucky, College of Medicine, Lexington, Kentucky, United States Background: Nicotine use is a substantial burden to public health. Further, craving and relapse to smoking appear to vary as a function of sex. In women, increases in 17β-estradiol (E2) and progesterone (P4) are associated with addiction vulnerability and resilience, respectively. P4 has been examined clinically as a smoking cessation agent and shows promise for women. However, studies incorporating men have shown unclear or limited to no efficacy. To date, there are no studies evaluating the mechanisms by which P4 may yield sex-specific efficacy, and no studies have been conducted to model the effects of P4 in a nicotine rodent model. Further, steroid hormones including P4 have anti-inflammatory properties. We found that neuroimmune signaling within the nucleus accumbens core (NAcore) is driven by nicotine seeking and consumption in a sex-specific fashion, whereby female rats are more susceptible to nicotine-induced neuroimmune consequences as compared to males. However, it is not yet known if P4 can reduce nicotine-induced neuroinflammatory signaling mechanisms. Methods: Gonad-intact male and female Long Evans rats (N = 6-8/group) underwent nicotine self-administration (0.06 mg/kg/infusion) for 10 sessions, followed by 15 sessions of nicotine self-administration in which rats received either daily systemic P4 (1.75 mg/kg, SC in 0.1 mL sesame oil) or vehicle (0.1 mL sesame oil) 2 h prior to sessions. Rats were then perfused, and NAcore microglia were quantified via 3DMorph. ANOVA or t-tests were used for statistical analyses. Uterine horns from females were also dissected to confirm hormonal treatments. Results: Here, we show that daily P4 treatments decreased nicotine consumption in female (but not male) rats (ANOVA, p < 0.05). We further show that in females, P4 reduced nicotine-induced NAcore microglial activation as compared to vehicle, measured by increased territorial volume (p < 0.05) and % occupied volume (p < 0.05), indicating that P4 reduces nicotine-induced activation of microglia. Male NAcore microglial morphology is currently being evaluated. Finally, uterine horns were significantly lighter in weight (g) in P4 as compared to vehicle-treated females, demonstrating that P4 treatment impacted the female reproductive system. Conclusions: Together, inhibiting nicotine-induced chronic activation of NAcore microglia may underlie the sex-specific therapeutic efficacy of P4 seen clinically. However, given that P4 does not reduce smoking in men, our results may justify examination of other possible therapeutics that have anti-inflammatory properties for smoking cessation, which may yield higher efficacy across both biological sexes. Next, we will test necessity of P4 to inhibit NAcore neuroimmune activation to exert therapeutic effects via our newly validated chemogenetic approach. Keywords: Microglial Activation, Nicotine Addiction, Nucleus Accumbens Core, Progesterone, Sex Differences Disclosure: Nothing to disclose. P694. Microglial Acid-Sensing Regulates Ethanol Consumption and Respiratory Depression in Mice Katherine McMurray, Renu Sah University of Cincinnati, Cincinnati, Ohio, United States Background: Alcohol use disorders (AUDs) are prevalent and debilitating. Alcohol has a wide range of effects on the body, engaging systems important for regulation of behaviors, emotions and physiology, all of which all contribute to the cycle of alcohol use and abuse. Alcohol use represents a potent threat to physiological homeostasis (e.g. neutral pH) through its ability to induce acidosis and neuroinflammation. Maintaining physiological homeostasis is critical for an organism’s survival, and threats to homeostasis elicit behavioral, emotional and physiological responses directed toward this goal. Acidosis appears shortly after alcohol use, can last up to 24 hours in plasma and brain, and is positively correlated with withdrawal severity. Most alcohol research has focused on mechanisms underlying the rewarding and aversive aspects of alcohol use as a motivated behavior. However, alcohol’s effects on behavior and physiology could also result from a drive to restore pH homeostasis. Therefore, the role of acid-sensing in regulating behavioral and physiological responses to alcohol, and the specific acid-sensing receptors mediating these effects, need to be investigated. A prominent acid sensor is the T-cell death associated gene 8 (TDAG8) receptor, located on microglia. Our lab recently identified a role for TDAG8 in panic-relevant behavior and physiology. TDAG8 mediates these effects through neuroinflammatory signaling within the subfornical organ (SFO). The SFO is a sensory circumventricular organ that has a leaky blood brain barrier and helps maintain physiological homeostasis by regulating behaviors, respiration and cardiovascular function. Lesion studies show SFO also regulates ethanol consumption, likely though its projections to other regions regulating ethanol consumption like the bed nucleus of the stria terminalis and central amygdala. Given the high comorbidity between panic disorder and AUDs, and the growing evidence for neuroinflammatory regulation of alcohol use/abuse, is it likely that TDAG8 activation in SFO may be a novel neuroimmune mediator of behavioral and physiological responses to alcohol use and regulator of alcohol consumption. To determine whether TDAG8 is a shared mechanism across panic and AUD, we tested the hypothesis that TDAG8 knockout would reduce voluntary ethanol consumption and microglial activation in the SFO. As acidosis is a respiratory stimulant, we also tested the hypothesis that TDAG8 knockout would exacerbate respiratory responses to ethanol. Methods: Male and female TDAG8 knockout (TDAG8-/-) or wild-type (TDAG8+/+) littermates were used (8-16 weeks old; n = 5-12). To determine if ethanol increases TDAG8 expression, TDAG8-promoter driven GFP expression was quantified by immunohistochemistry (IHC; anti-GFP) in transgenic mice 24 h after injection with 2 g/kg ethanol. To examine the effect of TDAG8 on voluntary ethanol consumption, we used the 4-day drinking in the dark (DID) paradigm. We used the same DID procedure to measure sucrose consumption, water consumption and ethanol consumption by separating the tests by one week. Mice were sacrificed 90 m after the last DID procedure (ethanol exposure). To determine effects of TDAG8 on ethanol-evoked neuroinflammation, fixed brain tissue was stained for microglial marker IBA-1 and microglia soma perimeter was then quantified. In separate cohorts of mice, ethanol’s effects on locomotion and respiration were examined using the open field test (locomotor stimulation, 1.25 g/kg ethanol), balance beam test (ataxia, 2 g/kg ethanol) and plethysmography (respiratory depression; 4 g/kg ethanol), respectively. Statistical analysis was preformed using Students t test, ANOVA, 2-way ANOVA or 3-Way repeated ANOVA as needed. Results: Ethanol increased TDAG8-promoter driven GFP expression within the SFO (p < 0.05), but not another circumventricular organ expressing TDAG8, the OVLT. During DID, both male and female TDAG8 deficient mice drank less ethanol than their WT littermates (genotype p < 0.05). There was no effect on sucrose or water consumption. TDAG8-/- mice also had reduced microglial soma perimeters compared to TDAG8 + / + mice (p < 0.05), which was specific to the SFO. Genotype had no effect on ethanol-evoked locomotor stimulation, but knockout animals demonstrated increased footslips on the balance beam test (p < 0.05). TDAG8-/- mice also had increased respiratory depression in response to a high dose of ethanol (p < 0.05). Conclusions: Together, these data point to acid-sensing receptor TDAG8 as a novel regulator of ethanol consumption and aversive outcomes (ataxia, respiratory depression) associated with alcohol use. Further, they suggest that these effects could be mediated through neuroimmune signaling within the SFO. Given the association of TDAG8 with both AUD- and panic-associated outcomes, this suggests SFO-TDAG8 and associated neuroimmune effectors may provide a unique and novel shared mechanism for AUD and panic disorder. Overall, these data support the idea that some behavioral and physiological responses to alcohol use may result from a drive to restore physiological homeostasis after a pH challenge. They also suggest acid-sensing receptors may represent a novel treatment target for alcohol use disorders. Keywords: Alcohol Use Disorder, Mouse Models, Neuroinflammation, Acid-Sensing Disclosure: PsyBio Therapeutics, Inc: Grant (Spouse). P695. Distinct Endothelial and Neuroimmune Responses in the Prefrontal Cortex is Associated With Relapse to Drinking in Alcohol Dependent Female and Male Rats Hannah Nonoguchi, Rajitha Narreddy, Timothy Kouo, Michael Jin, Mahasweta Nayak, Chitra Mandyam University of California, San Diego, San Diego, California, United States Background: The prefrontal cortex (PFC) is important for the development of alcohol addiction. Neuronal activity in the PFC is regulated by immune responses, and neuroimmune responses are assisted by disruption of the blood-brain barrier (BBB). The detrimental effects of chronic alcohol consumption, abstinence and relapse to alcohol drinking on neuroimmune response and BBB integrity in the PFC have been minimally explored. Moreover, the sex specific effects on these markers in the context of alcohol consumption and relapse are unknown. Here we seek to answer these questions. Methods: Adult female and male rats were made ethanol dependent by chronic intermittent ethanol vapor (CIE) and ethanol drinking (ED) procedure. Rats were euthanized after relapse session and plasma isolated from trunk blood and brain tissue homogenate of the PFC were analyzed for cytokines and chemokines using a 9-plex panel from Meso Scale Discovery. BBB disruption was analyzed with tight junction and adherens junction proteins claudin-5 and VE-cadherin via Western blotting and VEGF by ELISA. This allowed us to compare sex differences in the levels of individual cytokines as well as associations among cytokines and expression of tight junction proteins between the plasma and PFC. Results: CIE increased ED in both female and male rats, with females having higher ethanol consumption during CIE and relapse to ethanol drinking sessions compared with males. In parallel, CIE females had higher levels of interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-10 and VEGF in the plasma compared to CIE males. CIE females had higher levels of TNF-α and VEGF in the PFC compared with control females. However, CIE males had lower levels of IFN-γ, IL-1β and IL-4 compared with control males. Furthermore, in the PFC, abstinence reduced the expression of VE-cadherin in both sexes. However, relapse to drinking increased levels of VE-cadherin in CIE females beyond the control condition and this effect was not observed in CIE males. Claudin-5 expression was not altered in both sexes. Conclusions: These results reveal significant sex differences in pro- and anti-inflammatory cytokine levels in plasma and PFC that were associated with BBB disruption after relapse to ethanol drinking, and emphasize their possible role in alcohol addiction. Keywords: Alcohol Dependence, Neuroimmune Activation, Blood-Brain Barrier Disclosure: Nothing to disclose. P696. Preliminary Evidence for an Elevated Neuroinflammatory Signal in Opioid Use Disorder: Findings from a PET TSPO Imaging Study Eric Woodcock, Gustavo Angarita-Africano, David Matuskey, Jim Ropchan, Nabeel Nabulsi, Yiyun Huang, Ansel Hillmer, Richard Carson, Robert Malison, Kelly Cosgrove Wayne State University School of Medicine, Detroit, Michigan, United States Background: Preclinical studies show that repeated opioid administration is associated with elevated levels of glial markers, e.g., Iba1 and GFAP, in the rodent brain. Postmortem analyses of deceased opioid users show elevated levels of glial markers and alterations in neuroinflammatory signaling pathways. Yet, direct evidence that chronic opioid use may be neuroinflammatory in living individuals with opioid use disorder (OUD) has not been published to date. In our previous work, we showed that a single morphine dose acutely increased 18 kDa translocator protein (TSPO) availability, a neuroimmune marker, by 25-32% across brain regions in healthy adult volunteers via a [11 C]PBR28 Positron Emission Tomography (PET) imaging study. In the current study, we build on this work to investigate neuroimmune status of OUD patients and well-matched control subjects using [11 C]PBR28 PET imaging. Methods: Individuals currently using opioids and who met DSM5 criteria for OUD (n = 6) were recruited locally, admitted to a locked inpatient unit, and inducted on buprenorphine. Individuals with OUD were titrated to an individualized dose and stabilized for 5+ days to achieve steady-state plasma buprenorphine levels prior to a 120-minute [11 C]PBR28 PET scan. During the inpatient stay, non-medical opioid abstinence was verified with daily urine screens. Concurrently, healthy control subjects (n = 18) were recruited, screened, and underwent identical [11 C]PBR28 PET scanning procedures. On scan day, subjects also completed a computerized cognitive battery, self-report measures, and provided a plasma sample (assayed for biomarkers). During each PET scan, arterial blood was acquired to measure the metabolite-corrected arterial input function. Total volume of distribution (VT), i.e., TSPO availability, was estimated in 10 brain regions of interest (ROIs) using multilinear analysis-1 (MA-1; t=30). Group differences in regional [11 C]PBR28 VT were evaluated using linear mixed effects models with rs6971 genotype (‘high’ vs. ‘moderate’ affinity binders) and diagnostic group (OUD vs. controls) entered as fixed factors, and regional VT as the within-subject repeated factor. Partial correlations, controlling for rs6971 genotype, investigated relationships between regional [11 C]PBR28 VT and subjective and behavioral data among OUD patients. Results: Groups were well-matched for age, gender, ethnicity, body weight, body mass index, rs6971 genotype, and cigarette smoking status (ps > 0.15). Individuals with OUD were 32.0 ± 6.2 years old, 50% female, mostly white (83.3%), and reported using 6.3 ± 5.2 bags of heroin per day for 6.7 ± 2.1 years on average. Four individuals with OUD reported intranasal heroin use, whereas two reported injection use. Mean length of inpatient stay was 12.3 days (range: 10-15 days) and mean daily buprenorphine dose during stabilization was 7.7 mg (range: 4-16 mg). Linear mixed effect models showed that individuals with OUD exhibited significantly higher TSPO availability by 20% on average (13-24% across ROIs), relative to controls, F(1,236)=5.06, p = 0.036. Partial correlations, controlling for rs6971 genotype, indicated that longer inpatient stay prior to scanning, i.e., longer opioid abstinence and time in buprenorphine treatment, was correlated with lower TSPO availability in the prefrontal cortex (PFC; r = −0.99, p = 0.001). Also, partial correlations, controlling for rs6971 genotype, indicated that lower TSPO availability in the PFC was ‘trend-level’ correlated with less opioid craving on scan day (r = 0.80, p = 0.059). Consistent with the literature, individuals with OUD generally exhibited lower levels of peripheral cytokines/chemokines than controls with GM-CSF and IL-8 reaching statistical significance (ps < 0.05; TNF-α and IL-10 were ‘trend-level’: ps<0.10). Finally, there were no group differences in cognitive task performance (ps > 0.05). Conclusions: Our preliminary findings show the first in vivo evidence that individuals with OUD, in the first 1-2 weeks of treatment, exhibit elevated levels of neuroimmune signaling as evidenced by PET TSPO imaging. Longer inpatient stay prior to scanning was correlated with lower, i.e., more ‘normal’, TSPO levels in the PFC, suggestive of neuroimmune recovery with treatment. Further, lower TSPO levels in the PFC were ‘trend-level’ correlated with less subjective opioid craving. Taken together, our findings suggest that individuals with OUD may exhibit a neuroinflammatory phenotype upon entry to treatment, and that neuroimmune recovery may be possible with prolonged opioid abstinence which may translate to less opioid craving. Our findings highlight the neuroimmune system as a potential therapeutic target in OUD. Keywords: Opioid Use Disorder, Neuroinflammation, PET Imaging, Neuroimmune Mechanisms, Buprenorphine Maintenance Disclosure: Nothing to disclose. P697. Influence of Sleep Quality on Alcohol Use During a Quit Attempt Among Treatment-Seeking Individuals With Alcohol Use Disorder Wave-Ananda Baskerville, Steven Nieto, Erica Grodin, Diana Ho, Artha Gillis, Karen Miotto, Lara Ray University of California Los Angeles, Los Angeles, California, United States Background: Sleep problems are highly prevalent among individuals with alcohol use disorder (AUD). The relationship between alcohol use and sleep quality is conceptualized as bidirectional, in that sleep problems can be induced by chronic alcohol consumption and alcohol may be used to self-medicate sleep problems. Sleep difficulties may begin during early stages of abstinence from alcohol and commonly persist into long-term abstinence. The occurrence of sleep difficulties is clinically important given that sleep difficulties commonly precede alcohol relapse. Although there is established research on sleep difficulties as a risk factor for relapse, research focused on early stages of abstinence are lacking. Thus, the present study tests the relationship between sleep quality and alcohol use during a quit attempt in treatment-seeking individuals with an AUD. Methods: The present study utilized data from a two-week early efficacy paradigm trial to screen medications for AUD using naltrexone and varenicline. Fifty-three eligible participants were randomized to either naltrexone (n = 15), varenicline (n = 19), or matched placebo (n = 19). Randomized participants reported on their alcohol use during the 30-days prior to randomization using the Timeline Followback and their sleep quality using the Pittsburgh Sleep Quality Index (PSQI). Following a weeklong medication titration period, participants were asked to attend an in-person visit on Day 8 to begin a 7-day practice quit attempt and complete electronic daily dairy assessments (DDA) to report on previous day alcohol consumption, sleep quality, mood, and craving. On Day 14, participants reported on their alcohol consumption and sleep quality. A logistic regression analysis was conducted to test the relationship between baseline sleep quality (continuous variable) and abstinence (binary variable: abstinent versus not abstinent) during the practice quit attempt. A simple linear regression analysis was conducted to investigate the relationship between baseline sleep quality and total drinks during the 7-day practice quit. Results: An unadjusted logistic regression analysis revealed no relationship between baseline sleep quality and abstinence during the practice quit attempt, B = (-.009), SE = 0.012, Wald = 0.541, p < .462. An unadjusted linear regression analysis showed that baseline sleep quality was not associated with total drinks during the practice quit attempt, B = −.025, SE = 0.044, p = 0.575. Conclusions: While the bidirectional relationship between sleep quality and alcohol drinking is well documented, these preliminary results suggest that sleep quality is not associated with drinking behavior during the practice quit attempt among treatment-seeking individuals with AUD. Future statistical analyses are underway testing alcohol craving and negative mood as moderators of the relationship between sleep quality and drinking behaviors during the practice quit attempt. Keywords: Alcohol Dependence, Sleep Disturbances, Alcohol Abstinence, Drinking Disclosure: Nothing to disclose. P698. Transcranial Magnetic Stimulation of Dorsolateral Prefrontal Cortex Reduces Craving and Increases Salience Network Connectivity in People Who Smoke Cigarettes Nicole Petersen, Timothy Jordan, Michael Apostol, Edythe London, Andrew Leuchter Semel Institute for Neuroscience and Human Behavior at UCLA, Los Angeles, California, United States Background: Repetitive Transcranial Magnetic Stimulation (rTMS), a noninvasive brain stimulation technique, was recently approved by the FDA as a treatment for Tobacco Use Disorder. Although the efficacy of rTMS as a smoking cessation treatment has been demonstrated, the mechanism by which this occurs is not known, and such mechanistic information could improve and optimize treatment outcomes. This data reported here are part of a registered clinical trial, NCT03827265. Methods: Thirty-two people who smoke cigarettes daily were included. Before data collection, each was asked to remain abstinent from smoking cigarettes overnight (or at least 12 hours prior to stimulation) to heighten cigarette craving, and abstinence was verified by a carbon monoxide level of <10 ppm in expired air. Participants received 3,000 pulses of 10-Hz rTMS (5-second trains with 10-second inter-train intervals over 15 minutes) delivered to the left dorsolateral prefrontal cortex. They were not presented with any smoking-related or craving-inducing cues, and instead were instructed to relax and remain still. Spontaneous craving symptoms were measured by self-report using the Urge to Smoke scale and Shiffman-Jarvik Withdrawal Scale (craving subscale) before and after rTMS, and resting-state functional connectivity was measured by BOLD fMRI. Independent component analysis was used to identify the default mode and salience networks, and connectivity maps were compared before and after rTMS. Results: Single-session rTMS significantly reduced self-reported craving measurements on both the Urge to Smoke and Shiffman-Jarvik Withdrawal scales (Urge to Smoke: p = 0.01, d = 0.3; Shiffman-Jarvik: p = 0.02, d = 0.39). Effects of rTMS on default mode network connectivity did not survive familywise error correction, but robust and widespread effects on salience network connectivity were found. rTMS significantly increased connectivity between the salience network and clusters with maximum intensity in primary visual cortex (k = 8,551), right putamen (k = 7,020), posterior cingulate cortex (k = 556), supplementary motor area (k = 117), left putamen (k = 52), sensorimotor cortex (k = 47), thalamus (k = 45), anterior cingulate cortex (k = 35), and hippocampus (k = 21). Conclusions: Brain stimulation targeting the dorsolateral prefrontal cortex alters connectivity of the salience network. This finding suggests that the salience network is a justifiable target for future investigations of its usefulness as a biomarker of rTMS efficacy. The salience network also may itself provide clinically useful targets for stimulation to reduce cigarette craving, as its network hubs are accessible with rTMS. Keywords: Neuromodulation, Non-Invasive Neuromodulation, Resting State Networks Disclosure: Nothing to disclose. P699. Impact of a Substance Use Treatment Program for Women on Negative and Positive Urgency and Relationships to Striatal Responsivity to Reward Robin Aupperle, Jennifer Stewart, Rayus Kuplicki, Mallory Cannon, Emily Choquette, Martin Paulus Laureate Institute for Brain Research, Tulsa, Oklahoma, United States Background: Methamphetamines and opioids are among the most commonly used illegal substances, accounting for approximately 10% disability adjusted life years worldwide. The biological processes that contribute to the initiation and maintenance of substance use disorders are still poorly understood. Identifying such processes could help to better develop targets for interventions to improve outcomes of these disorders. Negative and positive urgency, i.e., responding impulsively to experiences of negative and positive affect respectively, have been proposed as behavioral processes contributing to the maintenance of substance use disorders. Altered ventral striatal dopamine release and activity during reward anticipation has been associated with substance use disorders and individual differences in impulsivity. This investigation examined impact of a comprehensive substance use intervention on positive and negative urgency and striatal anticipation to reward to determine whether these processes could be considered targetable disease modifying processes for addiction. Methods: Participants (N = 168, Mean age = 32.71) were women recruited from the Women in Recovery (WiR) program, a court-ordered mental health diversion program for drug-related offenses. WiR includes comprehensive treatment services, including empirically supported treatment for SUD and mental health and access to pharmacological therapy, parenting classes, GED classes, and occupational assistance. WiR participants remain in the treatment program for an average of 17 months. A total of 74% (N = 125) of participants successfully graduated from the WiR program; current analysis focused on N = 75 women who completed the UPPS-P and functional magnetic resonance imaging (fMRI) at baseline and one-year after enrolling in the WiR program. Participants mean age = 32.73 (SD = 7.23); 69% reported less than or equal to a high school education; 53% and 24% reported methamphetamine and opiates as their drug of choice respectively. Linear mixed models were used to assess changes in (a) the negative and positive urgency sub-facets of the UPPS-P and (b) left and right nucleus accumbens (NAcc) activity during anticipation of reward (+5 vs +0) and loss (-5 vs -0) on the monetary incentive delay task during fMRI. For NAcc activity, average percent signal change was extracted from Brainnetome Atlas NAcc regions of interest. Regression analyses were used to examine relationships between negative/positive urgency and striatal reactivity at baseline, as well whether the extent of change in striatal reactivity predicted the one-year outcomes in positive and negative urgency (covarying for baseline urgency). Average motion during fMRI, age, and education were also utilized as covariates across all analyses. Study was registered at clinical trials.gov (NCT02601495). Results: From baseline to one-year outcome, women reported significant decreases in both positive (F(1, 79) = 19.38, p < 0.001, d = −0.99) and negative urgency (F(1, 80) = 52.89, p < 0.001, d = −1.63) but no significant changes were observed for striatal reactivity to reward or loss anticipation (ps > 0.10). While striatal reactivity did not relate to urgency measures at baseline (ps > 0.10), greater increases from baseline to one-year outcome in right and left striatal reactivity during reward anticipation related to greater decreases in positive (right: t(70)= -2.59, p = 0.012, β = −5.69; left: t(70)= -3.03, p = 0.003, β = −6.78) and negative urgency (right: t (70) = −2.50, p = 0.015, β = −3.86; left: t(70) = −2.62, p = 0.011, β = −4.13). Conclusions: Results indicate that comprehensive and extended substance use treatment is effective at reducing negative and positive urgency. Although there were no overall changes in NAcc activation to reward or loss anticipation with treatment, results indicate that the extent to which treatment increases engagement of NAcc when anticipating non-drug rewards be an important neural process contributing to changes in positive and negative urgency. Taken together, this longitudinal study of a comprehensive intervention for individuals with substance use disorder supports the idea that positive and negative urgency may be disease modifying processes on a behavioral level that is linked to changes in neural reactivity to reward anticipation. Future longitudinal and experimental research is needed to identify specific treatment strategies that directly target these processes. Keywords: Striatum, Reward Anticipation, Substance Use Disorder, Functional Magnetic Resonance Imaging, Impulsivity Disclosure: Nothing to disclose. P701. Dynamics of Dopamine Increases During Methylphenidate Challenges in Humans Measured With PET Dardo Tomasi, Peter Manza, Jean Logan, Ehsan Shokri Kojori, Michele-Vera Yonga, Danielle Kroll, Dana Feldman, Katherine McPherson, Catherine Biesecker, Evan Dennis, Allison Johnson, Kai Yuan, Wen-Tung Wang, John Butman, Gene-Jack Wang, Nora Volkow National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: The dynamics of dopamine (DA) are relevant to reward signaling including drug rewards, but access to methods that quantify DA dynamics in the human brain have been limited. Here, we show that a simple approach using positron emission tomography (PET) can be used to non-invasively assess the dynamics of dopamine increases induced by methylphenidate (MP) in the human striatum. In simulations, we tested the hypothesis that the time-varying differences in standardized uptake value ratio (SUVr) between [11 C]raclopride scans collected with and without a MP-challenge reflect the dynamics of dopamine increases induced by MP in the striatum. To test the hypothesis that the intensity of the ‘high’ reflects the rate of dopamine increases in the striatum, we carried out a within-subject [11 C]raclopride PET study with a double-blind placebo-controlled design in healthy adults. We studied dynamic dopamine increases using oral-MP (results in slow brain delivery) and intravenous (IV)-MP (results in fast brain delivery) challenges, in association with measured subjective responses to MP using self-reports of ‘high’ throughout the scan. Methods: To simulate effects of time-varying DA concentrations on [11 C]raclopride binding of D2/3 receptors we used the linear extension of the simplified reference region model (LSSRM). We tested twenty healthy adults (36.1 ± 9.6 years old; 9 females) who underwent 90-min long PET scans collected in 3 randomly ordered sessions (placebo, oral-MP, and IV-MP) while simultaneously recording their self-reported ‘high’ ratings (0-10) under resting conditions, using oral-MP and IV-MP. In each session, each participant received an oral pill (60mg-MP or placebo) 30 min before injection of the PET tracer ([11 C]raclopride), followed 30 min after the tracer by an IV administration (0.25 mg/kg-MP or placebo, manually injected as a ~30-second bolus). List mode PET emission data were acquired continuously for 90 min and initiated immediately after manual injection of [11 C]raclopride (dose = 15.7 ± 1.9 mCi; duration 5-10 seconds). A 3-dimensional ordered-subset expectation-maximization (OSEM) algorithm was used to create PET time series consisting of 48 time windows (30 frames of 1 min, followed by 12 frames of 2.5 min, and 6 frames of 5 min). Standardized uptake values (SUVs) for [11 C]raclopride were calculated after normalization for body weight and injected dose and spatially normalized to MNI space. Relative SUV time series, SUVr(t), were computed in MNI space by normalizing each SUV volume by its mean SUV in cerebellum. Results: In simulated data we found that the dynamics of endogenous DA increases shape the time-varying SUVr changes (delta SUVr) elicited by MP. To assess the sensitivity of DA’s time-to-peak (TTP) increase to MP dose we simulated the normal variability of delta SUVr dynamics within and across individuals (1000 simulations). Specifically, random variations in LSSRM parameters (5%) also caused minimal variability in fitted TTP (SD = 0.55 min). Differently, random variations in MP dose (10%), input function, and LSSRM parameters (5%) showed that fitted TTP was significantly associated with variations of the MP dose (R2 = 0.88), but not with variations of the other parameters. Similarly, we assessed the association between fitted TTP and the ‘true’ DA rate TTP using 4% random variability in all parameters (1000 simulations) and found that fitted and ‘true’ DA rate TTP had excellent correlation (R2 = 0.94). These simulations suggest that fitted TTP can accurately predict DA rate TTP [coefficient of variation ~ 0.1]. The feelings of reward elicited by MP in the participants were stronger for IV- than for oral-MP (P < 0.0002), and the gamma cumulative distribution function accurately fitted the dynamics of delta SUVr as predicted by our model. Fitted DA release TTP in putamen was significantly correlated with the difference in peak ‘high’ ratings between MP and placebo, independently for oral-MP and IV-MP, such that shorter TTP was associated with higher self-reports of “high” from MP (oral: R(13)= 0.76, IV: R(18)= 0.69; P < 0.003, two-sided). Conclusions: Dynamic PET is a unique tool to assess the rate of DA increases induced by potentially addictive drugs (or other rewarding stimuli) in the human brain. Drug-related DA dynamic measures can also be used to advance our understanding of their associations with brain function and behavior simultaneously collected with PET-MRI scanners in humans (or laboratory animals). In simulations, we show that time-varying ΔSUVr parallels the dynamics of DA increases induced by MP in the striatum. In a double-blind placebo-controlled PET study with a within-subjects design we show that the intensity of the ‘high’ reflects the dynamics of DA increases in the striatum. These findings provide strong evidence that the speed of DA increases in the striatum, which is influenced by the rate of drug uptake in the brain and is modulated by the route of drug administration, accounts for why a drug like MP can be used safely for oral ADHD treatment, whereas it can result in addiction when injected. Our findings that the faster the rate of DA increases, the more intense the “high”, would also explain why very large oral doses of MP can also be rewarding. Keywords: Dopamine Release, Methylphenidate, Reward Disclosure: Nothing to disclose. P702. Text Mining of Substance Cessation Support Groups on Reddit Reveals Robust Subjective Experience Phenotype Uniquely Enriched for Anxiety, Disgust, and Gratitude Genevieve Yang, Hung-mo Lin, Rita Goldstein Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: Online communities and the posts they generate represent an unprecedented resource for studying subjective emotional experiences, capturing population types and sizes not typically available in the laboratory. Here we mined such a platform to explore a putative specificity of the emotional experience of substance cessation and its cross-substance overlap. Revealing transdiagnostic clues that could ultimately be used for mental health outreach was an important motivation for this exploration. Specifically, we aimed to characterize the emotions associated with cessation of three major substances and compare them to emotional experiences reported in non-substance cessation posts. Methods: Two million pseudonymous posts made respectively in the Fall of 2020 (discovery dataset) and Fall of 2019 (replication) were obtained from 394 forums at Reddit.com. We tracked emotion word frequencies in posts from three substance cessation forums (for alcohol, nicotine, and cannabis topic categories), contrasting them to general forums. Emotion word frequencies were further tracked on multiple distinct categories of emotions and represented as a multidimensional emotion vector for each forum. We quantified the degree of emotional resemblance between different forums by computing cosine similarity on these vectorized representations. For substance cessation posts with self-reported time since last use, we explored changes in the use of emotion words as a function of abstinence duration. Results: Compared to posts from general forums, substance cessation posts showed more expression of anxiety, disgust, pride, and gratitude words. ‘Anxious’ emotion words were attenuated for abstinence durations > 100 days compared to shorter durations (t12 = 3.08, two-tailed, P = 0.00930). The cosine similarity analysis identified an emotion profile preferentially expressed in the cessation posts across substances, with lesser but still prominent similarities to posts about social anxiety and ADHD. These results were replicated in the 2019 (pre-COVID-19) data and were distinct from control analyses using non-emotion words. Conclusions: We identified a unique subjective experience phenotype of emotions associated with the cessation of three major substance types, replicable across two time periods. We noted changes to this experience as a function of duration of abstinence. Although to a lesser extent, this phenotype quantifiably resembled the emotion phenomenology of other relevant subjective experiences (social anxiety, ADHD). Taken together, these transdiagnostic results suggest a novel approach for future identification of at-risk populations, allowing for the development and deployment of specific and timely interventions. Keywords: Natural Language Processing (NLP), Addiction Phenotypes, Alcohol, Nicotine Addiction, Cannabis Use Disclosure: Nothing to disclose. P703. Effects of Intermittent- vs Long-Access Cocaine Self-Administration on Addiction-Like Behaviors and NAc Glutamatergic Plasticity Carrie Ferrario, Amanda Catalfio, Megan Wickens, Tracy Fetterly, Allison Nieto University of Michigan Medical School, Ann Arbor, Michigan, United States Background: Long Access self-administration procedures (LgA, 6 h + /day) have become the gold-standard in the addiction field because they produce addiction-like behaviors, and changes in brain not seen following shorter drug access that models drug taking (1-2 h/day). However, Zimmer and colleagues recently developed an intermittent access self-administration procedure (IntA) to better model the patterns of cocaine use seen in addicts. IntA produces more robust addiction-like behaviors compared to LgA (cocaine-motivation, reinstatement, and cocaine-seeking) despite much less drug intake. Whether these distinct patterns of cocaine exposure produce similar or different alterations in nucleus accumbens (NAc) function is poorly understood, and there is limited understanding of the behavioral effects of IntA across sex. Methods: We used whole-cell patch clamping to evaluate how IntA vs LgA cocaine alters NAc excitatory transmission. Given the role of NAc CP-AMPA receptors in cocaine-seeking following LgA, we measured sensitivity to the CP-AMPAR antagonist Naspm, and sEPSCs. We also examined sex differences in IntA effects on psychomotor sensitization, and how increasing the intermittency of self-administration affects motivation for cocaine and psychomotor sensitization using newly established deep-learning based approaches. Results: Initial results suggest that IntA produces enhancements in NAc glutamate transmission that are distinct from those seen following LgA. IntA produced more robust psychomotor sensitization compared to LgA, and greater sensitization in females than males. Finally, increased intermittency of cocaine use enhanced motivation for the drug. Conclusions: Overall, the data suggest that IntA may better model addiction and thus provide additional information about translationally relevant targets for the treatment of substance use disorder. Keywords: Cocaine Addiction, Nucelus Accumbens, Motivation Disclosure: Nothing to disclose. P704. Prenatal Circadian Rhythm Disruption Induces Sex-Specific Substance Use and Mood-Related Phenotypes in Mice Lauren DePoy, Colleen McClung University of Pittsburgh, Pittsburgh, Pennsylvania, United States Background: 20% of Americans are at risk for environmental circadian rhythm disruptions (CRD) due to shift work. Shift workers experience substantial negative health outcomes, but females are especially affected with greater vulnerability for substance use (SU) and adverse outcomes associated with pregnancy. These outcomes not only occur during pregnancy, but offspring are affected at birth and later in life. In mice, prenatal CRD (pCRD) recapitulates these risks, increasing adverse pregnancy outcomes and altering behavior in adult offspring. However, it is unknown whether pCRD affects SU in mature offspring. Methods: C57Bl/6 J dams were sham handled or disrupted, by reversing the light/dark cycle 4 times during gestation. Reward- and mood-related behaviors were measured in adult offspring. Cocaine reward was measured using conditioned place preference (5 mg/kg cocaine). Contingency degradation was used to measure decision making. Mice were trained to respond on two levers for food, then the likelihood that one of those levers will be reinforced was degraded. Another cohort was trained to respond for food before jugular catheterization. After recovery, mice were trained to respond on a different lever for cocaine. Acquisition, the reinforcing and motivational properties of cocaine, extinction and cue-induced reinstatement were measured. Throughout, 2-way ANOVAs were used for data analysis (disruption group by sex), with additional repeated measures as appropriate (i.e. session, lever, dose). Significant interactions were followed up with Sidak posthoc tests. p < 0.05 was considered significant and p < 0.1 trending. Samples sizes of 6-14, depending on the experiment. Results: Interestingly, females exposed to pCRD developed an anhedonic-like phenotype with decreased food self-administration (interaction, p = 0.007), cocaine intake (disruption, p = 0.008) and reinforcing properties of cocaine (interaction, p = 0.01). On the other hand, pCRD males showed a SU-like phenotype with increased cocaine preference (interaction, p = 0.08), higher order food self-administration (disruption, p = 0.099) and cocaine reinforcement (interaction, p = 0.08). Furthermore, while male pCRD mice maintained goal-directed decision making, responding more on a reinforced lever, female pCRD mice did not, indicating habit formation. Together, these results suggest that male and female mice exposed to pCRD respond differently for rewarding outcomes. In order to determine whether these divergent behavioral outcomes are unique to reward I next measured anxiety-like behavior. As expected, preliminary results in the open field test and elevated plus maze (interaction, p = 0.005) paralleled reward-related behavior. Anhedonic-like female pCRD mice showed increased anxiety-like behavior and pCRD males showed decreased anxiety/increased risk-taking behavior. Conclusions: These results suggest that pCRD may predispose individuals to distinct psychiatric disorders based on sex, mood disorders in females and SU disorders in males. By understanding how disrupted rhythms during pregnancy affect behavior in adulthood, we can develop novel therapeutic approaches for SU and mood disorders in adults. Keywords: Circadian Rhythms, Substance Use, Sex Differences Disclosure: Nothing to disclose. P705. Effects of Acute Cannabis Exposure on Simulated Automotive Driving Behavior: A Longitudinal, Double-Blind, Placebo-Controlled Study Shashwath Meda, Michael Stevens, Brian Pittman, Ralitza Guerguieva, Erwin Boer, Gregory Book, Nicholas Ward, Catherine Boyle, Godfrey Pearlson Hartford Healthcare, Yale University, Hartford, Connecticut, United States Background: Acute cannabis intoxication may have significant effects on complex day-to-day activities such as automobile driving that require integrating multiple cognitive and psychomotor functions. The recent legalization of both recreational and/or medicinal marijuana in many states has created an urgent need to better understand the dose dependent effects of Tetrahydrocannabinol (THC) on driving behavior. The present study employed a longitudinal, double-blind, placebo with 2 active doses to investigate the differential effects of THC levels in cannabis on a variety of driving-related behaviors in a controlled, naturalistic, simulated environment. The current study attempts to overcome a limitation of prior cannabis driving studies by challenging driving within naturalistic driving scenarios that better approximate real-world risky situations. Based on previous findings from both simulated and real-world driving, we hypothesized subjects to have increased lane deviations. In addition, we postulated that the driving challenges would be relatively more sensitive than prior studies in identifying dose-sensitive drugged driving differences, such as altered risky overtaking decisions or inconsistent speed during safe passing, fewer fine steering and/or gas pedal adjustments when lane keeping is perturbed by environmental factors, and a relative inability to safely follow a lead car under the influence of cannabis, compared to placebo. Methods: Data are presented on N = 38 subjects (N = 25 male, frequent cannabis users, mean age 24.25 + 7.01), each exposed to a placebo, low and high dose of cannabis on three separate days. On each day, following a single acute inhaled 0.5 g dose of either 0%, 5.9% or 13% of THC via a desktop ‘Volcano’ vaporizer, subjects drove a virtual driving simulator (RTI Sim Vehicle platform) three times inside an MRI scanner and once out of scanner, randomized, and dispersed throughout an eight hour daily period. During each driving session, a variety of real time behavioral risk measures corresponding to lane-keeping (LK) following simulated wind gusts, lead car following (CF) and safe overtaking (OT) were parameterized using custom Matlab scripts to extract event-locked driving performance information, resulting in a total of 29 dependent variables. Data were analyzed using a generalized linear mixed model framework in SPSS v24 which included dose, time (time since dose in mins), instrument (desktop PC versus MRI), dosetime, doseinstrument, age and sex as primary fixed factors. In addition, subjectdose were modelled as random effects to better capture the variation in slopes across individuals and dose. The above model was further refined on a variable by variable basis by removing the fixed interaction terms if they were found to be non-significant. Results: The original model revealed no significant fixed interaction terms. The lane keeping paradigm showed that under the influence of cannabis, the number of finer steering adjustments was reduced in a dose dependent manner (F = 7.57;p < 0.001). In the car following task, there was an increase in correlation lag between the subject’s car and a lead car (F = 2.99;p < 0.05) and fewer fine gas pedal corrections (F = 3.59;p < 0.03). In addition, albeit non-significant, a trend of decreased time to a potential collision was noted for higher doses (F = 2.50;p < 0.08). Consistent with the greater overall complexity of the overtaking task, drugged drivers showed more widespread abnormalities on several driving metrics. These included decreased median total time to pass (F = 3.26; p < 0.04), lower median minimum speed during overtake (F = 3.66; p < 0.02), lower median speed entering the oncoming lane (F = 3.64; p < 0.03). In addition, several comparable metrics involving speed and judgement of when it was safe to pass obstacles reached trend levels of statistical significance, i.e., decreased median gap to passed cars (F = 2.56;p < 0.08), median maximum overtaking speed (F = 3.64;p < 0.1) and median speed leaving oncoming lane (F = 2.41;p < 0.09). Most of the above dose-dependent impairments tended to diminish significantly throughout the day as cannabis effects waned, although to a varying degree across metrics. Conclusions: From the current study, we draw three main conclusions: A) As expected, we observed widespread dose-related behavioral impairments in naturalistic scenarios that challenge automatic driving behavior. There were relatively more and diverse impairments on these tasks compared to prior reports. B) We were able to detect impairments in several driving behaviors related to fine steering/gas pedal adjustments, altered speed during passing and safe car following that have not been previously reported. C) These new effects were seen only during the high dose and not the low dose, offering more clarity on dose dependency. In general, individuals while driving under the influence of cannabis demonstrated rather ‘lethargic’ or delayed responses to stimulus events, putting them at risk of responding more slowly to sudden driving challenges. Keywords: Cannabis, Simulated Driving, Behavioral Pharmacology, Substance Abuse Disorders, Alcohol and Substance Use Disorders Disclosure: Nothing to disclose. P706. Cannabis Induced Brain-Behavior Correlates of Simulated Automobile Driving: A Double-Blind, Placebo-Controlled fMRI Study Shashwath Meda, Michael Stevens, Erwin Boer, Gregory Book, Nicholas Ward, Catherine Boyle, Godfrey Pearlson Olin Neuropsychiatry Research Center, Hartford, Connecticut, United States Background: Driving is a complex everyday activity that requires the use and integration of different cognitive and psychomotor functions, many of which are known to be affected by cannabis. Given the public health and legal implications of drugged-driving and rapidly increasing use of cannabis nationwide, there is an urgent need to better understand the drug’s effects on such functions and their underlying brain mechanisms in the context of driving. This longitudinal, double-blind placebo-controlled study investigated the effects of cannabis on driving brain-behavior relationships in a controlled, simulated environment using functional MRI (fMRI). Based on prior findings on cognitive impairments while driving under the influence of cannabis, and our own prior work on alcohol-impaired driving, we hypothesized that brain regions/circuits related to executive functioning (prefrontal/frontal cortex), attentional control (fronto-parietal, anterior cingulate), information processing (parietal, temporal), motor coordination (cerebellum) and visual (primary and extrastriate visual cortex) would be prominently affected in our current study. Methods: N = 29 frequent cannabis users (22 male, mean/SD age 23.90 + 5.26 and 7 female, mean/SD age 21.85 + 1.77 were administered 0.5 grams of 13% THC or placebo flower cannabis via a Stortz+Bickel ‘Volcano’ vaporizer using paced inhalation, on separate days at least 1 week apart. On each study day, participants drove a virtual driving simulator (steering wheel, brake, gas pedal) inside an MRI scanner approximately 30 minutes post-dosing. Each fMRI driving session presented a naturalistic simulated environment that unobtrusively engaged drivers with scenarios that tested specific driving skills and response. There were three, approximately 10 min epochs where drivers engaged in tasks of lane-keeping/weaving (LK), lead car-following (CF), and safe overtaking from a parked starting place (OT). fMRI data were prepared for analyses using the Human Connectome Project (HCP) pipeline v4.2. Independent Component Analysis (ICA) identified 40 valid neural networks from both drugged and placebo fMRI sessions. Regional connectivity estimates were averaged within 360 discrete cortical parcels identified by the HCP and another 27 sub-cortical regions. For each subject, a ‘placebo minus drug’ map of spatial connectivity difference was computed to quantify changes in functional connectivity due to THC. A similar difference score was computed for the 29 behavioral datapoints that described complex driving behavior. Principal component analysis (PCA) reduced these scores to 9 latent factors (3 for each task) that preserved between 80-92% of the driving behavior variance. FSL randomize was used to assess non-parametric statistical associations between connectivity maps and behavioral data. Results were thresholded and displayed at an FWE < 0.05 level adjusted for searching all parcels. Results: For car-following, Factor 1 correlated with connectivity values in anterior BA 9/46, intra parietal, and superior temporal (STS) parcels. Factor 2 correlated with posterior ACC and visual cortex connectivity. Factor 3 correlated with visual cortex and ventral diencephalon. Similarly, for lane-keeping, Factor 1 correlated with STS/inferior parietal, Factor 2 with inferior frontal and nucleus accumbens, and Factor 3 with ventral area 9-46/10 and nucleus accumbens. Lastly, for the overtaking task, factor 2 correlated with connectivity values from inferior frontal, 9-46, and cerebellum parcels, while Factor 3 associated with inferior frontal, BA9, and parahippocampus. Conclusions: As hypothesized, cannabis-related changes in connectivity of lateral prefrontal, cingulate, parietal cortex, and cerebellum were associated with drug-related driving task differences. Additional, non-hypothesized regions, including nucleus accumbens were also affected by recent cannabis use. These preliminary results depict complex yet informative links between many key brain areas sensitive to acute cannabis exposure and a profile of driving behaviors within a simulated environment that are also affected by the drug. These drug-related brain circuit alterations also differed significantly from those seen in our prior work on alcohol-impaired drivers. The current links underscore the likelihood that cannabis’ acute effects on the brain networks engaged for driving are related to real-world risks to public safety by underlying driving related behavior. Keywords: Cannabis, Simulated Driving, Functional MRI (fMRI) Disclosure: Nothing to disclose. P707. Association Between Immune Dysregulation and Response to Naltrexone-Bupropion Combination in Methamphetamine Use Disorder: Findings From the ADAPT-2 Study Kimberlyn Maravet Baig-Ward, Manish K. Jha, Abu Minhajuddin, Brittany Mason-Mah, Cherise Chin Fatt, Thomas Carmody, Sidarth Wakhlu, Steve Shoptaw, Jane Foster, Madhukar Trivedi* The University of Texas Southwestern Medical Center, Dallas, Texas, United States Background: There are no approved pharmacological treatments for methamphetamine use disorder (MUD). We recently showed that while response to naltrexone-bupropion combination was 5-fold higher than placebo, only 13.6% of individuals were able to attain response. Here, we evaluated the association of immune dysregulation with response to naltrexone-bupropion treatment. Methods: Participants of the Accelerated Development of Additive Treatment for Methamphetamine Disorder [ADAPT-2] study with available week-6 post-treatment initiation plasma samples were included. Immune markers were assayed with the Bioplex Pro™ human cytokine standard 27-plex kit (Bio-Rad Laboratories, Hercules, CA, USA). Principal component analysis was used to identify latent factors of immune function. Logistic regression was used for association between immune factors and response (defined as 3 out of 4 negative urine sample over a 2-week evaluation period). Covariates included age, sex, race, and ethnicity. Post hoc analyses evaluated percentage of individuals with negative urine drug sample based on the levels of immune markers (high versus low, median split). Results: N = 49 and N = 119 participants were randomized to naltrexone-bupropion and placebo, respectively. The first two principal components were retained and explained over 75% variance in the immune markers. First principal component (PC1) markers included interferon γ, interleukin (IL) 1β, IL-17A, and IL-2. Second principal component (PC2) markers included IL-4, IL-9, macrophage inflammatory protein 1 beta, and tumor necrosis factor alpha. At week-6, higher levels of PC2 were associated with lower likelihood of response at week-6 to naltrexone-bupropion [odds ratio (OR) = 0.28, 95% confidence interval (CI) = 0.09, 0.90; p = 0.032). No similar association were noted for placebo group. Notably, the association between higher levels of PC2 at week-6 and lower likelihood of response to naltrexone-bupropion combination at week-12 was also significant (OR = 0.17, 95% CI = 0.04, 0.70; p = 0.013). Low vs. high levels of PC2 were associated with higher proportion of methamphetamine-negative urine samples with naltrexone-bupropion by week-2 and this difference was maintained until week-12. Conclusions: We found that alterations in immune markers were significantly associated with poor response to naltrexone-bupropion combination. Future studies are needed to understand the cellular and molecular underpinnings of this association and clinical significance. Keywords: Methamphetamine Use Disorder, Naltrexone, Bupropion, Stimulant Use Disorder Disclosures: Acadia Pharmaceuticals, Alkermes Inc, Axsome Therapeutics, Biogen MA Inc, Circular Genomics Inc., GH Research Limited, Janssen, Legion Health Inc, Merck Sharp and Dohme Corp., Mind Medicine (MindMed) Inc., Myriad Neuroscience, Neurocrine Biosciences Inc, Noema Pharma AG, Orexo US Inc, Otsuka, SAGE Therapeutics, Titan Pharmaceuticals Inc,: Consultant (Self),, Alto Neuroscience Inc, GreenLight VitalSign6, Legion Health Inc, Heading Health, Signant Health: Advisory Board (Self). P708. A Single-Dose of Ketone Ester Decreases Brain Glucose Metabolism in Alcohol Use Disorder Corinde Wiers, Anthony Young, Juliana Byanyima, Xinyi Li, Robert Doot, Sianneh Vesslee, Rishika Reddy, Zhenhao Shi, Reagan Wetherill, Timothy Pond, Nora Volkow, Henry Kranzler, Jacob Dubroff University of Pennsylvania, Philadelphia, Pennsylvania, United States Background: Acute alcohol intake decreases brain glucose metabolism and increases brain uptake of acetate, a metabolite of alcohol. Individuals with alcohol use disorder (AUD) show elevated brain acetate metabolism at the expense of glucose, and the shift in energy utilization persists beyond acute intoxication. We recently reported that nutritional ketosis and administration of ketone bodies as an alternative energy source over glucose reduce alcohol withdrawal and alcohol craving in AUD. However, the regional effects of nutritional ketosis on brain glucose metabolism have not been studied in AUD. Methods: Four participants (n = 2 male individuals with AUD, 2 non-dependent controls [1 male, 1 female]) underwent two separate study visits, once after an overnight fast (baseline visit), and once after an overnight fast and 1.5 hr. following the consumption of 395 mg/kg (R)-3-hydroxybutyl (R)-3-hydroxybutyrate Ketone Ester (KE) solution (TdeltaS Global Inc.), in randomized order. We measured blood glucose and ketone levels. Brain glucose metabolism was assessed using positron emission tomography with [18 F]fluorodeoxyglucose ([18 F]FDG). Voxel-wise maps of the cerebral metabolic rate of glucose (CMRglc; μmol/100 mL/min) were computed in PMOD v3.7 (PMOD Technologies, Zurich, Switzerland), and further analyzed with SPM12 (Wellcome Trust Centre for Neuroimaging, London, UK). Results: A single dose of KE elevated blood ketone (beta-hydroxybutyrate) levels compared to the baseline visit (F3,9 = 24.4, p < 0.001). Although KE administration did not change blood glucose levels (F3,9 = 1.6, p = 0.27), it decreased whole-brain CMRglc by 18.5% (mean baseline=15.1 + - 1.8 SD, mean KE = 12.3 + - 1.8 SD, F1,3 = 32.1, p = 0.01). In SPM analyses the largest KE-induced CMRglc reductions were in the frontal cortex (peak MNI = [-22, -2, 64], k = 5892, t = 16.2, pFWE<0.001), including the bilateral inferior frontal gyrus (peak left [-52, 4, 24], peak right = [58, 14, 24], k = 132, t = 11.3, pFWE =0.015); occipital cortex (peak = [-14, -86, 0], k = 206, t = 16.0, pFWE=0.001); temporal gyrus (peak = [-58, 0, -34], k = 243, t = 13.1 pFWE <0.001); and anterior cingulate cortex (peak = [-8, 16, 26], k = 410, t = 8.4, pFWE<0.001). KE did not increase CMRglc in any brain region. Sample sizes in this pilot study were too small to compare the AUD and control participants on CMRglc. Conclusions: These findings provide preliminary evidence of an effect of KE administration in reducing brain glucose metabolism in humans, consistent with a shift from glucose to ketones as a brain energy source. Average reductions in CMRglc of 18.5% are similar to global average reductions documented with 0.5 g/kg alcohol administration. Data collection using this paradigm is ongoing, as are studies of the effects of KE on alcohol consumption. Documenting the clinical and neurometabolic effects of nutritional ketosis will yield fundamental knowledge of its potentially beneficial effects as a novel treatment for AUD and aid in identifying its underlying neural mechanisms. Keywords: Alcohol and Substance Use Disorders, Positron Emission Tomography, Ketones Disclosure: Nothing to disclose. P709. Social Determinants of Health Associated With How Cannabis is Obtained and Used in Patients With Cancer Rebecca Ashare, Esther Turay, Brooke Worster, Salimah Meghani SUNY Buffalo, Buffalo, New York, United States Background: Despite increased rates of cannabis use among patients with cancer, there are gaps in our understanding of barriers to accessing cannabis. Social determinants of health (SDoH) are associated with access to healthcare, but few studies have evaluated how SDoH relate to cannabis access and use among cancer patients. Methods: We examined whether access to and modes of cannabis use associated with lower risk differed across SDoH among patients receiving treatment from a large National Cancer Institute (NCI) designated cancer center. This anonymous cross-sectional survey was developed in collaboration with the NCI Cannabis Supplement consortium. We evaluated the association of race (Black/African-American vs White), gender, income, and age with mode of cannabis use, source of obtaining cannabis (dispensary, friend/family, unlicensed dealer/seller, etc.), what influences their purchase (price, safety, etc.), and medical cannabis certification status. Results: Overall, 1,053 patients completed the survey and 352 (33.4%) reported using cannabis since their cancer diagnosis. Patients who identified as Black/African-American were less likely to be medically certified (p = 0.04). Males and Black/African-Americans were more likely to report smoking cannabis (vs other forms, ps<0.01) and to purchase cannabis from an unlicensed dealer/seller (p < 0.01). Lower-income patients were more likely to be influenced by price and ease of access (ps < 0.05). Although cannabis users were younger than non-users, age was not associated with any outcomes. Conclusions: The current data shed light on how critical drivers of health disparities (such as race, gender, and income) are associated with where patients with cancer obtain cannabis, what forms they use, and what influences their purchase decisions. More research is needed to understand the health outcomes associated with these differences in cannabis use and access-related factors such as the role of clinician-patient communication on certification, education around safe cannabis use, and the impact of statewide policies intended to increase access to cannabis. Keywords: Cannabis, Cancer, Social Determinants of Health Inequity Disclosure: Investigator-initiated grant from Novo Nordisk, Inc: Grant (Self). P710. Applying Behavioral Economics to Screen Medications for Alcohol Use Disorder Steven Nieto, James MacKillop, Alicia Izquierdo, Lara Ray University of California - Los Angeles, Los Angeles, California, United States Background: Maladaptive decision-making processes are a hallmark of alcohol use disorder (AUD). A behavioral economic approach to AUD synthesizes concepts and methods from psychology and microeconomics to understand cognitive processes that contribute to overconsumption of alcohol. The excessive delay discounting of temporally distant rewards and increased alcohol demand is characteristic of individuals with AUD and provide support for dysfunctional decision-making processes. The multiple-choice procedure (MCP) was developed to efficiently investigate the relationship between drug preferences and alternative reinforcers. Although behavioral economic paradigms are relevant to alcohol-related pathology, they have not been leveraged to assess medication signals in the human laboratory. Methods: A total of 40 men and women with current AUD and reporting intrinsic motivation to change their drinking, were randomly assigned to receive naltrexone (50 mg QD), varenicline (1 mg BID), or matched placebo for two-weeks. After a week-long medication titration period, participants began a 7-day practice quit attempt, during which they had daily virtual (online and phone) visits where they reported on their alcohol use. Participants completed an alcohol cue-reactivity paradigm and a behavioral economic battery following study drug administration on the final day of the practice quit attempt (Day 14). The battery included the MCP, wherein participants chose between a standard alcohol drink immediately or a monetary reinforcer in one week, followed by a state-based hypothetical purchase task to assess alcohol demand, and then a delay discounting task. A chi-square test was conducted to examine the relationship between medication group (placebo, varenicline, and naltrexone) and alcohol choice (alcohol now versus money later) on the MCP. One-way analysis of variance (ANOVA) tests were used to examine medication effects on alcohol demand and delay discounting rates. Results: A chi-square test revealed no relationship between medication group and alcohol choice via the MCP (χ2 = 0.13, p = 0.94). Unadjusted one-way ANOVAs showed that varenicline and naltrexone, relative to placebo, did not influence alcohol demand indices (i.e., intensity [F = 0.10, p = 0.91], Omax [F = 1.02, p = 0.37], and breakpoint [F = 1.15, p = 0.33]), or change delay discounting rates (overall ln[k]; F = 1.13, p = 0.32). Conclusions: Previous work demonstrates that varenicline and naltrexone have modest efficacy in reducing alcohol consumption. In this 2-week human laboratory trial, these medications did not influence behavioral economic indices suggesting that longer treatment periods may be necessary to influence decision-making processes that subserve AUD. Future statistical analyses will examine medication effects by considering the interplay between behavioral economics and alcohol cue-reactivity. Keywords: Behavioral Economics, Alcohol Use Disorder - Treatment, Medication Development Disclosure: Nothing to disclose. P711. Attention-Deficit/Hyperactivity Disorder and Self-Control Moderate Effects of the COMT Inhibitor Tolcapone on Alcohol Consumption and Control-Related Brain Activation Joseph Schacht, Konstanin Voronin, Michaela Hoffman, Raymond Anton University of Colorado School of Medicine, Aurora, Colorado, United States Background: Individuals with Alcohol Use Disorder (AUD) have higher levels of trait impulsivity than those without AUD (Sanchez-Roige et al., 2014), and are disproportionately likely to also have Attention-Deficit/Hyperactivity Disorder (ADHD) (van Emmerik-van Oortmerssen, 2012). A shared deficit in AUD, ADHD, and impulsivity is cognitive control. A key neurobiological influence on cognitive control is dopamine signaling in the prefrontal cortex (PFC), where catechol-O-methyltransferase (COMT) is the primary regulator of dopamine tone. The COMT inhibitor tolcapone, which potentiates elicited cortical dopamine release and improves performance on a variety of cognitive control tasks, could particularly benefit individuals with AUD and either ADHD or low self-control. Methods: This study was a secondary analysis of a recently published randomized controlled trial of tolcapone (Schacht et al., 2022) in which 90 non-treatment-seeking individuals with AUD (59% male, 88% of European-American descent, mean age = 26.5 [SD = 5.1], mean drinks per day = 7.0 [SD = 2.3]) were prospectively genotyped for the COMT rs4680 (val158met) single nucleotide polymorphism and randomized, based on their rs4680 genotype, to tolcapone (titrated to 200 mg t.i.d.) or placebo for 8 days. To assess cortically dependent cognitive control, participants completed a visual spatial working memory (SWM) task at baseline and after 7 days of study medication; data from this task have not previously been reported. ADHD diagnosis, as assessed by the World Health Organization Adult ADHD Self-Report Scale (WHO-ASRS), and Barratt Impulsiveness Scale (BIS-11) self-control subscale score were analyzed as moderators of the effect of tolcapone, relative to placebo, on the number of drinks consumed during the medication period. ADHD diagnosis was also analyzed as a moderator of tolcapone effects on cortical activation during the SWM task. Results: ADHD diagnosis significantly moderated the effect of medication group on drinks per day during the medication period (F(1, 80) = 6.42, p = 0.013), such that, among individuals with ADHD (n = 15), tolcapone, relative to placebo, reduced the number of drinks consumed per day (F(1, 80) = 5.09, p = 0.027; mean difference = 3.2 drinks per day, 95% CI = 0.4-6.0), while among those without ADHD, there was no significant difference between tolcapone and placebo. BIS-11 self-control subscale score also moderated the effect of medication group on drinks per day during the medication period at a trend level, such that tolcapone, relative to placebo, reduced drinking more among individuals with lower self-control (F(1, 80) = 3.77, p = 0.056). ADHD diagnosis also significantly moderated the effect of medication on cortical activation in the SWM task; between baseline and Day 7, task-related activation decreased, likely due to task habituation, but among individuals with ADHD, activation of the left dorsolateral PFC (DLPFC) was less reduced in the tolcapone group than the placebo group (z > 2.3, cluster-corrected p < 0.05). Greater DLPFC activation on Day 7 was associated with less drinking during the medication period at a trend level (r(77) = −0.21, p = 0.069), suggesting that sustained activation of this area during a task requiring cognitive control was associated with greater ability to resist drinking. Conclusions: These data suggest that tolcapone may be particularly effective in reducing drinking and increasing cortical function among individuals with AUD who also have ADHD and/or low levels of self-control. Among these individuals, tolcapone may “rescue” cognitive control by increasing cortical dopamine release when individuals make efforts to control their behavior. We are currently evaluating this hypothesis further in a sample of individuals with comorbid AUD and ADHD (R01AA026859). Keywords: ADHD, Impulsivity, Alcohol Use Disorder - Treatment, COMT Inhibitor, Cognitive Control Disclosure: Nothing to disclose. P712. Cannabis Challenge Effects on fMRI-Measured Brain Activity During Time Estimation Krishna Patel, Godfrey Pearlson, Catherine Boyle, Michael Stevens* Yale University School of Medicine, Hartford, Connecticut, United States Background: Cannabis is widely popular recreational drug in the US. The drug is known to alter the subjective experience of time. However, its effects on time estimation at a brain level are still largely unexplored. Our goal was to investigate acute effects of cannabis on an fMRI time estimation task by evaluating brain activation differences between cannabis and placebo conditions. We hypothesized that participants’ time estimation accuracy and corresponding BOLD response would be altered during the active cannabis condition, compared to placebo. Methods: In this placebo-controlled, double-blind, randomized trial, a total of N = 44 participants had 3 dose visits, at each of which they received either high-dose cannabis (0.5 gm of ~12.5% THC flower), low dose cannabis (0.5 gm of ~5.7% flower) or 0.5 gm placebo, using paced inhalation from a Volcano vaporizer via vaporizer. Drug material was supplied by NIDA/RTI. For the current study we analyzed fMRI data from the first placebo and high dose fMRI sessions throughout each dosing day, in which participants performed a time estimation task. Participants were asked to respond with a mouse click as to which box of two boxes displayed for different intervals was displayed on the screen longer. Both sub-second and supra-second temporal intervals were tested, with a range of easy to hard discriminations. Prior studies have indicated that these different intervals often differentially recruit cerebellar or basal ganglia regions into interval timing networks, respectively. Both were of interest in this study given the abundance of cannabinoid receptors in these regions. We used the Human Connectome Project processing pipeline to prepare fMRI data for GLM modeling of activation using the FSL FEAT toolbox. This model estimated the unique effect sub-second (short) and supra-second (long) interval discrimination, their average effect, and their difference. From these contrasts, the mean activation amplitudes within 387 brain parcels from the Human Connectome cortical atlas were extracted. Robust statistics in R software estimated a paired t test equivalent using the bootdpci function to assess the difference between placebo and the high dose drug conditions for each contrast. Results: Results for the short sub-second and long supra-second intervals were highly similar, so these were collapsed into a single condition for reporting purposes. Brain regions whose activity significantly differed between p < 0.05 and p < 0.001 for the average of short and long duration stimuli included premotor cortex, somatosensory and motor cortex, posterior cingulate cortex, visual area, medial temporal cortex, paracentral and midcingulate cortex, anterior cingulate and medial prefrontal cortex, inferior frontal cortex, tempo-parieto-occipital junction, premotor cortex, somatosensory motor cortex, posterior cingulate cortex, medial temporal cortex, orbital and polar frontal cortex, hippocampus. Only premotor cortex survived False Discovery Rate corrections for searching all 387 parcels across the entire brain for the average of short and long temporal estimation conditions. Next, we were interested whether cannabis might have a greater or lesser effect with different length intervals. In contrast to the main effect of cannabis on task activity, differences p < 0.05 were observed mostly in visual cortex, including parcels in the ventral stream visual cortex, dorsal stream visual cortex, and early visual cortex. Long versus short-interval brain activity also differed to cannabis in anterior cingulate and medial prefrontal cortex parcels (p < 0.05). Conclusions: The current study observed multiple brain activation differences for the initial post-dose, acute high-dose cannabis vs. placebo conditions. The strongest effect was seen for a premotor cortical region, which was the only parcel to survive multiple comparison correction for searching the whole brain. However, the other effects were noteworthy. A post hoc power analysis showed that adding 10 additional subjects to this sample would achieve significance with multiple comparison correction for medium effect sizes at alpha=0.05. This study identifies for the first time which brain regions engaged for mental time estimation are altered by recent cannabis use. Future studies that examine all doses and tasks would elucidate how the effects unfold longitudinally post-dose and determine if any effects are dose-dependent. Keywords: Cannabis, fMRI, Time Estimation Disclosure: Nothing to disclose. P713. COVID-19 Pandemic Impact on Alcohol Consumption and Pharmacodynamic Responses: Insights From Neurofunctional Domain Analysis Vijay Ramchandani, Bethany Stangl, Jeremy Luk, Melanie Schwandt, Tommy Gunawan, Shyamala Venkatesh, Courtney Vaughan, Rhianna Vergeer, Noa Leiter, Emma McCabe, Mikayla Bergwood, Andrew Waters, Paule Joseph, Reza Momenan, David Goldman, Nancy Diazgranados National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: The COVID-19 pandemic continues to impact individuals, communities, and the global economy. Social isolation, health anxiety, and financial stress can all impact alcohol-related outcomes among individuals across the spectrum of alcohol use disorder. Changes in alcohol use and related problems have been reported; however, there has been substantial heterogeneity in magnitude and directionality of these effects. A major determinant of changes in alcohol use is in the pharmacodynamic response to alcohol, which can be considered in the context of the neurofunctional domains of incentive salience or reward, negative emotionality or stress-reactivity, and executive function or loss of control over drinking. These domains are characterized in the Addictions Neurochemical Assessment (ANA) framework that is aligned with the 3-domain cycle of addiction. The objective of this study was to examine changes in alcohol use and consequences related to the COVID-19 pandemic in a sample of individuals with and without alcohol use disorder (AUD), and to examine these changes in relationship to alcohol response measures aligned with the ANA framework. Methods: Individuals across the spectrum of alcohol use and alcohol use disorder, who had previously participated in the NIAAA natural history study between 2015 and 2020, were contacted via phone for study participation. After consent, participants completed an initial survey to obtain a pre-pandemic baseline and to capture the initial impact of the pandemic. Participants completed subsequent surveys ranging from weekly to every six months for two years beginning June 2020. Assessments included demographics, the Alcohol Use Disorder Identification Test (AUDIT) to measure alcohol consumption and problems, and a COVID-19 stress scale. Incentive salience measures included the Self-Report of the Effects of Alcohol Scale (SRE), Anticipated-Biphasic Alcohol Effects Scale (A-BAES), and Penn Alcohol Craving Scale (PACS); negative emotionality measures included the Perceived Stress Scale (PSS) and the Positive Affect Negative Affect Scale (PANAS); and executive function measures included the Impaired Control Scale (ICS). Data were analyzed using general linear models covarying for history of AUD, sex, age, household income, enrollment phase, and time since participation in the natural history study. Results: The study sample included 391 participants (mean age = 44.8 years; 48.0% female). Data from the AUDIT scale indicated a wide variation in pandemic-related changes in alcohol use, with 29% of participants reporting increases, 33% reporting decreases, and 38% reporting no change in AUDIT-Consumption scores. Participants were categorized into three groups based on their AUDIT score change from pre-pandemic to during the pandemic: no change, decrease, or increase. Participants who had an increase in their AUDIT scores from pre-pandemic reported significantly higher scores on incentive salience (PACS, p < 0.001; A-BAES sedation, p < 0.001; and trending for SRE, p = 0.06), negative emotionality (PSS, p < 0.001, Negative Affect Scale, p < 0.001), and executive function (ICS failed attempts to control drinking, p < 0.005) than those that had a decrease or no change in AUDIT scores. Individuals with a History of AUD was significant in all models (all p’s<0.01). Household income was significant in negative emotionality and executive function models (all p’s <0.01). Age was negatively associated with negative emotionality, while males showed significantly higher scores on incentive salience measures. Finally, those enrolled in the early pandemic phase scored higher on negative emotionality. Conclusions: This study indicated significant shifts in alcohol consumption, both increases and decreases, associated with the pandemic, although a substantial proportion of individuals showed no change in alcohol consumption as well. An increase in AUDIT scores during the pandemic was associated with higher scores in measures across all three neurofunctional domains, compared to groups that showed a decrease or no change in their AUDIT scores. Individuals with a history of AUD showed significant main effects, highlighting potential biobehavioral correlates of vulnerability to problem drinking and addiction vulnerability. The neurofunctional domain lens may provide additional insights into the underlying pharmacodynamic mechanisms that impact alcohol responses and risk for AUD. Keywords: Pharmacodynamics, the COVID-19 Pandemic, Incentive Salience, Negative Emotionality, Executive Function Disclosure: Nothing to disclose. P714. Sex Dependent Effects of Cannabis: Comparison of Self-Administration and Positive Subjective Drug Effects Between Female and Male Volunteers Stephanie Lake, Margaret Haney, Ziva Cooper UCLA Center for Cannabis and Cannabinoids, Jane and TerrySemel Institute for Neuroscience and Human Behavior, Los Angeles, California, United States Background: In recent years, rates of cannabis use and cannabis use disorder (CUD) have increased significantly among females; epidemiological studies point to an accelerated progression from first cannabis use to development of CUD in females compared to males. Preclinical studies suggest possible biological sex-based differences in the reinforcing effects of cannabinoid receptor 1 (CB1) agonists such as delta-9-tetrahydrocannabinol (THC), the primary psychoactive component of the cannabis plant, which may provide insight into why females may be more vulnerable to developing CUD. However, these preclinical findings have yet to be translated to humans. This study sought to explore sex differences in cannabis self-administration and self-reported positive subjective drug ratings in healthy male and female volunteers. Methods: Data from two within-subject randomized controlled studies comparing the subjective and reinforcing effects of smoked active cannabis (~25 mg THC) versus placebo cannabis (0 mg THC) were pooled for this analysis. Healthy non-treatment seeking males (n = 55) and females (n = 13) were matched for current cannabis use and included in this analysis. During two outpatient laboratory sessions, participants smoked experimenter administered placebo (0% THC) or active (~5.5% THC) cannabis (one drug condition per session). Subjective mood and drug effects were measured via the Subjective Effect-Visual Analog Scale (SE-VAS) and Cannabis Rating Form (CRF) at several timepoints post-cannabis administration. In the afternoon, participants had an opportunity to self-administer up to 3 puffs (cost: $1/puff) of the cannabis type that was smoked earlier in the session. Using generalized linear mixed models, subjective mood and drug effects were examined as a function of cannabis strength (active versus placebo), sex (female versus male), and time, and self-administration was analyzed as a function of cannabis strength (active versus placebo) and sex (female versus male). Results: Active cannabis increased positive subjective ratings including ‘Liking’, ‘Take again’, and ‘Good Effect’ relative to placebo cannabis (p < 0.01), and females reported significantly higher ratings relative to males on these measures after active cannabis administration (p < 0.05). Placebo cannabis was self-administered by 12 (19.0%) males and 2 (15.4%) females (p = 0.89). Active cannabis was self-administered by proportionally more females than males (53.8% versus 36.3%), but a significant sex difference was not detected (interaction p = 0.18). Conclusions: Under active cannabis conditions, females reported higher ratings of positive subjective drug effects relative to males. Despite these differences, females were not significantly more likely to self-administer active cannabis. These findings suggest that there may be other sex- or gender-based factors that modify the relationship between heightened sensitivity to certain positive subjective drug ratings and reinforcing effects of cannabis. Given the small number of females in this secondary exploratory analysis, future research with a higher number of females is needed to test sex-based differences in cannabis’ acute subjective drug, mood, and reinforcing effects. Keywords: Cannabis, Cannabis Use Disorder, THC, Sex Differences Disclosure: Canopy Growth Corporation: Consultant (Self) P715. Baseline Affective Symptomatology Moderates Acute Subjective Effects of High Potency THC and CBD Cannabis Concentrates Renée Martin-Willett, Carillon Skrzynski, Hollis Karoly, Joshua Elmore, L. Cinnamon Bidwell University of Colorado, Boulder, Boulder, Colorado, United States Background: As cannabis legalization spreads throughout the US, products with high concentrations of ∆9-tetrahydrocannabinol (THC) become more commonly consumed. However, relatively little is known about the effects of these highly potent products and their relationship to baseline levels of affect, and the impacts of Cannabidiol (CBD) dominant concentrates on affect and intoxication are also not well understood. Thus, the current study took an observational approach to assess the relationship between baseline affective symptoms and use of high THC or high CBD cannabis concentrates as they relate to acute mood and intoxication responses to cannabis. Methods: Using a naturalistic at-home administration study design, participants were randomly assigned to ad libitum administration of either a THC-dominant (84.99% THC and THCa, <1% CBD) or CBD-dominant (74.7% CBD, 4.1% CBDa, 4.5% THC and THCa) concentrate product. 54 adults (48% female; Mean age 29.87 (9.52 SD)) were assessed at a baseline appointment and then five days later in a mobile pharmacology lab before, immediately, and one hour after ad libitum administration of their assigned concentrate. Models were run regressing each outcome on time, condition, baseline affective symptom measures, and their two and three-way interactions. Tests of simple effects/slopes were conducted for models with significant interactions. In the case of simple effects, levels of baseline affect were held at one standard deviation (SD) above (‘high’), below (‘low’), or at the mean (‘average’) of scores. Results: Participants at the follow-up appointment demonstrated cannabinoid levels in blood that were commensurate with their assigned concentrate group (THC dominant or CBD dominant). There was a significant two-way interaction between condition and baseline depression levels on acute changes in positive mood (F = 9.47, p < 0.005) such that higher levels of depression were significantly associated with higher levels of elation for the THC-dominant group but not for the CBD-dominant group. There was also a three-way interaction between condition, baseline anxiety, and time on acute changes in tension (F = 5.55, p < 0.01) such that tension decreased in the CBD-dominant group at average and high anxiety levels, but not low anxiety levels or among the THC-dominant group. Finally, there was a significant three-way interaction between condition, baseline anxiety levels, and time on acute intoxication (F = 3.72, p = 0.03). Specifically, THC users at all anxiety levels were more intoxicated than CBD users immediately post-use, but at one hour post-use, THC users were comparably intoxicated to CBD users at high anxiety levels but more intoxicated than CBD users with average and low anxiety. These data were drawn from a fully powered study of the effects of concentrate use on psychological, functional, and motor outcomes, but the current study was an exploratory moderation analysis based on post-hoc observations. Conclusions: This is one of the first studies to examine whether baseline affective symptoms moderate the acute effects of ad libitum use of high potency cannabis concentrates, including both THC- and CBD-dominant products. These exploratory analyses suggest an important relationship between a user’s baseline levels of anxiety or depression as they pertain to one’s subjective drug experience. As concentrate products continue to gain popularity on the legal market, these preliminary findings may be especially relevant towards informing future research on cannabis concentrate misuse or withdrawal. Keywords: Cannabis Use, Cannabis Concentrates, Affective Psychopathology Disclosure: Nothing to disclose. P716. Consuming Oral Cannabidiol (CBD) Prior to a Standard Alcohol Dose Prolongs Physiological and Subjective Effects of Alcohol Without Impacting Impairment, Affect or Craving: A Pilot Human Laboratory Study Hollis Karoly, Meggan Drennan, Mark Prince, Leila Zulic, Gregory Dooley, William DeJong, Michael Milburn Colorado State University, Fort Collins, Colorado, United States Background: Cannabidiol (CBD) is a cannabinoid commonly found in the cannabis plant. CBD thought to be safe, non-intoxicating and well-tolerated in humans. In recent years, CBD has garnered considerable attention as a potential treatment for numerous medical and psychiatric conditions, including substance use disorders. In particular, rodent studies have demonstrated that CBD appears to reduce alcohol consumption and decrease other markers of alcohol dependence in murine models of alcohol use disorder (AUD). The mechanism(s) through which CBD may serve to reduce alcohol intake is not well understood, and it is also presently unknown whether CBD decreases alcohol consumption and influences other AUD phenotypes in humans. Further, the existing human laboratory work exploring the acute effects of CBD and alcohol is limited and conflicting. The present placebo-controlled, crossover study aims to assess the effects of oral CBD on alcohol-induced psychomotor performance, self-reported affect, alcohol craving and subjective responses to alcohol in a sample of N = 36 heavy-drinking human participants. Methods: Heavy drinkers (males and females) who do not regularly use cannabis and were not CBD-naïve were recruited to participate in this 3-session pilot study. At each session, eligible individuals consumed either 30 mg CBD, 200 mg CBD, or placebo prior to drinking a standardized dose of alcohol designed to bring their blood alcohol content (BAC) to .06 g/dL. Participants were blind to which dose they received at each session. All three sessions were identical except for the CBD dose administered. Participants completed sessions in randomized order. At each session, following the CBD (or placebo) administration and alcohol consumption period, participants completed measures of psychomotor impairment, affect, alcohol craving, subjective responses to alcohol and had their breath alcohol content (BrAC) measured. Blood was drawn at the start of the experiment, at 25 minutes after CBD administration and once more 60 minutes after the alcohol administration period. Differences in these outcomes across the three CBD conditions and by sex were explored using multilevel structural equation models. Separate models were run for each of the following dependent variables of interest: BrAC, alcohol craving, low arousal positive affect, high arousal positive affect, alcohol-induced sedation, alcohol-induced stimulation and psychomotor impairment. Due to the research design, where each participant completed each condition (placebo, 30 mg CBD and 200 mg CBD) and within each condition there were 7 repeated measures of interest, MSEMs were run with a latent growth curve on the within-level to capture change over time. Condition was treated as a within-person variable and sex was treated as a between-person variable. To examine whether condition (30 mg CBD, 200 mg CBD, placebo) influenced changes over time, we regressed the latent slope parameter defined by the latent growth curve model on a within-person condition variable. To determine if sex moderated the condition on change relationship we created a random slope of condition predicting the latent slope variable and regressed that random slope on the between-person sex variable. This is referred to as the random coefficient prediction method. We also used this method to determine if the overall change over time was moderated by sex by regressing the random latent slope parameter on the between-person sex variable. We also examined CBD blood levels during each session before CBD ingestion, 25 minutes post-ingestion and 1-hour post-alcohol consumption to ensure that participants showed expected increases in blood-CBD following ingestion of each CBD dose. Results: Plots of CBD-blood during the experiment were consistent with the CBD doses administered, such that individuals showed increased blood-CBD over the course of the experiment, and CBD blood-levels were higher for the 200 mg CBD condition (average peak CBD = 96.39 ng/ml) than the 30 mg CBD condition (average peak CBD = 8.19 ng/ml). BrAC and high arousal positive affect were significantly reduced over the course of the descending limb of the BAC curve. All conditions showed significant differences in change in in BrAC over the descending limb of the BAC curve. Specifically, the placebo condition had the steepest negative BrAC slope, followed by 30 mg CBD, and 200 mg CBD had the flattest BrAC slope. A significant difference emerged between 200 mg CBD and placebo on changes in stimulation, with placebo having a steeper negative slope. There was a significant difference between 30 mg and placebo for sedation, with placebo having a steeper negative slope. No significant sex differences or moderating effects of sex emerged for any outcomes Conclusions: Results from this pilot study indicate that pre-treatment with oral CBD prior to consuming a dose of alcohol appears to extend the physiological effects of alcohol (i.e., BrAC) and prolong the subjective effects of alcohol (specifically, stimulation and sedation). Notably, in this study, CBD did not impact alcohol positive affect or craving, suggesting that craving-reduction is likely not a mechanism through which CBD serves to reduce alcohol intake. Although preliminary, these findings suggest that CBD should be further explored for its potential to aid individuals who wish to reduce their drinking, and that extending the physiological and subjective effects of alcohol may be one mechanism through which CBD could help to reduce alcohol consumption. Keywords: Cannabidiol, Alcohol, Subjective Effects Disclosure: Nothing to disclose. P717. Short-Term Anxiolytic and Harm-Reducing Effects of Cannabidiol in Cannabis Flower L. Cinnamon Bidwell, Renée Martin-Willett, Marco Ortiz Torres, Gregory Giordano, Jonathon Lisano, Carillon Skrzynski, Kent Hutchison, Angela Bryan University of Colorado, Boulder, Boulder, Colorado, United States Background: Cannabis is increasingly used to self-treat anxiety; however, data are mixed on anxiolytic effects. Additionally, the primary cannabinoids ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD) have varying pharmacological actions that may render different short-term effects on anxiety. Methods: These are the first data on the primary hypotheses from a fully powered NIDA-funded R01(DA044131; PI Bidwell; ClinicalTrials.gov Identifier: NCT03491384) that uses experimental methodologies to examine the impact of different cannabinoids in individuals with anxiety. Using an at-home administration procedure compliant with federal law, the present study examined the acute effects of three cannabis flower chemovars with different THC to CBD ratios to test whether chemovars with a higher CBD content produce different effects in anxious cannabis users. Participants who reported at least mild to moderate anxiety (Score of ≥ 5 on the Generalized Anxiety Disorder-7 (GAD-7) scale) and current use of (or intent to use) cannabis to cope with anxiety were recruited for the study [N = 201 adults (Male = 91, Female = 110); Mean cannabis use days past 14 days = 6.36 (SD = 5.24)]. Participants were randomly assigned to ad libitum administration of one of three chemovars (THC dominant: 24% THC, 1% CBD; THC + CBD: 9% THC, 10% CBD; CBD dominant: 1% THC, 23% CBD) and assessed in a mobile pharmacology lab before (pre-use) and immediately after (post-use) ad libitum administration of their assigned chemovar. A sex-matched group of non-users with mild to moderate anxiety (GAD-7 ≥ 5; n = 41) were recruited and evaluated over these same time points. Using a mixed model ANOVA design, with four groups, two assessment points (pre- and post-use, and an average ICC = 0.5 between assessment points, a total n = 240 (60 per group) allowed us to detect a groupXtime interaction effect as small as f = 0.12. Results: Plasma cannabinoids as well as subjective mood and intoxication effects were assessed at each time point. Participants who used the CBD-dominant and THC + CBD chemovars had less THC and more CBD in plasma after cannabis use compared to participants who used the THC-dominant chemovar. The CBD dominant chemovar was associated with acute reductions in anxiety and tension as compared to the THC dominant and the THC + CBD chemovars. In addition, the use of all three strains was associated with intoxication and positive subjective mood effects. Results pointed to graduated drug reward effects across strains, with the highest levels of intoxication and positive mood being present in the THC dominant chemovar, the next highest in the THC + CBD chemovar, and the lowest levels present in the CBD dominant chemovar. Similarly, adverse effects, including paranoia, demonstrated a graduated effect across strain groups, with the highest levels in the THC dominant chemovar, the THC + CBD chemovar falling in the middle, and the CBD dominant showing significantly lower levels than the other two strain groups. Non-users did not show significant change in either objective or subjective mood and intoxication effects between the study time points. Conclusions: In one of the first studies to examine the differential effects of ad libitum use of cannabinoids on measures relevant to individuals with anxiety who use cannabis, the CBD dominant chemovar was associated with short-term anxiolytic effects as compared to a THC + CBD and a THC dominant chemovar. Participants using the CBD dominant and THC + CBD chemovars also reported lower THC plasma levels and yet still some level of intoxication and positive drug effects, which is intriguing from a harm reduction perspective. Our study contributes novel data on the effects of various THC to CBD ratios using chemovars that are widely available in state-regulated markets, with an emphasis on the effects of CBD. Further research is needed to clarify the anxiolytic and harm reduction potential of CBD in cannabis products. Keywords: Cannabis, Delta9-Tetrahydrocannabinol, CBD, Anxiety, Generalized Anxiety Disorder Disclosure: Nothing to disclose. P718. THC Modulates Pain Sensitivity Among Persons Receiving Opioid Agonist Therapy for Opioid Use Disorder: A Within-Subject, Randomized, Placebo-Controlled Human Laboratory Study Joao De Aquino, Catherine Xie, Julia Meyerovich, Mohini Ranganathan, Peggy Compton, Brian Pittman, Mehmet Sofuoglu Yale University School of Medicine, West Haven, Connecticut, United States Background: The opioid and cannabinoid receptor systems are inextricably linked, overlapping at the anatomical, functional, and behavioral levels. Seminal preclinical studies have reported that cannabinoid agonists can induce opioid-sparing effects (i.e., a reduction in the effective dose of an opioid agonist). In the grip of the opioid crisis, a growing number of states has authorized the medicinal use of cannabinoids — in some cases, even replacing opioids — to treat two often co-occurring conditions: Pain and opioid use disorder (OUD). These rapid changes in public policy are in stark contrast with the lack of experimental data on the effects of cannabinoid agonists among persons with OUD — leaving clinicians, patients, and stakeholders without high-quality evidence to guide their decisions. To address this consequential knowledge gap, we have designed the first experimental human study to investigate the acute effects of the main constituent of cannabis, delta-9-tetrahydrocannabinol (THC), among persons with OUD receiving methadone — the most common opioid agonist therapy for OUD. Methods: We conducted a Phase I within-subject, crossover, human laboratory study including 27 persons with OUD who were receiving methadone therapy. Across three test sessions, each lasting for 5 hours, participants were randomly assigned to receive single doses of oral THC (10 mg or 20 mg), administered as dronabinol; or placebo. Before each test session, abstinence from non-medical substance use was confirmed by a urine laboratory immunoassay. Pain sensitivity in response to THC administration was measured by the Cold Pressor Test (CPT) at 4 °C and by the McGill Pain Questionnaire (MPQ). The abuse liability of THC was measured by the Drug Effects Questionnaire (DEQ). Cognitive performance was indexed by the Hopkins Verbal Learning Test (HVLT). We used mixed-effects models to examine the main effects of THC dose, and interactions between THC (10 mg, 20 mg) and methadone doses (low-dose methadone defined as < 90 mg/day; and high-dose methadone defined as ≥ 90 mg/day). At a significance level of 0.05, this exploratory study had 80% power to detect medium effect sizes (d ≥ 0.56). Results: Participants were aged 47.3 ± 12.2 years old. Approximately 76% of participants self-identified as male, and 24% self-identified as female. The average methadone dose was 96.6 ± 35.1 mg/day. Approximately 48% of participants were receiving low-dose methadone (62 ± 22.7 mg/day), and 52% were receiving high-dose methadone (118 ± 19.7 mg/day). Preliminary results show a main effect of THC dose on pain sensitivity, indexed by the MPQ total pain score (F(2,104)=5.33, p = 0.006): Pain sensitivity was lower under 10 mg THC (d = 0.65) and 20 mg THC (d = 0.39) than under placebo. Post-hoc analyses indicated that the lower MPQ total pain scores under THC were due to a reduction of sensory pain (F(2,104)=5.93, p = 0.003), rather than of affective pain. Although there were no main effects of THC dose, nor THC by methadone dose interactions for CPT outcomes, 20 mg THC was associated with higher CPT pain tolerance among persons receiving low-dose methadone (d = 1.01) than among persons receiving high-dose methadone. Further, we observed a main effect of THC dose for abuse liability (F(2,463)=4.05, p = 0.02), as 20 mg THC produced greater DEQ stimulatory effects (d = 0.57) than placebo. Lastly, we found a main effect of THC dose for cognitive performance (F(2,39)=3.72, p = 0.03): Compared to placebo, 20 mg THC reduced HVLT delayed recall (d = 0.47). No interactions between THC and methadone doses were observed for abuse liability and cognitive outcomes. Conclusions: The study had several notable findings. First, likely due to cross-tolerance between opioid and cannabinoid agonists for analgesia, relatively high doses of THC may be required to produce acute analgesic effects among persons receiving therapeutic doses of methadone. Yet, persons receiving lower doses of methadone may be more likely to derive analgesic effects from THC. Second, our findings support the notion that, in this population, THC modulation of acute pain is mediated primarily by sensory nociception, rather by affective pain processes. Third, the analgesic effects of THC among persons with OUD receiving methadone therapy may occur at the expense of abuse liability and cognitive deficits — similar to analgesia, the cross-tolerance between opioid and cannabinoid agonists for reinforcing and cognitive effects is incomplete. Collectively, these data have key implications for future studies investigating opioid-sparing effects of cannabinoids among persons receiving opioid agonist therapy for OUD. It is critical to examine the risk/benefit ratio of sparing opioids by administering cannabinoids when opioid agonist doses are lower in OUD treatment — during initiation and taper. Future studies are warranted to ascertain whether, during these vulnerable periods in OUD pharmacotherapy, the trade-off between therapeutic and adverse effects of cannabinoids can be optimized to reduce the harm from opioids, including overdose deaths. Keywords: Cannabis, Opioid Agonist Treatment, Analgesia, Mu-Opioid Receptor Agonist, Cannabinoid Receptor Disclosure: Nothing to disclose. P719. The Trajectory of Cannabis Withdrawal in Cannabis and Tobacco Co-Users: A Preliminary Investigation Je Sern Yeap, Isabela Lara Uquillas, Tony George, Romina Mizrahi, Rachel Rabin* McGill University, Verdun, Canada Background: The cannabis withdrawal syndrome, consisting primarily of mood and behavioral symptoms, is well-established in people with frequent cannabis use (CU) and follows a distinct trajectory. Withdrawal symptoms begin 24 hours after cessation, peak within 7 days, and dissipate after 28 days of abstinence. In animal models, reduced endocannabinoid activity, indexed by low levels of endocannabinoids (anandamide and 2-arachidonoylglycerol, 2-AG), has been linked to the presence and severity of cannabis withdrawal-like symptoms. However, the neurobiological substrates underlying cannabis withdrawal in humans have not yet been identified. Tobacco co-use is highly prevalent among people with CU. Notably, relative to cannabis-only users, co-users have increased difficulty quitting cannabis demonstrated by their higher and accelerated relapse rates. This may reflect that greater proportions of co-users, compared to cannabis-only users, experience cannabis withdrawal and experience it at greater severity levels. Our previous work has shown that when individuals with CU were parsed according to tobacco dependence severity (rather than the presence/absence of tobacco use), associations with cannabis withdrawal severity were even more robust. Notably, the few studies assessing the relationship between tobacco co-use and cannabis withdrawal have been cross-sectional. Thus, it is unknown whether among co-users cannabis withdrawal severity is transiently elevated or remains persistently elevated for the duration of the withdrawal syndrome (i.e., 28 days). Aim 1: To examine if cannabis withdrawal is elevated and remains persistently elevated in severity in people with CU and high tobacco dependence following 28 days of cannabis abstinence relative to individuals with CU and low tobacco dependence. Aim 2: To test associations between endocannabinoid levels and cannabis withdrawal severity and tobacco use following overnight abstinence. Methods: Men with CU (N = 20) were parsed according to their tobacco dependence severity using the Fagerstrom Test for Nicotine Dependence (FTND), which is optimal for classifying low (FTND ≤ 4) and high tobacco dependence (FTND ≥ 5). Participants underwent 28 days of cannabis abstinence facilitated by contingency management and weekly supportive therapy. Abstinence was biochemically-verified with twice weekly urine analysis and self-reported cannabis use. Cannabis withdrawal severity was assessed weekly using the Marijuana Withdrawal Checklist (MWC). Further, in an independent sample of men with CU (N = 8), following overnight cannabis abstinence, we evaluated relationships between serum-derived anandamide and 2-AG levels and cannabis withdrawal severity and tobacco consumption. Results: The majority of participants (n = 14) achieved biochemically-verified cannabis abstinence, five participants significantly reduced their cannabis use by >70%. Among these 19 individuals, n = 12 were classified with low tobacco dependence and n = 7 were classified with high tobacco dependence. Controlling for age, between-group differences revealed a significant difference in baseline withdrawal severity (p < 0.01) that persisted across all 28 days of abstinence (Group x Time interaction effect, F(4,64) =4.40, p < 0.01, η2 = 0.22). Correlations between cannabis withdrawal severity and endocannabinoid levels revealed negative associations between cannabis craving and anandamide (r = −0.78; p = 0.02) and 2-AG (r = −0.84; p = 0.01). Moreover, we found that tobacco consumption (i.e., cigarettes per day) negatively correlated with anandamide levels (r = −0.71; p < 0.05). Conclusions: Our preliminary data showed that following 28 days of cannabis abstinence, people with CU with high tobacco dependence exhibited significantly elevated cannabis withdrawal symptoms which remained persistently elevated compared to people with CU and low tobacco dependence. The unique withdrawal trajectory observed in individuals with CU and high tobacco dependence is of great clinical importance given that longer episodes of elevated withdrawal severity would prolong the time that these individuals remain at high risk for cannabis relapse, hindering long-term recovery. Reduced endocannabinoid levels may underlie increased cannabis withdrawal severity in people with CU, and tobacco co-use may further decrease endocannabinoid levels in a dose-dependent manner. Thus, greater tobacco consumption in individuals with CU may lead to more severe withdrawal symptoms. Our findings have important treatment implications for developing interventions for cannabis-tobacco co-users. Keywords: Cannabis Use Disorder, Tobacco, Withdrawal, Abstinence, Endocannabinoids Disclosure: Nothing to disclose. P720. Influence of Suvorexant Maintenance on Commodity Demand Following Cocaine Challenge: A Cross-Over, Double-Blind Comparison Justin Strickland, Kevin Hatton, Lon Hays, Abner Rayapati, Joshua Lile, Craig Rush, William Stoops Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: A promising target for the regulation of drug use motivation is the orexin system. Preclinical evidence indicates a connection between drug-regulated behaviors and orexin producing cells in the lateral hypothalamus and brain limbic areas (e.g., the nucleus accumbens and ventral tegmental area). Existing in vivo evidence from preclinical rodent models shows that antagonism of the orexin system attenuates the behavioral effects of, and motivation for, varied drug classes. A broadly consistent preclinical finding is this orexin antagonism or knockdown, especially of the orexin1 receptor, increases demand elasticity (i.e., indicating lower drug use motivation) while leaving consumption at unconstrained price (i.e., demand intensity) unchanged. Suvorexant is a dual orexin1 and orexin2 receptor antagonist that is clinically approved and of interest for clinical translation. This analysis focused on drug demand data from a within-subject human laboratory study that determined the influence of suvorexant maintenance on cocaine pharmacodynamics. Methods: Eight (4 women; 2 White, 5 Black, 1 Multiethnic) non-treatment seeking participants with cocaine use disorder were enrolled in a cross-over, double-blind, randomized residential study (one participant with partial data due to the COVID-19 pandemic). Participants were maintained on 0, 5, 10 and 20 mg oral suvorexant/day in randomized order with experimental sessions completed after at least three days of maintenance on each target suvorexant dose. Experimental sessions included administration of a sample dose of 0, 10, and 30 mg/70 kg of intravenous cocaine. Analyses focused on standardized behavioral economic demand tasks completed 15 minutes after the cocaine sampling dose. Commodities in these tasks included the sampled cocaine dose as well as chocolate, cigarettes, and alcohol. The dependent measures were observed demand values (i.e., intensity, Pmax, Omax, and breakpoint). Mixed-effect models evaluated demand outcomes with Suvorexant Dose (0, 5, 10 and 20 mg/day) and Cocaine Dose (0, 10 and 30 mg/70 kg) as within-subject factors. Results: A main effect of Cocaine Dose was observed for all cocaine demand measures (p values < 0.001) reflecting a dose-related increase in demand for the blinded cocaine dose. A main effect of Suvorexant Dose was observed for cocaine Pmax (p = 0.035) reflecting an increase in cocaine demand (higher Pmax) for the 10 mg cocaine dose when participants were maintained on 10 mg suvorexant (p = 0.009) and 20 mg suvorexant (p = 0.039) relative to placebo. Cocaine challenge did not alter demand for alcohol, but a main effect of Suvorexant Dose was observed for alcohol Pmax (p = 0.021) and Omax (p = 0.022) reflecting increases in alcohol demand when participants were maintained on the 10 mg suvorexant dose regardless of sampled cocaine dose that session. Cocaine challenge did not alter demand for cigarettes, or chocolate (p values > 0.107) nor were effects of suvorexant maintenance observed for cigarette or chocolate demand. Exploratory analyses indicated no differential effects based on participant gender. Conclusions: Suvorexant maintenance increased demand for a low dose of cocaine, as well as for alcohol, in a dose-related manner. These findings contrast with preclinical findings, which may be explained by differences in acute versus subchronic dosing or the lack of orexin receptor subtype selectivity of suvorexant. These data also support the validity of demand procedures for measured blinded drug demand by showing an expected dose-related increase in demand for blinded doses of cocaine and low responding for placebo. Future research should test more selective orexin antagonists in combination with cocaine and other substances to determine dual versus single orexin receptor targeting effects in clinical populations. Keywords: Dual Orexin Receptor Antagonist, Behavioral Economics, Cocaine, Addiction, Alcohol Disclosure: Nothing to disclose. P721. Voluntary Adolescent Binge Alcohol Exposure Enhances the Ability of Cocaine Microinjected Into the Poster VTA to Stimulate Dopamine Release in the Nucleus Accumbens Shell Zachary Rodd, Sheketha Hauser, R. Aaron Waiess, Eric Engleman, Richard Bell Indiana University School of Medicine, Indianapolis, Indiana, United States Background: Most adolescents will consume alcohol. The biological consequences of the normal experimentation with alcohol and drugs of abuse during adolescence is enhanced by binge alcohol consumption. Adolescent Binge Alcohol Exposure (ABAE; alcohol BEC levels > 80 mg%) produces persistent alterations in the adult brain that increases the likelihood of adult alcohol consumption and self-administration of other drugs of abuse. Previous research has indicated that when cocaine is microinjected into the posterior VTA, there is an increase in extracellular levels of dopamine (DA) within the Nucleus Accumbens Shell (AcbSh). The current experiment examined the effects of ABAE on the ability of cocaine microinjected into the posterior VTA to stimulate DA release in the AcbSh. Methods: Alcohol preferring (P) rats were given 24-hr free-choice access to water, 15%, and 30% EtOH from post-natal day (PND) 28-48. The control group (CON) was only given access to water during this time. From PND 48-89, rats were paired housed in standard shoebox cages. On PND 90, rats were surgically implanted with a guide cannula aimed at the posterior VTA and AcbSh (ipsilateral). All animals were handled and monitored for health. On PND 98, all rats had a microdialysis probed inserted into the AcbSh the night before the microinjection-microdialysis experiment. ABAE and CON were microinjected with 0 (aCSF), 25, 100, or 250 pmol cocaine HCL into the posterior VTA (30 microinjections over a 5 min period; 5 sec pulse microinjection – 15 sec timeout). Dialysis samples from the AcbSh were collected every 20 minutes. Sampling occurred 2 hours before cocaine microinjections and 4 hours after. Results: Adolescent P rats consumed more than 7 g/kg/day EtOH, which has been shown to produce BECs > 80 mg%. In CON rats, 100 and 250 pmol cocaine microinjected into the posterior VTA increased DA in the AcbSh (50 and 82%) for 40 or 60 minutes. ABAE P rats microinjected with 25, 100 or 250 pmol cocaine into the posterior VTA increased DA in the AcbSh (126%, 192%, and 164%, respectively) in the AcbSh for 80 or 100 minutes. Conclusions: The data demonstrate that ABAE produced neuroadaptations within the posterior VTA that increased the sensitivity to, and responsiveness to, cocaine. The cross-sensitization to other drugs of abuse produced by ABAE indicates that ABAE produces neuroadaptations that mediate a common system that is activated by drugs of abuse. Future research must determine the biological neuroadaptations produced by ABAE that mediate these effects. Keywords: Adolescence, Adolescent Alcohol, Cocaine Sensitivity, Dopamine, Microdialysis Disclosure: Nothing to disclose. P722. Novel Positive Allosteric Modulators Augment 5-HT2A Receptor Functionality Christina Merritt, Andrew Bolinger, Noelle Anastasio, Jia Zhou, Kathryn Cunningham Center for Addiction Research, University of Texas Medical Branch at Galveston, Galveston, Texas, United States Background: The serotonin 5-HT2A receptor (5-HT2AR) is a modulator of cortical circuitry and serotonergic psychedelics (e.g., psilocybin) are inviting attention based upon the postulation that 5-HT2AR agonists may “liberate” cortical dysfunction in disease states. The selective chemotype targeting of the 5-HT2AR is challenging given the similarity of the orthosteric sites across the 5-HT2R family. Targeting 5-HT2AR allosteric site(s), which differ from the orthosteric site for endogenous 5-HT, creates new opportunities to optimize 5-HT2AR signaling in disorders marked by cortical dysfunction. We have crystalized a chemical biology strategy to potentiate 5-HT2AR functional responses with positive allosteric modulators (PAMs). Herein, we report the first-generation series of 5-HT2AR PAMs, unlocking an entirely new and innovative line of investigation. Methods: Newly designed and synthesized molecules were screened for efficacy to enhance 5-HT-induced intracellular calcium (Cai2+) efflux in stable Chinese hamster ovary (CHO) cell lines expressing the human (h) 5-HT2AR relative to h5-HT2BR or h5-HT2CR cell lines. Selected 5-HT2AR PAMs (CTW0404, CTW0419) were evaluated in a series of in vitro adsorption, distribution, metabolism and excretion (ADME) and secondary competition binding. Drug metabolism and pharmacokinetics (DMPK), and blood-brain barrier penetration analyses were conducted in male Sprague-Dawley rats. In rats trained to discriminate the 5-HT2AR agonist 2,5-dimethoxy-4-iodoamphetamine [(-)-DOI] from saline in a two-lever, water-reinforced task, we assessed the PAMs in substitution and potentiation tests. Rats were also monitored for the head twitch response following treatment with 5-HT2AR PAMs as a proxy for hallucinogenic potential. In silico molecular docking with receptor X-ray crystal structures was used to identify potential PAM binding sites. Results: CTW0404 and CTW0419 failed to exhibit intrinsic activity in the three cell lines, but did potentiate 5-HT-evoked Cai2+ in the 5-HT2AR-, but not the 5-HT2BR or 5-HT2CR, cells. In vitro ADME analyses demonstrated that CTW0404 and CTW0419 possess drug-like qualities, including long half-lifes (T1/2) and limited cytochrome P450 (CYP450) enzyme inhibition. CTW0419, but not CTW0404, is predicted to be a p-glycoprotein substrate. In vivo DMPK studies detected both compounds in rat brain with a greater brain to plasma ratio for CTW0404, relative to CTW0419. Neither CTW0404 nor CTW0419 substituted for the discriminative stimulus of (-)-DOI nor did these compounds evoke head twitches. In silico molecular docking has identified potential binding site to less conserved sites, rather than the orthosteric binding pocket. Conclusions: We have designed, synthesized, and evaluated a novel series of 5-HT2AR PAMs with promising profiles in vitro and in vivo. Our ongoing studies are focused on providing the basis for choosing new molecular entities with optimized profiles, necessary for medication candidate selection and preclinical development. Keywords: 5-HT2A Receptor, Positive Allosteric Modulators, Drug Discovery Disclosure: Nothing to disclose. P723. Region-Specific Effects of Psilocin on Neuronal Activity in Male and Female Rodents Melissa Herman, Gavin Schmitz, Devin Effinger University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States Background: The psychedelic compound psilocybin is currently characterized as a drug of abuse and is listed as a Schedule I controlled substance by the United States Drug Enforcement Agency. Recent work suggests that psilocybin, or it’s active metabolite psilocin, may have therapeutic potential in the treatment of psychiatric conditions like anxiety, depression, and substance use disorders. Despite the clinical potential of drugs like psilocybin as novel therapeutics, the underlying cellular actions and brain region-specific effects of these drugs remain poorly understood. Methods: Electrophysiological recording in prefrontal cortex (PFC) and central amygdala (CeA) neurons were performed in male and female C57BL6/J mice (Jackson) and or 5-HT2AR-eGFP-CreERT2 mice (provided by Bryan Roth). Fiber photometry recordings using genetically-encoded calcium sensors (jGCaMP7f) injected into the CeA were performed in male and female Sprague Dawley rats. Paired or unpaired t-tests will be used to compare means for single-factor analyses. Two-way ANOVAs (with sex and drug exposure as between-subjects factors) were used to compare differences among conditions and groups. Repeated measures ANOVAs were used where appropriate. Results: Focal application of psilocin (10 uM) produced variable changes in firing in PFC neurons from C57Bl6/J mice but consistent increases in firing were observed in 5-HT2A PFC neurons from 5-HT2AR-eGFP-CreERT2 mice. No sex differences in the effects of psilocin on PFC firing were observed. In contrast, focal psilocin application produced consistent decreases in firing in non-specified and 5HT2A CeA neurons from C57Bl6/J and 5-HT2AR-eGFP-CreERT2 mice, respectively. Psilocin effects on firing in both the PFC and CeA were mediated by the 5HT2A receptor. Calcium dynamics in the CeA of male and female rats produced consistent increases in time-locked CeA reactivity to an aversive air puff stimulus. Administration of psilocin (2 mg/kg, sc) increased stimulus-locked CeA reactivity in female but not male rats as compared to vehicle controls. Male rats displayed consistent reductions in stimulus-locked CeA reactivity that persisted for 28 days. Sex differences in stimulus-locked behavioral responses (active and passive) and effects of psilocin on stimulus-locked behavioral response were also observed. Conclusions: Collectively, these findings provide important brain region- and context-specific effects of the psychedelic compound psilocin, providing an improved mechanistic understanding of the neurobiology of these compounds which is essential for therapeutic consideration. Keywords: Amygdala, Medial Prefrontal Cortex, Psychedelics, Psilocybin, Psilocin Disclosure: Nothing to disclose. P724. (S)-Ketamine Reinforcement and Abuse Liability in Rats is Mediated by Mu Opioid Receptor Activation Marjorie Levinstein, Meghan Carlton, Tommaso Di Ianni, Emilya Ventriglia, Arianna Rizzo, Juan Gomez, Reece Budinich, Yavin Shaham, Raag Airan, Carlos Zarate, Jordi Bonaventura, Mike Michaelides National Institute on Drug Abuse, Baltimore, Maryland, United States Background: (S)-ketamine is an enantiomer of (R,S)-ketamine and an FDA-approved medication for treatment-resistant depression and major depressive disorder with acute suicidal ideation or behavior. (S)-ketamine is regarded as a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, but it also binds to and activates the mu opioid receptor (MOR) in vitro. However, the contribution of MORs to (S)-ketamine’s in vivo pharmacological actions and intravenous self-administration (IVSA), a method used to screen drugs for potential abuse liability, is not well understood. Methods: We implanted adult rats (n = 8) with jugular vein catheters. Rats underwent (S)-ketamine IVSA (0.5 mg/kg/infusion) and their IVSA dose-response curve was determined. Rats were then exposed to additional IVSA sessions at the unit dose (0.25 mg/kg/infusion, corresponding to the peak of the ascending limb of the dose-response curve) that were preceded by either saline (vehicle), naltrexone (3 mg/kg, SC), or the NMDA antagonist MK-801 (0.1 mg/kg, SC) pretreatment 15 min prior to IVSA. Functional ultrasound imaging (fUSI), a neuroimaging modality used to measure brain-wide changes in cerebral blood volume (CBV) was then performed. Rats (n = 10) were pretreated with saline or naltrexone (3 mg/kg, SC) followed by a single IV injection of saline or 0.5 mg/kg (S)-ketamine 15 min later while imaging the mPFC and NAc. We used positron emission tomography (PET) and [18 F]fluoroethyl-diprenorphine (FE-DPN) to examine in vivo MOR occupancy by a reinforcing dose of IV (S)-ketamine. Rats (n = 12) were anesthetized and then injected IV with a bolus of [18 F]FE-DPN (~650 µCi) followed immediately by PET imaging acquisition while receiving an IV infusion of saline (n = 6) or (S)-ketamine (n = 6, 20 mg/kg/h). Next, we exposed these rats to 8 daily sessions of IV saline or (S)-ketamine infusions (20 mg/kg/h, 60 min) and scanned them on day 9 using PET and [18 F]FE-DPN. To confirm that the BPND changes were due to lower MOR density, the rats were euthanized 24 h after PET and MOR density was assessed using [3H]DAMGO. Adjacent brain sections were exposed to DAMGO- (10 µM) or (S)-ketamine-stimulated (10 µM) [35 S]GTPγS autoradiography. Kappa opioid receptor (KOR) and NMDA receptor densities were assessed using [3H]U69,593 and [3H]MK801. Rats were then exposed to 8 daily IV saline (n = 9) or (S)-ketamine (n = 10) (20 mg/kg/h, 60 min) infusions. On day 9, rats started daily 3-h training sessions for heroin IVSA (5 days at 100 µg/kg/infusion, 5 days at 50 µg/kg/infusion). After training, we performed a dose-response assessment of heroin IVSA. Results: Rats acquired (S)-ketamine IVSA and showed an inverted U-shaped dose-response function with highest responding to a 0.25 mg/kg/infusion unit dose. Naltrexone (t(7)=6.47; P = 0.03) and MK-801 (t(7)=6.65; P = 0.03) significantly decreased (S)-ketamine infusions at this dose. Naltrexone pretreatment significantly decreased (S)-ketamine-induced CBV increases in the nucleus accumbens (NAc) core (corrected P = 0.03) and shell (corrected P = 0.036). Compared to saline, acute (S)-ketamine significantly decreased [18 F]FE-DPN BPND across several brain regions, indicating that reinforcing doses of (S)-ketamine occupy MOR in these regions in vivo. Repeated IV (S)-ketamine led to significant decreases in [18 F]FE-DPN BPND in NAc (P = 0.018), thalamus (P < 0.0001), superior colliculus (P = 0.001), ventral midbrain (P = 0.0042), medulla (P = 0.0032) and periaqueductal gray (P < 0.0001). Consistent with PET, (S)-ketamine exposure significantly decreased [3H]DAMGO binding in mPFC (t(8) = 2.681, P = 0.028), NAc (t(8) = 2.45, P = 0.04), and thalamus (t(8) = 2.4, P = 0.043) compared to control. DAMGO increased [35 S]GTPγS binding in both saline and (S)-ketamine-infused rats but its effect on [35 S]GTPγS binding in (S)-ketamine-infused rats was significantly lower compared to saline-infused rats in NAc (t(8) = 4.066, P = 0.0036). 10 µM (S)-ketamine increased [35 S]GTPγS binding in saline-treated rats in mPFC (t(3) = 6.05, P = 0.009), CPu (t(3) = 4.971, P = 0.016) and NAc (t(3) = 3.4, P = 0.042). (S)-ketamine did not significantly increase [35 S]GTPγS binding in rats exposed to repeated (S)-ketamine infusions. (S)-ketamine did not affect KOR or NMDA receptor density. Repeated IV (S)-ketamine significantly increased subsequent heroin IVSA and intake. Both saline and (S)-ketamine-infused rats acquired heroin IVSA, but rats infused with repeated (S)-ketamine had significantly more heroin infusions compared to saline-infused rats (2-way RM ANOVA: session x group interaction: F(9, 153) = 2.96; P = 0.002). Heroin infusions for saline- and (S)-ketamine-infused rats followed the typical inverted U-shaped function, but (S)-ketamine-infused rats had significantly greater infusions than saline-infused rats, specifically at the peak (6.25 and 12.5 µg/kg) unit doses (Mixed-effects ANOVA: dose x group interaction: F(6,97) = 2.98; P = 0.01) indicating that repeated exposure to reinforcing doses of (S)-ketamine increased subsequent heroin IVSA. Conclusions: Our findings indicate that (S)-ketamine, at reinforcing, self-administered doses, rapidly binds to and activates MORs in the mPFC, NAc and other brain regions in vivo. Upon repeated exposure, (S)-ketamine induced MOR desensitization and increased heroin intake, likely due to tolerance. Our results suggest that MOR activation contributes to (S)-ketamine self-administration, and possibly its abuse liability. Keywords: Esketamine, Mu-Opioid Receptors, Heroin Self-Administration Disclosure: Nothing to disclose. P725. Fos-Expressing Neuronal Ensembles in Rat Nucleus Accumbens Encode Initial Cocaine Seeking in Rats Bo Sortman, Samantha Rakela, Christina Gobin, Brandon Warren University of Florida, Gainesville, Florida, United States Background: Synchronized activity of neuronal ensembles within the (NAc) has been shown to drive cocaine seeking in well-trained rats. However, it is unclear how rapidly this circuitry is recruited to control cocaine-seeking behavior. Here, we tested the necessity of NAc neuronal ensembles in initial cocaine seeking behavior using the Daun02 inactivation procedure. Methods: We trained male and female transgenic Fos-LacZ rats to self-administer cocaine for 3 hr daily sessions until rats met acquisition criteria (>30 active lever presses, >75% responding on active lever). Upon meeting acquisition criteria, we subjected rats to an additional 30 min cocaine self-administration session to reactivate neuronal ensembles associated with cocaine-seeking behavior. Ninety min later, we infused Daun02 to selectively inactivate NAc Fos expressing ensembles activated by cocaine self-administration. We tested the rats’ cocaine seeking behavior 2 days later in a 30 min non-reinforced recall test. Results: Rats readily learned to self-administer cocaine and all rats reached acquisition criteria within 9 days. The average number of sessions to reach acquisition criteria was 4.1 sessions. There were no differences between groups that would go on to receive vehicle or Daun02 on induction day. We found that inactivation of NAc neuronal ensembles with Daun02 reduced cocaine-seeking after initial cocaine self-administration on test day (t32 = 2.2, p = 0.03). Daun02 infusions decreased Fos-expression after the test, indicating ablation of Fos-expressing neuronal ensembles by Daun02. Conclusions: These results suggest that NAc neuronal ensembles are formed during initial learning of cocaine self-administration and required for initial cocaine-seeking behavior. Keywords: Stimulants, Cocaine Self-Administration, Memory and Learning Disclosure: Nothing to disclose. P726. Differential Sensitivity to Punishment by Signaled Versus Unsignaled Footshock in Male and Female Rats Self-Administering MDPV Michelle Doyle, Lindsey Peng, Christina George, Kenner Rice, Gregory Collins* University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States Background: When allowed to self-administer 3,4-methylenedioxypyrovalerone (MDPV), or structurally related synthetic cathinones, approximately 30% of rats (“high-responders”) develop unusually high levels of dysregulated drug-taking similar to the binge-like patterns of cathinone (e.g., “bath salts”) use that has been reported in humans. The goals of the current studies were threefold: 1) to assess whether high-responder rats were less sensitive to punishment of MDPV-maintained responding than low-responder rats; 2) to evaluate if the punishing effects of footshock were altered when a stimulus signaling an impending footshock was introduced; and 3) to determine if these effects varied as a function of sex or the unit-dose of MDPV available for self-administration. Methods: Adult male (n = 20) and female (n = 20) Sprague-Dawley rats were surgically prepared with an indwelling venous catheter and trained to self-administer MDPV (0.032 mg/kg/inf) under an FR1:TO5-sec schedule of reinforcement during daily 90-min sessions. Once responding stabilized under an FR5:TO5-sec schedule of reinforcement, rats were categorized as high- or low-responders based on the proportion of active lever responses that occurred during the post-infusion timeouts (≥20% = high responder, <20% = low responder). Inhibition functions were first generated for unsignaled footshock (0.05-0.9 mA; paired with ~50% of infusions, and increasing intensity across sessions) in rats self-administering 0.01, 0.032, and 0.1 mg/kg/infusion MDPV. Subsequently, rats were then trained on a signaled footshock procedure, in which a stimulus (flashing light) was introduced after two responses to signal to the rat that completion of the FR5 would result in an infusion paired with a footshock (intensities individualized to each rat’s IC50); footshocks could be avoided by withholding responding for 30 sec. After rats acquired the signaled shock procedure, footshock intensities were systematically varied (0.05-0.9 mA; increasing across sessions) to generate inhibition functions for signaled footshock in rats self-administering 0.01, 0.032, and 0.1 mg/kg/infusion MDPV. Results: When footshocks were unsignaled, sensitivities to the punishing effects of footshock were not different between high- and low-responder rats (mean IC50s- high-responders: 0.30 mA; low-responders: 0.36 mA); however, high-responder rats received significantly more total current than low-responder rats (mean: 45 mA vs 18 mA, respectively). When footshocks were unsignaled, high-responder rats were less good at withholding responding during signaled trials and avoided fewer footshocks than low-responder rats. Compared to low-responder rats, high-responder rats responded more in the presence of the signal indicating impending shock and avoided a smaller proportion of shocks. Under both signaled and unsignaled conditions, the effectiveness of footshocks to punish responding was inversely correlated with the unit-dose of MDPV available for infusion. Though the pattern of effects did not differ, females were more sensitive to punishment by footshock than males. Conclusions: Even though the punishing effects of footshock did not differ between high- and low-responder rats when they were unsignaled, providing information (a signal) that continuing to respond would result in punishment resulted in clear differences between the phenotypes with high-responders avoiding punishment at a significantly lower frequency than low-responder rats. Together, these studies suggest that signaled procedures might provide a more sensitive measure of punishment, and that the high levels of drug-taking observed in the high-responder rats represent a compulsive-like pattern of responding, which may be related to the DSM criteria for substance use disorder, continued use despite adverse consequence. Keywords: MDPV Self-Administration, Punishment, Sex Differences Disclosure: Nothing to disclose. P727. Metformin Effects on Cocaine Conditioned Reward in Male and Female Rats Edith Hernandez, Mahamed Mohamud Abdulahi, Sarah Ufearo, Sade Spencer University of Minnesota Medical School, Minneapolis, Minnesota, United States Background: Cocaine use disorder (CUD) remains a significant problem in the United States as cocaine-associated overdoses rates have risen to dramatic levels in recent years. There is no current FDA-approved treatment for CUD leading to high attrition rates for those in recovery, and therefore high rates of relapse and overdose. We aim to investigate the therapeutic potential of metformin, a Type II Diabetes drug, to influence the rewarding effects of cocaine in a conditioned place preference (CPP) model. Metformin acts as an indirect activator of adenosine monophosphate activated protein kinase (AMPK), a metabolic energy sensor responsible for maintaining intracellular homeostasis after a stressor depletes ATP levels. When activated by phosphorylation, pAMPK rebalances the ratio of AMP to ATP throughout the body as well as in the brain. Recent studies have shown a decrease in pAMPK within the nucleus accumbens core (NAc) after repeated cocaine use. Our lab recently demonstrated that metformin microinjection into the NAc can reduce cue-induced reinstatement of cocaine seeking. The current experiments extend this study to the systemic administration of metformin as a more translational approach to drug delivery. Methods: Cocaine conditioned place preference (CPP) was performed in adult male and female Sprague Dawley rats in a 2-chamber box (MedAssociates). Rats were conditioned for 5 days in twice daily 30-minute sessions, control and treatment, using an unbiased research design. Rats displaying >80% preference for either chamber during a baseline pre-test were excluded from the analysis. Pretreatment of metformin (175 mg/kg) or saline occurred 30 minutes before conditioning with cocaine (10 mg/kg, 20 mg/kg) or vehicle (saline). All injections were given intraperitoneal. After conditioning, time spent in each chamber was assessed during 15 minutes of free exploration with no drug exposure. Results: Sex differences were observed in the conditioned rewarding effects of cocaine with female rats (n = 11; p = 0.0002 for cocaine vs saline paired chamber during post-test) but not male rats (n = 9; p = 0.19 for cocaine vs saline paired chamber during post-test) acquiring CPP for a 20 mg/kg dose of cocaine. Data were analyzed by two-way repeated measures analysis of variance (ANOVA) followed by Sidak’s multiple comparisons. Preliminary data show a lack of CPP in males (n = 4; p = 0.83) and females (n = 8; p = 0.86) for a 10 mg/kg conditioning dose of cocaine. A pretreatment of 175 mg/kg metformin had no effect on CPP for 20 mg/kg cocaine in female rats. In ongoing studies, we will add animals to increase power and complete dose response analysis for cocaine and metformin. Conclusions: Dose-dependent effects of cocaine conditioning have been observed in female and male rats. We are interested in determining if a similar dose-dependent effect for metformin pretreatment may be observed. Our previous research suggests increased sensitivity to the effects of metformin in female rats. We find no effect of metformin pretreatment on acquisition of cocaine CPP in our preliminary results, but aim to further assess metformin effects on expression or reinstatement of this behavior. The continuation of this study will inform the potential development of metformin as a pharmacotherapy for cocaine use disorder. Keywords: Cocaine, Adenosine Monophosphate Activated Protein Kinase (AMPK), Conditioned Place Preference, Cocaine Sex Differences, Metformin Disclosure: Nothing to disclose. P728. Interactions Between Gabapentinoids and Opioids in Rats Shawn Flynn, Charles France University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States Background: Recent epidemiological studies suggest significant misuse of gabapentinoids (gabapentin and pregabalin) in people with opioid use disorder, and that co-use of gabapentinoids and opioids increases the risk of opioid-related death; post-mortem studies have identified gabapentinoids in up to 40 percent of fatal drug overdoses, primarily those involving opioids. The number of gabapentinoid prescriptions is also on the rise, with the majority of these prescriptions being for off-label indications. Despite these alarming trends, little research has evaluated potentially harmful interactions between gabapentinoids and opioids. Individuals with opioid use disorder that use gabapentinoids often report that gabapentinoids enhance the euphoric/positive effects of opioid drugs. This study examined the effects of gabapentinoids on heroin-induced ventilatory depression and its reversal by naloxone, and on self-administration of heroin under a progressive ratio schedule of reinforcement. Methods: Five male Sprague Dawley rats were pretreated with gabapentin (10-100 mg/kg), pregabalin (1-32 mg/kg), or saline i.v. 30 minutes prior to receiving increasing doses of heroin while ventilation was measured using whole-body plethysmography. Three infusions of heroin were administered at 3-minute intervals resulting in cumulative doses of 0.1, 0.32, and 1.0 mg/kg, respectively. Five minutes following the third heroin infusion animals received saline or naloxone (0.0056-0.01 mg/kg). The primary outcome of this study was minute volume, the total volume of air ventilated per minute. In a second experiment seven male and seven female Sprague Dawley rats were trained to self-administer heroin (0.01 mg/kg/infusion) first under a fixed ratio 1 timeout 5 seconds schedule of reinforcement where one lever press resulted in an infusion of drug followed by a 5-second timeout, and then under a progressive ratio schedule of reinforcement in which the response requirement increased for each infusion earned. Following acquisition of heroin self-administration and stable responding for the training dose of heroin under the progressive ratio schedule of reinforcement saline was substituted for heroin until behavior stabilized at fewer than 5 infusions per session. Then, animals were randomized to a dose of heroin (0.0032-0.1 mg/kg/infusion). Animals self-administered each dose of heroin for at least 3 days until consecutive days differed by no more than 1 infusion earned. The following day animals were pretreated with gabapentin (1-10 mg/kg) or pregabalin (0.1-1 mg/kg) i.v. 15 minutes prior to the self-administration session. Heroin unit dose and pretreatment condition were evaluated in a pseudo-random order until heroin dose-effect curves were determined under each pretreatment condition. The number of infusions earned and the breakpoint (the number of responses required for the last infusion earned) at each dose was compared across pretreatment conditions and sex¬. All procedures were approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee. Results: Heroin dose-dependently reduced minute volume with 1 mg/kg suppressing minute volume to <50% of baseline in all animals. Pretreatment with gabapentin or pregabalin did not significantly alter the effects of heroin on minute volume. Naloxone dose-dependently reversed the ventilatory depressive effects of heroin with 0.0056 mg/kg restoring ventilation to baseline levels. Following pretreatment with gabapentin (100 mg/kg) or pregabalin (32 mg/kg) this dose of naloxone was no longer sufficient to restore normal ventilation when administered 5 minutes after the largest dose of heroin. A larger dose of naloxone (0.01 mg/kg) was required to return minute volume to baseline levels. Animals self-administered heroin in a dose-dependent fashion with breakpoints significantly greater than for saline. In most animals 0.032 mg/kg/infusion of heroin maintained the highest breakpoint and number of infusions earned. Gabapentin increased the reinforcing effects of heroin in some, but not all animals. Conclusions: Pretreatment with gabapentinoids did not alter the ventilatory depressive effects of heroin but reduced the potency of naloxone to reverse heroin-induced ventilatory depression. Gabapentinoids increased the reinforcing effects of heroin in some subjects, consistent with reports in humans that gabapentinoids can enhance the positive/euphoric effects of opioids. Future studies will expand these findings to other opioids such as fentanyl and confirm the selectivity of the enhancement of the reinforcing effects of heroin for opioids by determining the effects of gabapentinoids on self-administration of fentanyl and cocaine. The current study suggests that there might be significant interactions between gabapentinoids and opioids related to both opioid overdose and substance misuse that warrant further investigation. Keywords: Opioid Abuse, Opioid Overdose, Drug-Drug Interaction, Gabapentin, Pregabalin Disclosure: Nothing to disclose. P729. Effects of Methocinnamox, a Long-Acting Mu Opioid Receptor Antagonist, on Fentanyl Choice in Monkeys Responding Under a Food Versus Drug Choice Procedure David Maguire, Charles France Univ. of Texas Health Science Center, San Antonio, Texas, United States Background: Methocinnamox (MCAM), a long-acting mu opioid receptor antagonist, attenuates the reinforcing and ventilatory-depressant effects of opioids in rats and nonhuman primates, suggesting it could be an effective treatment for opioid use disorder and overdose. Previous studies examined effects of MCAM on heroin and fentanyl self-administration under fixed-ratio schedules of drug reinforcement. The current study extends characterization of the potential therapeutic utility of MCAM to examine effects on responding under a food versus drug choice procedure wherein changes in reinforcing effects are evaluated by changes in behavioral allocation from responding for drug to responding for a non-drug alternative such as food. Most studies to date administered MCAM within 1 hr of the test session. This study also compared pretreatment times to determine whether the duration of action of MCAM depends on temporal proximity of MCAM administration to test sessions. Methods: Five rhesus monkeys (2 females and 3 males) lever-pressed for food or i.v. infusions of fentanyl under a concurrent fixed-ratio 30 schedule of reinforcement. Daily 4-hr sessions were divided into blocks with each block comprising 2 forced trials followed by up to 6 choice trials. Responding on one lever delivered a 300-mg sucrose pellet, whereas responding on the other lever delivered a unit dose of fentanyl that increased across blocks each session from 0.1 to 3.2 µg/kg/infusion. MCAM (1.0 mg/kg) was administered i.v. either 1 or 20 hr prior to test sessions, and, for comparison, the opioid receptor antagonist naltrexone (0.1 mg/kg) was administered i.v. 15 min prior to a test session. Results: Choice of fentanyl increased with increasing unit doses of fentanyl from less than 10% infusion choice with unit doses of 0.1 and 0.32 µg/kg/infusion to greater than 90% infusion choice with unit doses of 1.0 and 3.2 µg/kg/infusion. Naltrexone decreased choice of fentanyl on the day of treatment, shifting the fentanyl dose-effect curve for percent infusion choice rightward and downward; naltrexone was no longer effective 24 hours later with choice returning to control. MCAM also decreased choice of fentanyl; however, effects of MCAM lasted for 2 to 3 days following a single injection. The time to recovery of responding following MCAM administration did not differ across pretreatment times. Conclusions: Fentanyl dose-dependently increased choice of drug and decreased choice of food with increasing unit doses. Naltrexone and MCAM treatment decreased choice of fentanyl and prompted a reallocation of responding toward food. These results confirm that effects of MCAM in decreasing responding for opioids under other conditions is selective insofar as in this study responding for food increased when responding for fentanyl decreased. Effects of naltrexone lasted less than 24 hr, whereas effects of MCAM lasted for several days. The duration of effect of MCAM did not appear to differ between pretreatment times. These data are consistent with previous studies showing long-lasting and selective attenuation of opioid self-administration. Demonstration that MCAM promotes the reallocation of responding from drug to a non-drug reinforcer provides additional support for the notion that MCAM might be a safe and effective treatment for opioid use disorder. Keywords: Opioid Abuse, Opioid Antagonist Treatment, Nonhuman Primates, Choice Procedure, Fentanyl Disclosure: Nothing to disclose. P730. Zinc Modulates Cocaine-Related Behaviors and Dopamine Transmission as a Function of Sex Oscar Solís, Emilya Ventriglia, Fallon Curry, Juan Gomez, Michael Michaelides National Institute on Drug Abuse, NIH, Baltimore, Maryland, United States Background: Zinc (Zn2+) is an essential life element that is implicated in neurophysiological homeostasis, and its dysregulation is associated with various disorders, such as substance use disorder (SUD). Within a subset of glutamatergic neurons, synaptic Zn2+ is co-released with glutamate. The striatum receives a dense innervation of Zn2 + -containing neurons (zincergic). Previous studies showed that synaptic Zn2+ is an endogenous modulator of striatal dopamine neurotransmission. Moreover, Zn2+ potentiates the effects of cocaine by binding the dopamine transporter (DAT), resulting in the enhancement of cocaine-mediated behaviors. For instance, we reported that the absence of synaptic Zn2+ reduces conditioned place preference, locomotor sensitization and self-administration to cocaine. Interestingly, previous evidence demonstrated that estrogen reduces total and synaptic Zn2+ in the brain. In line with these findings, Zn2+ exerts sexually dimorphic effects on locomotion and at skilled motor learning tasks. However, the interaction between synaptic Zn2+ and sex on dopamine transmission has not been characterized. In addition, we investigated the role of Zn2+ in the dorsal striatum (DS) in the development and expression of behavioral sensitization by cocaine. Finally, we studied the proportion of zincergic neurons that project to the striatum. Methods: To study if Zn2+ alters dopamine transmission in the striatum in a sex dependent manner, we conducted autoradiography assays in brain tissue from C57BL/6 J female and male mice (n = 5/group). We used the cocaine analog [3H]WIN-35,428 and [3H]SCH23390 to quantify binding to the dopamine transporter (DAT) and dopamine D1 receptor (D1R) respectively, in the presence of physiological concentrations of Zn2 + . Then, to study the role of Zn2+ on cocaine-induced locomotor sensitization, we bilaterally microinjected saline or TPEN (Zn2 + chelator) into the striatum of C57BL/6 J female and male mice (n = 8/group), followed by cocaine injections (10 mg/kg; i.p.) for 5 days. After 2 weeks of withdrawal, mice were challenged with cocaine (10 mg/kg; i.p.) to study the expression of the locomotor sensitization. Finally, in order to study the proportion of glutamatergic/zincergic neurons that project to the striatum. We combined retrobeads and fluorescent in-situ hybridization (RNAscope), this approach allows to trace the neurons projecting to the striatum, and of those, what proportion are zincergic in the cerebral cortex, amygdala, and paraventricular thalamus of C57BL/6 J mice (n = 4). Results: Our autoradiography analysis revealed that [3H]WIN-35,428 binding to DAT was significantly lower in the DS of male compared to female mice (t = 3.08, p = 0.015). Interestingly, physiological concentrations of Zn2 + (10 µm) significantly increased [3H]WIN-35,428 binding to DAT in both female (t = 8.490, p = 0.001) and male mice (t = 4.98, p = 0.001). Notably, this effect was larger in males than in female mice (t = 4.06, p = 0.003). We observed no significant differences in [3H]SCH23390 binding to the D1R neither between female and male mice (t = 0.94, p = 0.37) nor in the presence of Zn2 + (t = 0.01, p = 0.82). We also found that bilateral Zn2+ chelation in the DS of mice significantly decreased the development and expression of the locomotor sensitization in male (p = 0.001 by post hoc Tukey comparison) but not in female mice. Finally, by using RNAscope, our preliminary results show that in the cerebral cortex, zincergic neurons account for 55% of glutamatergic neurons that project to the striatum. Conclusions: Our findings suggest that part of the effects of Zn2+ on dopamine transmission is by binding the DAT and not the D1R, and this effect is dependent on sex. In addition, our results suggest that Zn2+ augments the locomotor activity induced by cocaine in male mice. Finally, we show that a population of cortical neurons that project to the striatum bear the machinery to release Zn2+ in the striatum. Further studies will address the zincergic circuits that modulate cocaine-seeking behaviors. Keywords: Cocaine, Dopamine Transporter, D1 Dopamine Receptors, Zinc Disclosure: Nothing to disclose. P731. Prenatal Heroin Exposure Alters Brain Morphology and Connectivity in Adolescent Mice Lauren Slosky, Kathryn J. Hornburg, Gary Cofer, James J. Cook, Yi Qi, Fiona Porkka, Nicholas B. Clark, Andrea Pires, Jeffrey R. Petrella, Leonard E. White, William C. Wetsel, Lawrence S. Barak, Marc G. Caron, G. Allan Johnson University of Minnesota, Minneapolis, Minnesota, United States Background: The United States is experiencing a dramatic increase in maternal opioid misuse and, consequently, the number of individuals exposed to opioids in utero. Prenatal opioid exposure has both acute and long-lasting effects on health and wellbeing. Effects on the brain, often identified at school-age, manifest as cognitive impairment, attention deficit, and reduced scholastic achievement. Understanding the neurobiological basis for these deficits is critical to identifying affected individuals and developing effective interventions. Here, we examine how in utero exposure to the highly addictive and widely misused opioid heroin affects brain development into early adolescence in a mouse model. Methods: Pregnant C57BL/6 J mice received escalating (1-10 mg/kg, 10 ml/kg, s.c.) doses of heroin twice daily on gestational days 4-18. Control mice received twice daily vehicle injections (physiological saline, 10 ml/kg, s.c.) or no intervention. Litters remained in their home cages with the dam and were not weaned. The brains of offspring were assessed on postnatal day 28 using 9.4 T diffusion magnetic resonance imaging (MRI) of postmortem specimens at 36 micron resolution. Whole brain metrics and the metrics of 166 bilateral regions (332 ROIs) were compared between randomly selected heroin exposed (NHeroin = 14 (10 male, 4 female) and control (Ncontrol = 14 (8 male, 6 female)) offspring. Two approaches were employed in the comparison of the brains of heroin exposed and control mice: (1) A comparison of scalar phenotypes was conducted to identify changes in global and regional brain volumes, and (2) Connectome/tractography analysis was performed to identify differences in global and regional connectivity. To compare group-mean volumes for every atlas-defined brain region, we used the Kruskal–Wallis analysis of variance with the Benjamini-Hochberg false discovery rate correction. To compare group-mean global and regional connectomes, we used a dimension reduction strategy and multivariate analysis of variance with the Bonferroni correction. Results: Offspring from heroin-exposed and control dams did not differ in body weight (heroin-exposed mean ± SD: 13.2 ± 2.75 g; control mean ± SD: 13.8 ± 1.25 g; two-sided Wilcoxon Rank Sum test, p = 0.4079). Whole brain volume, however, was reduced in heroin-exposed mice (mean ± SD: 411 ± 34.6 mm3), as compared to controls (mean ± SD: 447 ± 9.41 mm3; two-sided Wilcoxon Rank Sum test, p = 0.0063). After standardizing for whole brain volume, we identified bilateral heroin-associated volume changes in 29 regions (Benjamini-Hochberg adjusted p-values <0.05). Regions with bilaterally reduced standardized volumes in heroin-exposed offspring relative to controls include the ectorhinal and insular cortices. Regions with bilaterally increased standardized volumes in heroin-exposed offspring relative to controls include the periaqueductal gray, septal region, striatum, and hypothalamus. Leveraging microscopic resolution diffusion tensor imaging and precise regional parcellation, we generated whole brain structural MRI diffusion connectomes. Global brain connectivity was unchanged between the heroin-exposed and control offspring. In individual connectome profiles of all 332 ROIs, a single region met the Bonferroni criteria: the left septal region, a region that acts as a hub for limbic regulatory actions. Conclusions: Consistent with clinical evidence, our findings suggest that prenatal opioid exposure may have effects on brain morphology, connectivity, and, consequently, function that persists into adolescence. Future studies are needed to dissect how stress, postnatal mother-pup interactions, and offspring sex interact with gestational opioid exposure to bring about changes in brain structure and behavior. This work expands our understanding of the risks associated with opioid misuse during pregnancy and identifies biomarkers that may facilitate diagnosis and treatment. Keywords: Prenatal Drug Exposure, Opioids, Magnetic Resonance Imaging, Mouse Models, Brain Structural Connectivity Disclosure: Nothing to disclose. P732. Activation of 5-HT1B Receptors Attenuates the Acquisition of Nicotine Reward in Adolescent Male Rats Arturo Zavala, Tiffany Gonzalez California State University - Long Beach, Long Beach, California, United States Background: Nicotine addiction continues to be a significant concern, particularly in adolescent populations. Activation of serotonin (5-HT)1B receptors decreases the rewarding and reinforcing effects of stimulant drugs, such as cocaine and methamphetamine. However, the result of 5-HT1B activation on nicotine reward has not been examined. In the present study, we examined the hypothesis that administering CP 94,253, a 5-HT1B receptor agonist, would reduce nicotine preference in adolescent male rats using a 10-day Conditioned Place Preference (CPP) procedure, a well-established animal model of drug reward. Methods: On postnatal day (PD) 28, baseline preference for a two-sided apparatus was assessed during a 15 min session. In two-day cycles, rats received an injection of CP 94,253 (0 or 5.6 mg/kg, IP) 15 min before the administration of nicotine (0, 0.2, or 0.6 mg/kg, n = 12-14) on one day and saline (n = 12-14) administration on the other day before being confined to one side of the two-chamber apparatus for 15 min. This two-day cycle was repeated over the next 6 days. On day 10, the preference for the nicotine-paired chamber was assessed for 15 min. Results: Rats exhibited nicotine-induced CPP when conditioned with either 0.2 or 0.6 mg/kg of nicotine (p < 0.05). Administration of CP 94,253 (5.6 mg/kg) before nicotine (0.2 or 0.6 mg/kg) resulted in a decreased preference for the nicotine-paired compartment (p < 0.05). Conclusions: The present findings demonstrate that activation of 5-HT1B receptors with CP 94,253 attenuated the acquisition of nicotine-induced CPP in male adolescent rats. Overall, these findings further add to a growing body of literature that points to the 5-HT1B receptor as a pharmacological target for treating psychostimulant addiction. Future studies should examine the effects of 5-HT1B receptors on nicotine reward in female rats. Keywords: Nicotine Addiction, Adolescence, Reward Disclosure: Nothing to disclose. P733. The Impact of Eating a High Fat or Ketogenic Diet on Sensitivity of Rats to Dopaminergic Drugs Katherine Serafine, Madeline Elsey, Nina Beltran The University of Texas At El Paso, El Paso, Texas, United States Background: Eating a high fat diet leads to negative health consequences such as obesity and type 2 diabetes. Recent evidence also suggests that eating a high fat diet can impact drug sensitivity. For example, eating a high fat laboratory chow enhances the sensitivity of rats to the behavioral effects of drugs, including drugs that act on dopamine systems, such as methamphetamine and quinpirole (a dopamine D2/D3 receptor agonist). Previous preclinical research has primarily focused on exploring the effects of traditional high fat diets that are high in fat and carbohydrates on drug sensitivity. In contrast, a ketogenic diet is high in fat, but very low in carbohydrates. While high fat/high carbohydrate diets lead to weight gain, ketogenic diets can promote weight loss. It is not known if eating a ketogenic diet might enhance sensitivity of rats to the behavioral effects of drugs that act on dopamine systems, similarly to what has been shown with traditional high fat/high carbohydrate diets. This project tested the hypothesis that rats eating a high fat/high carbohydrate diet would be more sensitive to the behavioral effects of methamphetamine and quinpirole, but that rats eating a ketogenic chow would not differ from rats eating a standard (low fat) laboratory chow. Methods: To test this hypothesis, male (n = 24) and female (n = 36) Sprague-Dawley rats eating high fat chow (60% kcal from fat), ketogenic chow (90.5% kcal from fat) or standard chow (17% kcal from fat) were tested once weekly with cumulative doses of either methamphetamine (0.1-3.2 mg/kg; i.p.) or quinpirole (0.0032-3.2 mg/kg; i.p.). To assess drug sensitivity, methamphetamine-induced locomotion and sensitization were examined, as well as quinpirole-elicited yawning. Average locomotor activity counts and yawning were analyzed using two-way repeated measures ANOVAs with diet and dose as factors, and Tukey or Sidak post hoc comparisons where appropriate. Results: Rats eating high fat chow were more sensitive to the locomotor-stimulating effects of smaller and intermediate doses of methamphetamine (e.g., 0.32, 1.0 mg/kg) than rats eating standard chow. Rats eating ketogenic chow were also more sensitive than rats eating standard chow to the locomotor-stimulating effects of methamphetamine, but only at the largest dose tested (3.2 mg/kg). Rats eating high fat chow were also more sensitive to quinpirole-induced yawning than rats eating standard chow. In contrast, quinpirole-induced yawning was comparable between rats eating a ketogenic chow and rats eating standard chow. Conclusions: These results suggest while eating a high fat/high carbohydrate diet can enhance the sensitivity of individuals to dopaminergic drugs, this effect might not translate identically to ketogenic diets. These results add to the growing literature suggesting that ketogenic diets and traditional high fat/high carbohydrate diets differ with regard to their impacts on health. Keywords: Methamphetamine, Dopamine, High Fat Diet, Ketogenic Diet, Rats Disclosure: Nothing to disclose. P734. Sex Differences in GABA Regulation of Dopamine Release in the Nucleus Accumbens and Its Role in Cocaine Use Disorder Brooke Christensen, Addison Van Namen, Erin Calipari Vanderbilt University Medical Center, Nashville, Tennessee, United States Background: While sex differences in the pervasiveness and prognosis of neuropsychiatric disorders have long been known to exist, there are few instances where approaches to pharmacological treatment of these disorders differ between the sexes, which likely contributes to ineffective treatments in women. In substance use disorder (SUD) women exhibit increased propensity to use drugs, a faster transition to addiction from first use, greater problems maintaining abstinence, and relapse at a higher rate than men. At the center of sex-differences in addiction vulnerability is the mesolimbic dopamine system. While work has focused on sex differences in the anatomy of dopamine neurons and relative dopamine levels, an important characteristic of dopamine release from axon terminals in the nucleus accumbens (NAc) is that it is rapidly modulated by local regulatory mechanisms independent of somatic activity. GABA released from local microcircuitry in the NAc has been shown to play a critical role in regulating dopamine release at the terminals through ionotropic GABA-A and Gi-coupled GABA-B receptors and has also been implicated in cocaine-induced processes. Here we define basal sex differences in dopamine release regulation via GABA in the NAc and show how this is dysregulated by chronic cocaine exposure. Methods: To dissociate dopamine terminal regulation from somatic regulation we utilize ex vivo fast scan cyclic voltammetry in striatal brain slices. Dopamine release was evoked from terminals, and GABA receptor modulation of this signal was determined via the application of picrotoxin (GABA-A antagonist), muscimol (GABA-A agonist), saclofen (GABA-B antagonist), and baclofen (GABA-B agonist) bath application to slices. This was done at baseline in both males and females as well as after chronic cocaine exposure. Results: First we found that both GABA-A and GABA-B receptors modulate dopamine release on a rapid time scale directly at the terminals in the NAc. There were sex differences in this regulation, with much greater regulation of dopamine release by GABA receptors in males as compared to females. Importantly, chronic cocaine exposure resulted in receptor-specific plasticity in this microcircuit regulatory mechanism. Conclusions: The results of these studies will contribute to the understanding of how sex fits into the comprehensive framework for dopamine release regulation and how dysregulation of these processes influences the trajectory of CUD in both males and females. Keywords: Nucleus Accumbens, Cocaine Use Disorder, GABA-A Receptors, GABA-B Receptors, Fast Scan Cyclic Voltammetry Disclosure: Nothing to disclose. P735. Effect of Sex and Age on Nicotine Vapour Pharmacokinetics, Reward, Withdrawal, and Functional Connectivity in Rats Jude Frie, Patrick McCunn, Ahmad Hassan, Karling Luciani, Chuyun Chen, Rachel Tyndale, Jibran Khokhar* Western University, London, Canada Background: Nicotine use is constantly evolving, with vaping now making up a significant portion of consumed tobacco products. Though vaping likely represents a safer alternative to smoking, it is not without risks, many of which are not well understood. A particular area that requires attention is the effects vaping has on populations that are especially vulnerable to nicotine, such as women and adolescents. Here we evaluate the sex- and age-dependent vulnerabilities to nicotine vapour in a rat model of nicotine vapour exposure. Methods: Passive nicotine exposures were conducted via JUUL e-cigarettes via a custom-built OpenVape apparatus. Animals were evaluated for reward-like behaviour in a place conditioning paradigm, locomotion in an open field, precipitated withdrawal following i.p. mecamylamine injection (1.5 mg/kg), nicotine and nicotine metabolite brain and plasma pharmacokinetics, and functional magnetic resonance imaging. Results: Nicotine and nicotine metabolite plasma and brain levels were similar between adults and adolescents, although females did show higher plasma nicotine and cotinine plasma levels at 10 minutes compared to adolescent females. Adult females had greater nicotine and nicotine metabolite concentrations than adult males in both blood plasma and brain supernatant. This trend was similar in adolescent female brain supernatant but not plasma where results were not significantly different. Female, but not male adults, displayed conditioned place preference (CPP) at a high dose of nicotine vapour. Female adolescents did not acquire CPP at any dose tested. Both adult and adolescent males displayed similar levels of precipitated nicotine vapour-induced withdrawal. Female rats did not display any precipitated nicotine withdrawal. Passive nicotine vapor exposure resulted in hyperlocomotion in both adult and adolescent males, but not females. Functional MRI revealed a single network consisting of 12 edges and 13 nodes that displayed reduced connectivity when controlling for age and sex. An additional significant group by sex interaction effect was found with 5 edges and 6 nodes showing further reduced connectivity in females compared to males. Conclusions: Our findings suggest that nicotine vapour pharmacokinetics as well as behavioural and neural impacts are affected by both sex and age, with unique reward, withdrawal and pharmacokinetic profiles, as well as functional connectivity changes in regions involved in nicotine cue reactivity, withdrawal, and dependence. Keywords: Electronic cigarette (e-cigarette), Nicotine Metabolism, Resting State Functional Connectivity, Nicotine Exposure, Nicotine vapor Disclosure: Nothing to disclose. P736. Effects of Sex and Estrous Cycle on Intravenous Oxycodone Self-Administration and Stress-Induced or Cue-Induced Reinstatement of Oxycodone Seeking in Rats Nicole Hinds, Ireneusz Wojtas, Daniel Manvich* Rowan University School of Osteopathic Medicine, Stratford, New Jersey, United States Background: The abuse of prescription and illicit opioids has culminated in a national healthcare crisis in the United States. Oxycodone is among the most widely prescribed and misused opioid pain relievers and has been associated with a high risk for transition to compulsive opioid misuse. While sex differences have been reported for the abuse-related effects of other opioids (e.g., heroin) in both humans and experimental animals, only a small number of studies to date have examined sex differences specifically with respect to oxycodone, with conflicting results reported. Here, we sought to assess potential sex differences in the reinforcing effects of oxycodone, as well as stress-induced or cue-induced oxycodone-seeking behavior, using IV oxycodone self-administration and reinstatement procedures. We also assessed whether these measures varied in females as a function of estrous cycle phase. Methods: In experiment 1, adult male (n = 5) and female (n = 4) Long-Evans rats were first trained to self-administer 0.03 mg/kg/infusion oxycodone according to a fixed-ratio 1 schedule of reinforcement in daily 2-hr sessions (5-6 days per week). A dose-response function was then established (0.003 – 0.03 mg/kg/infusion) with doses presented in descending order. In experiment 2, a separate group of adult male (n = 8) and female (n = 17) Long-Evans rats were trained to self-administer 0.03 mg/kg/infusion oxycodone for 8 days, followed by 0.01 mg/kg/infusion oxycodone for 10 days. Responding was then extinguished in daily 2-hr sessions during which active-lever responses had no scheduled consequences. Animals then underwent sequential reinstatement tests following exposure to either intermittent, unpredictable footshock (test #1) or reintroduction of an oxycodone-paired cue light (test #2), with reinstatement tests separated by additional extinction sessions. Vaginal smears were collected daily from female subjects for histological confirmation of estrous cycle phase (proestrus, estrus, metestrus, diestrus). Results: In the dose-response experiment, IV oxycodone produced a typical inverted U-shape function when response rate was used as the dependent measure, with 0.01 mg/kg/infusion representing the “peak”-effective dose in both sexes. Oxycodone functioned as a more effective reinforcer in females than males as evidenced by an upward shift of the dose-response curve, particularly at the two lower doses tested (0.003 and 0.01 mg/kg/infusion; main effect of sex, p < 0.05). This upward shift in responding was not accompanied by a significantly greater number of oxycodone infusions earned in females. In the second experiment, females again exhibited higher rates of responding than males at both the 0.01 and 0.03 mg/kg/infusion doses of IV oxycodone. In females, within-subjects analysis revealed that rates of responding for IV oxycodone were significantly higher during metestrus/diestrus as compared to proestrus/estrus (main effect of estrous phase, p < 0.05). In reinstatement tests, neither males nor females displayed appreciable footshock-induced reinstatement of oxycodone seeking, while females exhibited a robust cue-induced oxycodone-seeking response (main effect extinction vs. reinstatement, p < 0.05) that was not observed in males and which was independent of estrous cycle phase. Conclusions: We report here that IV oxycodone functions as a more effective reinforcer in female rats as compared to male rats. Females also exhibited a more robust drug-seeking response following re-exposure to oxycodone-associated discrete cues than males under the present testing conditions, suggesting that sex differences may not only apply to oxycodone taking, but also to oxycodone seeking. We also reveal for the first time that the reinforcing effects of IV oxycodone in females are modulated by estrous cycle phase, an effect that is likely mediated by fluctuations in ovarian hormones although additional experiments are required to confirm this supposition. Taken together, our results confirm and extend previous work suggesting that females may be more vulnerable than males to the abuse-related effects of oxycodone and may also be more susceptible to certain modalities of relapse, although the magnitude of these risks may vary across the menstrual cycle. Keywords: Oxycodone, Sex Difference, Estrous Cycle, Reinstatement, Self-Administration Disclosure: Nothing to disclose. P737. Adolescent Nicotine Vapor Exposure Increases Nicotine Vapor Seeking Behavior in Adult Male Rats Liliana Maynez-Anchondo, Miguel Urbina, Olga Rohrer, Ian Mendez* The University of Texas at El Paso School of Pharmacy, El Paso, Texas, United States Background: In recent years there has been an increase in nicotine vapor consumption via electronic nicotine delivery systems, particularly in adolescents. While the health effects of nicotine vapor continues to be investigated, its distinct effects on the brain and behavior remain unclear. Previous studies have used intravenous self-administration to investigate nicotine seeking and taking behaviors. The primary objective of this study was to use a nicotine vapor self-administration (NVSA) system to assess changes in motivation for nicotine vapor intake during adulthood, in rats with a history of adolescent and/or early adulthood nicotine vapor exposure. Methods: Male Sprague-Dawley rats (N = 24) were passively exposed to 0 mg/mL vehicle control (50/50 propylene glycol/vegetable glycerin, PG/VG) or 24 mg/mL nicotine vapor for 10 daily 90-minute sessions during late adolescence (PND 58-67) and/or early adulthood (PND 159-168), resulting in 4 groups defined by early life passive nicotine vapor exposure (adolescent exposure-early adulthood exposure, 0-0, 24-0, 0-24, 24-24, n = 6/group). To assess nicotine vapor’s short-term effects on motivation for reward, lever pressing for food pellets was assessed using a progressive ratio schedule of reinforcement, immediately after each adolescent and early adulthood passive vapor exposure session. Approximately 25 weeks after early adulthood exposure, motivation for nicotine reward was assessed by testing the rats for 6 mg/mL NVSA across 4 daily 1-hour sessions. Responding in both the progressive ratio task and NVSA was not assessed until acquisition of the task was identified with a criteria of less than 15% variability across 2 consecutive days. Mixed model ANOVA was used to compare effects of passive nicotine vapor exposure on lever pressing for food in the progressive ratio task, as well as nicotine vapor deliveries and active nosepoke entries during NVSA. One-way ANOVA, Fishers LSD, and independent samples t-test were used for post-hoc analyses. Results: Analysis of lever presses for food rewards immediately following passive nicotine vapor exposure during adolescence revealed a significant interaction of treatment group and day (F(9,189)=2.61, p < 0.01, ηp2 = 0.11), with t-tests showing higher responding in the 24 mg/mL nicotine vapor group, relative to 0 mg/mL vehicle control group, on test days 1,5,6, and 7 (ps < 0.05). No statistical differences were seen between treatment groups when responding for food rewards following passive vapor exposure during early adulthood. For NVSA, no effect of treatment groups were observed for total nicotine vapor deliveries. However, analysis of total active nosepoke entries identified a main effect of treatment group (F(3,19)=3.31, p < 0.05, ηp2 = 0.34), with Fishers LSD showing the 24-0 and 0-24 nicotine vapor exposure groups being significantly different from the 0-0 vehicle control group across test days (ps < 0.05). The 24-0 group was also identified as significantly different from the 24-24 group across test days (p < 0.05). One-way ANOVAs comparing treatment groups on each of the 4 NVSA test days revealed significant increases in reward seeking nosepokes for the 24-0 (test days 1 and 2) and 0-24 (test day 1) exposure groups, relative to the 0-0 group (p < 0.05). Notably, rats in the 24-0 group also showed more reward seeking nosepokes than rats in the 24-24 group on test day 2 (p < 0.05). Conclusions: Our findings demonstrate that adolescent-only and early adulthood-only exposure to nicotine vapor causes immediate and long-term increases in motivation for rewards and may promote e-cigarette use later in life. Interestingly, when nicotine vapor exposure occurred during both adolescence and early adulthood, no increases in nicotine vapor seeking behavior were observed. Additional studies on the neurobiological and behavioral effects of nicotine vapor will be necessary for the development of effective treatment strategies, educational programs, and public policies aimed at curbing recreational use of e-cigarettes. Keywords: Nicotine Vapor, Self-Administration, Motivation Disclosure: Nothing to disclose. P738. Sex-Specific Cholinergic Regulation of Dopamine Release Mechanisms Through Nicotinic Receptors in the Nucleus Accumbens Lillian Brady, Kimberly Thibeault, Jennifer Tat, Jordan Yorgason, Erin Calipari Vanderbilt University, Smyrna, Tennessee, United States Background: For many psychiatric disorders, such as anxiety, depression, and substance use disorder (SUD), sex is a critical biological variable and women represent a particularly vulnerable population. The mesolimbic dopamine system is involved in the expression of sex-specific behaviors and is a critical mediator of many psychiatric disease states. While work has focused on sex differences in the anatomy of dopamine neurons and relative dopamine levels, an important characteristic of dopamine release from axon terminals in the nucleus accumbens (NAc) is that it is rapidly modulated by local regulatory mechanisms independent of somatic activity. One of the most potent regulators of dopamine terminal function is through α4β2 containing nicotinic acetylcholine receptors (nAChRs). In this series of studies, we define the molecular mechanism underling these unique sex differences in dopamine terminal regulation and show how it relates to motivated behaviors. Methods: We measured sub second release of dopamine using ex vivo fast-scan cyclic voltammetry in NAc brain slices from males (n = 7 – 10 slices), naturally cycling (intact, n = 7 - 10) and ovariectomized females (n = 7 – 10). We assessed the effects of a range of agonists and antagonists of nAChRs on dopamine release with nicotine, Dhβe, α-conotoxin PIA, and Methyllcaconitine (MLA). Additionally, we measured the potentiating effects of 17β-estradiol (E2) on dopamine release mechanisms with and without application of the estrogen receptor antagonists MPP and PHTPP or the nAChR antagonist Dhβe. Lastly, we defined the sex-specific effects of ChAT interneuron stimulation on a dopamine-dependent reinforcement task using Gq-DREADDs injected into the NAc of male (N = 13) and female (N = 15) ChAT-cre + /-. Results: We find that nAChR regulation of dopamine release is not present in intact females; however, ovariectomy rescues this regulation in females – indicating that ovarian hormones play a significant role in this process. Critically, we define the molecular mechanism underling these distinctive sex differences in dopamine regulation. Through a series of experiments, we find that acute E2 actions on dopamine terminals increases dopamine release via actions on α4β2-nAChRs. Specifically, we find that the acute potentiating effects of E2 on dopamine release are blocked by antagonism of α4β2-nAChRs, but not α6- or α7-nAChRs or estrogen receptors. Finally, using chemogenetic approaches in awake and behaving animals, we link these sex differences to sex-specific motivated behaviors. Conclusions: Here we show that this textbook mechanism of dopamine terminal regulation is not present in females under most conditions. Second, through these studies, we discovered a novel unstudied mechanism by which nAChRs are a substrate for estradiol effects on the brain, independent of canonical estrogen receptor signaling mechanisms. Keywords: Dopamine, Sex Differences, Nicotinic Acetylcholine Receptors, Nicotine/Substance Use Disorder Disclosure: Nothing to disclose. P739. Evaluation of Kappa and Mu Opioid Receptor Occupancy by CVL-354 Using PET in Nonhuman Primates Sridhar Duvvuri, Philip Iredale, Georgette Suidan, Srinivas Chakilam, Giri Gokulrangan, Nabeel Nabulsi, Yiyun Huang, Daniel Holden, Richard Carson Cerevel Therapeutics, Cambridge, Massachusetts, United States Background: CVL-354 is a potent kappa opioid receptor (KOR) antagonist with significant in vitro selectivity over the Mu opioid receptors (MOR) as measured in cell lines expressing each subtype. Subsequent in vivo receptor occupancy studies in mice showed that CVL-354 displaced [3H]PF-04767135 a KOR preferring radioligand with an ID50 of 0.1 mg/kg and [3H]carfentanil (here denoted CFN), a MOR specific ligand, with an ID50 of 4 mg/kg, confirming KOR selectivity. The objective of this study was to measure receptor occupancy at both Kappa and Mu receptors with varying doses of CVL-354 using radiotracers [11 C]LY2795050 and [11 C]CFN in non-human primates. The hypothesis was that CVL-354 would block receptors in a dose-dependent manner, with a higher affinity for Kappa than Mu. Data from this study would then facilitate dose selection for a clinical study with same tracers. Methods: For KOR imaging, two rhesus macaques (1 male and female) were scanned once at baseline and several times following administration of varying doses of CVL-354. Similarly, for MOR imaging, two female rhesus macaques were scanned once at baseline and one- or two-times following administration of varying doses of CVL-354. On all scan days, [11 C] LY2795050 or [11 C]CFN were injected as a 3-minute bolus in <10 mL. On all blocking scans, CVL-354 bolus + constant infusion was initiated ~15 minutes before the start of the [11 C] LY2795050 or [11 C]CFN injection. This delivery consisted of a 2-minute bolus followed by a maintenance infusion through the end of the scan. Dynamic scan data were reconstructed with a filtered back projection algorithm with corrections for attenuation, normalization, scatter and randoms. Binding potential (BPND) estimates of [11 C]LY2795050 and [11 C]CFN were produced using the SRTM method with the cerebellum as a reference region. Receptor occupancy (RO) was calculated using the BPND estimates. PK samples were taken throughout the delivery of CVL-354 and later submitted for analysis. Plasma concentrations were used to calculate IC50 for both KOR and MOR RO using the standard Emax model. Results: Among evaluated regions of interest (amygdala, brainstem, caudate, cerebellum, cingulate cortex, frontal cortex, globus pallidus, hippocampus, insula, nucleus accumbens, occipital cortex, pons, putamen, centrum semiovale, substantia nigra, temporal cortex and thalamus), CVL-354 produced dose-dependent occupancy at both KOR and MOR as measured by [11 C]LY2795050 and [11 C]CFN, respectively. However, as expected, the molecule was at least 10-fold more potent at the KOR. Conclusions: These data confirm dose-dependent target binding of CVL-354 to both KOR and MOR receptors in nonhuman primates. The results also confirm greater selectivity for the KOR. CVL-354 consistently demonstrated greater affinity towards KOR than MOR (10-40 fold) across the various preclinical evaluations of receptor affinity/activity Keywords: PET Imaging, Kappa Opioid Receptor Antagonist, Non Human Primate Disclosure: Cerevel Therapeutics: Employee (Self). P740. Preclinical Assessment of the Effects of Cannabidiol on Alcohol Dependence Giordano de Guglielmo, Selen Dirik, Angelica Martinez, Caitlin Crook, Ran Qiao, Michelle Doyle, Schweitzer Paul, Kallupi Marsida University of California - San Diego, La Jolla, California, United States Background: Cannabidiol (CBD), a non-psychoactive constituent of the cannabis plant, has received attention for its potential to decrease drug and alcohol use given its anti-inflammatory, antioxidant, and neuroprotective effects. Methods: Here we used a multidisciplinary approach, combining state of the art behavioral models, immunohistochemistry, and electrophysiology to evaluate the effects of chronic (60 mg/kg/day) CBD treatment in several alcohol-related behaviors and on the alcohol-induced neurodegeneration in alcohol dependent male and female rats. We used two different animal models to induce alcohol dependence in rats: the chronic intermittent ethanol vapor exposure (CIE) model and the recently developed ethanol vapor self-administration model (EVSA). The new EVSA model highlights the volitional aspects of alcohol dependence. Results: We found that chronic CBD treatment prevents the development of alcohol dependence in the EVSA model, by reducing alcohol-induced neurodegeneration in the nucleus accumbens shell (NAcSh) and the dorsomedial striatum (DMS). In animals treated after the establishment of alcohol dependence (CIE model), CBD reduces alcohol drinking, somatic and emotional signs of withdrawal. Finally, the treatment was also effective in reducing alcohol seeking and stress-induced reinstatement, possibly by reverting the reduction of neuronal excitability induced by alcohol in the basolateral amygdala (BLA). Conclusions: These results extend to the current literature and indicate a profile of potential benefit of CBD for the treatment of alcohol dependence. Keywords: Alcohol Dependence, Cannabidiol, Basolateral Amygdala, Neurodegeneration Disclosure: Nothing to disclose. P741. A Novel, Short-Acting Kappa Opioid Receptor Antagonist Blocks the Analgesic Effects of U50,488 and Attenuates Symptoms of Spontaneous Oxycodone Withdrawal in Rats Georgette Suidan, Megan Neal, Gillian Driscoll, Philip Iredale, Sridhar Duvvuri, Srinivas Chakilam, Giri Gokulrangan, Scott Carrier, Elena Chartoff Cerevel Therapeutics, Cambridge, Massachusetts, United States Background: A large body of preclinical evidence has demonstrated that the neuropeptide dynorphin (DYN), which acts at kappa opioid receptors (KOR), is a key player in opioid withdrawal (Koob, 2009; Bruchas et al., 2010). Chronic opioid exposure increases DYN and KOR activation and is thought to produce negative affective states including anhedonia, anxiety-like, and aversive behaviors. Importantly, KOR antagonism has been shown to reduce opioid withdrawal signs and escalation of opioid self-administration in rodent models. However, KORA compounds used in these preclinical models (e.g., norBNI, JDTic) have extremely long KOR antagonist actions—on the order of weeks. As such, the development of a selective and short-acting KOR antagonist has the therapeutic potential to facilitate discontinuation of opioid use with a pharmacological profile better suited for clinical development. CVL-354 is a potent, selective and short-acting KOR antagonist that was tested in a rat model of spontaneous oxycodone withdrawal. Methods: Adult male Sprague Dawley rats were used. To determine dose and time course effects of CVL-354 KOR antagonism, rats were treated with CVL-354 (0.0 – 1.0 mg/kg, SC) followed by the KOR agonist, U50,488 (30 mg/kg, SC) and nociceptive responses were measured in the Tail Flick (TF) and Hot Plate (HP) thermal pain assays. To determine the effects of CVL-354 on oxycodone somatic withdrawal signs, rats were subcutaneously implanted with iPRECIO minipumps (Alzet, model SMP 200) programmed to deliver an escalating dose regimen of oxycodone or saline 2x/day for 14 days. For oxycodone, the escalating dose regimen was 0.5, 1.0, 2.0, 4.0, and 8.0 mg/kg/infusions (2 infusions/day; 7:00-9:00AM/PM), with the 0.5 mg/kg dose administered for 2 days and the remaining doses administered for 3 days each. After the 14-day escalating dose oxycodone (or saline) regimen, spontaneous opioid withdrawal signs emerged, including diarrhea, ptosis, wet dog shakes, teeth chattering, and body flattening. To determine the effects of CVL-354 on somatic withdrawal, rats were administered CVL-354 (0.0, 0.1, 0.3, 1.0 mg/kg, SC; n = 7-19) at 6-h (Wdrl d0) and 24-h (Wdrl d1) after cessation of drug delivery. Opioid withdrawal can also alter locomotor activity, with directionality dependent on factors such as novelty and area of the test arena. As such, we tested the effects of CVL-354 on oxycodone withdrawal-induced alterations in locomotor activity in Open Field chambers (Med Associates) on Wdrl d1, immediately after somatic withdrawal measurements (n = 7-11). Activity was digitally recorded and later scored using DeepLabCut (Mathis et al., 2018). As a control for attenuation of spontaneous withdrawal signs, we administered the a2 noradrenergic agonist lofexidine (0.64 mg/kg) to separate rats. At the end of behavioral testing on Wdrl d1, rats were euthanized via rapid decapitation to collect plasma for ELISA-based corticosterone analysis (n = 6-13). Statistical analyses used were: One-way ANOVA (somatic withdrawal, corticosterone) and two-way ANOVA with repeated measures on time (open field test, tail flick and hot plate tests). Dunnett’s posthoc tests comparing treatment to control groups were done when appropriate. Results: A dose range of 0.1-1.0 mg/kg CVL-354 blocked U50,488-induced analgesia in the TF and HP tests at 1- and 4-h, but not at 24-h. Within this same dose range, CVL-354 reduced spontaneous oxycodone somatic withdrawal signs (p < 0.01). In neither the pain nor the somatic withdrawal assays did CVL-354 have an effect on its own. Lofexidine, the current standard of care for mitigation of acute opioid withdrawal symptoms, also significantly reduced somatic withdrawal signs suggesting predictive validity of this model (p < 0.01). Locomotor activity was significantly decreased during spontaneous oxycodone withdrawal compared to activity in control rats (p < 0.05). Lofexidine exacerbated withdrawal-induced decreases in locomotor activity (p < 0.01), whereas CVL-354 had no effect. Finally, spontaneous oxycodone withdrawal resulted in significantly elevated levels of unbound plasma corticosterone compared to control rats (p < 0.01). Interestingly, lofexidine pre-treatment significantly potentiated plasma corticosterone levels compared to oxycodone withdrawn rats pre-treated with vehicle (p < 0.01), whereas CVL-354 (0.3 mg/kg) pre-treatment showed a trend to decrease oxycodone withdrawal-induced plasma corticosterone levels. Conclusions: These data demonstrate that CVL-354 has KOR antagonist actions in thermal pain assays for at least 4, but less than 24, hours, and that it is effective at reducing somatic withdrawal signs in a model of spontaneous oxycodone withdrawal model. Intriguingly, lofexidine treatment suppressed locomotor activity and exacerbated levels of the stress biomarker, corticosterone. CVL-354 did not produce these negative effects, suggesting that KOR antagonism may provide better overall efficacy for mitigation of opioid withdrawal symptoms. Keywords: Kappa Opioid Receptor, Kappa Opioid Receptor Antagonist, Opioid Dependence, Pharmacotherapy, Animal Model, Withdrawal Disclosure: Cerevel Therapeutics: Employee, Stock/Equity (Self). P742. The Ghrelin System as a Potential Target for Treatment of Alcohol Use Disorder: Pharmacological Evidence Rani Richardson, George Koob, Leandro Vendruscolo, Lorenzo Leggio National Institutes of Health, Baltimore, Maryland, United States Background: Alcohol use disorder (AUD) is a chronic and relapsing neuropsychiatric disease that is a highly prevalent public health issue. Binge drinking is very common, harmful, and is an important step in the AUD spectrum. Yet, there are only a few Food and Drug Administration (FDA)-approved medications for the treatment of AUD. It is, therefore, important to develop and identify new medications to treat AUD. Towards that end, we investigated the effects of novel compounds that target the ghrelin system on binge-like drinking in a mouse model of AUD. Ghrelin is a stomach-derived peptide hormone with known roles in regulating appetite and food intake. Ghrelin receptors (GHSR-1a) are expressed both in the brain and the periphery. Recent studies show that the ghrelin system is also implicated in alcohol-related behaviors. For example, ghrelin administration increases alcohol intake, whereas blockade of GHSR-1a with specific antagonists such as JMV 2959 decreases alcohol intake. Most of these studies utilized male subjects only and did not measure binge-like drinking. Methods: “Drinking in the Dark” (DID) is a model of binge drinking that has been validated in C57Bl6 mice. The DID procedure takes advantage of the most active circadian period in mice, which is 3 hours into the dark cycle. During this time, we replace the animal’s water bottle with a sweetened alcohol solution or a non-alcohol containing sweetened solution for 2-4 hours each day, leading to pharmacologically significant blood alcohol levels. In the present study, we manipulated the ghrelin system by 1) blocking the ghrelin receptor and 2) sequestering biologically-active ghrelin. First, we investigated six GHSR-1a blockers for their ability to block binge drinking. The prototype GHSR-1a antagonist JMV 2959 has been shown to decrease drinking in 2-bottle choice and operant models but has not been tested in binge-drinking. Other compounds tested include PF-5190457, PF-6870961, HM-04, YIL-781, which are novel and have not been tested in a model of binge drinking. PF-5190457 is a GHSR-1a inverse agonist and represents the only GHSR-1a blocker that has moved forward to human research in AUD. Of note, in humans, it was recently discovered that PF-5190457 administration leads to a major hydroxy-metabolite, PF-6870961, which has specific activity as a GHSR-1a antagonist. Other GHSR-1a antagonists we tested are HM-04 and YIL-781, and the recently discovered endogenous GHSR-1a antagonist LEAP-2. We hypothesized that pharmacological blockade of GHSR-1a would decrease binge-like alcohol intake in mice of both sexes. Additionally, we performed another experiment that used an anti- ghrelin vaccine to sequester biological activity of the endogenous receptor ligand, acyl-ghrelin. We hypothesized that blocking acyl-ghrelin would decrease binge-like alcohol intake in mice of both sexes. We used 48 male and 48 female C57Bl6 mice of 11 weeks of age, (8-11 male and female mice per compound, per solution). The sample size was chosen based on a power analysis and our previous work. A within-subjects, Latin-square design was employed. Alcohol intake was measured in g/kg of body weight and results were analyzed by two-way ANOVA. Results: Our results showed that systemic administration of JMV 2959 (p < 0.0001), PF-5190457 (p = 0.0002), PF-6870961 (p = 0.0109), and HM-04 (p = 0.0059) reduced alcohol intake in mice of both sexes. YIL-781 reduced intake in male (p = 0.0108) but not female (p = NS) mice. LEAP-2 had no effect on alcohol intake (p = NS). For the cohort of animals who received sweet solution with no alcohol content. PF-5190457, PF-6870961, YIL-781, and LEAP-2 had no effect on intake. JMV 2959 reduced intake of this solution (p = 0.003) as did HM-04 for females (p = 0.0029). HM-04 had no effect on intake of this solution in males (p = NS). In another experiment, anti-ghrelin vaccine administration had no effect on binge drinking compared to sham vaccine administration (p = NS). Finally, to investigate the sedative and locomotor effects of the drug, we performed 2 validated tests of locomotion. The rotarod and circular corridor tests were used to assess motor coordination/ataxia and spontaneous locomotion, respectively. The majority of the drugs had a significant effect on locomotion (p < 0.05). Conclusions: Ghrelin receptor blockade preferentially reduces alcohol intake, and there is little difference in intake between males and females in response to ghrelin receptor blockade. Additionally, circulating acyl-ghrelin has no effect on alcohol intake. Our findings provide novel information that supports the role of the ghrelin system in binge drinking and identifies that system as a potential target for pharmacological interventions against AUD. It appears that the receptor blockade is a more promising approach than modulating the acyl-ghrelin molecule itself. This could be due to the intrinsic activity of the ghrelin receptor, which can be up to 50% without the acyl-ghrelin molecule bound. We also addressed a gap in the literature regarding the paucity of information on the effects of sex on the ghrelin system. Future studies will need to elucidate the biological mechanisms by which the ghrelin system affects alcohol drinking and their modulation by sex and individual differences. Finally, this work has translational relevance as PF-5190457 is the first GHSR-1a inverse agonist that has moved to the clinic for medication development. Keywords: Alcohol Use Disorder - Treatment, Binge-Drinking, Ghrelin, Alcohol Consumption, Ghrelin Receptor Disclosure: Nothing to disclose. P743. Behavioral and Neurobiological Consequences of Chronic Vaporized Δ9-Tetrahydrocannabinol (THC) Self-Administration in Female Rats Catherine Moore, Catherine Davis, Yuma Kitase, Balaji Vijayakumar, Lauren Jantzie, Elise Weerts Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Vaping of cannabis and cannabis extracts containing Δ-9-tetrahydrocannabinol (THC, the primary psychoactive constituent of cannabis) is on the rise. Developing a model of THC vapor self-administration in rodents is critical for increasing our understanding of the behavioral and biological consequences of chronic vaporized THC use. Methods: Female Sprague-Dawley rats (N = 48; 24 per group) were allowed to self-administer either THC vapor (50 mg/ml) or vehicle vapor (VEH: 100% propylene glycol) on an intermittent (e.g., every other day) basis. Initially, rats self-administered 3 s vapor puffs under a fixed ratio 1 (FR1) schedule for 20 sessions. Subsequently, the FR was gradually increased from 1 to 5 (≥5 sessions at each FR). Next, the concentration of THC in the e-liquid was adjusted from 5-200 mg/ml in randomized order (≥5 sessions at each concentration) to assess whether number of THC vapor puffs obtained were titrated in a concentration dependent manner. To evaluate differences in anxiety-like behavior between THC vapor and VEH vapor self-administering rats (N = 10/group), subjects were tested in an open field test 24-hrs following their last vapor self-administration session. At the termination of behavioral studies, rats (N = 3-4/group) were euthanized, and Diffusion Tensor Imaging (DTI) was performed ex vivo on fixed brains. Total length of time of intermittent vapor self-administration prior to brain collection was 8 months. Results: Under an FR1 schedule of reinforcement, VEH animals acquired a greater number of vapor puffs per session compared with THC animals (Mean ± SEM: THC puffs: 8.8 ± 1.1, VEH puffs: 12.2 ± 1.1; F(1,34)=4.51, p < 0.05). However, when the effort required to obtain vapor puffs increased from FR1 to FR5, THC animals maintained their levels of puffs obtained, while the amount of puffs obtained by VEH animals decreased as the FR requirement increased (Drug x FR interaction; F(4,136) = 6.39, p < 0.01). As the THC concentration was adjusted from 5-200 mg/ml, THC animals titrated the number of puffs obtained, self-administering a greater number of puffs at lower drug concentrations (Drug x Concentration interaction; F(1,34) = 22.5, p < 0.001). Rats who self-administered THC vapor displayed increased anxiety-like behavior in the open field test when assessed 24-hrs post-session (t(18)=2.99, p < 0.01). Analysis of DTI metrics revealed decreased microstructure and impaired diffusivity in major white matter tracts, including the corpus callosum, in rats that self-administered THC vapor compared with VEH vapor (fractional anisotropy, FA; Mean ±SD: Control = 0.63 ± 0.02, THC = 0.57 ± 0.03, p = 0.015). Conclusions: THC vapor maintained self-administration behavior in female rats. Chronic THC vapor self-administration resulted in increased anxiety-like behavior and reduced white matter integrity. Preclinical models of vaporized THC self-administration are important for translational value and informing our understanding of the effects of cannabis constituents and consequences of chronic THC vapor exposure. Keywords: THC, Vapor, Diffusion Tensor Imaging (DTI), Cannabis, Drug Self-Administration Disclosure: Nothing to disclose. P744. Repeated Binge-Like Intake of Methamphetamine and the Cathinone Derivative Methyledioxypyrovalerone (MDPV) Produce Differential Effects on Prefrontal Neuroinflammation and Cognitive Flexibility Amanda Acuna, Erin Nagy, Paula Overby, Jonna Jackson-Leyrer, M. Foster Olive Arizona State University, Tempe, Arizona, United States Background: Methamphetamine (METH) abuse is associated with impairments in executive functioning, including working memory, impulse control, and cognitive flexibility, which may result from drug-induced dysfunction of the prefrontal cortex (PFC). Recent evidence suggests that METH produces increases in immune signaling, microglial activation, and neurotoxicity in this region. The current study sought to investigate the ability of repeated binge-like intake of METH to induce neuroinflammation in the PFC, and the potential role of inflammatory processes in METH-induced deficits in cognitive flexibility. For comparison purposes, we also examined the effects of the synthetic cathinone derivative methyledioxypyrovalerone (MDPV), which exerts potent cocaine-like monoamine reuptake blockade and has been reported to induce neuroinflammation and cognitive dysfunction. Methods: Separate groups of male and female rats were allowed to intravenously self-administer either METH (0.05 mg/kg/infusion), MDPV (0.05 mg/kg/infusion), or saline in three binge-like access sessions, each 96-hr in length and separated by 72-hr of forced abstinence in the home cage. Three weeks following the end of the third 96-hr session, brain tissue was harvested for immunohistochemical assessment of microglia and astrocyte density and morphology, or multiplex ELISA analysis of cytokine levels. To assess drug-induced changes in cognitive flexibility, both prior to and after drug access, additional groups of male rats underwent assessment of cognitive flexibility in an Attentional Set Shifting Task (ASST). The effects of the non-steroidal anti-inflammatory drug parecoxib on post-drug ASST performance was also examined. Results: Rats self-administering METH demonstrated increased levels of CXCL1, CXCL2, fractalkine, IFN-gamma, IL-1a, IL-1b, IL-2, IL-6, IL-18, leptin and MCP-1) in the PFC as compared to animals self-administering saline. In rats self-administering MDPV, only increases in levels of IL-6 in the PFC were observed, which were specific to male rats. No differences in PFC astrocyte density were observed, although we found an unexpected decrease in the density of microglia in this region. PFC microglia showed reduced territorial volumes and ramification, suggesting an activated inflammatory state. With regards to cognitive function, animals with a history of METH intake showed deficits in extradimensional shift strategy as compared to prior to METH intake. However, animals treated with parecoxib following METH intake showed no difference in set-shifting ability between pre- and post-METH assessments. Finally, animals with a history of MDPV intake showed no drug-induced deficits in ASST performance, while saline control animals showed a slight improvement in performance during post-intake testing. Conclusions: These results show that neuroimmune activation in the PFC persists several weeks into abstinence following binge-like METH intake, with parallel deficits in PFC-mediated cognitive flexibility. Effects effects of MDPV on neuroimmune activation appear to be less robust. The anti-inflammatory agents parecoxib appeared to attenuate METH-induced cognitive impairments. These findings suggest that targeting neuroimmune response to METH may prove to be effective in facilitating recovery of cognitive function following chronic METH intake. Keywords: Methamphetamine Self-Administration, Synthetic Psychoactive Cathinones, Neuroinflamation, Cytokines, Binge Disclosure: Nothing to disclose. P745. Ethanol Potentiates Fentanyl-Induced Respiratory Depression Renata Marchette, Emma Frye, Lyndsay Hastings, Janaina Vendruscolo, Aidan Hampson, Nora Volkow, Leandro Vendruscolo, George Koob Neurobiology of Addiction Section, NIDA IRP, Baltimore, Maryland, United States Background: Drug overdose deaths involving opioids continue to increase in the United States, now topping 100,000 annually (Centers for Disease Control, 2021). Opioid overdose deaths are primarily due to the respiratory depressant effects of opioids, which inhibit both peripheral and central areas responsible for maintaining respiratory rhythm and flow. Alcohol misuse, which at high doses can result in respiratory depression and death, is also frequently reported in deaths involving heroin and fentanyl with estimates of co-involvement of around 30% in 2017. This is clinically relevant because while naloxone may be very effective in reversing opioid-induced overdoses, its efficacy might be lower for the reversal of overdoses from combined alcohol and fentanyl use. Therefore, the investigation of the interactions of fentanyl and alcohol on respiratory depression and lethality is relevant to help develop targeted interventions for co-occurring alcohol/opioid overdoses. Aim: To characterize the effects of concomitant administration of fentanyl and ethanol on ventilation measures. Methods: Using whole body plethysmography, we analyzed ventilation parameters on a breath-by-breath basis. We have previously observed that fentanyl (12, 25, and 50 ug/kg) reduces minute ventilation, frequency of breathing, and peak inspiratory flow while increasing the duration of the inspiratory time, end-inspiratory-pause and apneic pauses in male and female rats. Therefore, we chose the intermediate fentanyl dose for the current study. Twelve female and 12 male Long-Evans rats underwent intravenous (i.v.) catheter surgery. After habituation to plethysmography chambers, the rats were tested in a within-subjects, Latin-square design with four tests one week apart. In each session, all rats received 5 mL/kg, i.v. of sterile water, fentanyl (25 ug/kg), ethanol (1.18 g/kg), or a combination of fentanyl and ethanol over 1 min. At the end of the experiment, we collected blood for fentanyl and ethanol measurement. Results: Only the combination of fentanyl and ethanol resulted in mortality (~42% females, ~33% males) and the administration of naloxone did not rescue them. When compared to water, treatment with fentanyl, ethanol, and the fentanyl and ethanol combination led to a reduction in minute ventilation (F3,30 = 14.84, p < 0.0001), frequency (F3,30 = 4.03, p = 0.02), peak inspiratory flow (F3,30 = 30.66, p < 0.0001), and an increase in inspiratory time (F3,30 = 17.47, p < 0.0001) and apneic pauses (F3,30 = 5.63, p = 0.003). The fentanyl and ethanol combination led to a more pronounced reduction in minute ventilation than fentanyl alone (p = 0.007) and greater inspiratory time and apneic pauses than fentanyl alone (p = 0.03) or ethanol alone (p = 0.02). There were no significant sex differences in any of the variables analyzed. Conclusions: Ethanol alone induces robust respiratory depression, but a combination of fentanyl and ethanol increases the risk of death. Further investigations will explore what drives the increases in apneic pauses caused by the combination of ethanol and fentanyl and other mechanisms of drug interactions that make their concomitant use lethal in animal models. This research contributes to the neurobiological understanding of simultaneous substance misuse, which will be valuable to future investigations of compounds that seek effective alternatives and complementary approaches to reverse respiratory depression and prevent overdose deaths. Keywords: Opioid Overdose, Ethanol, Whole-Body Plethysmography, Fentanyl, Respiratory Depression Disclosure: Nothing to disclose. P746. A Translational Rodent Model of Gestational Opioid Exposure: Effects of Morphine Compared to Buprenorphine on the Maternal Brain, Maternal Behavior, and Offspring Outcome Susanne Brummelte, Abigail Myers, Lauren Richardson, Chela Wallin, Surbhi Neole, Nejra Kulaglic, Shane Perrine, Mariana Angoa-Perez, Donald Kuhn, Scott Bowen Wayne State University, Detroit, Michigan, United States Background: Due to the opioid epidemic, the number of pregnant women receiving Medications for Opioid Use Disorder (MOUDs), including buprenorphine (BUP) has increased drastically within the last several years. Clinically, BUP produces preferable outcomes for exposed infants as compared to methadone or opioid misuse. However, there is a dearth of knowledge about BUP’s effects on maternal caregiving behaviors and underlying neural networks that are critical during the transition to motherhood. BUP’s mechanism of action (partial mu-agonist/kappa antagonist) varies significantly from morphine’s (full mu-agonist), which may result in a different impact on the maternal brain during a critical neuroplasticity period. Methods: We used a translational rodent model to mimic chronic opioid (mis)use (morphine exposure, 3-6 mg/kg/day, b.i.d.) or opioid maintenance drug (BUP exposure, 1 mg/kg/day) to investigate the behavioral and neurochemical consequences of gestational opioid exposure on dams and their offspring. Opioid or saline administration to female rats (N = 50) via subcutaneous injections began 7 days prior to mating and continued daily throughout pregnancy until postpartum day 2 (PD2) or was discontinued on gestational day 19 to allow for drug clearance before parturition. Dams’ maternal behaviors were monitored through detailed observations of pup-directed and non-pup directed behaviors and dams and pups underwent a series of behavioral tests, including a pup retrieval test, a hunting task, and a two-chamber pup-odor preference test. Pups (both males and females) and dams were sacrificed on postnatal day 2, and brains and trunk blood were collected for subsequent analysis. Data were analyzed with factorial or repeated measures ANOVAs. Results: Our findings indicate that BUP exposure, continued and discontinued, resulted in more maternal care deficits (i.e. less time spent on pup-directed behaviors), increased postpartum pup mortality (9% for BUP, 4.8% for morphine, 1.8% for Veh; p = 0.04 (BUP vs. Veh) and maternal deficiencies in the pup retrieval and pup-odor preference tests, but not in the hunting task, as compared to our control (and partly morphine) groups. Conversely, care behavior and survival rates of the morphine groups varied little from controls. Preliminary results further suggest that each opioid also resulted in a unique change in the microbiome profile of the maternal gut. Using high performance liquid chromatography, we will further analyze neurotransmitter levels and their metabolites in the maternal brain (collected on PD2) to investigate potential neurological effects of these opioids on the maternal brain network. Conclusions: Our results suggest that BUP exposure during pregnancy negatively influences pup survival rates, which may be due to BUP’s unique pharmacological action on the maternal brain interfering with maternal caregiving behaviors. More research is critical to elucidate how BUP mechanistically interacts with the neural network during the transition to motherhood to help alleviate possible negative consequences for mothers and their offspring. Keywords: Opioid Dependence, Pharmacotherapy, Animal Model, Withdrawal, Pregnancy, Maternal Behavior Disclosure: Nothing to disclose. P747. Corticotrophin-Releasing Hormone in the Prelimbic Medial Prefrontal Cortex is Critical for Driving Stress-Enhanced Alcohol Drinking in a Mouse Model of AUD Jennifer Rinker, Sudarat Nimitvilai, John Woodward, Howard Becker, Patrick Mulholland Medical University of South Carolina, Charleston, South Carolina, United States Background: Alcohol use disorder (AUD) is characterized as a chronic and relapsing neuropsychiatric disorder, with stress as one of the main contributing factors that drives relapse. Corticotrophin-releasing hormone (CRH) is a stress neuropeptide that has long been the focus of alcohol research in the context of extended amygdala circuitry. And while alcohol activates the CRH system, these studies often exclude the influence of stress, which might contribute to why recent CRH antagonists in clinical trials have failed to show consistent positive effects. Our understanding of the role of CRH in stress and alcohol is limited to its function in the extended amygdala and the hypothalamic circuitry, however CRH is expressed fairly ubiquitously throughout the cortex and many subcortical regions, including key regions implicated in regulating alcohol consumption like the medial prefrontal cortex (mPFC). Methods: Here we utilize a number of transgenic mouse lines to interrogate the role of CRH in the prelimbic (PL) region of the mPFC in regulating stress-enhanced alcohol consumption. First, we characterized the expression, distribution and molecular phenotype of CRH + neurons in the PL in order to better understand how changes in CRH neuron activity in response to alcohol and stress my be influencing PL activity. We then characterized the effects of chronic stress (forced swim stress, FSS) and alcohol (chronic intermittent ethanol, CIE) exposure on CRH neuron physiology in the PL using Crh-Cre x Ai14 reporter mice and slice electrophysiology recordings. We next utilized Crh-Flox mice to conditionally knockdown Crh expression in the PL and examine the effects on stress-enhanced alcohol consumption using the CIE-FSS model that models chronic stress effects on drinking. Results: Expression of CRH is found nearly evenly distributed throughout layers 2-5 of the PL and infralimbic (IL) cortices of the mPFC, with slightly (but not significantly) denser expression in PL compared to the IL (p = 0.12), and an increasing gradient from anterior to posterior PL. Interestingly we also characterized expression in the paraventricular thalamus (PVT), and saw an increase in expression of CRH across the anterior to posterior axis, and thus will be the focus of future studies in the lab. While, CRH has been reported to be co-expressed in both GABAergic and glutamatergic neurons in the PL, we found that approximately 92% of neurons were GABAergic. The GABAergic-CRH subtype in the PL shows a fast-spiking phenotype and are modestly sensitive to going into depolarization block at higher current injection steps (~33% of neurons recorded). Interestingly, when exposed to the CIE-FSS paradigm, GABAergic-CRH neurons become hyperexcitable, showing decreases in rheobase (p < 0.01) and significant leftward shifts in the dose response curve for current-evoked action potential firing (p < 0.05). Unexpectedly, ~96% of GABAergic-CRH neurons in PL are sensitive to depolarization block at current steps higher than 100pA, which represents a significant shift in excitability in this population. Further, we knocked down Crh in the PL through Cre-Lox excision in the Crh-Flox mice and, as expected, we saw greater than 55% knockdown in functional CRH protein product as confirmed by western blot. Using this knockdown approach, we show a complete blockade of the stress-enhanced escalation of alcohol consumption in Crh-knockdown mice exposed to CIE-FSS compared to Crh-intact CIE-FSS mice and unstressed controls (p < 0.05, and p < 0.01, respectively). Conclusions: Taken together, we have identified the PL mPFC as a critical hub for CRH effects on stress and alcohol interactions. Chronic alcohol and stress recruit CRF in many regions throughout the brain, but GABAergic-CRH neurons in the PL mPFC are particularly sensitive to these effects becoming hyperexcitable in response to chronic stress and alcohol exposure. Hyperexcitability and activity in local CRH circuitry in the PL are crucial for driving stress-enhanced alcohol consumption, as knockdown of Crh in the PL completely blocks this effect. While intact CRH signaling in the PL mPFC is crucial, future experiments will examine synaptic changes induced by excessive CRH signaling in response to chronic stress and alcohol to identify additional druggable targets for treatment of this sensitive population. Additionally, it might be worthwhile to examine CRH antagonists again as potential treatments for AUD with an emphasis on individuals suffering from co-morbid exposure to extreme stress. Keywords: Alcohol Dependence, Acute and Chronic Stress, Corticotropin-Releasing Hormone, Corticotropin-Releasing Factor Disclosure: Nothing to disclose. P748. Alcohol and Orexin Effects on VTA-CeA Circuitry and Anxiety-Like Behavior in Rats Elizabeth Avegno, Shealan Cruise, Nicholas Gilpin Louisiana State University Health Sciences Center, New Orleans, Louisiana, United States Background: Humans with alcohol use disorder (AUD) often experience negative affect during withdrawal, and depressed mood and anxiety are positively correlated with relapse during abstinence. The neural adaptations that occur during the transition to dependence are not entirely understood, but may include a gradual recruitment of brain stress circuitry by mesolimbic reward circuitry that is activated during early stages of alcohol use. We have previously demonstrated that chronic alcohol increases the activity of a circuit between the ventral tegmental area (VTA) to the central nucleus of the amygdala (CeA), regions important for mediating acute alcohol reinforcement and alcohol withdrawal-associated behaviors, respectively, raising the possibility that activation of this circuit mediates increases in anxiety-like behavior during alcohol withdrawal. The mechanism by which the VTA-CeA circuit becomes activated is unknown, but may occur via orexin-mediated disinhibition. Here, we explored the role of the VTA-CeA circuit in mediating aversive or anxiety-like behavior in alcohol-dependent and naïve rats, as well as the role of orexin signaling in mediating circuit activation and behavior. We hypothesized that (1) VTA-CeA activation is aversive and (2) contributes to increased anxiety-like behavior during alcohol withdrawal, (3) that VTA-CeA circuit activation occurs via an orexin-mediated mechanism, and (4) that driving the VTA orexin system alone is sufficient to produce an anxiety-like phenotype. Methods: Adult male and female Long-Evans rats were used in all experiments. To evaluate the role of VTA-CeA in behavioral assays, we used a dual virus approach to isolate CeA-projecting VTA neurons with an intra-CeA injection of a retro-cre virus (pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40) and transfect cells with a cre-dependent excitatory DREADD (pAAV-hSyn-DIO-hM3D(Gq)-mCherry), inhibitory DREADD (pAAV-hSyn-DIO-hM4D(Gi)-mCherry), or inactive control (pAAV-hSyn-DIO-mCherry; n = 3-4/group). To characterize the role of intra-VTA orexin on behavior, orexin A (50 nM) was site-specifically administered in the VTA prior to anxiety-like behavioral assays (n = 7-8/group). Alcohol dependence was induced using a chronic ethanol vapor exposure model, and rats were tested during acute withdrawal. Activity of CeA-projecting VTA rat neurons was measured using retrograde tracing and slice electrophysiology, and anxiety-like behavior was assessed using light/dark box and elevated plus maze behavioral assays. To investigate the mechanism behind altered VTA-CeA physiology in alcohol dependence, we manipulated VTA orexin 1 receptor activity pharmacologically in the above experiments. Results: Preliminary data suggest a role for the VTA-CeA circuit in mediating aversive and anxiety-like behavior in rats. Using a chemogenetic approach to activate the VTA-CeA circuit in otherwise experimentally naïve rats, we demonstrate that circuit activation alone may be aversive, as tested in a conditioned place aversion assay. Additionally, chemogenetic inhibition of VTA-CeA projections may rescue increased anxiety-like behavior during alcohol withdrawal, as measured in a light/dark box behavioral assay. Ongoing experiments are replicating these findings, as well as utilizing pharmacological strategies to investigate the mechanism underlying activation of CeA-projecting VTA neurons. We demonstrate that intra-VTA orexin A administration is sufficient to produce an anxiety-like phenotype in otherwise experimentally naïve rats (p = 0.0236; two-tailed t-test), and preliminary electrophysiological data suggest that VTA-CeA neurons may become activated via an orexin receptor-dependent mechanism. Conclusions: Crosstalk between brain reward and stress systems plays a critical role in behavioral dysregulation induced by alcohol dependence. These studies expand on our published findings demonstrating increased activity of the VTA-CeA circuit in alcohol-dependent, withdrawn rats by exploring the possibility that this circuit mediates some aspects of behavioral dysregulation associated with alcohol dependence. Results of these studies have the potential to expand our understanding of circuitry involved in aversive and anxiety-like behavior, as well as informing therapeutic strategies for individuals with AUD. Keywords: Alcohol and Substance Use Disorders, Orexin, Ventral Tegmenal Area, Central Amygdala Disclosure: Nothing to disclose. P749. Circuit- And Subregion-Specific Effects of Chronic Ethanol Exposure on Medial Prefrontal Cortex Inputs to the Rostromedial Tegmental Nucleus Kathryn Przbysz, Joel Shillinglaw, Shannon Wheeler, Elizabeth Glover* University of Illinois at Chicago, Chicago, Illinois, United States Background: Chronic ethanol induces physiological neuroadaptations in the medial prefrontal cortex (mPFC) that are thought to play an important role in driving maladaptive behaviors that impede recovery. Previous research has focused primarily on mPFC neurons arising from the prelimbic (PL) subregion of the mPFC despite the fact that the infralimbic (IL) subregion is also heavily implicated in addiction. Moreover, the circuit-specificity of ethanol’s effects on the mPFC remain relatively unexplored. The rostromedial tegmental nucleus (RMTg) plays an important role in alcohol-related behaviors and recent work from our lab reveals the presence of dense input from layer V PL and IL mPFC neurons. The present study was designed to investigate the subregion- and circuit-specific effects of chronic ethanol exposure on projection-undefined vs RMTg-projecting mPFC neurons. Methods: Adult male Long-Evans rats were stereotaxically injected with fluorescent retrobeads into the RMTg. After one week of recovery, rats were rendered dependent using a standard 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Rats were euthanized and slices prepared for whole-cell patch-clamp recordings approximately one week after CIE exposure. Recordings were made from both tracer-labeled (RMTg-projecting) and unlabeled (projection-undefined) PL and IL neurons. Injection sites were confirmed after recordings by microscopic inspection of slices containing the RMTg. Results: Current clamp recordings in the PL mPFC revealed that projection-undefined neurons were significantly more excitable than their RMTg-projecting counterparts (p < 0.05). Interestingly, CIE exposure produced a significant increase in excitability in RMTg-projecting PL mPFC neurons but not projection-undefined population (p < 0.05). CIE exposure also significantly increased sEPSC frequency in RMTg-projecting but not projection-undefined PL mPFC neurons (p < 0.001). In contrast, we observed no significant differences in excitability in either RMTg-projecting or projection-undefined IL mPFC neurons between CIE and AIR controls. While CIE exposure had no effect on sEPSCs, it significantly increased sIPSC frequency in RMTg-projecting neurons but not projection-undefined IL mPFC neurons (p < 0.05). Conclusions: Taken together, our data indicate that CIE exposure produces distinct subregion- and circuit-specific effects. These CIE-induced alterations in RMTg-projecting PL and IL mPFC neuron physiology suggest that deficits in mPFC-dependent behaviors due to chronic ethanol may be linked to functional changes in RMTg-projecting PL and IL mPFC neurons. Keywords: Prelimbic Cortex, Infralimbic Cortex, Reward And Aversion, Alcohol and Substance Use Disorders Disclosure: Nothing to disclose. P750. Physiological Changes in Noradrenergic Neurons in a Mouse Model of Opioid Use Disorder Zoe McElligott, Anthony Downs University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States Background: Noradrenergic systems are implicated in playing a role in both the aversion to opioid withdrawal and in opioid reward learning. Previously we have shown that an opioid withdrawal model of opioid use disorder (OUD) enhances norepinephrine (NE) release dynamics in the bed nucleus of the stria terminalis (BNST), a brain region playing important roles in both the rewarding and aversive properties of reinforcing drugs, receiving the densest innervation of NE in the brain, mainly from the medullary A2/Nucleus of the solitary tract (NST). Changes in noradrenergic neurons have been studied in models of opioid use disorder, but have mainly been the pontine A6/locus coeruleus neurons and not those found in the medulla. To begin to explore how opioid exposure and withdrawal may alter the physiology of A2/NST neurons we used a genetic reporter mouse that expresses EGFP in noradrenergic neurons. Methods: Methods were approved by the University of North Carolina IACUC. Female adult (at least 10 weeks in age) dopamine-beta-hydroxylase (DBH)-cre mice were crossed to L10a-EGFP mice (L10a) to label NE neurons. Mice were administered one of 3 different treatment paradigms consisting of 2 injections (s.c.) timed 2 hours apart (first injection between 10-11 AM) across 3 days. The first group was administered a saline injection followed by a naloxone injection (SN). The second group was administered a morphine (10 mg/kg) injection followed by a saline injection (MS). The third group was administered a morphine (10 mg/kg) injection followed by a naloxone injection (1 mg/kg). On day 4, 24 hours following the last injection, mice were euthanized, brains were sliced, and recordings were performed (as; Torruella Suarez et al., 2020; Downs et al., 2022). Results: To explore the impact of opioid withdrawal on noradrenergic function in the A2/NSS noradrenergic neurons. DBH-cre mice were crossed to the L10a line so that noradrenergic (A2) neurons could be differentiated from neighboring neurons in the NTS. To explore adaptations in glutamatergic transmission on to the A2/NST neurons, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) at -80 mV in the presence of picrotoxin (25 uM). While we did not see a change in the amplitude of the events across the groups (one-way ANOVA), there was a trend for a decrease in the frequency of events in the animals that had been exposed to morphine (p = 0.0730). When the area of the currents was further examined (total charge transfer), there was a significant reduction (one-way ANOVA, F(2,28) = 11.84, p < 0.001) in the current area in the MN group as compared to the SN and MS groups (Tukey’s multiple comparisons p < 0.01 and p < 0.001 respectively.) Correspondingly, there was a significant reduction in the decay of these currents in the MN group (one-way ANOVA, F(2,28) = 13.79, p < 0.0001) as compared to the SN and MS groups (Tukey’s multiple comparisons p < 0.0001 and p < 0.01 respectively). We next examined miniature excitatory post synaptic currents (mEPSCs) both at baseline and in the presence of NASPM (50 uM) a blocker of calcium-permeable AMPA receptors (CP-AMPARs). While there was a main effect of the group (F(2,14) = 4.804, p < 0.05) on the amplitude, we found no differences in the average amplitude of the mEPSCs between the groups both in the presence and the absence of NASPM. When we examined event frequency however, there was a main effect of NASPM treatment (two-way repeated measures ANOVA, F(1,28) = 4.441, p < 0.05), however the only group with a significant difference between the baseline frequency and post-NASPM frequency was the MN group (Sidak’s multiple comparisons test, p < 0.05). When we examined decay kinetics we found that there was a significant interaction (two-way repeated measured ANOVA, F(1,14) = 9.225, p < 0.01) between the groups before and after NASPM application such that there was a significant difference only in the MN group following NASPM (Sidak’s multiple comparisons test, p < 0.01). Finally, we examined properties of excitability in the A2/NST neurons. We first examined spontaneous discharge from these neurons and found that there was a significant effect of treatment on the frequency of A2/NST action potentials (one-way ANOVA, F (2, 17) = 4.467, p < 0.05), where the MN group was significantly higher than the SN group (Tukey’s multiple comparisons, p < 0.05) and a trend for a difference between with the MS group (Tukey’s multiple comparisons, p = 0.0818). Cells were then injected with current to maintain them at -75 mV, and a increasing steps of current (delta 20 pA, to a total of 400 pA) were examined to determine the number of action potentials that fire at each current step. Here we found a current by treatment group interaction (F(40, 220) = 3.101, p < 0.0001), and a main effect of treatment group (F(2, 11) = 8.866, p < 0.01), such that MN neurons fired more action potentials compared to the other groups and there were no differences between the MS and SN animals. Conclusions: Together, these data suggest that there are adaptations to morphine exposure and withdrawal that occur in the A2/NST neurons, and that the effects are most dramatic in our model of repeated opioid withdrawal, as compared to mice exposed to morphine without naloxone, or naloxone alone. Our data here suggest that the MN model results in profound plasticity on glutamatergic synapses promotion the insertion of CP-AMPA receptors, and regulates the intrinsic excitability of these neurons. Future studies will explore these phenomena in male mice, and examine how long lasting and/or transient these changes are. Keywords: Norepinephrine, Opioid Addiction, Opioid Dependence, Pharmacotherapy, Animal Model, Withdrawal Disclosure: Nothing to disclose. P751. Altered Cognitive Control and Neurophysiological Signaling Following Rodent Prenatal Alcohol Exposure Sarah Olguin, Valentina Licheri, James Cavanagh, Jared Young, Jonathan Brigman University of New Mexico Health Sciences Center, Albuquerque, New Mexico, United States Background: It is well established that alcohol consumption during pregnancy can lead to poor outcomes in offspring. However, Fetal Alcohol Spectrum Disorders (FASD) are still the most common type of neurodevelopmental syndrome. Based the National Institutes of Mental Health (NIMH) recent focus on examining Research Domain Criteria (RDoC) via quantification of behavior more likely to reflect a specific neurophysiological circuit. Individuals with FASD have consistently been found to have difficulties with the domain of cognitive control. We have recently shown that the touchscreen 5 Choice Continuous Performance Task (5C-CPT) can measure both attention and cognitive control in rodents, but also demonstrates similar cross-species neural signatures when coupled with EEG-recording. Here we examined the impact of a moderate prenatal alcohol exposure model on 5C-CPT, as well as measures of effort and reward learning, performance and EEG signatures in frontal, parietal and motor cortices. Methods: Male and female mice (12-16 per sex/trt) were obtained from the New Mexico Alcohol Research Center where PAE is established via a drinking-in-the-dark model (10% EtOH with 0.066% saccharine for 4 h/day throughout gestation; BAC: ~90 mg/dL; controls=0.066% sweetened water, “SAC”). Beginning at 8-10 weeks, mice were trained to touch white square target stimuli. Once responding rapidly and accurately mice underwent stereotactic surgery and fitted with dura-resting skull screws targeting medial prefrontal, parietal, and motor cortices. After recovery and reminder to criterion, non-target trials (5 stimuli presented; withholding of response was rewarded) were added at a 2:1 ratio for 5 days followed by recording at a 5:1 ratio for 12 days. ANOVA was used to examine main effect of treatment, sex, and interaction effects for dependent variables including behavioral measures of accuracy, impulsivity and false alarm rates as well as EEG measures such as power. Results: During training to target trials, all mice required more sessions to reach criteria as stimulus presentation decreased [Session: p < 0.0001]. However, there was no significant differences on sessions to reach criterion between sex or treatment with no interaction [Sex: p = 0.79; Trt: p = 0.97; Sex x Trt x Session: p = 0.89]. When nontarget trials were introduced for the 5C-CPT at a 2:1 ratio for 5 sessions there was a significant difference for sex and treatment, but not for session, and no interaction for hit rate [Session: p = 0.96, Sex: p = 0.04; Trt: p = 0.002; Sex x Trt:, p = 0.06]. The false alarm rate was significantly different for sex and treatment with an interaction, with no main effect of session [Session: p = 0.74, Sex: F(1,140)=17.49, p < 0.0001; Trt: F(1,140)=32.07 p < 0.0001; Sex x Trt: F(1,140)=4.49, p = 0.04]. During EEG recording on a difficult the 5:1 variant, PAE mice were significantly more likely to respond to nontargets than SAC controls [Session: p < 0.0001, Sex: p = 0.59; Trt: p = 0.001] with no interaction [Sex x Trt: p = 0.25], indicative of poor response inhibition to these unpredictable stimuli. EEG Time-frequency analysis revealed strong high frequency enhancement in correct rejection trials compared to target correct trials with an interaction of sex x treatment [ROI: p = 0.20; Sex: p = 0.54; Trt: p = 0.65; Sex x Trt: p = 0.04]. In addition, we found a strong low frequency enhancement in target-post-error response trials compared to target-post-target [ROI: p = 0.005] with no effect of sex, treatment, nor an interaction [Sex: p = 0.31; Trt: p = 0.22 Sex x Trt: p = 0.11]. Conclusions: Utilizing a PAE model that impairs cognitive control, we found that no alterations attention to target-only trials. However, PAE consistently impaired performance when animals were required to withhold responding to rare nontarget trials. Importantly, EEG results showed an increase in frontal theta in both PAE and SAC animals regardless of sex for post-error correction as well as an increase in posterior beta during choice conflict indicative of increased mental effort. Analysis of posterior beta revealed a sex x treatment effect with increased power during correct rejection trials in female PAE mice as compared to target correct trials indicative of increased mental effort required to differentiate trial types. These results demonstrate 1) that 5C-CPT can detect post-error signal changes consistent with findings in human EEG studies and 2) that alterations in cortical activity via EEG may be used as a potential biomarker of PAE. Keywords: Cognitive Control, EEG Biomarkers, Touchscreen Disclosure: Nothing to disclose. P752. Prelimbic Neuron Calcium Activity Predicts Perceived Hedonic Value Across Drinking Solutions and Alcohol Dependent States in Mice Patrick Mulholland, Munir Kutlu, Erin Calipari, Jennifer Rinker Medical University of South Carolina, Charleston, South Carolina, United States Background: Binge alcohol (ethanol) drinking and per capita alcohol consumption are increasing, as are alcohol-related hospitalization rates and deaths. The prefrontal cortex (PFC) encodes anticipatory and goal-directed behaviors, and prolonged alcohol misuse impairs function of the PFC and shifts behaviors toward those that bias alcohol-seeking over natural rewards. Because of the importance of neural activity in the prelimbic (PrL) cortex during active ethanol drinking and adaptations in the PrL produced by alcohol dependence that drive excessive and compulsive ethanol-seeking behaviors, the first goal of this study was to determine population activity in PrL glutamatergic projection neurons surrounding drinking for different rewarding solutions and how alcohol dependence alters PrL activity during voluntary alcohol drinking and alcohol-bias for natural rewards. A second focus of this study was to determine how adulterating alcohol with quinine changes the neural activity pattern of PrL neurons before and after induction of dependence. Methods: To achieve these goals, we used fiber photometry recordings of GCaMP6f calcium transients to define PrL neural activity patterns surrounding licking bouts while C57BL/6 J mice were actively drinking water, alcohol (20%, v/v), and sucrose (1%, w/v) in their home cages. Next, we determine how chronic intermittent ethanol (CIE) exposure altered alcohol drinking and choice behaviors for alcohol over natural rewards in male and female mice. We also assessed neural activity patterns surrounding binge alcohol drinking in the presence of quinine. Because machine learning (ML) can predict neural activity from behavior, we applied two different ML algorithms – support vector machine (SVM) and extreme gradient boost (XGBoost) classifiers – to determine if the signature of neural activity preceding drinking behavior can predict the hedonic value across three different solutions and the alcohol dependence-induced change in the PrL signal. All data were analyzed using mixed linear models. Results: Mice consumed more sucrose than water and alcohol, and licking bouts occurred during prolonged up-states in the GCaMP6f signal that were higher for sucrose and alcohol compared to water. Consistent with evidence of increased neural activity during goal-directed behaviors, we observed an increase in the PrL GCaMP6f population signal preceding drinking bouts. The GCaMP6f signal for sucrose was significantly higher than alcohol and water, and the signal for alcohol was significantly higher than water. Contrary to our hypothesis that PrL→NAcore circuitry would encode for alcohol over water, the GCaMP6f signal surrounding licking bouts for alcohol were similar to those for water. Using the population signal, the SVM significantly predicted when mice were drinking water, alcohol, and sucrose with high accuracy (p < 0.001) within individual drinking sessions, and the XGBoost classifier demonstrated modest, but highly significant (p < 0.001) accuracy for predicting the three drinking solution from one another across drinking sessions. CIE exposure increased alcohol drinking and produced a bias toward alcohol over sucrose. Over time, the signal preceding water, alcohol, and sucrose drinking bouts decreased in non-dependent mice, but the signal preceding alcohol bouts remained elevated in dependent mice. The addition of quinine to the alcohol drinking solution also decreased the signal in controls, but not in CIE exposed mice, and the SVM significantly predicted the quinine-induced change in the calcium signal prior to, but not after CIE exposure. Conclusions: In this study, we provide experimental and computational evidence from supervised machine learning classifiers that PrL population activity patterns track the perceived hedonic value of rewarding solutions in C57BL/6 J mice voluntarily drinking in a home cage model. One striking finding from these studies is that ramping of PrL activity predicts drinking behavior for three solutions with differing hedonic value. Moreover, we showed that mice predominantly consumed fluid when the PrL population signal was in a prolonged upstate, and features of the pre-bout ramping signal could accurately discriminate between the three drinking solutions. Consistent with a declining VTA signaling for sucrose licking within a single drinking session, we observed a decrease in the peak calcium signal for all solutions across time. Another main finding from our study is that the pre-bout PrL signal parallels escalated and compulsive drinking behavior in a mouse model for the study of AUD. Our results provide evidence for a functional signature in the PrL cortex that aligns with the salience of reward-related behaviors likely signaling the intention to drink, even when mice are rendered dependent upon alcohol. Finally, we identified an aberrant intention signal in the PrL cortex of dependent mice that likely serves as a mechanism driving compulsive-like alcohol drinking and alcohol-biased choice behaviors. Keywords: Alcohol, Medial Prefrontal Cortex, Compulsive Behavior, Fiber Photometry, Machine Learning Disclosure: Nothing to disclose. P753. Heroin-Seeking Behavior is Regulated by Phospholipase Cgamma1 in the Nucleus Accumbens Ethan Anderson, Makoto Taniguchi, Christopher Cowan Medical University of South Carolina, Charleston, South Carolina, United States Background: Chronic opioid use leads to long-lasting increases in drug-seeking behavior; however, the causal molecular and cellular mechanisms responsible are not fully understood. One mechanism may involve the brain-derived neurotrophic factor (BDNF) signaling pathway through its activation of phospholipase Cgamma1 in the nucleus accumbens (NAc). BDNF mRNA levels are oppositely regulated following chronic heroin exposure vs withdrawal, suggesting it may play a role in opioid-related behavior during both these phases. Here we show that endogenous PLCgamma1 in the rat nucleus accumbens (NAc) can both limit and enhance the development of relapse-like heroin seeking depending on the phase of the self-administration paradigm when PLCgamma1 is manipulated. Methods: We first infused a shRNA expression viral vector that reduces PLCgamma1 levels (AAV-shPLCgamma1) bilaterally into the NAc of both male and female rats using stereotaxic surgery. Three weeks later we allowed the rats to self-administer heroin for at least 12 days. Following a 7-day abstinence period, we measured context-associated heroin seeking during extinction conditions. In a separate experiment, we first allowed rats to acquire heroin self-administration for 12 days, then rats underwent a stereotaxic surgery to infuse AAV-shPLCgamma1. After 3 weeks of abstinence, we again measured context-associated heroin seeking during extinction conditions. Finally, we also examined the effects of AAV-shPLCgamma1 infused before the acquisition of sucrose-taking to determine if the effects on heroin-seeking were generalizable to non-drug rewards. All experimental protocols in animal studies were approved by the Medical University of South Carolina’s Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Results: We found that reducing PLCgamma1 in the NAc prior to the start of aquiring self-administration behavior led to an eventual increase in heroin-seeking behavior. Since we reduced PLCgamma1 levels in the NAc prior to the initiation of heroin self-administration, we next examined whether reducing PLCgamma1 after cessation of self-administration would also increase heroin-seeking behavior. Surprisingly, reducing PLCgamma1 levels after the self-administration phase suppressed context-associated heroin seeking, an effect opposite to when PLCgamma1 was reduced prior to the acquisition of heroin self-administration. In contrast to the significant effects on heroin-seeking, no obvious effects were observed for sucrose-seeking behavior. In addition, no obvious sex differences were observed in these experiments. Conclusions: These results strongly suggest that PLCgamma1 plays different roles during different phases of heroin self-administration: acquisition, abstinence, and context-associated heroin seeking. Moreover, our findings suggest that therapeutics targeting PLCgamma1 function might be helpful for treating relapse vulnerability in individuals suffering from opioid use disorder. Keywords: Heroin Self-Administration, Extinction and Reinstatement, Nucleus Accumbens Disclosure: NeuroEpigenix, LLC: Founder (Self). P754. Parabrachial Extended Amygdala Circuit Activity is Heightened Following Repeated Stress: Implications for Alcohol Abstinence Anel Jaramillo, Laith Kayat, Nicholas Petersen, Bretton Nabit, Samuel Centanni, Danny Winder Vanderbilt University, Nashville, Tennessee, United States Background: Forced abstinence in a mouse preclinical model of chronic ethanol vapor exposure (CIE) increases anxiety-like behavior and produces neuronal adaptations in the bed nucleus of the stria terminalis (BNST), a region critical for affective behavior. Lateral parabrachial nucleus (LPBN) projections expressing calcitonin gene related protein (CGRP, Calca gene) drive bed nucleus of the stria terminalis (BNST) in vivo activity in synchrony with anxiety-like behavior. We hypothesize an anxiogenic role for LPBN(CGRP) in alcohol abstinence and stress, associated with heightened BNST activity. Methods: All procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and institutional guidelines approved by the Institutional Animal Care and Use Committee at Vanderbilt University. Male and female CalcaCRE mice (n = 5-10/sex/ virus group) received bilateral injections of CRE-dependent hM3D(Gq) DREADDs or CRE-dependent mCherry fluorophore in the LPBN. Anxiety-like behavior was measured with the elevated plus maze (EPM) following LPBN(CGRP) hM3D(Gq) activation by CNO (clozapine-n-oxide, 3 mg/kg, IP; 30 min pretreatment). To investigate the role of LPBN(CGRP) activation on stress-induced anxiety and BNST activity, CalcaCRE male mice (n = 3/exposure/treatment) received bilateral injections of CRE-dependent hM3D(Gq) DREADDs in the LPBN and the calcium indicator GcAMP7 in the BNST. Following 4 days of repeated forced swim test stress paired with LPBN(CGRP) hM3D(Gq) activation, anxiety-like behavior was measured with NSFT. To identify cell-specific changes in LPBN(CGRP)-- > BNST neurotransmission BNST-containing brain slices were bath applied CNO (10uM) and changes in GCaMP fluorescence were measured with slice photometry recordings. Post hoc thick slice immunohistochemistry was performed to characterize LPBN(CGRP) projections innervating BNST cells. Lastly, to investigate LPBN(CGRP) inhibition on anxiety-like behavior CalcaCRE male and female mice received bilateral injections of CRE-dependent hM4D(Gi) DREADDs in the LPBN and were exposed to chronic intermittent ethanol vapor exposure (CIE; n = 4-8/sex/ vapor exposure). In early CIE withdrawal (4-6 hr) mice were given CNO to activate LPBN(CGRP) inhibitory hM4D and tested in EPM. To investigate stress and anxiety during prolonged abstinence (2 wks), mice underwent 4 days of (FSS) and anxiety-like behavior measured by novelty suppressed feeding task (NSFT). Results: LPBN(CGRP) activation increased EPM time immobile in hM3D(Gq) relative to mCherry group (2-way ANOVA virus p = 0.03). LPBN(CGRP) activation paired with FSS increased body rotations across time on day 3 (2-way ANOVA time x treatment p < 0.02) with no changes in NSFT behavior. Ex vivo slice recordings demonstrate LPBN(CGRP) activation decreased global BNST spike frequency through recruitment of inhibitory and excitatory neuronal populations. This LPBN(CGRP)-induced decrease in BNST activity was potentiated in FSS relative to no stress controls (3-way ANOVA, wash, stress, treatment p < 0.02). Post hoc analysis demonstrates colocalization of pituitary adenylate cyclase activating (PACAP)-expressing LPBN(CGRP) projections in the BNST. In early withdrawal LPBN(CGRP) inhibition increased EPM immobility in CIE relative to air vapor exposure (2-way ANOVA vapor p = 0.05; treatment p = 0.01). In prolonged withdrawal, LPBN(CGRP) inhibition increased total body rotations relative to saline controls on FSS day 2 (2-way ANOVA, p = 0.004). A history of LPBN(CGRP) inhibition increased NSFT immobility relative to saline controls (2-way ANOVA treatment p = 0.05). Conclusions: These data demonstrate LPBN(CGRP) modulates stress and anxiety-like behavioral responses associated, in part, with heightened BNST activity. Additionally, the data demonstrate a history of repeated LPBN CGRP neuron inhibition is anxiogenic in prolonged abstinence suggesting long lasting LPBN(CGRP)-induced changes in behavior. Given pharmaceutical treatments for migraines targeting CGRP inhibition are bioavailable, these studies inform a potential role for CGRP treatments in alcohol-abstinence. Keywords: Alcohol Abstinence, Anxiety and Stress, Anxiety Circuitry, BNST, Parabrachial Nucleus Disclosure: Nothing to disclose. P755. Cell-Type Specific Ca2 + Dynamics in the Nucleus Accumbens Underlying Natural Reward Seeking Behavior Reda Chalhoub, Camille Carthy, Jordan Hopkins, Peter Kalivas Medical University of South Carolina, Charleston, South Carolina, United States Background: The Nucleus Accumbens core (NAc) plays a central role in reward seeking and cue-reward associations. Its constituents Dopamine Receptor type 1 and 2 expressing GABAergic medium spiny neurons (D1- and D2- MSNs) integrate cortical and limbic inputs to regulate reward seeking behavior. The downstream effects of these adaptations on D1- and D2- MSNs, and the information by these neuronal populations during reward seeking, remains largely unknown. Methods: We recorded single-cell calcium Ca2+ dynamics in D1- and D2- MSNs in freely behaving Drd1- and Drd2-cre transgenic mice using a miniature microscope (nVista) and virally expressed Cre-dependent Ca2+ indicator (syn- GCaMP6f). Mice were trained to self-administer sucrose in daily 2 h-sessions during which a single nose-poke (FR1) resulted in an intravenous cocaine injection and presentation of drug-contingent cue. Natural reward seeking was assessed after 10 days of abstinence, during which cues associated with the natural reward were present, but the reward was omitted. The same behavioral paradigm was also used to assess the effect of direct time-locked optogenetic stimulation of D1- and D2- MSNs (20 Hz, 1 s) expressing ChR2(H134r) on reward seeking behavior. All recordings were motion corrected and normalized to background, and PCA/ICA algorithm was used to extract individual cell locations and traces. Individual neuronal traces were aligned to relevant behavioral events, and further analyzed using custom MATLAB codes. Results: While time-locked optogenetic stimulation of Dl- or D2-MSNs (n = 4 per group, p < 0.05) respectively potentiated or halted reward seeking activity, overall recorded calcium activity shows an increase in D2-MSNs activity, compared to D1-MSNs following reward seeking (ANOVA, n = 150-200 cells, p < 0.01). At a single cell level, both Dl- (n = 5, ~200-250 cells) and D2- (n = 4, ~180-200 cells) MSNs showed a highly heterogenous response around a seeking epoch (5 seconds before to 10 seconds after a seeking nosepoke). 30-40% of D1- or D2-MSNs exhibit a stable divergent (equally distributed between excitatory and inhibitory ensembles) time-locked response associated with cued-seeking across trials. This divergent activity profile around seeking epochs is absent early in training and develops progressively over training in both population of neurons. Conclusions: This data challenges the functional dichotomy of the direct/indirect pathways hypothesis and suggest a dynamic interaction between both cell types to regulate reward seeking. Keywords: Reward, Nucleus Accumbens, in Vivo Calcium Imaging Disclosure: Nothing to disclose. P756. Paternal Cocaine Self-Administration Enhances Fear-Related Memory and Inhibits Amygdala Synaptic Activity Only in Male Offspring Matthew Rich, Samantha Worobey, Sharvari Mankame, Zhiping Pang, Sarah Swinford-Jackson, Chris Pierce Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, United States Background: Cocaine self-administration by male rats induces epigenetic modifications in sperm resulting in neuronal and behavioral alterations in offspring including decreases in cocaine taking, enhanced anxiety, spatial memory deficits, and reductions in hippocampal synaptic activity. Given the high degree of overlap between the brain systems resulting in pathological responses to cocaine and stress, we examined whether sire cocaine taking would influence fear-associated behavioral and physiological effects in drug-naïve male and female progeny. Methods: Adult male Sprague-Dawley rats were trained to self-administer cocaine for 60 days while a second group of male rats received yoked saline infusions. Rats then mated with drug-naïve female rats to create an F1 generation of saline- or cocaine-sired offspring. Both male and female F1 rats were then subjected to amygdala-dependent auditory fear conditioning and cue extinction. Separate groups of rats underwent electrophysiological analyses of amygdala synaptic activity. Fear-associated memories were generated by exposing rats (n = 8-9 per group) to 3 presentations of an audio tone (conditioned stimulus) that co-terminated with a 2 second, 0.75 mA footshock. Each presentation was separated by a 1-minute intertrial interval, during which we recorded the animal’s freezing response. 24 hours later, rats were placed in a novel context (different floor, odor, and visual cues) and underwent cue extinction, which consisted of 18 tone-only presentations. Freezing was measured in response to each tone presentation, averaged across 3 consecutive trials. On Day 3, rats received 18 more tone-only presentations in the extinction context. For electrophysiology experiments, we performed whole-cell patch clamp recordings in lateral amygdala principal neurons (n = 15-21 neurons, 5-7 rats per group) and compared baseline measures of synaptic activity (spontaneous activity, AMPA:NMDA, paired-pulse ratio [PPR]) by stimulating the external capsule, which carries information from cortical sources such as the medial prefrontal cortex and auditory cortex. We used a spike-timing dependent plasticity protocol ([0.1 ms presynaptic pulse followed by a 1 ms postsynaptic spike, separated by 10 ms] delivered at 1 Hz for 100 sec) to induce long-term potentiation at these synapses. Fear conditioning experiments were analyzed using two-way repeated measures analysis of variance (RM-ANOVA) with the within-subjects factor being time (CS trial) and the between-subjects factor being sire. Electrophysiological comparisons were made with either unpaird t-test or RM-ANOVA. Results: Similar to other studies, these experiments revealed a sex-dependent effect of sire, whereby cocaine-sired males but not female rats generated stronger fear-associated memories. There were no differences in the acquisition of auditory fear, with equivalent levels of freezing observed in both saline-sired and cocaine-sired offspring. On Day 2, both cocaine- and saline-sired male rats exhibited a high level of initial freezing that was subsequently extinguished across the session. However, cocaine-sired male rats spent a significantly higher percentage of time freezing relative to saline-sired rats. The total Day 2 extinction data, analyzed by an unpaired t-test, indicated a significant difference between groups [t(100)=4.537, p < 0.0001]. On Day 3, there was again slightly higher initial freezing in the cocaine-sired offspring, however by the end of the session both groups exhibited similar degree of extinction. Taken together, these data are indicative of a stronger cue-evoked fear memory in cocaine-sired male rats that persists longer and is therefore more resistant to extinction. These phenotypic differences were absent in female offspring; saline- and cocaine-sired females had equivalent levels of freezing across all phases of behavioral testing. In male rats that had not been subjected to fear conditioning, we found no differences in baseline measures of synaptic activity, but did observe differences in LTP induction. In saline-sired rats, there was a robust LTP induction as indicated by an increase in EPSC amplitude compared to baseline recordings, that persisted for more than 45 minutes post-induction. However, there was no increase in EPSC amplitude in amygdala synapses from cocaine-sired animals. 2 way ANOVA indicates a main effect of sire [F(1,12)=13.90, p < 0.0029], suggesting a deficit in LTP induction in cocaine-sired animals. Similar to our behavioral findings, there were no differences in LTP induction in saline vs. cocaine-sired female offspring. Conclusions: In summary, male, but not female, cocaine-sired rats exhibit resistance to extinction of cue-conditioned fear memories and this resilience may be related to deficits associated with cortical-amygdala synaptic LTP. Ongoing experiments are working to further delineate the molecular and physiological mechanisms responsible for the deficits in fear extinction and LTP in cocaine-sired male rats. Keywords: Slice Electrophysiology, Fear Conditioning and Extinction, Cocaine Self-Administration, Amygdala, Transgenerational Disclosure: Nothing to disclose. P757. Activation of Thalamic Inputs to the Dorsomedial Striatum Drives Reinforcement, Place Preference, and Locomotor Activity Michael Hochstein, Doris Chang, Christopher Bouslog, Kidus Amelga, Kari Johnson Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States Background: Glutamatergic thalamic inputs to the striatum produce dopamine release via actions on dopaminergic terminals, and this form of dopamine release is independent of somatic firing of dopamine neurons. There is increasing evidence that dopamine release evoked by somatic firing vs. terminal regulation mechanisms produces unique contributions to behavior. Striatal dopamine release plays important roles in the acute and chronic effects of several classes of psychoactive drugs, but whether psychoactive drugs can differentially impact dopamine-mediated behaviors driven by dopamine neuron firing vs. terminal mechanisms such as thalamically-evoked dopamine release has not been elucidated. Our previous work demonstrated that closed-loop optogenetic stimulation of thalamic inputs to the dorsomedial striatum (DMS) is sufficient to reinforce operant responding in mice. We further assessed reward, reinforcement, and locomotor stimulation driven by this pathway using optogenetic stimulation. Methods: Adult male and female C57BL/6 J mice were injected with AAV9-hSyn-hChR2(H134R)-eYPF (or control virus expressing eYFP) targeted to the anterior intralaminar nuclei of the thalamus to allow optogenetic stimulation of thalamic neurons using channelrhodopsin-2 (ChR2). Optical fibers were implanted in the DMS to selectively activate terminals of DMS-projecting thalamic neurons. In experiment 1, mice were trained to press a lever to receive a one-second train of optogenetic stimulation (455 nm, ~7 mW, 20 Hz, 5 ms pulse width) of thalamostriatal terminals on a continuous reinforcement schedule. Following ten 30-minute training sessions, the effect of within-session changes in stimulation frequency was assessed using 4 stimulation frequencies (presented in ascending order) for 10 minutes each. In experiment 2, we determined whether mice would seek optogenetic stimulation in a real-time place preference test. Optogenetic stimulation (20 pulses, 20 Hz, delivered every 5 s) was delivered in one chamber of a place preference apparatus and the amount of time mice spent in the stimulation-paired side was measured. In experiment 3, we assessed the effects of thalamostriatal stimulation on locomotor activity in an open field. Data were analyzed using unpaired t test or one-way, two-way, or three-way repeated measures ANOVA. Results: In experiment 1, all mice (n = 6) readily acquired lever-pressing behavior when lever presses were reinforced with 20 Hz optical stimulation of thalamic terminals in the DMS. Across ten training sessions, mice escalated lever pressing of the active lever but not the inactive lever (two-way repeated measures ANOVA, lever x session interaction: F(9, 45) = 9.40, p < 0.001). In response to within-session changes in stimulation frequency, mice updated their response rates, with lower stimulation frequencies producing lower rates of responding (one-way repeated measures ANOVA, main effect of stimulation frequency: F(3, 12) = 6.735, p = 0.0065). In experiment 2, mice expressing ChR2 (n = 18) spent more time in the chamber paired with optogenetic stimulation of thalamostriatal terminals during 3 consecutive sessions, but did not exhibit conditioned place preference when the stimulation was discontinued. In contrast, mice expressing YFP (n = 9) did not exhibit real-time place preference for optogenetic stimulation (three way RM ANOVA, virus x stimulation side interaction, F(1,25) = 5.087, p = 0.0331). We also assessed whether thalamostriatal activation can impact locomotor activity in an open field. Compared with mice expressing YFP, optogenetic stimulation in ChR2-expressing mice enhanced locomotor activity in a 20-min open field test by increasing both velocity and distance traveled (distance traveled ChR2 vs. YFP, unpaired t test: p = 0.0263; velocity: p = 0.036; n = 9/group). Effects on locomotion were rapidly modulated during repeated light-on versus light-off epochs in a single open field session (distance traveled, two-way RM ANOVA virus x stimulation interaction: F(1,16) = 11.40, p = 0.0039; velocity, virus x stimulation interaction: F(1,16) = 11.43, p = 0.0038). Thalamostriatal activation selectively enhanced horizontal locomotion, as we did not observe changes in the performance of other natural behaviors (i.e., rearing and grooming). Optogenetic stimulation of thalamostriatal terminals also did not affect avoidance of the center of the open field (ChR2 vs. YFP, unpaired t test: p = 0.69). Conclusions: Together with our previous findings, these results suggest that activation of thalamostriatal neurons can support both reward and reinforcement learning, while also promoting locomotion. We have also established a model of optogenetic intracranial self-stimulation of thalamostriatal terminals that will allow investigation of the effects of psychoactive drugs on reward and behavioral reinforcement driven by this pathway. Future studies will investigate the potential roles of this circuit in reward, reinforcement, and psychomotor stimulation produced by psychoactive drugs. Keywords: Dorsomedial Striatum, Thalamus, Reward Circuitry, Reinforcement Learning, Locomotor Activity Disclosure: Nothing to disclose. P758. Activity in the Lateral Habenula Predicts Avoidance of Fentanyl Withdrawal Associated Contexts and is Modulated During Oral Fentanyl Self-Administration Kevin Coffey, William Nickelson, Amina Khan, John Neumaier University of Washington, Seattle, Washington, United States Background: Opioid abuse has reached epidemic proportions in the United States, with the synthetic opioid fentanyl involved in almost half of the 40,000 opioid related deaths each year. Fentanyl produces rapid and potent euphoria while withdrawal is severe, causing significant physical and emotional turmoil. Both the positive and negative emotional experiences associated with fentanyl use are strong motivators of relapse. Though many brain structures and circuits are involved in opioid seeking, a compelling node for study is the Lateral Habenula (LHb), a small brain structure with an outsize impact on decision making. The LHb receives convergent input from across the basal ganglia and the limbic system and projects strongly to several key midbrain nuclei involved in decision making. This circuitry makes the LHb poised to integrate cognitive and emotional stimuli with both positive and negative valance, and to influence behavior. Using fiber photometry, this study aims to explore the role of the LHb in two unique components of relapse, 1) avoidance of negative emotional states associated with withdrawal modeled by conditioned place aversion to naloxone precipitated withdrawal, and 2) drug seeking in response fentanyl associated cues, modeled by oral fentanyl self-administration and cued reinstatement testing. Methods: Fiber Photometry: 500nL of AAV1-Syn-jGCaMP7f was injected into the LHb and an optical fiber (400 µm, NA0.66) was placed directly above the injection. An isosbestic 405 nm LED as well as the signal generating 465 nm LED were used to eliminate artifacts. Event related calcium transients (ΔF/F) were analyzed around behavioral events including chamber transitions, lever presses, head entries, etc. Conditioned Place Aversion (CPA): On day 0, rats were allowed free access to both sides of a CPA test chamber for 15 minutes. On days 1-5, rats underwent rapidly dose-escalating fentanyl (or vehicle) injections (2x daily; 200, 400, 600, 800, 1000 µg/kg ip). On day 6, animals received their morning fentanyl injection, then 2 hours later received a saline injection and were confined to one side of the CPA chamber for 1 hour. After returning to their home cage for 3 hours, animals received an injection of naloxone (0.3 mg/kg ip) and were confined to the other side of the chamber for 1 hour. On day 7, animals were allowed free access to both sides of the CPA chamber for a 15-minute test session. Oral Fentanyl Self-Administration (SA): Three days prior to SA, rats were pre-exposed to 0.1% saccharin solution overnight in their home cage. Male and female rats (n = 8,8) were trained to lever press (FR1) for liquid fentanyl (70 μg/mL) in Hydropac H20 (0.15 ml/kg/delivery) for 3 hours a day, 5 days a week, over 3 weeks. The first 5 days of SA employed a “saccharin fade” where saccharin concentration quickly fell (0.1%, 0.08%, 0.06% 0.04%, 0.02%). Upon active lever press, liquid was delivered paired with a compound stimulus (CS) consisting of illumination of a lever light (1 s) coincident with a 4.5 kHz tone (10 s) and extinction of the house light (10 s), during which a time-out was imposed. Beginning week 4, rats underwent extinction training for 7 days. After extinction, animals underwent cued reinstatement testing signaled by a CS presentation. Repeated measures ANOVA were used to assess change in behavior across SA, and ANOVA were used to compare extinction to reinstatement. Results: Conditioned Place Aversion: Naloxone reliably produces withdrawal in both male and female rats that have undergone escalating fentanyl injections (f(1,29)=7.71, p < 0.01). During CPA testing, LHb activity increases during trials where approaches from the saline to the naloxone paired chamber are halted, and trials where rats successfully exit the naloxone paired chamber. Oral Fentanyl Self Administration: Both male and female rats self-administer escalating quantities of fentanyl across 3-weeks of self-administration (F(14,154)=12.76, P < 0.01). The latency between lever pressing and head entry also decreased significantly over weeks (F(14,154)=4.96, P < 0.01). Conditioned cues were sufficient to induce reinstatement of fentanyl seeking in males and females following 7 days of extinction training, as evidenced by increased lever pressing (F(1,24)=16.81, P < 0.01) and head entries (F(1,24)=15.1, P < 0.01), and decreased head entry latency (F(1,24)=-7.15, P = 0.013). LHb activity increases after lever presses and decreases during rewarded head entries. If head entries go unrewarded, LHb activity rebounds rapidly. Conclusions: The lateral habenula is engaged during conditioned place aversion testing and oral fentanyl self-administration, though the exact nature of that activity and whether it is necessary for the expression of these behaviors is still unclear. While recording from the LHb holistically, we noticed some heterogeneous responses across animals that may be due to subtle variations in GCaMP7f expression profiles, or a mix of increases and decreases in activity. In the future we aim to record from individual neurons in the LHb while optically tagging their projections to the midbrain so we can capture circuit specific increases and decreases in activity, and ultimately manipulate those circuits to try to reduce conditioned place aversion and reinstatement to fentanyl seeking. Keywords: Fentanyl, Oral Self-Administration, Fiber Photometry, Lateral Habenula, Opioid Withdrawal Disclosure: Nothing to disclose. P759. Amygdala-Cortical Circuit Determinants of Social Isolation-Induced Alcohol Consumption Reesha Patel, Aniek von Hoek, Makenzie Patarino, Kelly Kim, Felix Taschbach, Hao Li, Christopher Lee, Raymundo Miranda, Kanha Batra, Laurel Keyes, Avraham Libster, Romy Wichmann, Marcus Benna, Kay Tye Salk Institute for Biological Studies, La Jolla, California, United States Background: Although there are correlations between social isolation and increased alcohol consumption, as further supported by the surge in alcohol sales and use during the COVID-19 pandemic, little is known about the neurobiological mechanisms underlying these phenomena. What brain changes are induced by social that trigger alcohol drinking? Methods: To address this question, we used a combination of behavior, ex vivo patch electrophysiology, optogenetics, cellular resolution calcium imaging, and machine learning. Results: We found that social rank predicts alcohol drinking (r2 = 0.25; p = 0.01; n = 26), where subordinates drink more than dominants (p < 0.01; n = 3/group), and that social isolation further increases alcohol drinking in all mice (**p < 0.001; n = 14), while decreasing sucrose drinking (p < 0.05; n = 6). We then found that social isolation increases neural excitability in the basolateral amygdala (BLA) (p < 0.05; n = 13-14 cells), and stimulating BLA terminals in the medial prefrontal cortex (mPFC) is sufficient to increase consumption of alcohol (*p < 0.001; n = 6/group). To reveal how social isolation modifies BLA representations of alcohol, we used longitudinal cellular-resolution calcium imaging and machine learning. We found that alcohol responsive amygdala functional clusters turnover following social isolation (n = 309 neurons/ 6 mice). To determine the impact of amygdala functional turnover on representation of alcohol, we used a generalized linear model (GLM) and found population-level amygdala dynamics was sufficient to decode alcohol verses water consumption, and social isolation increases GLM decoding performance. In contrast, social isolation decreases GLM decoding performance of sucrose verses water consumption, consistent with the diametrically opposing effects of social isolation on alcohol and sucrose consumption. To then determine how amygdala inputs can modify mPFC representations of alcohol, we combined optogenetics and imaging and found that amygdala-cortical terminal activation abolishes positive valence responses to sucrose without altering negative valence responses to shock (n = 708 neurons/ 6 mice), suggesting that the amygdala-cortical circuit induces a negative affective or loneliness-like state by inhibiting positive encoding cortical neurons which may motivate alcohol use. Conclusions: Together, we identified a cellular substrate of social isolation and resolved a role for the amygdala-cortical circuit in social isolation-induced escalated alcohol drinking. Keywords: Basolateral Amygdala, Social Isolation, Alcohol, Prefrontal Cortex Disclosure: Nothing to disclose. P760. Cocaine Induced Neurophysiological Alterations in Corticostriatal Circuits to Reward Predictive Cues Following Outcome Devaluation Mark Niedringhaus, Leighelle Adrian, Timothy Sloand, Elizabeth West Rowan University, Stratford, New Jersey, United States Background: The ability to alter behavior in response to changes in consequences is necessary for navigating an ever-changing environment. Substance use disorders (SUDs) are characterized by a continuation of maladaptive behavior despite negative consequences. Thus, characterizing the underlying processes that modulate the ability to change or stop behavior in response to updated expected outcomes is critical for understanding the neurobiological alterations in SUDs. A history of cocaine exposure leads to impaired ability to adjust behavioral responding to a decrease in expected outcome value following a devaluation procedure in rats. Here, we investigated how cocaine exposure followed by withdrawal leads to aberrant, differential patterns of neural activity in subregions of the rat frontal cortex (prelimbic, PrL vs infralimbic, IL) and striatum (ventral vs dorsal striatum) to reward predictive cues following a decrease in expected outcome value. Methods: Mildly water deprived Long-Evans rats either underwent self-administration for cocaine (i.v., 1 mg/kg/press, cocaine group) or self-administration for water (0.2 ml into a water trough) and saline (i.v.) as a control group. Rats were either implanted with unilateral microwire arrays in the PrL and nucleus accumbens core (NAc) subregion of the ventral striatum (n = 13 cocaine, n = 8 control) or unilateral microwire arrays in the IL and dorsolateral striatum (DLS) subregion of the dorsal striatum (n = 6 cocaine, n = 7 control). Rats underwent self-administration daily for 2-hour sessions over 14 days (~22 mg of cocaine per session). After 3 weeks of abstinence, rats underwent Pavlovian conditioning for 10 days. They were presented with two distinct cue light patterns as conditioned stimuli (CS+), each predicting a different reward (grain or sucrose pellet). One CS + predicted a sugar pellet and the other CS + predicted a grain pellet (10 trials each). Two other light patterns did not predict a reward (CS-). After 10 days of conditioning, rats underwent a devaluation procedure for the sugar pellets to induce a conditioned taste aversion (LiCl, i.p., 0.3 M; 7.5 ml/kg). Post-devaluation, rats were tested on the same Pavlovian task to evaluate their ability to avoid the CS + previously paired with the devalued outcome vs nondevalued outcome in the absence of the rewards (under extinction). Results: PrL, IL, NAc and DLS electrophysiological recordings showed neuronal populations that were phasic to CS + [excited or inhibited] or nonphasic (no response to CS+). In the PrL, there were no differences in the overall responding to NonDevalued and Devalued cues in cocaine and control rats. In the NAc, rats with a history of cocaine showed more phasic neurons to the devalued CS + (29% inhibited and 23% excited) compared to NonDevalued CS + (22% inhibited and 13% excited). In contrast, control rats show similar % of phasic to both Devalued (10% inhibited and 7% excited) and NonDevalued (18% inhibited and 7% excited) CS + . In the DLS, we found a higher % of excited phasic neurons compared the control group in the DLS to both Devalued CS + (cocaine 37% and control 14%) and NonDevalued CS + (cocaine 26% and control 14%). In the IL, we found similar pattern as the DLS in that the cocaine group showed higher % of excited phasic neurons compared to the control group in response to both Devalued CS + : (cocaine 18% and control 3%) and NonDevalued CS + (cocaine 18% and control 7%). Conclusions: Here, we show that the NAc neurons respond more to the devalued CS + compared to NonDevalued CS + in cocaine rats compared to control rats, while we found elevated neural activity, particularly excited neurons, in the IL and DLS after a history cocaine to reward predictive cues. In contrast, we did not observe differences in PrL neural activity to NonDevalued and Devalued CS + after a history of cocaine. Together these findings suggest overactive neural encoding, particularly to the devalued CS + in the NAc, DLS, and IL. One possibility is this overactive neural responding to reward predictive cues post-devaluation contributes to the inability to disengage from the cues that predict the devalued outcome. Future studies will aim to determine if these changes in neural activity in these regions after history of cocaine are causally linked to impaired ability to shift behavior following outcome devaluation. Keywords: Dorsolateral Striatum, Infralimbic Cortex, Prelimbic Cortex, Nucleus Accumbens, Pavlovian Conditioning Disclosure: Nothing to disclose. P761. Stress-Induced Escalation of Cocaine Intake is Modulated by Sex and Endocannabinoid Signaling Jayme McReynolds, Andrew Gaulden, Erin Tepe, Sierra Rollins, Cecilia Hillard, John Mantsch University of Cincinnati, Cincinnati, Ohio, United States Background: Clinical evidence has identified stress as an important contributing factor to substance use disorder (SUD). This is particularly problematic as stress is unavoidable in daily life. Therefore, understanding the neurobiological mechanisms that underlie the contribution of stress to SUD is critical. One characteristic of SUD is a loss of control over drug intake that is modeled, in the rat, by conditions that result in escalating patterns of drug self-administration (SA). Repeated daily stress at the time of SA induces an escalation of cocaine intake in a glucocorticoid-dependent manner. This stress-induced escalation of SA is likely influenced by sex, as there are notable sex differences in both SUD and stress reactivity, and likely involves neurobiological mediators that connect stress-responsive and reward systems in the brain, such as the endocannabinoid system (eCB). We hypothesize that repeated stress at the time of SA induces a persistent increase in eCB signaling, particularly in regions critical for both cocaine taking and seeking, that results in escalation of cocaine use and increased susceptibility to reinstatement and that these effects are influenced by sex. Methods: Male and female SD rats were trained to SA cocaine (0.5 mg/kg/inf) on a FR 4 schedule in 4 ×30 min SA sessions separated by 5-min drug-free periods. Some rats received intermittent electric footshock stress in the SA chamber during the 5 min drug-free period over 14 days. Rats underwent extinction training and were tested for increased susceptibility for reinstatement to various stimuli. We examined the involvement of endocannabinoid signaling in stress-escalated cocaine intake by administration of a cannabinoid receptor type 1 (CB1R) antagonist systemically or directly into the nucleus accumbens (NAc) shell or ventral tegmental area (VTA) prior to a SA session. Changes in CB1R binding and density were examined in the NAc shell and VTA 24 h after the last SA session in rats that underwent cocaine or saline SA under stress and non-stress conditions. Rats were also tested for the effects of the CB1R antagonist systemically or directly into the prelimbic cortex (PrL) on cocaine-primed reinstatement (10 mg/kg, i.p.) to test for involvement of endocannabinoid signaling in augmented reinstatement following stress-induced escalation of cocaine intake. Rats are currently being tested for the role of eCB signaling on changes in cocaine-induced dopamine dependent upon drug or stress history by using in vivo fiber photometry measurement of a dopamine biosensor. Results: Electric footshock stress administered daily at the time of self-administration induced an emergent escalation of cocaine intake in both male and female rats (n = 16-18/group). However, female rats tend to escalate faster and to a greater degree. Systemic administration of the CB1R antagonist attenuates cocaine intake only in stress-escalated rats in males but attenuates cocaine intake in both no stress and stressed female rats though there is a greater sensitivity to the antagonist in the stress-escalated rats (n = 6-10/group). In male rats, this effect is localized to both the NAc shell and VTA as direct administration of the antagonist attenuates cocaine intake in stress-escalated rats (n = 6-9/group). A history of stress at the time of cocaine SA increases the time to reach extinction criterion in both sexes though the effect is greater in female rats. Rats with a history of stress at the time of SA show augmented reinstatement to a priming injection of cocaine (n = 11-14/group), re-introduction of the footshock stress during the 5-min drug-free period (n = 9-12/group), and to an injection of the alpha-2 adrenergic receptor antagonist yohimbine (n = 14-17/group). Furthermore, the recruitment of eCB signaling to influence drug-related behavior is long-lasting as systemic or intra-PrL administration of the CB1R antagonist prior to reinstatement attenuates cocaine-primed reinstatement only in rats with a prior history of stress at the time of SA (n = 8-9/group). Conclusions: Chronic stress induces a glucocorticoid-dependent escalation of cocaine intake that is the result of persistent neuroadaptations. These neuroadaptations likely result in long-lasting changes in the endocannabinoid system as repeated stress recruits endocannabinoid signaling in mesolimbic regions to drive drug use. Additionally, these stress-induced neuroadaptations are long-lasting and also occur in prefrontal cortical regions critical for drug-seeking behavior. All of these behaviors are also influenced by sex as females appear to be more sensitive to both the effects of stress of drug use and seeking and the role of endocannabinoid signaling in these effects. Understanding the unique mechanisms by which stress can drive drug use has implications for identifying and treating patients with SUD in whom stress is a contributing factor. Keywords: Acute and Chronic Stress, Endocannabinoids, Cocaine Self-Administration and Reinstatement, Sex Differences Disclosure: Nothing to disclose. P762. Methamphetamine Effects are Altered by Alcohol Bingeing: Sex Differences in Behavior and Neurochemical Measures Peter Serrano, Nicoletta Memos, Edgar Rodriguez, Jorge Avila, Michael Lewis Hunter College, CUNY, New York, New York, United States Background: Methamphetamine (MA) a highly addictive psychostimulant is frequently abused in combination with alcohol. More than 75% of those diagnosed with amphetamine dependence also have an alcohol use disorder (AUD). Despite different acute effects, both are associated with changes in key neurochemical in several brain regions associated with motivation and learning. Despite the co-morbidity little is known of the neurochemical and behavioral consequences of co-abuse. Moreover, little is known of their co-abuse in male and female populations. Using voluntary oral methamphetamine administration (VOMA) in both male and female mice, we have found major differences in behavioral and neurochemical systems in male and female mice. Using a modified escalating dose-access VOMA paradigm female mice exhibited greater behavioral and neurochemical deficits than males. [Previous studies have identified acute and chronic deficits in dopamine related markers following binge and VOMA models (Avila JA, 2018; Braren, Drapala, Tulloch, and Serrano, 2014; Xu, Zhu, and Angulo, 2005). Thus, we examined the effects of the dopamine transporter (DAT), the dopamine precursor tyrosine hydroxylase (TH) and the the dopamine receptor 1 and 2 (D1, D2) in hippocampus following chronic MA. The effect of neurotoxic or binge dosing of MA shows increased susceptibility in males than females as observed by more extensive striatal DA reduction and larger decreases in DAT (Bourque, Liu, Dluzen, and Di Paolo, 2011; Dluzen, Anderson, and Pilati, 2002; Wagner, Tekirian, and Cheo, 1993; Yu and Liao, 2000).] In addition, alcohol has been shown alter these systems with chronic self-administration. Previous research exploring the interaction of alcohol with the psychomotor stimulant cocaine has shown agonistic effects brain reward systems and activity (Lewis and June, 1994) and low dose alcohol and amphetamine effects on food intake. Effects of the combinations of alcohol with MA or other psychomotor stimulants on male and female mice is unknown. Methods: We used an animal model of voluntary oral methamphetamine administration (VOMA) that consisted of an acquisition phase from days 1-14 characterized by escalating doses of MA, and a binge phase from days 14-28 characterized by static doses of MA. Female VOMA mice displayed increased MA consumption during the binge phase of VOMA, demonstrating sex specific vulnerabilities to the maintenance of MA addiction. Mice were tested for spatial working memory performance on a radial 8-arm task following abstinence from VOMA. Other groups of mice were exposed to a binge model of alcohol self-administration prior to VOMA. Animals were trained to self-administration alcohol using the drinking in the dark (DID) paradigm. Male and female mice were given access to 15% (W/V) alcohol for two hours per day starting one hour into the dark portion of a 12/12 light dark cycle. Results: Results indicate that female VOMA mice had more working memory deficits correlated to higher MA consumption, a result not observed in male VOMA mice. Hippocampal and accumbal tissue were collected and analyzed using western blotting. Female VOMA mice had decreased GluA1, but not GluA2, in the hippocampus which may perpetuate synaptic destabilization and may underlie the observed increase in working memory deficits as well as increased GluA1 expression in the nucleus accumbens suggesting a female specific vulnerability toward abstinence induced drug craving and drug seeking. Furthermore, p-GSK3β signaling was decreased in the nucleus accumbens, hinting at enhanced downstream neurotoxicity and inflammation that is female specific. The effects of alcohol binge drinking prior to VOMA are expected to alter the intake of MA in both male and female mice in complex ways indicating enhancement of drug seeking behaviors. Effects on brain neurochemistry will follow. Conclusions: Our study reveals female specific neurochemical shifts in AMPA receptor signaling in the hippocampus and nucleus accumbens following abstinence from chronic MA consumption suggesting that these molecular modulations may underlie female susceptibility to MA-induced dysfunction. These data demonstrate a novel molecular signaling pathway that may be involved in enhanced vulnerability to drug craving and drug seeking that exacerbates memory deficits in vulnerable female populations following MA abuse. These data also validate VOMA as a model sensitive to sex differences in behavior and hippocampal neurochemistry following chronic MA exposure. We anticipate that prior bingeing on alcohol affects MA intake and neurochemical effect and that they differ in males and females. Keywords: Methamphetamine, Alcohol and Drugs, Nucleus Accumbens Glutamatergic Afferents, Hippocampal Function, Sex Differences Disclosure: Nothing to disclose. P763. VTA Dopamine Neurons Engage Subregion-Specific Striatal Dopamine Signals During Pavlovian Learning Liv Engel, Amy Wolff, Madelyn Blake, Val Collins, Sonal Sinha, Benjamin Saunders* University of Minnesota, Minneapolis, Minnesota, United States Background: Environmental cues, through Pavlovian learning, become conditioned stimuli that guide animals toward the acquisition of rewards (for example, food) by invigorating and directing seeking behavior. We have previously shown that brief optogenetic excitation of dopamine neurons, in temporal association with visual sensory cues, can instantiate those cues as conditioned stimuli that evoke conditioned movements. It remains unclear 1) how dopamine-neuron mediated, cue-evoked behavior is signaled by dopamine release downstream in striatal subregions and 2) how subregional signals evolve across stages of learning. Methods: Here, we made use of a genetically encoded dopamine biosensor (dLight) to monitor dopamine signaling in the nucleus accumbens core (NAC), dorsomedial striatum (DMS), and dorsolateral striatum (DLS) with fiber photometry, while tracking detailed movement features during optogenetic Pavlovian cue conditioning of VTA dopamine neurons. Results: Our results demonstrate a progressive recruitment of cue-evoked dopamine signaling across striatal subregions that correlates with different features of behavior. Cues paired with optogenetic activation of VTA dopamine neurons evoked dopamine release preferentially in the NAC early in training when behavioral responses were slower and directed toward the cue. Critically, these NAC signals got larger, rather than diminishing with extended training. As conditioning progressed, cue evoked signals also emerged in the DMS and DLS, when movement patterns became more vigorous and not directed at the cue. We found subregion specific heterogeneity in the relationship between cue-evoked signals and movement vigor. DMS cue-evoked signals predicted slower movements, while DLS signals predicted faster movements, and NAC signals were not correlated with movement. In additional studies, we found that brief optogenetic inhibition of DLS dopamine terminals at cue onset late, but not early, in training decreased the probability of cue-evoked locomotion and slowed the onset of movement initiation. Conclusions: Together our studies show dissociable, parallel functions for ventral and dorsal striatal dopamine signaling in guiding versus invigorating behaviors. Further, they suggest that large-scale plasticity across the striatal dopamine network emerges during Pavlovian learning to coordinate behavior. Keywords: Dopamine, Striatum, Fiber Photometry, Motivation, Cortico-Striatal-Thalamo-Cortical Circuits Disclosure: Nothing to disclose. P764. A Novel µ-Opioid Receptor Transgenic Cre Rat: Cellular and Behavioral Characterization Jennifer Bossert, Carlos Mejias-Aponte, Thomas Saunders, Lindsay Altidor, Michael Emery, Ida Fredriksson, Ashley Batista, Sarah Claypool, Kiera Caldwell, David Reiner, Vivek Kumar, Audrey Seashotlz, Elizabeth Hughs, Wanda Filipiak, Brandon Harvey, Christopher Richie, Francois Vautier, Michael Michaelides, Juan Gomez, Brigitte Kieffer, Stanley Watson, Huda Akil, Yavin Shaham NIDA, NIH, Baltimore, Maryland, United States Background: To specifically manipulate MOR-expressing neurons, we developed a transgenic rat to co-express Cre-recombinase and MOR under the endogenous Oprm1 gene promoter. We performed validation experiments to show expression patterns of both Oprm1 and Cre-recombinase and assess impact of targeting Cre to the Oprm1 gene on opioid-mediated pain responses and heroin self-administration (SA). Methods: We used RNAscope, fluorescence in situ hybridization chain reaction (HCR RNA-FISH), and autoradiography to verify that the knock-in manipulation had no effect on Oprm1 mRNA expression, and that iCre co-expresses with Oprm1. We test basal response to pain, morphine analgesia and tolerance. We trained male and female heterozygote (HET) rats and wildtype (WT) littermates to self-administer heroin and tested them in three relapse measures. We also tested the effect of nucleus accumbens (NAc) AAV1-EF1a-Flex-taCasp3-TEVP (Caspase3) injections on initiation and maintenance of heroin SA. Results: There were no differences between HET and WT rats in NAc MOR expression and function. Preliminary results showed co-localization of Oprm1 with iCre in HET rats. There were no differences in pain sensitivity or response to morphine, and no genotype-related differences for heroin SA, extinction responding, context-induced reinstatement, and heroin reacquisition. NAc Caspase3 lesions decreased MOR expression and function in HET but not WT. Additionally, the lesions had sex-specific effects on initiation and maintenance of heroin SA maintained by different drug doses and different fixed-ratio reinforcement schedules. Conclusions: The novel Oprm1-Cre transgenic rat can be used to study the role of brain Oprm1-expressing cells in opioid addiction- and pain-related behaviors, as well as other opioid-mediated learned and innate behaviors. Keywords: Opioid Abuse, Self-Administration, Pain Analgesia Disclosure: Nothing to disclose. P765. Nicotine Normalizes the Dynorphin-Dependent Expression of Negative Emotional States During Nicotine Abstinence Marsida Kallupi, Ami Cohen, Giordano de Guglielmo, Elena Crawford, George Koob, Paul Schweitzer, Olivier George UCSD, La Jolla, California, United States Background: Tobacco use disorder is the leading preventable cause of death worldwide. Negative emotional states during nicotine abstinence significantly contribute to the subsequent escalation of nicotine intake, but the neurobiological mechanisms that underlie this phenomenon are unclear. We hypothesized that the upregulation of dynorphin in the central nucleus of the amygdala (CeA) mediates the negative emotional states of abstinence, relapse, and escalation of nicotine intake through the inhibition γ-aminobutyric acid (GABA) transmission. Methods: We used intracellular recordings, immunohistochemistry, and viral-mediated downregulation of dynorphin in the CeA to evaluate the role of dynorphin in GABA transmission, hyperalgesia, conditioned place aversion, stress-induced reinstatement, and escalation of nicotine intake in male rats. The data were analyzed using one-way analysis of variance (ANOVA) for between-subject comparisons or repeated-measures ANOVA for within-subject comparisons, followed by the Newman-Keuls post hoc test as appropriate. For individual means comparisons, Student’s t-test was used. Results: Abstinence from nicotine increased dynorphin levels and dysregulated GABA transmission in the CeA, and nicotine (0.03 mg/kg/infusion) reversed these responses, normalizing dynorphin levels and GABA transmission back to control (pre-abstinence) level. Viral-mediated downregulation of dynorphin mRNA (by 75%) in the CeA prevented abstinence-dependent behaviors (hyperalgesia, aversion to withdrawal, and yohimbine-induced reinstatement of nicotine seeking) but not nicotine-dependent behavior (escalation of nicotine intake). Conclusions: These results demonstrate that nicotine normalizes dynorphin levels and dynorphin-dependent changes in GABA transmission during abstinence, and the upregulation of dynorphin in the CeA during abstinence is critical for the expression of the negative emotional states and stress-induced relapse but not nicotine intake. These results suggest that tobacco smoking may be partly driven by the ability of nicotine to normalize aberrant dynorphin/GABA transmission in the CeA during abstinence. Targeting the dynorphin system may represent a novel approach to reduce the negative emotional states of nicotine abstinence and reduce stress-induced relapse. Keywords: Dynorphin, Nicotine Addiction, GABA Transmission Disclosure: Nothing to disclose. P766. Context- and Lever Access-Dependent IEG Expression During Heroin Seeking After Forced Abstinence in Rats: Insights Into Functional Network Connectivity Jobe Ritchie, Jennifer Walters, Alexis Lacey, Justine Galliou, Sydney Swatzell, Taylor Brown, Jaclyn Roland-McGowan, Rita Fuchs* Washington State University, Pullman, Washington, United States Background: Opioid relapse after detoxification reflects the interplay of neural circuits that control goal-directed and habitual responses, attentional processing, and negative affect. While several relevant brain regions have been identified, functional connectivity among these brain regions during opioid-seeking behaviors has not been evaluated. The aim of this study was to map correlated activity-related protein/plasticity-related protein (ARP/PRP) expression across 43 brain regions as a function of exposure to heroin-paired contextual stimuli and lever access. Methods: Male Sprague-Dawley rats were trained to lever press for heroin infusions in a distinct environment (heroin-paired context) over 10 days. The rats were then exposed to a different environment without access to operant levers or heroin (unpaired context) over 14 days. On post-drug day 15, the rats (n = 7/group) received a 2-h drug-free test session in the heroin-paired context or the unpaired context, either with or without lever access. ARP/PRP expression (i.e., c-Fos and Zif268 immunoreactivity) was quantified in 43 brain regions in brain tissue collected immediately after the test session. Lever responses, cFos immunoreactivity, and Zif268 immunoreactivity were analyzed using analyses of variance with context and lever access as factors. Correlational and principal component analyses were used to evaluate relationships between normalized ARP/PRP expression values within and across brain regions after Fisher’s transformation. Functional network analyses were restricted to brain regions that exhibited significant ARP/PRP expression. Alpha was set at 0.05. Results: Heroin-seeking behavior was more persistent in the heroin-paired context than in the unpaired context at test (p < 0.05), and ARP/PRP protein expression varied as a function of testing context and lever access, with interaction between these variables observed in only eight brain regions. Furthermore, heroin-seeking behavior positively correlated with cFos expression in the medial septum and dorsal cornu ammonis 3 subregion of the hippocampus. Network-connectivity analyses revealed that exposure to the heroin-paired context augmented positively correlated ARP/PRP expression (p < 0.05), or coupling, among subregions of the frontal cortex, amygdala, and dorsal striatum compared to unpaired context exposure. The specific pattern of coupling suggested that subcircuits involved in executive, limbic, and motor function independently influence focused motivated behavioral output at multiple levels, including at the level of the M1 motor cortex. In contrast, unpaired context exposure generated coupling among a more diffuse set of brain regions, including subregions of the hippocampal formation and septum, which might reflect cognitive processes related to a wider response repertoire in the unpaired context. Lever access versus no-lever access elicited distinct correlated ARP/PRP expression (p < 0.05) among a smaller number of brain regions compared to context exposure. In particular, lever access resulted in coupling among brain regions that mediate affective, attentional, and stress responses, including the septum, lateral habenula, and central amygdala, possibly due to the emergence of negative affective states when lever responses were not reinforced during the test session. While lever access also elicited ventral pallidum-M1 coupling, no-lever access elicited negatively correlated ARP/PRP expression (p < 0.05), or decoupling, between these and other brain regions, suggesting functional connectivity that can bidirectionally control the initiation of operant behavior. Conclusions: These findings expand upon existing literature and provide impetus for future research investigating the causal contributions of several novel brain regions and circuits to heroin relapse. Keywords: Opioid, Self-Administration, cFos, Zif268, Heroin Seeking Disclosure: Nothing to disclose. P767. Dynorphinergic Control of Amygdalo-Striatal Circuits for Goal-Directed Behavior Raajaram Gowrishankar, Abigail Elerding, Sofia Shirley, Josie Van Tilburg, David Marcus, Kat Motovilov, Sean Piantadosi, Adam Gordon-Fennell, Charles Zhou, Khalid Abrera, Chunyang Dong, Lin Tian, Garret Stuber, Michael Bruchas University of Washington, Seattle, Washington, United States Background: Goal-directed action-outcome behavior is essential for survival and co-opted during pathological drug-seeking. Recent work suggests that neurons from the basolateral amygdala (BLA) projecting to the dorsomedial striatum (DMS) may contribute to goal-directed action; yet, how BLA-DMS activity instantiates goal-directed action, or how it is regulated during action-outcome behavior is unknown. Furthermore, ~50% of the neurons in the DMS express the endogenous opioid peptide dynorphin (dyn); however, how dyn in the DMS regulates action-outcome behavior is unknown. Here, we hypothesize that BLA-DMS projections coordinate action-outcome behavior, and their activity and subsequent behavior is refined by dyn-KOR signaling. Methods: All studies were conducted in equal numbers of male and female mice in accordance with NIH guidelines, approved by the IACUC at the University of Washington. No sex-dependent effects were observed. Below were the methods used: 1. Ex-vivo viral tracing and in situ hybridization of BLA projections to DMS (WT/Ai14/vglut1-cre/KOR-cre, n = 2-3 mice, 6 slices). 2. Ex-vivo electrophysiology of DMS neurons during optogenetic activation of BLA terminals (D1-tdtmto, n = 5 mice, 16 cells); KOR activation (U69,593 – 1uM) at BLA terminals during optogenetic activation (D1-tdtmto, n = 4 mice, 9-16 cells). 3. In-vivo fiber photometry during operant behavior of BLA-DMS terminalls (vglut1-Cre, n = 4 mice). 4. Time-locked optogenetic manipulations of BLA-DMS terminals in-vivo via activation using ChR2 (vglut1-cre or WT, n = 9 mice) or inhibition using PPO (vglut1-cre or WT, n = 9 mice). 5. In-vivo 2-photon imaging of BLA-DMS ensembles during head-fixed operant behavior (Ai14, n = 4 mice). 6. In-vivo fiber photometry during operant behavior of BLA-DMS terminals multiplexed with conditional dyn deletion (WT/pdyn-cKO, n = 7 mice) or dyn neuron stimulation (pdyn-cre, n = 4 mice). 7. In-vivo fiber photometry during operant behavior of dyn release in the DMS (WT/pdyn-cre, n = 4 mice). Results: We show that ~20% of BLA neurons project to the DMS (n = 2-3 mice, 6 slices) and are glutamatergic (vglut1+) using a combination of anterograde and retrograde viral tracing, and in situ hybridization. Additionally, BLA terminals preferentially activate DMS D1 + /dyn neurons (n = 16 cells, 2 Way ANOVA; p = 0.0014 – D1(+) vs. D1(-), F(1,203) = 10.50). During operant behavior where animals perform a nosepoke (action) to obtain a sucrose reward (outcome) multiplexed with fiber photometry, we find that as animals learn goal-directed action-outcome behavior (n = 4 mice, operant, Simple Linear Regression; p < 0.005, R2 = 0.48), BLA-DMS terminal activity emerges during action-outcome learning. We show that this activity is increased during action (n = 4 mice, z-score, t test; p < 0.0005 – untrained vs. trained), and inhibited during outcome (n = 4 mice, z-score, t test; p < 0.01 – untrained vs. trained). To determine whether BLA-DMS activity is causal to action-outcome behavior, we perform optogenetic manipulation of BLA-DMS terminals during operant behavior. We observe that photoactivation during outcome disrupts action (n = 9 mice, operant, t test; p < 0.05, t(8)=3.14) and photoinhibition enhances action (n = 9 mice, operant, t test; p < 0.005, t(8)=4.92). Furthermore, to determine if this pattern of activity is reflected at the BLA soma or is unique to BLA-DMS axon terminals, we use in vivo 2-photon Ca2+ imaging of BLA-DMS ensembles during head-fixed operant behavior. We observe that as mice learn operant behavior (n = 4 mice, operant, t test; p < 0.05, t(3)=3.4), distinct BLA ensembles (512 cells) are engaged during action vs. outcome, and that BLA-DMS ensembles (50/512 cells) are predominantly only action-activated. This suggests that the inhibition in BLA-DMS terminal activity during outcome occurs locally at BLA-DMS terminals. Using retrograde viral tracing and in situ hybridization, we find that ~60% of BLA-DMS neurons express KOR. We also observe that optogenetically-evoked activity via BLA terminals at DMS D1 + /dyn neurons is sensitive to KOR activity (n = 9-16 cells, t test; p < 0.001, t(21)=3.038). To delineate how BLA-DMS terminal activity may be regulated in the DMS by dyn-KOR signaling, we use conditional deletions of dyn or photoactivation of dyn release during in vivo photometry of BLA-DMS terminals and action-outcome behavior. We find that deletion of dyn in the DMS or KOR in the BLA, negatively impacts action-outcome learning and maintenance (n = 7 mice, operant, 2 Way ANOVA; p < 0.0005 – day x genotype, F(4,40) = 8.99 - WT vs. dyncKO) and BLA-DMS activity (n = 7 mice, z-score, t test; p < 0.0005 – WT vs. dyncKO). Conversely, we show that stimulating dyn release during outcome enhances action (n = 4 mice, operant, t test; p < 0.05, t(3)=3.2 – stim vs. no stim), and BLA-DMS activity (n = 4 mice, z-score, t test; p < 0.05 – stim vs. no stim). Finally, we show via in vivo photometry during action-outcome behavior using a novel dyn sensor (KLight1.3) that significant dyn release occurs during outcome retrieval and consumption (n = 4 mice, z-score, t test; p < 0.05 – baseline vs. outcome). Conclusions: Altogether, we show that BLA-DMS activity engenders goal-directed action-outcome learning and maintenance and that BLA-DMS terminal activity is distinct to the activity of DMS-projecting BLA soma. Furthermore, DMS dyn release during outcome, resulting in retrograde dyn transmission from the DMS onto KOR at BLA terminals enables dynamic changes in BLA-DMS activity and promotes action-outcome behavior. Keywords: Goal-Directed Behaviors, Dorsal Striatum, Basolateral Amygdala, Dynorphin, Kappa Opioid Receptor Disclosure: Nothing to disclose. P768. Time Varying Functional Connectivity Signatures of Acute Psychostimulant Administration Luis Colon-Perez University of North Texas Health Science Center, Fort Worth, Texas, United States Background: Psychostimulants, like cocaine and bath salts, activate the mesolimbic and mesocortical pathways via dopaminergic activity, leading to extended functional brain circuit alterations that can spread to the entire brain. We can explore the characteristics of acute psychostimulant activation and spread over the whole brain using computational neuroscience approaches and resting state fMRI. One approach that stands out is time-varying functional connectivity (tvfc) to determine the spatiotemporal characteristics of the brain under psychostimulants administration. To this end, we sought to explore the features of a single administration of a psychostimulant in the rat brain using resting state fMRI and tvfc. Methods: Rats were imaged under combined dexmedetomidine (0.02 mg/kg intraperitoneal)/isoflurane (0.5%) sedation (delivered in 70%N2/30%O2 at 0.1 L/min). Resting-state fMRI datasets were collected in a 4.7 Tesla Agilent system (Magnex Scientific) at 1 hour after i.p. administration of 3.0 mg/kg MDPV (n = 8), 15 mg/kg cocaine (n = 8), or saline (n = 7). Resting fMRI data were acquired with a 2-shot spin-echo echo planar imaging with 210 repetitions for a total acquisition time of ~7.5 mins (an image was acquired every 2 s). The resting state fMRI datasets were analyzed using time window approaches to estimate graph-theoretical biomarkers of network node flexibility. Notably, we began our efforts focusing on providing robust statistical benchmarks (i.e., surrogates) to provide convincing standards to improve the sensitivity and the interpretation of dynamical properties. We analyzed the global resting state using windows of 10 s, 20, 40, and 1 min, and overlapping between windows of n-4 time points, n-3, n-5, n-7, and n-10 time points per window, where n is the size of the time window. Then, we focused on the differences between drug and saline groups using a time window of 1 min and an overlap of n-1. The network metric assessed was flexibility, a node’s ability to switch modules between time windows. A module is defined by the modularity index, which measures the propensity of separating the network into modules with inter-module connectivity. Results: Comparison between drug and saline administered shows that the resting state data consistently shows a distinct pattern of flexible behavior in the brain. We compared three surrogates as null hypothesis times series, and they all showed similar behavior and different to the brain data. The surrogates were: phase scrambled fMRI time series without preserving static network metrics, phase scrambled fMRI time series preserving static features, and a limited bandpass series of random numbers resembling the temporal characteristics of fMRI time series. The global features between all experimental groups showed similar dynamical features indicative that the drug doses and studied time points preserve the dynamical features of the control group. However, individual node features showed a consistent, distinct profile among a subset of nodes in the temporal lobes, somatosensory cortices, and frontal cortices. Cocaine groups showed reduced flexibility; meanwhile, MDP showed increased flexibility compared to saline. These differences were significant between psychostimulants but not between individual psychostimulants and saline. Conclusions: This work shows that tvfc can offer unique features of dynamical function indicative of psychostimulant-induced activity. Despite the surrogates’ widely different time series features, they all showed similar properties. They were distinct from the fMRI data, indicating that the flexibility is quantifying an organized dynamical feature unique to brain function. Further studies are needed to establish the neural relevance of these flexible transitions and the mathematical formulation to interpret these results concerning brain function. Keywords: fMRI, MDPV, Time Varying Functional Connectivity, Network Analysis Disclosure: Nothing to disclose. P769. Contextual Opiate Tolerance: Brain-Wide Changes in Neuronal Activity and Control by Mediodorsal Thalamus and Anterior Cingulate Cortex Rafael Perez, Cheryl Chen, Marina Picciotto Yale University, New Haven, Connecticut, United States Background: Opioid analgesics are one of the most commonly prescribed medications in the U.S. However, opioids have considerable abuse liability due to their non-analgesic effects. As users increase their opioid intake in a particular context, they build tolerance against the effects of these drugs within the context. This phenomenon is known as associative analgesic tolerance. Since associative analgesic tolerance can lead to dose and use escalation, it increases opioid use disorder (OUD) risk. Therefore, a deeper understanding of the factors controlling associative tolerance may inform the development of treatments aimed at decreasing OUD risk in patient populations. While associative tolerance and its reversal have been described in animal studies, the biological mechanisms underlying this process are not understood. Here, we examined how associative opioid tolerance changes activity patterns throughout the entire brain and identified two target regions that orchestrate associative tolerance in male mice. Methods: In experiment 1, we established a training regimen that induces associative analgesic tolerance. Male and female C57BL/6 J mice (n = 20/ group) underwent associative tolerance training for 14 days. Each day, mice received injections of either fentanyl (25 ug/kg, s.c.) or saline before being placed in a context with distinct multi-sensory features. After 15 minutes within the context, basal nociception and fentanyl-induced analgesia were measured using the hotplate assay (latency to nociceptive response, max time = 30 seconds). Following this training phase, associative tolerance was measured by administering fentanyl in the fentanyl-paired, saline-paired, and a novel context before the hotplate test. In experiment 2, we identified which neuronal populations are engaged during associative tolerance across the entire brain. In this experiment we used male Ai14xArc-TRAP mice (n = 4/group), which allow for brain-wide, temporally defined tagging of active neuronal populations with the fluorescent marker tdTomato, were administered the TRAP activator tamoxifen on the last day of tolerance training to tag tolerance-active neurons. 4 weeks later, mice were injected with fentanyl and exposed to either the fentanyl- or saline-paired environment. Brains were extracted and processed using SHIELD to clear the brains and preserve endogenous fluorescence and then imaged using a custom- built high-speed selective plane illumination microscope (SPIM) to detect tagged neurons using an automatic cell counting algorithm. In experiment 3, we determined how context and/or fentanyl changed neuronal activity in specific areas implicated in contextual memory and pain processing in tolerant mice. Male mice (n = 3-4/group) underwent tolerance training before being exposed to one of the following conditions: saline-context and saline, saline context-and fentanyl, fentanyl-context and saline, or fentanyl context and fentanyl. Brains were extracted and analyzed via immunohistochemistry by staining for cFOS. Based on anatomical experiments, Based on anatomical experiments, in experiment 4, viral vectors encoding Designer Receptors Exclusively Activated by Designer Drugs (DREADDs; AAV5-hSyn-hM4D(Gi)-mCherry or AAV5-hSyn-mCherry, 300nL) were infused into the anterior cingulate (ACC) or dorsomedial thalamus (MDT) of male mice (n = 6-9/group). Mice underwent tolerance training and were injected with the DREADD activator CNO (3 mg/kg i.p.) before receiving fentanyl in the fentanyl context or saline in the saline context. Data were analyzed using one-way, two-way and three-way repeated measures ANOVA with post-hoc Sidak’s multiple comparisons tests when appropriate. Results: In experiment 1, we found that male and female mice that received fentanyl in the fentanyl-paired context showed tolerance, defined as a decrease in analgesic responses in the hotplate assay over the course of 7 daily exposures to fentanyl (male change = 11 seconds, F1, 133 = 753.7 p < 0.0001 female change = 10.5 seconds, 1, 19 = 110.3, p < 0.0001). However, tolerance was reversed in male, but not female, mice exposed to fentanyl in the saline-paired context or a novel environment (males p < 0.001, p > 0.05 females). In experiment 2, we found that exposure to fentanyl in the fentanyl-paired context increases activity in 80 identified brain regions. Post hoc literature analyses revealed that the activated areas cluster by function primarily into motor, sensory, learning, pain, and reward-associated groups. In experiment 3, we confirmed that the combination of fentanyl and the fentanyl context increased the number of cFOS-expressing neurons in the ACC and its upstream projection area, the MDT. Finally, chemogenetic silencing of either the MDT or ACC in the fentanyl-paired context suppressed fentanyl analgesic tolerance (ACC F1,14 = 5.279, P < 0.05 MDT F1,14 = 7.146 p < 0.05) but did not alter nociception when mice received saline in the saline-paired context (ACC p > 0.05, MDT p > 0.05). Conclusions: These findings establish a robust behavioral procedure for the study of associative opioid analgesic tolerance in mice. The findings also reveal how associative tolerance changes activity throughout the brain. Among the identified regions, the MDT and ACC appear to be important targets for the modulation of associative analgesic tolerance. Keywords: Opioid Tolerance, Environment, Whole-Brain Rodent Imaging, Anterior Cingulate Cortex (ACC), Thalamus Disclosure: Nothing to disclose. P770. Basolateral Amygdala Parvalbumin Expressing Interneurons Critically Regulate Reward Seeking Behaviors Kenneth Amaya, Pantelis Antonoudiou, Jamie Maguire Tufts University School of Medicine, Boston, Massachusetts, United States Background: The basolateral nucleus of the amygdala (BLA) is essential for both fear and reward learning alike. Recent advances have shown that BLA microcircuitry contributes to fear conditioning, particularly that parvalbumin-expressing interneurons (PV) are able to regulate both fear expression and fear-relevant BLA oscillatory states. However, despite BLA’s known role in regulating appetitive reward seeking, little is known about BLA oscillatory states during reward learning and even less is known about how BLA PV interneurons contribute to reward-related behaviors. Therefore, we aimed to fill this gap in knowledge by probing the role of PV interneurons during operant conditioning and, separately, characterizing BLA field potentials during reward learning to better understand population-level activity changes in BLA as reward is encoded and used. Methods: Subjects: Experiment 1: 24 adult heterozygous PV-Cre+ mice (12 female, 12 male) Experiment 2: 12 adult wild-type C57 mice (6 female, 6 male) Surgical Procedures: Basolateral amygdala (AP: -1.5 mm; ML: + /- 3.3 mm; DV: 4.5 mm) was targeted in both Experiments. Animals in Experiment 1 received bilateral infusions of a Cre-dependent inhibitory designer receptor exclusively activated by a designer drug (AAV9-DIO-hM4Di-mCherry) to target PV interneurons. Animals in Experiment 2 underwent unilateral implantation of a depth electrode targeting BLA (same coordinates). Following surgery, animals were allowed to recover 2 weeks prior to commencing behavioral training and testing. Behavioral Procedures: Behavioral procedures were carried out in 4 identical Med-Associates conditioning chambers enclosed in sound-attenuating chambers. Training began with a free-reward (0.1 mL 20% sucrose solution) magazine training session. Then, animals underwent operant conditioning where one of two nose-poke ports were reinforced (left or right, counterbalanced). Training sessions progressed from FR1 (3 sessions) to RR2 (2 sessions) to RR3 (2 sessions) before advancing to behavioral testing. Testing included a 5-min baseline test session in extinction followed immediately by a fully-rewarded RR3 session to re-establish responding levels. The following day, animals underwent satiety-induced devaluation where they were given free access to the reward then tested afterwards, in extinction, to assess whether responding changed as a result of the reward being reduced in value. Then, the next 3 days, the animals underwent a reversal where the identity of the reinforced operandum was changed. In Experiment 1, PV interneurons were inhibited during the test sessions only (Devaluation and Reversals). In Experiment 2, animals were split into behavioral groups for comparison (Deval/non-Deval; Reversal/non-Reversal) and local field potentials in BLA were recorded. Statistical analyses: Behavioral measures collected include nose-poke responses and magazine entries. Individual linear mixed models were used to analyze effects of dependent variables by fixed effects of group and session while accounting for random effects of differences in starting points for the variable in Session 1. Reported statistics include parameter estimates, 95% confidence intervals, and p-values. Behavioral analyses and data visualization were carried out in R (“lme4”, “ggplot2”). For Experiment 2, spectral analysis was performed on the recorded field potentials to characterize BLA network activity. Analyses and visualization were performed using custom Python scripts. Results: We demonstrated that BLA PV interneurons are necessary for goal-directed instrumental behavior following outcome devaluation. Further, we provided evidence that suppressing BLA PV interneuron activity is sufficient to drive improved performance after contingency reversals. Lastly, we characterize BLA field potential changes as a product of reward experience and operant conditioning. Conclusions: Taken together, these experiments investigate how local BLA activity governs appetitive reward seeking, both at the microcircuit and population activity levels. Specifically, showing that PV interneurons are necessary for flexible reward seeking is a first step towards a better understanding of how microcircuits within the amygdala mediate appetitive reward behaviors. We also examine BLA local field potential changes to characterize population-level dynamics as reward learning takes place. Given that PV interneurons can modulate BLA oscillatory states relevant to fear behaviors, it is possible that PV interneurons could perform a similar function for reward behavior. Thus, future studies will seek to identify a causal mechanism linking PV interneuron activity and BLA field potentials during appetitive reward seeking. Keywords: Parvalbumin Interneurons, Operant Behavior, Local Field Potentials, Amygdala-Based Networks, Reward Disclosure: Nothing to disclose. P771. Regional Differences in Striatal Dopamine Dynamics in Pavlovian Training With a Natural Reinforcer Armando Salinas, Jeong Lee, Yolanda Mateo, Shana Augustin, David Lovinger Louisiana State University Health Sciences Center Shreveport, Shreveport, Louisiana, United States Background: Dopamine is a critical neuromodulator for several neurobiological processes including learning and memory. Within the striatum, dopamine dynamics in the nucleus accumbens have been studied extensively and been classically associated with reward and reinforcement. In contrast, few studies have examined dopamine dynamics in the dorsal striatum in vivo. This is likely due to the difficulty in obtaining dorsal striatal dopamine signals in vivo with fast-scan cyclic voltammetry. Recently, genetically-encoded fluorescent biosensors for dopamine were developed and have facilitated monitoring of real-time dopamine dynamics in the dorsal striatum and other brain regions where detection was previously difficult. Methods: Male C57BL/6 J mice were stereotaxically infused with AAV encoding dLight1.3b into the nucleus accumbens, dorsomedial striatum, or dorsolateral striatum (4-6 mice per region). Three weeks later a fiber optic ferrule was implanted into the brain region of interest with fluorescence monitored intraoperatively to ensure placement in a location with adequate dLight expression. One month later, mice began a Pavlovian conditioning protocol with either a tone or white noise (CS+) being paired with a food reinforcer delivery (US+). Fiber photometry was performed during Pavlovian conditioning using a custom-built system and analysis was completed using custom MATLAB and python scripts. All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Results: Similar to previous work with fast-scan cyclic voltammetry, we found that in the nucleus accumbens, phasic dopamine transients were elicited by the US + early in training. As training progressed, the phasic dopamine transient became time-locked to the CS + and no longer responded to the US + . In the dorsomedial and dorsolateral striatum, we observed phasic dopamine increases in response to US + , and with extended Pavlovian conditioning phasic release increased in response to the CS + , but not the CS-, albeit to a lesser degree than in the nucleus accumbens. Interestingly, and unlike dopamine dynamics in the nucleus accumbens, in both dorsal striatal regions, the phasic dopamine increase to the US + persisted. This US + dopamine response was not present in unexpected omission trials suggesting that this response was not tied to motor responses but rather reflects a reinforcer-associated signal. Additionally, we found that dopamine transient decay kinetics differed between striatal subregions, with faster responses in dorsolateral striatum relative the other two regions (p < 0.01). Conclusions: This work suggests a role for dopamine in dorsal striatum subregions in processing of reinforcer-associated cues as well as reinforcer delivery, independent of motor activity. Keywords: Dopamine, Pavlovian Conditioning, Striatal Dopamine Signaling, Optical Biosensors Disclosure: Nothing to disclose. P772. Perirhinal Projections in Methamphetamine-Induced Recognition Memory Deficits and Relapse Katharine Nelson, Jordan Hopkins, Samuel Wood, Michael Scofield, Carmela Reichel Medical University of South Carolina, Charleston, South Carolina, United States Background: Methamphetamine (meth) self-administration reliably produces object recognition memory deficits due drug-induced plasticity within the perirhinal cortex (Prh). Prh projections are numerous and include the prefrontal cortex (PFC) and nucleus accumbens (NA)- two key nodes involved in relapse biology. We have previously shown that meth exposed animals (long access; LgA) perseverate responding to meth associated cues with little regard for a novel cue presented in the same environment, and the salience of the novel cue is enhanced with stimulation (mGluR5 positive allosteric modulator, GqDREADDs) of the Prh. This Prh stimulation restored meth induced deficits in recognition memory. We hypothesize that the Prh->PFC projection mediates recognition memory while the Prh->NA drives responding for novel cues. Methods: To evaluate the function of the Prh->PFC and Prh->NA we used a dual viral approach consisting of a retrograde adeno-associated virus encoding GFP-tagged Cre recombinase (rgAAV-GFP-Cre) in the projection area combined with excitatory or inhibitory cre-dependent DREADD in the cell body region [rAAV-hSyn-DIO-hM3D(Gq)-mCherry or rAAV-hSyn-DIO-hM4D(Gi)-mCherry (hM3Dq or hM4Di, respectively)]. First, we inhibited the Prh->PFC circuit in drug naive rats to induce a deficit in recognition memory. After three weeks for viral expression rats were tested for OR memory. Second, we activated the Prh->PFC to restore meth-induced deficits in recognition memory. Rats went through long access meth self-administration and object recognition memory tests. Brain tissue was processed for confocal microscopy to visualize mCherry and c-fos expression in the Prh. Similar experiments were conducted in the Prh->NA circuit in short (ShA) and LgA access meth animals in which relapse tests were conducted in the presence of the meth associated lever and an additional novel lever in the chamber as well as a novel cue light (novel cue group). Results: Inhibition of the Prh-PFC circuit prevented expression of OR memory, whereas activation of this circuit with an excitatory Cre-dependent DREADD in animals with a history of long access meth self-administration reversed the meth-induced OR deficit. Inhibition of the Prh-NAc circuit with CNO increased responding on the meth associated lever relative to the novel, whereas Veh resulted in similar responding on both levers. Unexpectedly, activation of the circuit following long access meth was without an effect. Conclusions: These data suggest a more complex circuitry governing OR memory than previously indicated with anatomical or lesion studies and informs future work aimed at understanding the role of the Prh and PFC and their connectivity in meth addiction. These findings indicate inhibition of PRH-NA in a ShA procedure is sufficient to shift behavior, however, activating the pathway in LgA “addicted” procedure is not sufficient to shift focus from meth associated cues to novel stimuli. Keywords: Methampheatmine Use Disorder, Drug Seeking, Episodic Memory Rodents, Novelty Seeking, Novel Object Recognition Disclosure: Nothing to disclose. P773. Striatonigral Mediated 2-Arachidonoylglycerol Mobilization Contributes to Ethanol Mediated Signaling and Behaviors Shana Augustin, Alexa Gracias, Rishitha Anumola, David Lovinger Northwestern University, Chicago, Illinois, United States Background: Alcohol Use Disorder (AUD) is a major public concern and deaths involving AUD have increased during the pandemic. Striatal endocannabinoids (eCBs) and cannabinoid type 1 (CB1) receptors are implicated in ethanol’s actions and may contribute to AUD. The principal striatal cells, striatopallidal and striatonigral medium spiny neurons (MSNs) can produce and release eCBs as needed in response to synaptic activity. Ethanol can alter 2-arachidonoylglycerol (2-AG) levels, which in turn may contribute to the reinforcing effects of ethanol. Furthermore, ethanol exposure preferentially affects striatonigral mediated synaptic plasticity and behaviors. Despite this, the role of striatonigral 2-AG mobilization in mediating alcohol effects remains unclear. Methods: In this study, we used targeted deletion of the 2-AG-synthesizing enzyme, diacylglycerol lipase α (DAGLα), in striatonigral MSNs to examine the role of striatonigral 2-AG mediated signaling in ethanol-induced eCB-CB1 inhibition, sedative effects of ethanol and voluntary ethanol consumption. To study ethanol effects on presynaptic eCB signaling dynamics in rodent brain slices in real-time with high-fidelity, we used acute brain slices expressing a genetically encoded fluorescent eCB biosensor (GRABeCB2.0) in primary sensorimotor cortex combined with slice photometry. Results: Mice lacking the 2-AG synthesizing enzyme in striatonigral MSNs (DAGLα D1 Cre+ mice) exhibited dramatically reduced evoked eCB release detected at corticostriatal terminals in the dorsolateral striatum compared to controls. Evoked eCB transients were blocked by a CB1 receptor antagonist, confirming the specificity of eCB mediated signal as measured by the fluorescent signal of GRABeCB2.0. Although evoked eCB release was altered in the DAGLα D1 Cre+ mice, the induction of eCB-dependent long-term depression was intact. Ethanol application inhibited eCB release, and this inhibition was also reduced in the DAGLα D1 Cre+ mice. Compared to control mice, male and female DAGLα D1 Cre+ mice showed decreased sensitivity to the sedative effects of ethanol. This decreased sensitivity was not accompanied by differences in blood ethanol content. DAGLα D1 Cre+ male mice showed decreased ethanol preference compared to controls in an intermittent two-bottle free choice escalating ethanol concentration consumption procedure. This decrease in ethanol preference was not accompanied by a decrease in ethanol intake, but an increase in ethanol-induced water consumption and total volume of fluid consumption. Female mice consumed more ethanol than male mice both for control and DAGLα D1 Cre+ mice. Also, this paradigm induced an escalation in ethanol consumption in both sexes and genotypes. Furthermore, there were no differences in sucrose and quinine intake and preference in a two-bottle free choice paradigm following ethanol consumption in the sexes and genotypes tested. Conclusions: Our findings show that selective deletion of 2-AG-mediated signaling in striatonigral MSNs is sufficient to alter ethanol-induced modulation of presynaptic eCB signaling dynamics and sensitivity to ethanol. My work aims to reveal a more accurate and detailed picture of eCB-CB1 signaling following ethanol exposure and the contribution of this signaling to behavioral effects of ethanol and AUD. Keywords: Endocannabinoids, Alcohol Use Disorder, Optical Biosensors Disclosure: Nothing to disclose. P774. Multi-Site Recordings of Neural Activity in the Infralimbic Cortex, Insular Cortex, and Basolateral Amygdala Across Heroin-Seeking Behavior Sean Farley, Jangjin Kim, Ryan LaLumiere The University of Iowa, Iowa City, Iowa, United States Background: Evidence indicates that infralimbic cortex (IL) promotes the extinction and inhibition of cocaine seeking, though its role in the extinction of opioid seeking is less clear. It is likely, however, that the IL interacts with other regions known to regulate drug seeking during this process. Indeed, anatomical findings reveal dense connections between the IL, the basolateral amygdala (BLA), and rostral agranular insular cortex (RAIC). Moreover, evidence from both human and rodent work indicates that the BLA and RAIC play strong roles in mediating drug craving and controlling drug-seeking behavior. Yet, how these regions interact with each other on a neurophysiological level across self-administration, extinction, and reinstatement for heroin seeking is unknown. Therefore, we sought to simultaneously record from this network in this behavioral paradigm to gain an understanding of the neural interactions among these regions during such behavior. Methods: Adult Sprague-Dawley rats were catheterized for intravenous heroin self-administration. The self-administration procedures used standard operant conditioning chambers with dual retractable levers. Self-administration began with a high-to-low dose tapered schedule, decreasing heroin dosage every two sessions until arriving at 0.083 mg/kg/infusion. Self-administration began under an FR1 schedule, where an active lever press produced a 50 uL infusion of heroin, onset of cue light and a 20 s timeout period. After rats achieved >100 presses in an FR1 session, self-administration transitioned to trial-based sessions in which the levers were only available in specific signaled trials during the session. When rats pressed on at least 80% of the trials, they were fitted with a three-site, 96-channel microdrive array (9 tetrodes/site), aimed at the IL, RAIC, and BLA. Microdrives were custom-designed and 3D-printed with stereolithography using standard and biocompatible photopolymer resins. Single-unit and local field potential (LFP) data was collected from each tetrode. Non-parametric, Wilcoxon signed-rank analysis was conducted on neural data (both spikes and LFPs) for the three recording sites comparing neuronal activity 2 s before/after 1) trial start and 2) lever press. We analyzed spike and LFP data from the IL, BLA, and RAIC during self-administration, the 1st and 5th extinction session, and heroin-primed reinstatement. Results: SINGLE-UNIT DATA: IL single-units had heterogeneous response profiles to the lever press during self-administration and extinction. During self-administration, some units had a short latency burst after the lever press, while others decreased firing. A third type of unit response profile was a ramping down of activity 500 ms before the lever press, then a recovery 500 ms after the lever press. This response profile was the most common during the first extinction session, but the number of units showing this diminished as extinction progressed. At reinstatement, IL single units showed a decreased in firing upon lever pressing. During self-administration, BLA single-units responses increased upon trial start and at reinforced lever press. However, these responses diminished during extinction and were not identified in reinstatement. During self-administration, RAIC single units exhibited heterogeneous responses at lever press. A nearly equal proportion of units ramped up or down within 1 s of the lever press. During early extinction, half of RAIC single-units’ activity significantly decreased upon the unreinforced lever press, but these responses were absent by the end extinction. LFP DATA: Spectral analyses revealed that RAIC alpha band (10-13 Hz) power significantly increased upon lever press during self-administration. During extinction sessions 1 and 5, theta band (4-10 Hz) power significantly dropped at the trial start signal, which was not observed during self-administration. However, significant increases in theta power occurred at the lever press during reinstatement. Throughout extinction, dynamic changes were observed in the IL. Upon unreinforced lever press, there was a significant decrease in the alpha band, followed by increases in the theta band (consistent with our previous findings). These band changes became more prominent as extinction progressed. IL theta power significantly increased upon lever press during the reinstatement session (also, consistent with our previous findings). During self-administration, BLA theta increased at trial onset, followed by an increase in power in the alpha band upon lever pressing. During extinction BLA theta increased after the unreinforced lever press and became more prominent throughout extinction and was maintained in reinstatement. Conclusions: Although this work is still being conducted, there appears to be a dynamic network in the IL-RAIC-BLA circuitry during drug seeking behavior. Subpopulations of IL and RAIC single units ramp their activity during goal-directed behavior, whereas BLA appears to encode predictive cues relevant to drug-seeking behavior. Oscillations in the theta and alpha bands were sensitive to changes in contingencies from self-administration to extinction in the IL and RAIC. Further data collection and in-depth connectivity analyses may reveal how the current corticolimbic circuit forms a functional network during the extinction of heroin seeking. Keywords: Recordings, Anterior Insula, Heroin Self-Administration, Basolateral Amygdala, Infralimbic Cortex Disclosure: Nothing to disclose. P775. Conditional Inducible Deletion of CB1 Receptors on Ventral Tegmental Area Astrocytes Maladaptively Potentiates Calcium Mobilization and Curtails Motivation for Food Reward Lanyuan Zhang, Andrew Kim, Kate Peters, Sonia Aroni, Ramesh Chandra, Natalie Zlebnik, Mary Kay Lobo, Joseph Cheer University of Maryland School of Medicine, Baltimore, Maryland, United States Background: Cannabinoid type I receptor (CB1R) is a critical regulator for dopamine (DA) neuron activity in the ventral tegmental area (VTA). In addition to the retrograde inhibition of neurotransmitter release, CB1R has been found to profoundly modulate neuronal plasticity by promoting astrocytic calcium (Ca2+) transients and gliotransmission in the hippocampus and the nucleus accumbens (NAc). However, whether VTA astrocytic CB1R modulate Ca2+ mobilization has not yet been explored. Furthermore, the consequences of ventral tegmental astrocytic CB1R deletion on mesolimbic dopamine release and motivated behaviors remain undetermined. Methods: In this study, we applied conditional mutagenesis to delete VTA astrocytic CB1R transcripts in adult mice to eliminate the confounding and potentially adverse effect of region-specific constitutive CB1R knockdown on neural development. And using fiber-photometry in freely behaving mice, we measured VTA astrocytic Ca2+ transients and NAc dopamine release in a progressive ratio task. Results: Conditional, inducible CB1R deletion in VTA astrocytes maladaptively elevated astrocytic Ca2+ transients, reduced motivation for food reward and inhibited reward-evoked DA release in the NAc. Conclusions: These findings establish CB1R on astrocytes in the VTA as critical regulators of mesolimbic dopaminergic projections recruited during the motivated pursuit of reward. Keywords: Astrocyte, CB1 Receptor, Ventral Tegmental Area (VTA), Dopamine, Incentive Motivation Disclosure: Nothing to disclose. P776. Chronic Stress Alters the Intrinsic Properties of Parvalbumin-Positive Interneurons in the Ventral Hippocampus Jennifer Donegan, Alyssa Marron, Lauren Hewitt, Alexandra Sanchez, Darrin Brager Dell Medical School at the University of Texas at Austin, Austin, Texas, United States Background: The ventral hippocampus (vHipp) regulates a diverse set of behaviors associated with motivation and emotion. Functional abnormalities in this region are observed in a variety of psychiatric disorders, including mood and anxiety disorders. We previously demonstrated that restoring the function of parvalbumin (PV)-positive inhibitory interneurons in the vHipp of rodents alleviates physiological and behavioral deficits, including alterations in dopamine cell activity, reduced social interaction, and cognitive inflexibility. Chronic stress is a major risk factor for mood and anxiety disorders. Therefore, the goal of the current experiments is to determine how chronic stress affects the synaptic connectivity and intrinsic membrane properties of PV-positive interneurons in the vHipp. Methods: To induce chronic stress, mice underwent chronic unpredictable stress (CUS), in which they were exposed to a total of 14 varied stressors, administered twice per day for 21 days. We used the mammalian GFP reconstitution across synaptic partners (mGRASP) technique to measure the synaptic connectivity between PV positive-interneurons and pyramidal cells in the vHipp. In a separate cohort of animals, we used whole-cell current clamp to measure the intrinsic properties of PV-positive neurons in the vHipp. Results: Our preliminary results suggest that CUS does not alter the number of synaptic contacts between PV neurons and vHipp pyramidal cells. However, PV neurons from CUS mice had a significantly lower input resistance compared to control mice. Additionally, CUS PV neurons fired fewer action potentials and had a smaller afterhyperpolarization compared to control PV neurons. Conclusions: These results suggest that the activity of PV-positive interneurons is altered by chronic stress. Understanding how chronic stress affects PV-positive interneurons in the vHipp may lead to the development of new cellular targets for the treatment of psychiatric disorders associated with vHipp dysfunction. Keywords: Parvalbumin Neurons, Ventral Hippocampus, Chronic Stress, Slice Electrophysiology, Synaptic Connections Disclosure: Nothing to disclose. P777. Corticotropin Releasing Factor (CRF) in Primate Extended Amygdala and Ventral Pallidum: Let’s Discuss Co-Transmission! Julie Fudge, Emily Kelly, Troy A. Hackett University of Rochester Medical Center, Rochester, New York, United States Background: The central extended amygdala (CEA) and ventral pallidum are involved in diverse motivated behaviors, and encompass a large region of the basal forebrain in primates. Corticotropin releasing factor (CRF), a canonical ‘stress molecule’ is enriched in the CEA, and is dynamically regulated. CRF’s role in behavior--particularly in higher species--has been hard to parse, in part because it modulates primary ‘fast’ transmitters in neural circuits. As a first step in clarifying CRF’s role in the CEA and ventral pallidum in a species that is anatomically close to the human, we delineated CRF’s distribution and co-expression with ‘fast’ transmitters’ in this complex region. Methods: To clarify CRF’s role in the context of its primary transmitters, we used a combination of immunocytochemical and spatial transcriptomics (RNAScope) approaches to 1) survey the distribution of CRF-positive neurons in subregions of the CEA and ventral pallidum, 2) examine the overall proportions of GABAergic and glutamatergic neurons by subregion, and 3) determine the proportion of CRF-mRNA neurons that co-express glutamate (Vglut2 mRNA) and GABA (Vgat mRNA) by subregion. Three young male macaques of similar age (3 years old) and housing were used. Results: Despite a dense cluster of CRF labeled cells in the BSTLcn, the majority of CRF-IR neurons were broadly dispersed throughout the rest of the CEA, including in the BSTLP, SLEA, CeLcn and CeM. This picture may be somewhat at odds with rodent models in which CRF-positive neurons are depicted as largely confined to dense clusters in BSTL or CeN. Surprisingly, the VP—both at classic rostral levels and its continuation into the ventromedial GPi-- also contained CRF-expressing cell bodies, detected both with protein and mRNA assays. We first examined VGAT and VGluT2 expression without respect to CRF expression. Overall, single-labeled VGAT-mRNA positive cells were the most prevalent cell type in the CEA and ventral pallidum (80%); however, VGluT2 mRNA was expressed in 20% of all neurons, 10% of which were VGAT/VGluT2 positive. Regional differences in the distribution of VGAT- and VGluT2- mRNA positive cells across CEA and ventral pallidal subregions were striking. With respect to the CRF neuronal population, CRF/VGAT-only neurons were found in highest proportions in lateral central bed nucleus, lateral central amygdala nucleus, and medial central amygdala nucleus (74%, 73%, and 85%, respectively). Lower percentages of CRF/VGAT-only labeled cells (53%, 54%, 17%, respectively) characterized the sublenticular extended amygdala, ventrolateral bed nucleus, and ventral pallidum. These regions had higher complements of CRF/VGAT/VGluT2 labeled neurons (33%, 29%, 67%, respectively). Across all subregions, relatively stable, low proportions of CRF/VGluT2 and CRF-mRNA single-labeled cells comprised the balance of the CRF labeled cells. Conclusions: CRF expression in the CEA and VP was in general widely dispersed, although a distinct CRF-positive cell clusters was apprecieated in the BSTLcn. This was appreciated in both protein and transcript assays. CRF co-expression with its primary transmitters was heterogeneous, depending on subregion. The dorsal bed nucleus and central nucleus (the ‘poles’ of the CEA) have relatively homogeneous populations of CRF- GABAergic neurons, mirroring their relatively restricted efferent systems. In contrast, the ventral lateral bed nucleus and sublenticular extended amygdala, and the ventral pallidum, have heterogeneous CRF cell types, including mixed GABA/glutamatergic subpopulations, and broader efferent paths. Keywords: Glutamate GABA Co-Release, Corticotropin-Releasing Factor (CRF), Nonhuman Primate Disclosure: Nothing to disclose. P778. Midfrontal Theta-Band Activity During Response Monitoring in Pediatric Patients With Attention-Deficit/Hyperactivity Disorder and Other Psychiatric Conditions Takakuni Suzuki, Pan Gu, Paul D. Arnold, Stephan Taylor, Yanni Liu, William Gehring, Gregory Hanna, Ivy Tso University of Michigan, Ann Arbor, Michigan, United States Background: The error-related negativity (ERN) is an event-related potentials (ERPs) characterized by a negative deflection in midfrontal electroencephalogram (EEG) recording after making an error in speeded response tasks. It is one of the most commonly studied ERP components and has been investigated as a potential physiological indicator of underlying transdiagnostic mechanisms, particularly in individuals with obsessive-compulsive disorder (OCD; abnormally large ERN) and attention-deficit/hyperactivity disorder (ADHD; abnormally small ERN). However, the ERN has been shown to reflect the combination of increased total power and partial increased intertrial phase coherence (ITPC) in theta-band (4-8 Hz) activity. Our group previously demonstrated that theta-band power was abnormally enhanced in OCD pediatric patients, but that the ITPC was comparable to HC. This study extends this knowledge on theta-band power and ITPC to other forms of psychopathology during response monitoring to enable a richer understanding into the transdiagnostic neurophysiological mechanism of various psychiatric conditions. Methods: EEG was recorded from 461 pediatric participants (Age M = 14.20, SD = 3.16; 57% female) with ADHD (N = 107), non-OCD forms of Anxiety disorders (AD; N = 185), and major depressive disorder (MDD; N = 67), as well as healthy control participants (HC; N = 196), while they completed the arrow flanker task. To extract theta-band power and ITPC from a midfrontal channel (Cz), time-frequency analyses using complex Morlet wavelet convolutions were conducted. Preliminary ANCOVAs, including sex, age, and accuracy as covariates, comparing each psychiatric condition group to HC were conducted separately. For each psychiatric group, two mixed ANCOVAs were conducted to investigate the effects of diagnostic status (vs. HC) and response type (error vs. correct) on post-response theta-band (1) power and (2) ITPC (total of 6 ANCOVAs). Results: Theta-band total power and ITPC were larger on error responses than on correct responses in all four groups (All F statistics larger than 208 [p < 0.001] for power and larger than 18 [p < 0.001] for ITPC). In the ANCOVAs of power, there was a Group x Response Type interaction in ADHD patients (F[1,301] =10.46, p = 0.001), such that correct response power was comparable to HC, but ADHD patients had lower error response power than HC participants. There was no other interaction effect of Group x Response Type and there was no main effect for group differences in total power among AD, ADHD, and MDD pediatric patients. For ITPC, there was no significant Group x Response Type interaction for any ANCOVA. However, there was a main effect of group in the ANCOVA comparing ADHD and HC (smaller ITPC in ADHD patients; F[1, 298] = 10.34, p = 0.001). There was no other main effect of group. Conclusions: This project investigated the potential theta-band power and ITPC abnormalities in pediatric patients with ADHD, AD, and MDD. AD and MDD patients exhibited comparable theta-band power and ITPC to HC. However, ADHD patients showed a complex theta-band power and ITPC pattern. They had comparable correct response power to HC, but abnormally smaller power on error responses. They also showed lower overall ITPC regardless of response type compared to HC participants. These data suggest that pediatric patients with ADHD have difficulty specifically in monitoring error responses, which could underlie their difficulty in noticing and correcting mistakes. At the same time, they also appear to have general response monitoring deficit as reflected by the generally lower ITPC, which might underlie the general impulsive tendencies of individuals with ADHD. The findings also underscore the utility of time-frequency analyses in investigating similarities and differences in neural mechanisms across various psychiatric conditions and in informing future neuromodulation treatment research. Keywords: EEG Electrophysiology, Time-Frequency, Attention Deficit Hyperactivity Disorder, Major Depressive Disorder, Anxiety Disorders Disclosure: Nothing to disclose. P779. Serotonin Receptors 2 A in the Rat MPFC are Necessary for Retrieval Induced Forgetting Maria Belen Zanoni, Mariana Imperatori, Michael Anderson, Pedro Bekinschtein, Noelia Weisstaub* Instituto de Neurociencia Cognitiva y Traslacional (INCYT), Buenos Aires, Argentina Background: Forgetting is a ubiquitous phenomenon that is actively promoted in many species. The very act of remembering some experiences can cause forgetting of others, in both humans and rats. We previously found that when rats need to retrieve a memory to guide exploration, it reduces later retention of other competing memories encoded in that environment. As with humans, this retrieval-induced forgetting (RIF) relies on prefrontal control processes, is competition-dependent (only occurs when memories compete) and is cue-independent (forgetting generalizes to a variety of cues). RIF is thought to be driven by inhibitory control signals from the prefrontal cortex that target areas where the memories are stored. Here we started disentangling the neurochemical signals in the prefrontal cortex that are essential to retrieval-induced forgetting. Specifically, this work aims to explore if and how the serotonergic system, participates in RIF and if the βarr2 signaling pathway is recruited when competition between memories takes place. Methods: Ethics statement: All experimental procedures were conducted in accordance with institutional regulations (Institutional Animal Care and Use Committee of the School of Medicine, University of Buenos Aires, ASP #49527/15) and government regulations (SENASAARS617.2002). All efforts were made to minimize the number of animals used and their suffering. Subjects: Male and female adult Wistar rats (weight range, 200–300 g) were used for the different experiments. When possible, we conducted within-subject or mixed experiments to reduce the number of total animals required. For each treatment, we reached a sample size of between 8-11 rats. Behavioral task: We modified the spontaneous object recognition procedure to include three phases equivalent to the ones present in human studies of retrieval-induced forgetting: encoding, retrieval practice, and test. In addition to this retrieval practice condition, there were two control conditions in which the intervening retrieval practice phase was replaced either by returning the rat to its home cage (time control) or by giving the rat the same number of exploration trials on entirely new objects (the interference control). Pharmacology: We used a pharmacological approach to manipulate the serotonin receptor 2 A (5-HT2aR) activity in the medial prefrontal cortex (mPFC) of cannulated rats, specifically during the phase when memories compete (retrieval practice). In different experiments, we infused an antagonist of the 5-HT2aR (MDL 11,939), specific inhibitors for members of the βarr2 signaling pathway, and an agonist of the 5-HT2aR (TCB-2). Quantification of behavior and statistical analysis: exploratory behavior was measured offline. We calculated a discrimination index as a proxy of memory and perform one-way ANOVA followed by Bonferroni’s post hoc comparisons in the experiments without intracranial infusion and two-way repeated-measures ANOVA followed by Bonferroni’s post hoc comparisons in the experiments with drugs or vehicle infusions. Results: We found that retrieval-induced forgetting of competing memories in rats requires prefrontal serotonin signaling through 2 A receptors. Blockade of medial prefrontal cortex 2 A receptors as animals encountered a familiar object impaired active forgetting of competing object memories as measured on a later long-term memory test (p < 0,0001). Moreover, infusion of a PI3K inhibitor, which is part of the Barr2 pathway, impaired RIF but did not affect memory in other ways (forgetting did not occur in any of the control conditions, p < 0,001). Consistently, injection of an agonist of the 5-HT2aR promoted RIF in animals that would not normally forget (p < 0,0001). Conclusions: We observed a bidirectional modulation of retrieval-induced forgetting by agonists and antagonists of 5-HT2aR in the medial prefrontal cortex. These findings establish the essential role of prefrontal serotonin in the active forgetting of competing memories, contributing to the shaping of retention in response to the behavioral goals of an organism. Future experiments will be designed to elucidate if this serotonin signaling targets the hippocampus to address RIF and if it is through the nucleus reuniens as the intermediate structure. Keywords: Retrieval Induced Forgetting, 5-HT2A Receptors, Behavioral Tasks, Inhibitory Control, Medial Prefrontal Cortex Disclosure: Nothing to disclose. P780. Inhibitory Circuit Correlates of Suicidal Ideation in Veterans Jennifer Barredo, Hannah Swearingen, Melanie Bozzay, Jake Winter, Jennifer Primack, Noah Philip Alpert Medical School, Brown University, Providence, Rhode Island, United States Background: Background: Problems with emotional regulation are associated with greater risk for suicidal ideation and behavior. Studies using functional magnetic resonance imaging (fMRI) and the stop signal task, an inhibitory control task, have linked lower activation in inhibitory brain circuits to poor emotional regulation. Lowered inhibitory circuit function may influence how ideation escalates and evolves over time, though few fMRI studies have examined this directly. Here, we examined the relationship between inhibitory function and ideation in a transdiagnostic sample of Veterans. Methods: We collected fMRI blood oxygen level-dependent (BOLD) activation during the stop signal task from Veterans (N = 16) with suicidal ideation referred from in-patient psychiatry or outpatient clinics at the Providence VA Health System. Self-reported ideation and behavior was collected prior to MRIs using the Columbia-Suicide Severity Rating Scale (C-SSRS). We identified inhibitory regions by contrasting BOLD during trials when participants successfully inhibited motor responses (stop trials) with non-stop trials (Go trials). Results were dual threshold multiple comparisons corrected (voxel p < 0.001, cluster p-FDR < 0.05). Results: Successful stopping evoked stronger fMRI activation in regions within circuits supporting inhibitory control (left supplementary motor area [SMA], left rostral anterior cingulate cortex [ACC], and right opercularis) and in right orbitalis. In all regions, recent ideation was negatively correlated with differences in stop and go trial activation (all r < -0.31). Conclusions: Our results support inhibitory function as a potential transdiagnostic correlate of ideation in Veterans. Notably, two regions (ACC, orbitalis) have also been linked to negative valance processing by previous fMRI studies examining correlates of ideation. Future studies should consider evaluating how interactions between inhibitory and valence circuits influence suicidal ideation. Keywords: Suicidal Ideation, Inhibitory Control, Veterans, fMRI Disclosure: Nothing to disclose. P781. The Mediating Role of the Addictions Neuroclinical Assessment Domain Factors in the Relationship Between Childhood Trauma and Alcohol Use Disorder With and Without Comorbid Mood Disorder Tommy Gunawan, Hannah Kim, Rajon Scott, Noa Leiter, Emma McCabe, Mikayla Bergwood, Jeremy Luk, Melanie Schwandt, Laura Kwako, Tonette Vinson, Yvonne Horneffer, David George, George Koob, David Goldman, Nancy Diazgranados, Vijay Ramchandani National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland, United States Background: The Addictions Neuroclinical Assessment (ANA) is a neuroscience-based clinical framework to better understand the etiology and heterogeneity of alcohol use disorder (AUD). ANA consists of three neurofunctional domains which are thought to underlie AUD: Executive Function (EF), Negative Emotionality (NE), and Incentive Salience (IS). History of childhood trauma (CT) is associated with disruptions in EF development, mood and affect, and reward processing in adulthood, as well as higher risk of AUD and comorbid psychiatric conditions. However, the mechanisms by which CT confers risk to AUD and other comorbidities is unclear. The present study seeks to examine the mediating role of the ANA domains in the relationship between CT and AUD with and without a comorbid mood disorder (MD). Methods: Participants (N = 300; 41.0% female; 74.0% with history of AUD; 40.7% with a history of MD) who were enrolled in the NIAAA Natural History Protocol completed the ANA battery, which consist of neurocognitive and self-report assessments of the three ANA domains. EF measures were response inhibition, short-term memory, inferential reasoning, task switching, mental rotation, attention, metacognition, and interoception. NE measures were distress tolerance, ostracism, amotivation, anhedonia, resilience, affect and alexithymia. IS measures were approach-avoidance bias, implicit alcohol associations, and alcohol demand. Ancillary measures assessed in the Natural History Protocol pertinent to the three domains were included in the analyses (alcohol sensitivity, anxiety, depression, aggression, impulsivity, craving, personality, delay discounting). CT was measured using the Childhood Trauma Questionnaire (CTQ). Psychiatric diagnoses were assessed using the Structured Clinical Interview for DSM‑5 Disorders. Participants were classified based on their history of AUD and mood disorder: Healthy controls (HC; n = 68, 23.9%), AUD only (n = 108, 36.0%), and AUD with comorbid MD (AUD + MD; n = 113, 37.7%). Those with MD only (i.e., without comorbid AUD) were excluded due to the small group size (n = 8, 2.7%). Factor analyses were used to identify the latent factors underlying each domain. Analysis of variance were used to compare CTQ scores and factor scores across the three diagnostic groupings. Structural equation models (SEM) were used to evaluate the relationships between CT, domain factors, and psychiatric diagnoses. Age, sex and race were included as covariates. Bonferroni correction was used to correct for multiple testing. Results: Five factors of EF were identified: Response Inhibition, Working Memory, Interoception, Rumination, and Impulsivity (TLI/CFI > 0.91, RMSEA = 0.05). Two factors of IS were found: Alcohol Sensitivity and Alcohol Motivation (TLI/CFI > 0.99, RMSEA = 0.03). Three factors of NE were elucidated: Internalizing, Externalizing, and Resilience (TLI/CFI > 0.94, RMSEA = 0.07). CTQ scores were associated with all factors (standardized estimates = 0.11 to 0.57, p’s < 0.02), except for alcohol sensitivity (p = 0.10) and response inhibition (p = 0.052). Compared to HC, individuals with a history of AUD (with and without comorbid MD) showed elevated dysfunction in all domain factors (Cohen’s d = 0.09 to 3.34; p’s < 0.01) except for resilience and response inhibition (p’s > 0.17), and endorsed greater levels of CT (Cohen’s d = 0.61, p < 0.001). Those with AUD + MD also showed greater levels of dysfunction in all domain factors (Cohen’s d = 0.17 to 3.53; p’s < 0.01) except for response inhibition and working memory (p’s > 0.82), and exhibited greater CT compared to those with AUD only (Cohen’s d = 0.46; p < 0.01). After controlling for all identified factors and covariates, only alcohol motivation mediated the relationship between CT and membership to the AUD only group (indirect effect = 0.31, p < 0.001), but not the AUD + MD group (indirect effect = 0.10, p > 0.20). Conclusions: We identified unique factors underlying each ANA domain. Individuals with AUD with and without comorbid MD showed elevated levels of dysfunction on most of these domain factors relative to HC, and individuals with AUD + MD showed additional levels of dysfunction compared to AUD only. CT was associated with most of these identified factors, but only alcohol motivation was a significant mediator between CT and risk of developing AUD without MD. This suggests that addressing heightened alcohol motivation in individuals with a history of CT may be especially pertinent in improving AUD treatment outcomes. Future analysis will focus on subtypes of CT (e.g., abuse, neglect) as well as additional psychiatric comorbidities commonly associated with AUD. Keywords: Childhood Trauma, Alcohol Use Disorder, Mood Disorder, Addictions Neuroclinical Assessment, RDoC Disclosure: Nothing to disclose. P782. Transdiagnostic Neural Correlates of Fear and Anxiety Sensitivity in a Focal Fear Sample Annmarie MacNamara, Shannon MacDonald Texas A&M University, College Station, Texas, United States Background: Fear and anxiety sensitivity (the belief that anxiety symptoms or arousal can be harmful/“fear of anxiety-related sensations”), are transdiagnostic constructs that may help parse heterogeneous diagnostic categories into more homogeneous, neurobiologically-based constituents, laying the groundwork for more targeted classification of the anxiety disorders. Both constructs have both been linked to aberrant processing of negative stimuli; as distinct contributors to psychopathology, fear and anxiety sensitivity were expected to show specific associations with neurocircuit response. Methods: Fifty-two adults (37 female; M = 23.65 years, SD = 9.69) who all shared a common “focal fear” diagnosis (i.e., specific phobia or performance-only social anxiety disorder), but varied in extent of additional comorbid anxiety and mood disorders, passively viewed negative and neutral pictures while fMRI BOLD was recorded. Analyses focused on identifying the unique neural correlates of fear and anxiety sensitivity, above and beyond the other dimension. Results: Greater fear was associated with reduced negative > neutral fMRI BOLD in the thalamus (t = 3.85, p < 0.05 FWE), suggesting avoidant processing of negative stimuli. On the other hand, higher levels of anxiety sensitivity were associated with increased negative > neutral fMRI BOLD in the insula (t = 3.93, p < 0.05 FWE), a brain region implicated in the representation of interoceptive information. Conclusions: Fear and anxiety sensitivity show unique associations with threat neurocircuitry, suggesting distinct means of responding to negative stimuli in the environment. These transdiagnostic constructs could serve as candidates for a more fine-grained means of understanding pathological anxiety. Keywords: Transdiagnostic, Negative Emotionality, fMRI, Adult Clinical Anxiety Disclosure: Nothing to disclose. P783. Race, Ethnicity, Education, Sex and Gender Effects on Neuropsychological Test Scores: Limitations of Current Evidence and Impact on Clinical Trials and Clinical Practice Phoebe Katims, Kristen Enriquez, Robert Bilder Semel Institute for Neuroscience and Human Behavior, Los Angeles, California, United States Background: Interpretation of neuropsychological (NP) tests depends on the quality of the normative standards available for the tests. The precision of measurement for each test variable depends on the psychometric properties of the test and how many people were used to standardize that test. Co-norming across tests is necessary when interpreting differences between scores on different tests at one time point (i.e., profile or discrepancy score analysis), or differences between scores on the same test repeated over time. The relevance of specific norms for an individual examinee further depends on multiple design features of the standardization studies, including: when the studies were conducted, sampling strategy, inclusion/exclusion criteria, age, sex/gender, education, race and ethnicity, socioeconomic status, and region. This paper examines the standardization studies of the most widely used NP tests, identifies their strengths and weaknesses, and makes recommendations for interpretive caveats based on these analyses. Methods: We reviewed the standardization strategies and coded information about the sampling frames, inclusion/exclusion criteria, stratification methods, sample sizes overall and within each stratum where relevant, methods for representing or analyzing race, ethnicity and other demographic characteristics. These methods were applied to the WAIS-IV, WMS-IV, CVLT3, D-KEFS, Pearson Advanced Clinical Solutions (ACS), Rey Complex Figure Test, WCST, Symbol Digit Modalities Test, RBANS, BVMT-R, HVLT, Halstead-Reitan (“Heaton et al.”) Norms for Boston Naming, Finger Tapping, Grooved Pegboard), MOANS, and MOAANS (Boston Naming, Trail Making Test, Judgement of Line Orientation). We calculated multiple indexes for each test, including standard errors and confidence intervals for scaled scores, and standard errors of measurement for repeated measures based on reported test reliabilities. Results: Most tests used age only as a stratification factor, providing “age corrected” scores for selected age bands. The sample sizes for the age strata range from 1 to ~200 but are usually less than 100 participants/stratum. Sex differences were rarely reported, and while larger studies estimated sex distributions from census statistics, some studies had markedly uneven distributions of sex. Education was not used as a stratification factor in any study, and only the ACS, Heaton and MOANS/MOAANS norms attempt corrections for education. The possible interactions of age and education on test scores are seldom reported and cell sizes for combinations of age and education may be too small to enable robust estimates of scores especially at lower levels of education and older ages. The possible impact of race and ethnicity are rarely interrogated except in ACS, Heaton and MOAANS norms, which all focus on “African American” participants. Discrepancies in scores across ACS, Heaton and MOAANS suggest marked sampling differences, and show the same raw scores may yield clinically meaningful differences in scaled scores depending on which norms are used. Most of the norms studied are at least 15 years old, and poorly represent current racial and ethnic characteristics of the United States. Conclusions: Existing norms have major limitations and may impact the clinical assessment of individuals and result in inappropriate treatment recommendations as well as inappropriate classification in clinical trials, which may include score “cutoffs” based on widely used normative standards. Particularly, race and ethnicity are poorly represented and existing norms present major conflicts for African American groups, with the same raw scores differing by a full standard deviation depending only on the source of normative data. Furthermore, these norms often fail to reflect demographic shifts in the United States, making underrepresentation of racial and ethnic minority groups more marked than before and leading to questions about whether results from these measures can be generalized. Additionally, sex differences are examined infrequently and it remains unclear to what extent sex or gender differences may affect some scores. Co-norming was done for only selected measures and those sample sizes are smaller so the precision of measurement of difference scores is often low; this is unfortunate because interpretation of clinical results and findings from clinical trials often involves examining differences between tests. Most norms use only age as a stratification factor, despite robust impacts of education on scores. Lack of standardization by educational background and selection of “representative” samples means that those of higher education will be given inappropriately higher standard scores and those of lower educational opportunity will be given inappropriately lower standard scores relative to their true abilities. There is an urgent need for new, preferably “dynamic” normative standards, that include sampling by socially and demographically meaningful metrics, to provide greater precision in assessment of neuropsychological scores and score discrepancies, and for evaluating the inclusion/exclusion criteria, and criteria for efficacy in clinical trials that use neurocognitive endpoints Keywords: Neuropsychology, Neurocognitive Assessment, Psychometric Properties, CNS Clinical Trials Disclosure: Atai Life Sciences: Honoraria (Self), Verasci: Honoraria (Self). P784. Tonic and Phasic Neurophysiological Relationships With Adverse Posttraumatic Outcomes Vary by Racial/Ethnic Group Nathaniel Harnett, Negar Fani, Sierra Carter, Leon Sanchez, Tanja Jovanovic, Grace Rowland, Lauren LeBois, Timothy Ely, Sanne van Rooij, Antonia Seligowski, Steven Bruce, Stacey House, Samuel McLean, Jennifer Stevens, Kerry Ressler Harvard Medical School, McLean Hospital, Belmont, Massachusetts, United States Background: Racial and ethnic groups experience differing levels of socioeconomic stress that can affect responses to traumatic stress. Emergent work demonstrates that both structural inequities and individual racism-related stressors underlie race-related differences in neural and physiological responses. In particular, core threat neurocircuitry such as the amygdala may be a key region impact by structural racism. Greater levels of negative life events throughout development appear to contribute to both lowered threat-elicited amygdala responses and subsequent skin conductance responses (SCRs) in racially minoritized individuals. Disparate levels of stressor exposure between groups may also partially explain race-related differences in posttraumatic symptoms after trauma exposure. However, limited research to date has investigated brain and physiological responses that may predict posttraumatic outcomes and whether these may differ by racial/ethnic group as a result of structural racial inequities. Understanding potential heterogeneity in brain-behavior relationships is crucial for the development of generalizable neural treatments and interventions for trauma and stress-related disorders. Methods: Participants (n = 283) were recruited as part of the AURORA Study, a multisite, longitudinal study of trauma and posttraumatic outcomes. Briefly, trauma-exposed participants were recruited from 22 Emergency Departments (EDs) from across the United States. Initial participant demographic and psychometric data were collected after admission to the ED which included trauma exposure type, participant marital status, income, education level, and employment. Participants’ home address was geocoded to derive an area deprivation index (ADI) to reflect neighborhood disadvantage. PTSD, depression, and anxiety symptoms were assessed with the PTSD Symptom Checklist for DSM-5 (PCL-5) and the PROMIS. Participants completed psychophysiological recording and functional magnetic resonance imaging (fMRI) approximately 2-weeks after trauma exposure. Skin conductance level (SCL) and SCR were assessed during a Pavlovian conditioning task. Amygdala reactivity to threat was assessed during passive viewing of fearful and neutral faces during fMRI and was indexed as the BOLD response for the fearful > neutral contrast. Amygdala functional connectivity was assessed using resting-state fMRI using a seed to voxel approach. Multiple comparisons at the voxel level were corrected using a clustering approach (alpha = 0.05) and Benjamini-Hochberg False Discovery Rate adjustments were made for other statistical tests. Results: Demographic data by racial/ethnic group are reported in Table 1. We observed significant differences in education level [X2 = 9.90, p = 0.007], income [X2 = 7.47. p = 0.023], and marital status [X2 = 6.46, p = 0.040]. No significant difference was observed in employment within the sample [X2 = 0.59, p = 0.745]. A significant difference in ADI was observed between the groups [F(2,280) = 31.73, p < 0.001]. There were significant racial/ethnic differences in tonic SCLs [F(2,126) = 6.41, p = 0.002] with Black participants showing significantly lower tonic SCL than White participants [t(61.40) = 3.25, p = 0.002, variance inequality adjusted]. Similarly, there were significant racial/ethnic differences in amygdala connectivity to dorsal anterior cingulate cortex, dorsolateral prefrontal cortex (PFC), insula, and cerebellum. However, there was no main effect of racial/ethnic group (p = 0.932) or racial/ethnic group by stimulus-type interaction (p = 0.832) on SCRs during threat acquisition. Similarly, there were no racial/ethnic differences in amygdala reactivity to threat (p > 0.05). Finally, multivariate models showed that racial/ethnic groups differed in the relationship between amygdala to dorsolateral PFC/dACC/insula connectivity and PTSD symptoms 3-months after trauma such that Hispanic individuals showed a negative relationship, white individuals showed an orthogonal relationship, and Black individuals showed a positive relationship between connectivity and symptom severity. Conclusions: The present findings suggest tonic levels of neurophysiological arousal differ between racial and ethnic groups in the early aftermath of trauma. However, phasic responses to threat remain highly similar. Importantly, race-related tonic differences also appear to influence prediction of subsequent PTSD symptoms. Together, these findings raise two important issues. First, they highlight how differences in exposure to life stressors may augment neural and behavioral reactions to subsequent traumatic stress as well as recovery from exposure. Second, these findings may suggest that neuromodulatory treatments thought to act on amygdala-PFC connectivity may have opposing effects depending on the life history of the patient. Further research is needed to better understand the neural mechanisms underlying the impact of structural racism on neurophysiological tone, and how to leverage such information for effective early interventions after trauma exposure. Keywords: Race Disparities, PTSD, Amygdala, Psychophysiology, Functional MRI (fMRI) Disclosure: Wiley Publishing: Honoraria (Self). P785. Mismatch Negativity Predicts Initial Auditory-Based Targeted Cognitive Training Performance in a Heterogeneous Veteran Population Across Psychiatric Disorders Yash Joshi, Christopher Gonzalez, Laura MacDonald, Addison Denning, Abigail Potter, Jenny Din, Jessica Minhas, Taylor Leposke, Juan Molina, Jo Talledo, Joyce Sprock, Neal Swerdlow, Gregory Light University of California, San Diego School of Medicine; VA San Diego Healthcare System, VISN 22 MIRECC, San Diego, California, United States Background: Auditory based targeted cognitive training (TCT) is a bottom-up, neuroplasticity-based computerized approach to cognitive remediation in which patients train on progressively more difficult auditory processing exercises designed to improve pitch and temporal acuity of low-level sensory information. This adaptive approach seeks to improve the speed, accuracy and fidelity of auditory information processing to generate upstream gains in cognition and function. Thus far, application of TCT has demonstrated utility in improving cognitive functioning and quality of life in those with schizophrenia spectrum disorders (SSD). Furthermore the event-related potentials mismatch negativity (MMN) and P3a, biomarkers of early auditory information processing known to be abnormal in SSD, predict TCT-associated cognitive gains in SSD populations. However, TCT may be broadly helpful for cognitive deficits associated with other psychiatric disorders as well, as presumably the neural circuits underlying TCT-associated gains in SSD populations are also present in others. In this pilot study we aimed to understand 1) how initial TCT performance is related to cognitive functioning in a heterogeneous population of patients with a range of psychiatric disorders, and 2) the utility of MMN and P3a in predicting initial performance on a TCT exercise which is representative of a typical ~30 h TCT training program. Methods: Age-matched Veteran participants (SSD n = 13, nonSSD n = 13, average age=51.9) with a range of psychiatric diagnoses engaged in mental health rehabilitation were recruited, and performance on a 1 hour TCT session was assessed. The SSD cohort was primarily composed of those with schizophrenia and schizoaffective disorder (85%), while the nonSSD group was primarily diagnosed with MDD (77%) and PTSD (69%). The TCT exercise consisted of Sound Sweeps, an auditory frequency discrimination task, in which participants completed “levels,” which consisted sets of 35 stimuli presentations. Stimuli presentation length were algorithmically modified based on correct or incorrect responses, in an adaptive manner. Initial TCT performance was operationalized as the score achieved over the first level of training, reported in milliseconds of stimuli presentation. Cognitive functioning was assessed by the MATRICS Consensus Cognitive Battery (MCCB). MMN and P3a were collected prior to completing 1 h of TCT. Results: On average, participants completed 13.1 levels (st.dev=5.3), with nonSSD participants completing more levels than SSD (nonSSD, average=15.5 levels, st.dev=5.3; SSD, average=10.7 levels, st.dev=4.4, p = 0.02). SSD participants scored lower on all MCCB domains compared to nonSSD participants, with significant differences in visual learning, problem solving and neurocognitive composite (all ps < 0.01). MMN amplitude between SSD and nonSSD subjects did not significantly differ in this cohort; P3a amplitude (p = 0.04) was significantly more impaired in SSD compared to nonSSD. Initial TCT performance was poorer in SSD compared to nonSSD participants, though did not achieve significance (nonSSD = 144.9 msec, st.dev=260.8; SSD = 276.2 msec, st.dev=349.0, p = 0.28). MCCB domain scores in working memory, verbal learning, visual learning, problem solving and neurocognitive composite were related to number of levels completed (r = 0.44, 0.60, 0.66, 0.55, 0.55 respectively; all ps<0.01). MCCB domain scores in attention, verbal learning, visual learning, problem solving and neurocognitive composite were related to initial TCT performance (r = −0.41, -0.45, -0.44, -0.39, -0.42 respectively; 0.023 < all ps <0.047). MMN was related to initial TCT performance (r = 0.52, p < 0.01), driven by SSD participants (r = 0.70, p < 0.01). Multivariable regression modeling revealed MMN and MCCB neurocognitive score were significant predictors of initial TCT performance (MMN, F = 8.2, p = 0.009; MCCB neurocognitive score, F = 6.0, p = 0.023) while P3a and diagnosis (SSD vs nonSSD) were not (Fs < 0.71, ps > 0.79). Conclusions: Neurophysiological biomarkers may have utility in predicting TCT performance not only in those with SSD but also other psychiatric disorders. These results inform future studies that aim to ameliorate cognitive impairment via TCT in a transdiagnostic manner. Further study of biomarker-guided TCT approaches across psychiatric populations is warranted. Keywords: Auditory Mismatch Negativity, Targeted Cognitive Training, Schizophrenia, PTSD, MDD Disclosure: Nothing to disclose. P786. Effects of Incentives on Spatial Working Memory in Patients With Schizophrenia and Patients With OCD Youngsun Cho, Doah Shin, Flora Moujaes, Charles Schleifer, Brendan Adkinson, Antonija Kolobaric, Morgan Flynn, Jie Lisa Ji, John Krystal, William Martin, John Murray, Grega Repovs, Christopher Pittenger, Alan Anticevic Yale Child Study Center, New Haven, Connecticut, United States Background: Motivational influences on cognition are critical for shaping goal-directed behaviors. Cognitive and motivational deficits co-occur across numerous psychiatric illnesses, presenting an opportunity to examine shared, as well as distinct, mechanisms across disorders. To begin to understand cross-diagnostic effects, we analyzed motivated spatial working memory (sWM) performance in adult patients with schizophrenia (SCZ), adult patients with obsessive-compulsive disorder (OCD) and typical adults (TYP) without psychiatric illness. Methods: An age-matched cohort of 27 patients with SCZ (m21, f6), 39 patients with OCD (m18, f21) and 34 typical adults (m21, f13) performed a previously published incentivized sWM task. This task was translated from similar tasks used with non-human primates that require spatial locations to be kept in mind. A joystick was used to indicate where participants remembered prior spatial locations. The possibility for monetary reward or loss was presented using cues (cued condition) or as a non-cued, contextual instruction given before a block of sWM trials (non-cued condition). A control condition that did not require working memory, but only motor movements with the joystick, was also included. sWM accuracy was calculated as the angular difference between presented and remembered spatial locations. Results: The TYP group performed significantly better than patients with SCZ and patients with OCD at manipulating the joystick without memory requirements (control condition) (main effect, p < 0.001). All subsequent analyses were covaried for performance in the control condition. Patients with OCD and TYP had significantly better baseline, neutral sWM performance, than patients with SCZ (ANCOVA, p < 0.001)). Reflecting the overall effects of incentives, there was a significant incentive (neutral sWM, reward, loss) x group (SCZ, OCD, TYP) interaction (p < 0.05). All groups improved sWM performance in response to reward and loss incentives (p < 0.05 for all pairwise comparisons). The interaction between incentive presentation (neutral sWM, cued, non-cued) and group trended towards significance (p = 0.065). Pairwise comparisons demonstrated that TYP differentiated between cued and non-cued presentation with significantly better sWM performance during cued presentations, compared to non-cued presentations (p < 0.005), while SCZ sWM performance did not differentiate between the two presentations (p > 0.05), and patients with OCD showed a trend towards differentiating between the two presentations (p = 0.06). When the effects of rewards and losses during cued presentation only were examined, all groups improved sWM performance when incentives were cued (main effect of cued incentive (neutral sWM, cued reward, cued loss), p < 0.01)) (non-significant interaction (cued incentive x group), p > 0.05). There was a significant interaction of non-cued incentive presentation (neutral sWM, non-cued reward, non-cued loss) and group (F(4,194)= 3.4, p < 0.05). Patients with SCZ had better sWM performance during non-cued loss (p < 0.005), but not during non-cued reward (p > 0.05). Patient with OCD and TYP had better sWM performance during both non-cued loss and non-cued reward (all p < 0.05). Conclusions: In general, incentives improved sWM performance in all groups. Differential effects of incentives on sWM performance in patients with SCZ, patients with OCD and TYP appeared related to the type of incentive presentation, and incentive valence. Future work may examine cognitive and motivational neural systems to better understand the systems in these groups that support sWM improvements in response to motivation. Keywords: Cognition, Reward, Schizophrenia (SCZ), OCD, Working Memory Disclosure: Nothing to disclose. P787. Willingness to Exert Social Effort is Selectively Related to Avoidance Motivation Chloe Savage, Greer Prettyman, Luis Fernando Viegas de Moraes Leme, Daniel Wolf University of Pennsylvania, Wynnewood, Pennsylvania, United States Background: Asociality, impairment in social motivation, is a disabling symptom of multiple neuropsychiatric disorders including schizophrenia, major depression, and autism. However, social motivation and willingness to exert social effort remain relatively understudied compared to motivation for monetary rewards. Choices about whether to exert social effort involve the desire for positive social interactions (social approach), but are also strongly influenced by a desire to avoid negative social experiences (social avoidance). In this study we investigated the relationships between social motivation, approach and avoidance tendencies, and clinical symptom domains associated with anxiety and depression. To quantify behavioral willingness to exert social effort, we developed a novel social effort discounting task (SEDT) and a parallel monetary effort discounting task (MEDT). We hypothesized that SEDT and MEDT would demonstrate similar relationships with general motivational tendencies, but that the social task would selectively relate to individual differences in self-reported avoidance tendencies and to social aspects of anxiety and depression. Methods: A sample of 500 participants (50% female, age range 18-30 mean 25) were studied via an online crowdsourcing platform (Prolific), yielding analyzable data from 413 individuals. Participants performed both the SEDT and MEDT, with two blocks of each task in counterbalanced order, and involving 200 total hypothetical choice trials. In both SEDT and MEDT, participants chose between a fixed low-effort/low-reward (EASY) and a variable higher-effort/higher-reward option (HARD) on each trial. In the SEDT, the reward unit was a “match” with a potential friend and the effort unit was a 25+ word invitation message; in the MEDT, the reward was a $1 gift card and the effort was a 25+ word request message. Task motivation was quantified using a linear discounting function SV = A-BE, where subjective value (SV) equals the amount of reward A discounted by the effort E. The free parameter B is estimated from the participant’s choices; the higher the B, the greater the negative impact of effort on SV and therefore the lower the motivation. Participants also completed a battery of self-report questionnaires including assessment of general motivation orientations for approach (Behavioral Activation Scale, BAS) and avoidance (Behavioral Inhibition Scale, BIS), as well as state motivation on the Motivation and Pleasure Scale (MAP), Chapman social and physical anhedonia scales, and general and social anxiety measures; their relationship to log-transformed SEDT and MEDT B values was examined. Results: SEDT and MEDT logB were strongly correlated (rho = 0.68), indicating roughly half the variance was shared reflecting general motivation. Higher logB (lower behavioral motivation) in both the SEDT and MEDT was inversely related to self-reported MAP global motivation (SEDT rho = −0.13, p = 0.006; MEDT rho = −.10, p = 0.04). Both task measures also correlated with trait approach motivation (BAS Drive, SEDT rho = −0.15, p = 0.002, MEDT rho = −0.14, p = 0.006). We also observed hypothesized task specificity. Trait avoidance motivation (BIS) correlated selectively with social task motivation (SEDT rho = −0.11, p = 0.03; MEDT rho=0.02, p = 0.65), and the relative strength of social vs. nonsocial task motivation (SEDT logB minus MEDT logB) correlated with avoidance motivation (rho = 0.10, p = 0.04). This relative strength subtraction measure also correlated significantly with social anxiety (rho = 0.10, p = 0.04) and trait social motivation (rho = −0.12, p = 0.01). Neither task related to depression severity. Conclusions: Consistent with our hypotheses, social and nonsocial task motivation were robustly related to each other and showed similar relationships to self-reported global state motivation and trait approach motivation. We also observed hypothesized differential relationships to avoidance motivation, trait social motivation and state social anxiety. Overall, our results indicate that the social EDT captures social-specific motivation above and beyond general motivation. These tasks can provide objective parallel behavioral measures of social and nonsocial motivation, facilitating investigation of the common and dissociable neurobehavioral underpinnings of social and nonsocial motivation and their disruption in neuropsychiatric disorders. Keywords: Social Reward, Motivation, Approach/Avoidance Disclosure: Nothing to disclose. P788. Neighborhood Violent Crime and Neural Correlates of Emotion Regulation in Patients With Major Depressive Disorder and Social Anxiety Disorder Cope Feurer, Jagan Jimmy, Melissa Uribe, Stewart Shankman, K. Luan Phan, Olusola Ajilore, Heide Klumpp University of Illinois at Chicago, Chicago, Illinois, United States Background: The capacity to regulate emotions in the face of aversive information plays a critical role in mental health. Reappraisal is a well-studied cognitive-linguistic regulation strategy known to engage regions that play a central role in top-down control and semantic processes. Accumulating data indicate the brain pathophysiology of debilitating internalizing conditions such as Major Depressive Disorder (MDD) and Social Anxiety Disorder (SAD) involve impairment in reappraisal mechanisms to varying degrees, highlighting individual differences in regulation capacity. Importantly, most of this work in adults has focused on individual-level factors (e.g., trauma history, symptom severity, problematic sleep). However, it is also important to understand how broader contextual factors (e.g., neighborhood characteristics) impact neurobiological correlates of emotion regulation. Specifically, there is increasing evidence that exposure to community violence or living in a neighborhood characterized by high levels of violent crime is associated with emotion regulation deficits in childhood and adolescence. However, the impact of community-level factors on the neural correlates of regulation in adults with psychopathology has been understudied. Therefore, the current study sought to address this gap in the literature by examining the relation between neighborhood violent crime and neurobiological indices of emotion regulation in patients with MDD and SAD from a large urban area. Methods: Participants were 63 un-medicated, treatment-seeking patients between the ages of 18 and 55 (Mean Age = 27.62; 63.5% female). Patients were administered the Liebowitz Social Anxiety Scale (LSAS) and Hamilton Depression Rating Scale (HAMD) to assess social anxiety and depression symptoms, respectively. Before treatment, participants completed a validated Emotion Regulation task in the scanner. The task comprised standardized images of general negative content, and regulation was evaluated by comparing the condition in which participants were instructed to decrease their emotional response to negative images via reappraisal (ReappNeg) relative to looking at negative images (LookNeg). Crime data were downloaded from the publicly available Chicago Data Portal and aggregated within census blocks to create indices of neighborhood violent and non-violent crime rates. Patients’ neighborhoods were defined by the census block of their home address at study enrollment. Finally, additional measures included the Area Deprivation Index, a geocoded index of neighborhood disadvantage, and the PCL-5, a measure of posttraumatic stress symptoms. Whole-brain regression analyses were conducted to evaluate the neural correlates of ReappNeg>LookNeg in which neighborhood violent crime was the covariate of interest and age and internalizing symptom severity (LSAS and HAMD composite score) were covariates of no interest. 3dClustSim was used to control for multiple comparisons, and clusters exceeding 558 voxels were considered significant (α < 0.05, p < 0.005). Results: Whole-brain regression analyses revealed an association between neighborhood violent crime and a cluster comprised largely of left inferior frontal gyrus (IFG; k = 627, z = 4.00, p < 0.001), such that greater violent crime associated with greater IFG engagement during reappraisal of negative stimuli. A similar cluster emerged for right IFG, though it did not survive correction for multiple comparison (k = 538, z = 3.91, p < 0.001). Follow-up tests of robustness indicated the main effect of neighborhood violent crime on left IFG activation was maintained when statistically controlling for patient neighborhood non-violent crime, neighborhood socioeconomic disadvantage, education, racial identity, and posttraumatic stress symptoms (all ps < 0.002). Conclusions: Current findings indicate that patients with MDD or SAD who live in neighborhoods with higher rates of violent crime exhibit more prefrontal engagement during emotion regulation. This association was, at least in part, independent of current symptomatology, neighborhood disadvantage or non-violent crime, trauma-related symptoms, and demographic characteristics, highlighting the unique contribution of environmental threat. Future research is needed to determine whether associations between neighborhood violent crime and greater IFG engagement during reappraisal also generalize to other internalizing conditions. Keywords: Functional MRI (fMRI), Emotion Regulation, Neighborhood Crime, Major Depressive Disorder, Social Anxiety Disorder Disclosure: Nothing to disclose. P789. Effects of Serotonin in Rats and Humans on Computations Underlying Flexible Decision-Making Jonathan Kanen, Qiang Luo, Andrea Bari, Nikolina Skandali, Barbara Sahakian, Johan Alsio, Benjamin Phillips, Rudolf Cardinal, Trevor Robbins University of Cambridge, Philadelphia, Pennsylvania, United States Background: Serotonin is critical for adapting behaviour flexibly to meet changing environmental demands; however, a unifying mechanistic framework accounting for its role in behavioural flexibility has remained elusive. Computational modelling may offer refined measures of behaviour which could additionally enhance comparisons across species and aid translational research. Methods: Probabilistic reversal learning, a well-established laboratory paradigm for studying behavioural flexibility, was tested in rats and humans following modulation of serotonin (5-hydroxytryptamine; 5-HT). Male rats were administered acutely either 1 mg/kg (n = 12) or 10 mg/kg (n = 18) of the selective serotonin reuptake inhibitor (SSRI) citalopram, or the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) which produces profound depletion of forebrain serotonin. In addition, rats (n = 7) were administered repeated 5 mg/kg citalopram for seven consecutive days compared with a vehicle group (n = 7). After seven days, the citalopram group received 10 mg/kg of citalopram twice per day for five consecutive days. Furthermore, healthy human male and female volunteers were administered an SSRI acutely (20 mg escitalopram; n = 65) in a double-blind randomised placebo-control design. Data were reanalysed using Bayesian hierarchical reinforcement learning models. Model parameters of interest were learning rates, for rewarded or punished outcomes, and “stickiness” which indexes a basic perseverative tendency, or choice repetition regardless of reinforcing feedback. In rats, stickiness was tied to a location (side or location of the cage) whereas in humans, stickiness was tied to a particular visual stimulus on a computer screen (stimulus stickiness). Results: Model comparison demonstrated the best fitting models included parameters for “stickiness,” learning rates (based on rewards and punishments separately), and reinforcement sensitivity which indexed the extent to which behaviour is driven by acquired value. We found that 5-HT depletion via 5,7-DHT significantly decreased side (location) stickiness (95% HDI: [-0.4635, -0.1134]) and the reward learning rate (85% HDI: [-0.0757, -0.0033]). There was no significant effect of 5,7-DHT on the reward learning rate, punishment learning rate, or reinforcement sensitivity [no group differences, all 0 ∈ 75% HDI]. A single dose of 1 mg/kg citalopram in rats also diminished the side (location) stickiness parameter (95% HDI: [-0.3330, -0.0441]). The reward learning rate was enhanced by the 1 mg/kg dose in rats (95% HDI: [0.0184, 0.3959]). There was no significant effect of 1 mg/kg on the punishment learning rate or reinforcement sensitivity (both 0 ∈ 75% HDI). A single high dose of citalopram in rats (10 mg/kg) decreased the reward learning rate (85% HDI: [-0.2888, -0.0009]) and enhanced reinforcement sensitivity (85% HDI: [0.0346, 0.5590]). There was no significant effect of 10 mg/kg on the punishment learning rate or side (location) stickiness. Seven-day administration of 5 mg/kg citalopram enhanced both the punishment learning rate (95% HDI: [0.0432, 0.6404]) and side (location) stickiness (75% HDI: [0.0135, 0.3054]). There was no significant effect of seven-day administration of 5 mg/kg citalopram on the other parameters (0 ∈ 75% HDI). Augmenting the preceding regimen with 10 mg/kg of citalopram twice per day, for five consecutive days, enhanced both the reward (95% HDI: [0.2699, 0.6780]) and the punishment (95% HDI: [0.2172, 0.7323]) learning rates, increased side (location) stickiness (90% HDI: [0.0075, 0.3414]), and decreased the reinforcement sensitivity (95% HDI: [-1.7233, -0.2540]). Administration of a single 20 mg dose of escitalopram to healthy humans decreased the reward learning rate (95% HDI: [-0.3612, -0.0392]), stimulus stickiness (85% HDI: [-0.3476, -0.0045]) and reinforcement sensitivity (80% HDI: [-3.1501, -0.1553]), and had no significant effect on the punishment learning rate (0 ∈ 75% HDI). Conclusions: Computational modelling of choices during probabilistic reversal learning revealed common effects of manipulating serotonin function across two species (rats and humans). These computational models revealed that the tendency to repeat behaviours irrespective of their outcome (stickiness) was decreased or increased by manipulations known to reduce (serotonin depletion in rats and acute low dose SSRI in both rats and humans) or boost (high repeated dose SSRI in rats) central serotonin function, respectively. Meanwhile, reward learning rates were also modulated bidirectionally across species and SSRI dose, while the punishment learning rates were altered (increased) only in instances of repeated SSRI dosing in rats. Collectively, these results support a role of serotonin in behavioural flexibility and plasticity across species that can be studied using computational measures that dissect choice, and has transdiagnostic relevance for neuropsychiatric disorders in which related subprocesses are perturbed. Keywords: Serotonin, Probabilistic Reversal Learning, Computational Reinforcement Learning Model Disclosure: Nothing to disclose. P790. Distinct Roles for Dopaminergic and Noradrenergic Signaling in Exploration During Decision Making Cathy Chen, Evan Knep, Dana Mueller, Becket Ebitz, Nicola Grissom University of Minnesota, Minneapolis, Minnesota, United States Background: The distinct roles of dopamine versus norepinephrine in catecholamine modulation of executive functions, especially decision making, have been surprisingly challenging to tease apart. In particular, whether there are distinct or shared roles for each neuromodulator in the explore-exploit tradeoff remains unclear. As differences in these decision making parameters are identified in numerous neuropsychiatric conditions, identifying neural mechanisms by which these parameters are altered will be important in guiding mechanistic hypotheses emerging from computational psychiatric approaches. Methods: Using the strengths of rodent models for repeated pharmacological testing, combined with our novel restless two-armed bandit task design, we tested the role of dopaminergic and noradrenergic transmission in decision making. We ran 32 mice (B6129SF1/J, 16 m/16 f) in a restless two-armed bandit task, which encourages both exploration and exploitation. We systemically administered a NE beta-receptor antagonist (propranolol), NE beta-receptor agonist (isoproterenol), a nonselective DA receptor antagonist (flupenthixol), and a nonselective DA receptor agonist (apomorphine) within subjects across sessions and examined changes in exploration as defined by a Hidden Markov Model approach, as well as analysis of overall performance, win-stay/lose-shift, and reinforcement learning model fits. Results: We found a bidirectional modulatory effect of dopamine receptor activities on the level of exploration. The dopamine receptor agonist apomorphine decreased exploration, while the dopamine receptor antagonist flupenthixol increased exploration. Beta-noradrenergic receptor activities also modulated exploration, but the effect is mediated by sex, possibly reflecting sex-dependent ceiling/floor effects of noradrenergic signaling. Conclusions: Overall, dopaminergic neuromodulation appears to exert more influence on exploration in decision making than does noradrenergic modulation, but sex differences played a stronger role in what effects are seen due to noradrenergic influences. This suggests that dopamine is a central modulator of the explore-exploit tradeoff, but sex differences in these effects may be due in part to underexplored noradrenergic influences. Keywords: Decision Making, Catecholamine, Explore-Exploit Dilemma, Sex Differences Disclosure: Nothing to disclose. P791. Instructed Motivational States Bias Reinforcement Learning and Memory Formation R. Alison Adcock, Alyssa H. Sinclair, Yuxi C. Wang Duke University, Durham, North Carolina, United States Background: Motivation influences goals, decisions, and memory formation. Imperative motivation links urgent goals to actions, whereas interrogative motivation integrates goals over time and space, thus supporting broader learning (Murty and Adcock, 2017; Dickerson and Adcock, 2018; Chiew and Adcock, 2019.) Whereas interrogative motivational states engage ventral tegmental area-hippocampus circuitry to support learning details and associations for flexible future behavior, imperative motivational states engage amygdala-cortical-medial temporal lobe circuitry to form sparse, decontextualized memories restricted to goal-relevant information. We posit that interrogative and imperative motivational states would have diverging consequences for reinforcement learning versus episodic, relational memory formation due to these underlying neural mechanisms. Methods: For two randomly assigned groups, we induced motivational states by manipulating only the cover stories for a reinforcement learning task: The Imperative group imagined executing a museum heist, whereas the Interrogative group imagined planning a future heist. Participants repeatedly chose from four “doors” to sample trial-unique paintings with variable rewards. The next day, participants returned to complete a surprise memory test. Participants performed old/new recognition of paintings and recalled painting-value and painting-door associations. We collected an initial sample (N = 100, 50% male, 50% female) and a pre-registered replication sample (N = 100, 50% male, 50% female). We analyzed reinforcement learning behavior using hierarchical Bayesian modeling, then classified trial-by-trial choices as either exploitation (choosing the door with the highest expected value), directed exploration (choosing the door with the highest estimated uncertainty), or random exploration (choosing one of the other two doors). Memory performance was assessed with linear models and mixed-effects logistic regression models. Results: Participants in the Imperative group made more exploitative choices (Sample 1: W = 1573.5, p = 0.026, Cohen’s d = 0.46, 95% CI [0.06, 0.85]; Sample 2: 1596, p = 0.017, Cohen’s d = 0.49, 95% CI [0.09, 0.89]) and earned more points during reinforcement learning (Sample 1: β = 0.13, 95% CI [-0.02, 0.28], t(96) = 1.74, p = 0.086; Sample 2: β = 0.28, 95% CI [0.11, 0.45], t(96) = 3.26, p = 0.002), demonstrating potential performance benefits during reward learning. Conversely, Interrogative motivation increased directed exploration during reinforcement learning (Sample 1: W = 872.5, p = 0.014, Cohen’s d = −0.71, 95% CI [-1.11, -0.30]; Sample 2: W = 961.5, p = 0.065, Cohen’s d = −0.46, 95% CI = [-0.85, -0.05]). There was no difference in random exploration between groups. At test, participants in the Interrogative group showed superior recognition memory for the incidentally-encoded paintings (Sample 1: β = −0.29, 95% CI [-0.51, -0.07], t(73) = −2.62, p = 0.011; Sample 2: β = −0.27, 95% CI [-0.51, -0.03], t(75) = −2.26, p = 0.027). Furthermore, we showed that reward modulated memory in the Interrogative group; high-value paintings were prioritized in memory (Sample 1: β = 0.13, z = 2.66, p = 0.008; Sample 2: β = 0.12, z = 2.96, p = 0.003). In the Imperative group, there was no effect of reward on memory (Sample 1: β = −0.01, z = −0.22, p = 0.825; Sample 2: β = −0.004, z = −0.10, p = 0.924). Conclusions: Overall, we found that a subtle instructional manipulation biased participants towards either an imperative or interrogative motivational state, thereby influencing both choice behavior and memory outcomes. The imperative/interrogative motivational framework unifies and reconciles findings from other proposed dichotomies in memory and decision-making research. Manipulating motivational states may enhance context-appropriate performance and memory formation, aligning cognitive processes and memory representations with current goals. Our results offer practical implications for educational practices, interventions for behavior change, and clinical treatments that aim to balance immediate performance goals with long-term learning. Keywords: Motivation, Reward, Decision Making, Memory, Reinforcement Learning Disclosure: Nothing to disclose. P792. A New Tool for Measuring Social Rejection Elicited Aggression Reveals Unique Relations Between Irritability and Aggression in Adolescents and Young Adults Megan Quarmley, Athena Vafiadis, Johanna Jarcho Temple University, Philadelphia, Pennsylvania, United States Background: Interpersonal violence is a leading cause of death for adolescents and young adults. One contributing factor may be an increased sensitivity to social rejection during this critical phase of development. Despite the strong connection between rejection and aggression in youth, little is known about the neurocognitive mechanisms that promote this relation. Mapping these mechanisms is a critical first step towards developing much needed interventions to curb aggressive behavior. Progress towards this goal has been hindered by a lack of ecologically-valid tasks that enable the measurement of in-the-moment social rejection elicited aggression. Here, we describe an initial study that demonstrates the efficacy of a novel task designed to evoke rejection elicited aggression. We replicate promising results in a second study that tests the task’s capacity to probe differences in rejection elicited aggression in adolescents and young adults who vary in their level of irritability, a transdiagnostic symptom associated with a proneness towards anger. Methods: Male and female participants (S1: N = 55, 20.36 ± 1.41 years; S2: N = 83, 12.39 ± 1.67 years, N = 90, 19.06 ± 1.64 years) completed the novel Virtual School With Aggression (VS-WA) task. Participants were told that they would be a new student who interacts with six age- and gender-matched peers online. They then completed a personal profile and avatar they believed would be sent to purported peers. They next learned the reputation of the peers (two nice, mean, unpredictable) via “Yelp-like” reviews left by prior purported participants. To maintain experimental control, participants actually interacted with a computer program that pre-determined the responses of the peers. During the first part of the task participants interacted with each peer 12 times (24 interactions per reputation). On each trial the participant received positive or negative feedback (nice: 100% positive; mean: 100% negative; unpredictable: 50% positive and negative), and responded with pre-specified text options. In the second part of the task, participants were given the opportunity to aggress against each purported peer in the context of a button pressing game. Participants learned that whoever pressed a button the most times in 5 sec would have the opportunity to send a noise blast at a volume of their choosing to each of the other peers. Noise blast options ranged from 1 (0 decibels) to 24 (120 decibel). Participants selected the volume for each peer 12 times (24 per reputation). Aggression was defined as the average volume selected for each peer type. Overall aggression was defined as the average volume selected for all peers. A repeated measures ANOVA, and follow-up paired samples t-tests, tested the hypothesis that the VS-WA task would elicit aggressive behavior that differed by peer reputation type (nice, mean, unpredictable). In Study 2, bivariate Pearson’s correlations tested the relation between age and aggressive behavior towards each peer type and overall aggression. The same analyses were performed for irritability using the Affective Reactivity Index (ARI) total score. A moderation analysis was conducted to test the effect of age on the relation between irritability and aggressive behavior towards mean peers. We focused on mean peers as they provided 100% negative feedback and therefore purely evoked social rejection elicited aggression. Johnson-Neyman analyses more precisely isolated regions of significance in moderation effects. Results: Across both studies, the VS-WA successfully evoked rejection-elicited aggression that differed depending whether peers were nice (S1: 7.06 ± 4.87; S2: 7.15 ± 5.10), mean (S1: 12.66 ± 7.14; S2: 14.92 ± 6.16), or unpredictable (S1: 10.72 ± 5.46; S2: 12.39 ± 5.13; ANOVAs S1: F(2, 108) = 20.57, p < 0.001, ε2 = 0.276; S2: F(2, 344) = 152.13, p.< .001, ε2 = 0.469). Participants were most aggressive to mean vs. unpredictable peers (S1: t(54) = 4.55, p < 0.001; S2: t(172) = 8.06, p < 0.001), and less aggressive towards nice vs. mean (S1: t(54) = 13.18, p < 0.001; S2: t(172) = 13.18, p < 0.001) and unpredictable peers (S1: t(54) = −4.12, p < 0.001; S2: t(172) = −12.70, p < 0.001). In Study 2, age was negatively correlated with aggression for each peer type (r’s < -.200, p’s < 0.01) and overall aggression (r = −.285, p < 0.001), whereas irritability was positive correlated with aggression for each peer type (r’s > 0.230, p’s < 0.01) and overall aggression (r = 0.280, p < 0.001). Age moderated the relation between irritability and aggression towards mean peers (R = 0.335, R2 = 0.113, F(3, 167) = 7.07, p < 0.001; interaction: ΔR2 = 0.032, b = 0.159, p = 0. 015). While there was no relation between irritability and aggression in younger adolescents, starting at 13.55 years of age, there was a positive relation between irritability and aggression that strengthened as age increased. Conclusions: We introduced and validated a novel task for studying rejection-elicited aggression that is optimized for studying neural mechanisms that promote this behavior. Across two studies we demonstrate the efficacy of the VS-WA, with level of aggression being sensitive to degree of peer-based rejection. Moreover, we showed the task can be used to probe age- and irritability-related differences in aggression. To that end we isolated unique age-based relations between irritability and rejection-elicited aggression. This effect has important implications for the development and implementation of interventions for irritable adolescents and young adults. Keywords: Agression, Social Rejection, Irritability, Adolescent Disclosure: Nothing to disclose. P793. A Transdiagnostic Assessment of Delay Discounting and Family History of Psychopathology Joshua Gowin, Jody Tanabe, Marcos Sanches, Matthew Sloan University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States Background: Delay discounting is a behavior conserved across species in which individuals prefer smaller immediate versus larger delayed rewards. It has been hypothesized that greater degrees of delay discounting are associated with psychiatric disorders in a transdiagnostic fashion. However, it is unclear if delay discounting behavior is a risk factor for these disorders, with individuals at higher risk demonstrating steeper delay discounting prior to the development of psychopathology. It remains critical to consider confounding variables such as socioeconomic status. Methods: The data is part of the Adolescent Brain and Cognitive Development (ABCD) study, which is a large, longitudinal study of 11,880 children. First, we looked at Spearman’s correlations between family history density of different psychiatric disorders and delay discounting behavior measured as the area under the curve. We then conducted mixed effects models to examine associations between family history density of different psychiatric disorders and delay discounting while accounting for sociodemographic factors. Results: Correlations between family history of psychopathology and delay discounting behavior were small in magnitude, ranging from ρ = −0.04 to 0.03. Without controlling for demographics, the model for family history of alcohol use problems had an estimate of -0.63 (95% CI -1.06, -0.20, p = 0.004), and this was one of the larger estimates. When adjusting for covariates, the effect of family history was reduced in the full sample (p = 0.019). In contrast, race was significant with an estimated effect of -6.72 for black relative to white individuals (95% CI -8.22, -5.23, p < 0.001). Hispanic relative to Non-Hispanic has a similar estimate (-3.69, p < 0.001). Males had steeper discounting than females (estimate = −2.68, p < 0.001). Parental education was significant, where having less than high school education was associated with steeper discounting than having a bachelor’s degree or higher (estimate = −4.61, p < 0.001). Household income was not significant. Conclusions: These results do not support the hypothesis that individuals at greater risk for psychopathology display steeper delay discounting behavior at ages 10-11. Sociodemographic factors played a larger role in predicting delay discounting behavior than family history of psychopathology. Keywords: Impulsivity, Delay Discounting, Adolescence, Heritability of Depression Disclosure: Nothing to disclose. P794. Prevalence and Correlates of Defense Mechanisms: A National Study Carlos Blanco, Leonie Kampe, Melanie Wall, Eve Caligor, Mark Olfson National Institute on Drug Abuse, Washington, District of Columbia, United States Background: Despite the clinical relevance of mechanisms of defense, there are no published studies of their prevalence, correlates and association with psychosocial functioning. The goal of this study was to estimate the prevalence and correlates of 12 defense mechanisms in the general adult population. Methods: Defense mechanisms were approximated from items used to assess personality traits in the National Epidemiological Survey on Alcohol and Related Conditions (NESARC), a representative sample of US adults (N = 36,653). We examined the associations between sociodemographic characteristics and prevalence of 3 types of defenses mechanisms (pathological, immature and neurotic). For each defense mechanism, we used the Short-Form 12 (SF-12) to compare psychosocial functioning among 3 groups: those who 1) endorsed the mechanism with impairment, 2) endorsed the mechanism without impairment, and 3) did not endorse the defense mechanism. Results: The prevalence of defense mechanisms ranged from 13.2% (splitting) to 44.5% (obsessive/controlling behavior). Pathological defenses were more strongly associated with immature defenses (OR = 5.4, 95% CI = 5.2-5.6) than with neurotic defenses (OR = 2.0, 95% CI = 1.9-2.0), whereas the association between immature and neurotic defenses had an intermediate value between the other two (OR = 2.2, 95% CI = 2.1-2.2). Pathological and immature defenses were associated with younger age, having been never married, lower educational attainment, and lower income. After adjusting the crude results for age and sex, individuals who did not endorse a given defense generally had higher scores on the mental health component of the SF-12 than those who endorsed the defense without self-recognized impairment who, in turn, had on average higher scores than those with self-recognized impairment. Conclusions: These results suggest that neurotic, immature, and pathological defense mechanisms are prevalent in the general population and associated with psychosocial impairment. Recognizing defense mechanisms may be important in clinical practice regardless of treatment modality. Keywords: Defense Mechanism, Psychopathology, Transdiagnostic Disclosure: Nothing to disclose. P795. Pleiotropic Influences of Neuropsychiatric Polygenic Risk on Common Laboratory Values in 660,000 US Veterans Tim Bigdeli, Peter Barr, Roseann Peterson, Georgios Voloudakis, Bryan Gorman, Cooperative Studies Program (CSP) #572, Million Veteran Program, Mihaela Aslan, Philip Harvey, Panos Roussos, Ayman Fanous* University of Arizona - Phoenix, Phoenix, Arizona, United States Background: Severe mental illness, including schizophrenia, bipolar, and major depression disorder are heritable, highly multifactorial disorders which cause significant disability, worldwide. Better understanding the pathophysiological mechanisms underlying disease risk and progression will be essential for alleviating the burden of these illnesses and comorbidities. The Million Veteran Program links genomic data, self-report survey data, and electronic health records from the Veterans Health Administration, which is the largest integrated health care system in the United States. Methods: We constructed and tested polygenic risk scores for schizophrenia, bipolar disorder, and major depression for association with median values for 70 laboratory tests in 660,000 participants. We applied genomic structural equation modeling to derive novel scores indexing shared and disorder-specific latent genetic factors. Results: Schizophrenia polygenic scores were robustly associated (P value < 10-8) with increased lipid levels, electrolyte imbalances, decreased liver and kidney function, and increased white blood cell count. Many associations were replicable among 125,000 African Americans, albeit with lesser statistical significance (P value < 10-5). We found that a cross-disorder latent genetic factor accounted for many findings, rather than any diagnosis-specific factors. These findings remained significant (P value < 10-6) when restricting analyses to individuals without diagnosed psychotic or affective illness, suggesting that these relationships are not simply consequences of medication side-effects. Conclusions: By applying a ‘reverse genetics’ approach to large-scale electronic health records, we add to a growing literature demonstrating the broad pleiotropic effects of currently indexable polygenic risk. Ongoing Mendelian randomization analyses seek to further parse biological from mediated pleiotropy. Keywords: Schizophrenia (SCZ), Bipolar Disorder (BD), Major Depressive Disorder, Genome-Wide Association Studies Disclosure: Nothing to disclose. P796. Novel PET Radioligand [18 F]PF-06445974 Can Measure LPS-Induced Phosphodiesterase 4B in Rat Brain Paul Parcon, Jeih-San Liow, Shawn Wu, Cheryl Morse, Carson Knoer, Victor Pike, Robert Innis National Institutes of Health, Rockville, Maryland, United States Background: Phosphodiesterase-4B (PDE4B) is an enzyme that metabolizes cAMP, thereby terminating the actions of this second messenger. Inhibition of PDE4B has antidepressant-like effects in animals, and inhibition of this enzyme in humans is anti-inflammatory in peripheral disorders (e.g., psoriasis and COPD). We developed [18 F]PF-06445974 as a radioligand that preferentially binds to the PDE4B subtype to study it in humans with major depressive disorder and in animals as a potential biomarker of neuroinflammation. In this study, we sought to determine whether intrastriatal injection of the inflammogen lipopolysaccharide (LPS) increases radioligand binding, which is an indirect measure of activation of the enzyme. That is, phosphorylation of PDE4 has been shown to markedly increase both its enzymatic activity and its affinity to binding of radiolabeled inhibitors. Methods: A total of 6 male Wild-type Sprague-Dawley rats were implanted with bilateral intrastriatal cannulas. After a week of recovery, 50 mg LPS in 5 µL of saline was infused into one hemisphere with the same volume of sterile saline on the contralateral side. At one and eight days after LPS, animals were injected with [18 F]PF-06445974 (20 MBq iv) and scanned for 90 min. Results: At one day after LPS injection, binding of [18 F]PF-06445974 in the lesioned striatum increased about 50% compared to the contralateral region. Areas distant from the lesion (e.g., cerebellum) were not affected by LPS injection. To confirm the specificity of the radioligand binding, rolipram (1 mg/kg iv) decreased binding to background levels. Finally, rescanning the animals one week after the lesion showed that radioligand binding had returned to baseline levels. Conclusions: We found that local injection of LPS into rat striatum increased radioligand binding about 50% compared to the contralateral side one day after the lesioning. The binding was selective for PDE4, as it was blocked by the PDE4-specific antagonist rolipram. In addition, the increase was transient and returned to baseline by one week after LPS injection. The increased binding may be caused by the phosphorylation/activation of PDE4B and suggest that PDE4B imaging may be a biomarker of neuroinflammation in the brain. Keywords: Neuroinflammation, Brain Based Markers for Depression, Positron Emission Tomography (PET), F-18 PET Imaging Disclosure: Nothing to disclose. P797. Exploring Relationships of Peripheral Inflammation With Negative and Positive Valence Systems Jessica Busler, Sarah Rose Slate, Katherine Coleman, Monica Foneska, Jake Taylor, Stanley Lyndon, Pamela Mahon Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States Background: Alterations in both negative (NVS) and positive valence (PVS) systems are characteristics of mood disorders and other disorders as evidenced by differences in experiences of constructs within these systems such as perceived stress, loneliness, sadness, positive affect, and emotional support. Abnormal inflammatory processes have also been implicated in mood disorders and can impact mood and cognitive symptoms. For example, inflammation has been shown to impact affective response inhibition and to impact on motivated behaviors such as reward valuation. It has also been shown that other PV symptoms (i.e. impaired motivation, impaired energy, and anhedonia) positively correlate with pro-inflammatory immunomarkers and that NV symptoms (i.e. anxiety and interpersonal sensitivity) negatively correlate with pro-inflammatory immunomarkers. Importantly, it has been suggested that women may be more vulnerable to inflammation-mediated depressive symptoms and behavior changes and display increased PV symptoms than men. However, sex comparisons in the relationship of peripheral inflammation to NVS and PVS factors including perceived stress, loneliness, sadness, positive affect, and emotional support are not well characterized. Therefore, we aimed to extend prior research in the link between valence systems and inflammation and examine the role of biological sex in the relationship between peripheral markers of inflammation [i.e. tumor necrosis factor-alpha (TNF-alpha) and Interleukin-6 (IL-6)] and function in NVS and PVS, measured by self-reports of positive affect, friendship, emotional support, loneliness, perceived stress, and sadness. Methods: Twenty-six participants (19 women and 17 men) were recruited at Brigham and Women’s Hospital, including both healthy participants and participants with bipolar disorder to enrich the sample for a range of function within NIMH Research Domain Criteria (RDoC) negative and positive valence systems. Participants were aged 35-63 (M = 47.94, SD = 8.53). Self-report positive affect, emotional support, friendship, loneliness, perceived stress, and sadness scores from NIH Toolbox were assessed to capture constructs in negative and positive valences systems. Peripheral markers of inflammation included pro-inflammatory cytokines TNF-alpha and IL-6 and were assayed from serum from a fasted morning blood draw. We conducted Pearson correlation analyses of each inflammatory marker with self-report measures in men and women separately. As a validity analysis, we conducted separate partial correlations in men and women controlling for age in the relationship between inflammatory markers with negative and positive valence systems. Results: We observed significant inverse correlations between TNF-alpha and loneliness (r = −0.704, p = 0.011), perceived stress (r = −0.588, p = 0.035), and sadness (r = −0.605, p = 0.017) scores in women. We also observed a significant positive correlation between TNF-alpha and emotional support scores (r = 0.616, p = 0.033) in women. Also in women, there was a trend in positive relationship between TNF-alpha with friendship (r = 0.517, p = 0.085) and positive affect (r = 0.455, p = 0.088) scores. No significant relationships emerged with IL-6 and we did not observe any significant associations in men or the combined sample of men and women. When controlling for age the relationship between TNF-alpha with loneliness (r = −0.658, p = 0.028) and sadness (r = −0.539, p = 0.047) scores remained significant in women and no other significant relationships emerged in men or the combined sample of men and women when controlling for age. Conclusions: These results suggest that inflammation is a key pathway to consider in the function of negative and positive valence systems in women. Specifically, TNF-alpha is inversely correlated with constructs within NVS and positively related to constructs in PVS in women. This is in line with previous research in depression showing that PV symptoms positively correlate with pro-inflammatory factors while NV symptoms negatively correlated with pro-inflammatory factors. Moreover, our findings of a link between inflammation and NVS and PVS in women but not in men extends and supports previous research indicating that women are more vulnerable to inflammation-induced mood alterations. Accordingly, our findings suggest a role for TNF-alpha as a mechanistic factor underlying sex differences in NVS and PVS functioning. Despite the promising nature of these findings, they are preliminary in nature and should be interpreted with caution given the small sample size. If confirmed, these findings have the potential to inform clinical care regarding the sex-informed use of anti-inflammatory medications to improve outcomes in disorders involving aberrant functioning in NVS and PVS. Keywords: Inflammation, Negative Valence System, Positive Valence, Cytokines, Mood Disorder Disclosure: Nothing to disclose. P798. CSF and Peripheral Inflammatory Biomarkers After COVID-19 Infection in a Pregnant Population Emma Smith, Frederieke Gigase, Vignesh Rajasekaran, Brett Collins, Joshua Hamburger, Benjamin Hyers, Rebecca Jessel, Andres Ramirez-Zamudio, Alan Adler, Kelly McMeen, Margaret McClure, Anna Rommel, Mara Graziani, Tammy Flores, Juliana Camacho Castro, Thalia Robakis, Julie Spicer, Robert Pietrzak, Veerle Bergink, Siobhan Dolan, Daniel Katz, Lotje DeWitte, M. Mercedes Perez-Rodriguez* Icahn School of Medicine at Mount Sinai, New York, New York, United States Background: In light of growing evidence of long-term sequelae after COVID-19 infection (termed post-covid conditions, long COVID or Post-Acute Sequelae of COVID [PASC]), there is an urgent need to investigate the potential long-term neuropsychiatric effects of SARS-CoV-2 infection. It has been estimated that between 10 and 30% of individuals who are infected with the virus, including those with mild disease, could experience post-covid sequelae. The central nervous system (CNS) is potentially susceptible to long-term changes from systemic inflammation that may occur during infection. While some evidence suggests that pregnant women may be more vulnerable to the acute effects of SARS-CoV-2 infection than the general population, the risk for potential long-term sequelae is unknown. This research aims to examine cerebrospinal fluid (CSF) and peripheral cytokine levels in pregnant individuals at delivery in relation to SARS-CoV-2 infection and vaccination status, to assess any potential long-term differences in inflammation. Methods: Participants were recruited from a prospective pregnancy cohort, Generation CSF, in the Mount Sinai Health System. CSF and plasma samples were drawn from 58 participants during labor and delivery (CSF n = 45, plasma n = 56, paired n = 43). A panel of fourteen cytokines was analyzed using a high sensitivity assay. In addition, SARS-CoV-2 infection and vaccination status were collected through self-report and extracted from medical records. Mann-Whitney U analysis was used to compare median cytokine levels between individuals who had tested positive for SARS-CoV-2 infection before or during pregnancy (n = 8) and individuals with no known prior SARS-CoV-2 infection (n = 50). Results: There were no significant differences in median cytokine levels in CSF and plasma between SARS-CoV-2 infected (before or during pregnancy) and non-infected groups. Subgroup analyses by vaccination status were not performed due to low sample sizes in some subgroups. Conclusions: This preliminary data analysis found no evidence of long-term central or peripheral inflammatory changes after SARS-CoV-2 infection in a pregnant population. This project is ongoing (Generation C-SF, target N = 500, R01MH127315) and further analyses in a larger sample including vaccination status are currently in progress. Keywords: Cytokines, Inflammation, CSF, COVID-19, Pregnancy Disclosure: Neurocrine Continental, Inc.: Consultant, Advisory Board (Self), Neurocrine Biosciences, Inc., Alkermes, Inc.: Consultant (Self), AICure, Millennium Pharmaceuticals, Inc. (Takeda Pharmaceutical Company Limited): Grant (Self). P799. The Transdiagnostic Comparative Efficacy of Non-Invasive Neurostimulation Interventions for Suicidal Ideation: Initial Results From a Systematic Review and Meta-Analysis Jenna Traynor, Jacob Koudys, Madeleine Weichel, Sapolnach Prompiengchai, Stephanie Bousleiman, Orly Lipsitz, Benjamin Walsh, Zafiris Daskalakis, Daniel M. Blumberger, Anthony Ruocco McLean Hospital, Harvard Medical School, Belmont, Massachusetts, United States Background: Recent research has suggested that non-invasive neurostimulation may reduce suicide ideation (SI). However, the efficacy of non-invasive neurostimulation interventions for SI has not yet been comprehensively reviewed and compared across methods and psychiatric disorders. Here, we present initial findings from the largest meta-analysis to date on the comparative efficacy of non-invasive neurostimulation for SI across psychiatric disorders. Methods: Embase, MEDLINE, PsycINFO, CINAHL, and Cochrane’s CENTRAL were used to search for peer-reviewed studies published from inception to June 2021. Human clinical trials examining the efficacy of non-invasive neurostimulation on psychiatric symptoms and reporting on a continuous outcome using a suicide-specific measure (e.g., Beck Scale for Suicide Ideation) or a suicide item from a standardized measure (e.g., suicide item on the HAM-D) were included. Random-effects meta-analyses of the standardized mean difference in SI scores from pre-to post treatment were used to estimate treatment effects. Where possible, effects of active v. sham treatment on SI were examined, as well as effects between psychiatric disorders and neurostimulation sites. Effect sizes are represented by Hedge’s g. Results: The search returned 10933 abstracts. 2591 studies underwent full text review. 39 studies were included in this preliminary analysis. Electroconvulsive Therapy (ECT): N = 14 ECT trials were identified, comprising 503 subjects with major depressive disorder (MDD). Only one trial was sham-controlled. There was a large and significant effect of ECT on SI (Hedge’s g = −1.56, SE = 0.17 [95% CI -1.89 -1.22], p < 0.001). A funnel plot suggested the presence of publication bias. Three studies compared ECT to other neurostimulation types, including low amplitude seizure therapy (LAP-ST; n = 1) and repetitive transcranial magnetic stimulation (rTMS; n = 2). No significant difference between ECT and LAP-ST on SI was reported in the one study that compared these interventions (p > 0.33). In contrast, a large and significant effect on SI, favoring ECT over rTMS, was found (Hedge’s g = −1.29, p < 0.001). Magnetic Seizure Therapy (MST): N = 2 trials examined the effects of MST on SI in 90 subjects with MDD. A large and significant effect of MST on SI was found (Hedge’s g = −0.82, SE = 0.20, 95% CI: -1.05, -0.58, p < 0.001). Due to the small number of trials, publication bias could not be assessed. Transcranial Magnetic Stimulation (TMS): N = 21 TMS trials were identified, comprising 928 subjects. A large and significant effect of TMS on SI was observed (Hedge’s g = −0.91, SE = 0.10, 95% CI: -1.10, -0.72, p < 0.001). Analysis of two adolescent trials suggested a medium but not significant effect of TMS on SI in this age group (Hedge’s g = −0.51, p = 0.06). In contrast, a large and significant effect on SI was found for 17 adult studies (Hedge’s g = −0.84, p < 0.001) and two geriatric studies (Hedge’s g = −2.01, p < 0.001). Across all TMS trials, a funnel plot suggested publication bias. Of the 21 TMS trials identified, 11 were sham-controlled, comprising 342 subjects. Overall, results favored active over sham TMS with a small, significant effect on SI (Hedge’s g = −0.36, SE = 0.08, [95% CI: -0.51, -0.21], p < 0.001). A funnel plot of sham-controlled TMS studies suggested a low chance of publication bias. Effect sizes across stimulation sites were similar and favored active over sham TMS with small and significant effects on SI: six trials stimulated the left dorsolateral prefrontal cortex (DLPFC)/prefrontal cortex (Hedge’s g = −0.41, p = 0.04) whereas four studies applied bilateral stimulation (Hedge’s g = −0.32, p = 0.009). One study stimulated the visual cortex and found no significant differences on SI in active v. sham TMS (Hedge’s g = −0.45, p = 0.12). Diagnostically, nine sham-controlled trials examined subjects with MDD. Results favored active over sham TMS for reducing SI in MDD (Hedge’s g = =0.40, p = 0.001). In contrast, two trials studied the effects of sham-controlled TMS in diagnoses other than depression (n = 1 borderline personality disorder and n = 1 posttraumatic stress disorder and/or traumatic brain injury). No significant differences between active v. sham TMS on SI in these diagnoses were found (Hedge’s g = −0.24, p = −.46). Transcranial Direct Current Stimulation (TDCS): N = 2 open-label TDCS pilot trials reported on an SI outcome. One investigated anorexia nervosa and the other, treatment-resistant obsessive-compulsive disorder. There was no significant effect of TDCS on SI in these samples (Hedge’s g = −0.06, p = 0.81). Conclusions: Initial results suggest that ECT, MST, and TMS may be efficacious for reducing SI. Although the strongest effects on SI were associated with ECT in MDD, only one ECT study was sham-controlled and there was a risk of publication bias across ECT studies. MST appears promising but the findings are limited by a lack of published data. Overall, sham-controlled TMS trials provided the highest quality evidence; active over sham TMS was associated with small but significant effects on SI, and no evidence of publication bias. Crucially, there is a paucity of neurostimulation trials in psychiatric groups other than MDD and this limits the generalizability of these findings to other groups that frequently experience SI. To date, analyses of two TDCS pilot trials in anorexia nervosa and OCD showed that TDCS did not significantly reduce SI in these groups. Meta analyses of remission and relapse rates are forthcoming. Keywords: Suicidality, Neurostimulation, Meta-Analysis Disclosure: Nothing to disclose. P800. CALM-IT: Feasibility, Reliability, Validity, and Clinical Relevance of a Novel Mobile Application Probing Inhibitory Control Lauren Henry, Elise Cardinale, Reut Naim, Simone Haller, Jennifer Meigs, Shannon Shaughnessy, Urmi Pandya, Olivia Siegal, Jessica Bezek, Ramaris German, Katharina Kircanski, Melissa Brotman National Institute of Mental Health, Bethesda, Maryland, United States Background: The identification of brain-based mechanisms underlying psychopathology is critical to the development of efficacious interventions. Inhibitory control, or the ability to modulate prepotent behavioral responses, is mediated through established neural circuitry, including ventrolateral, ventromedial, and dorsolateral prefrontal cortices; the orbitofrontal cortex; the inferior frontal gyrus; and the dorsal, posterior, anterior cingulate cortices. Research has linked inhibitory control deficits to increased risk for psychological symptoms and disorders. However, existing tools for assessing inhibitory control are repetitive, time intensive, expensive, and laboratory based, posing barriers to broad dissemination. We examined the utility of “CALM-IT,” a novel, gamified inhibitory control task based on the Go/No-Go paradigm delivered via mobile application platform to increase accessibility of an inhibitory control assessment in the clinical community. In four aims, we investigated the (1) feasibility, (2) reliability, (3) validity, and (4) potential clinical relevance of CALM-IT. Methods: Eighty-six youth (56.2% male) between 8 and 18 years old (M = 13.29, SD = 2.75) were recruited from the community. Youth with primary diagnoses of attention deficit hyperactivity disorder (ADHD; n = 25), anxiety disorder (n = 17), and disruptive mood dysregulation disorder (DMDD; n = 19); youth with clinically significant irritability not meeting the threshold for DMDD (n = 8); and youth without clinical diagnoses (n = 17) were included in the sample using the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime version (KSADS-PL). At home, youth completed CALM-IT on a mobile device across two sessions approximately one week apart (Mdays = 10.94, SDdays = 9.94). In the laboratory, youth completed canonical inhibitory control paradigms, including the Anti-saccade, AX Continuous Performance, Flanker, and Stop Signal tasks. On average, there were 429.51 days (SDdays = 359.64) between laboratory paradigm and CALM-IT completion. In addition, parents and youth completed a series of dimensional measures of psychopathology including assessments of irritability, anxiety, ADHD, and depression symptoms, respectively: Affective Reactivity Index (ARI), Screen for Anxiety Related Emotional Disorders (SCARED), Conners Comprehensive Behavior Ratings Scale (Conners), and Mood and Feelings Questionnaire (MFQ). Detailed information on study methods are available in the CALM-IT Registered Report. To determine feasibility, we calculated the percent of youth who completed CALM-IT with usable data, along with the percent of targets hit (indicating correct performance) as a measure of youth engagement. The intraclass correlation coefficient (ICC) between the two CALM-IT sessions provided an estimate of test-retest reliability. We operationalized inhibitory control performance during CALM-IT using d’, which assesses an individual’s ability to distinguish signal from noise. Specifically, we used d’ to determine the standardized difference between hits (successfully swiping at a target) versus false alarms (failing to inhibit response to hit a target). We then calculated a latent factor of inhibitory control from all four of the canonical laboratory-based assessments. Finally, we calculated the correlation between CALM-IT d’ and the latent factor of inhibitory control (validity) and the dimensional measures of psychopathology (clinical relevance). Results: Aim 1. Approximately 95.56% of CALM-IT levels produced complete and usable participant data. On average, youth hit 76.89% of targets (SD = 9.35%), demonstrating engagement with the task. Aim 2. Consistency was good for target hit rate (ICC = 0.85, p < 0.001) and moderate for d’ (ICC = 0.66, p < 0.001). Aim 3. On average, CALM-IT d’ = 0.00 (SD = 1.02). Greater inhibitory control as measured by CALM-IT d’ was associated with greater latent inhibitory control (r = 0.26, p = 0.05). Aim 4. Decreased inhibitory control as measured by CALM-IT d’ was associated with higher levels of youth-reported irritability on the ARI (r = −.27, p = 0.01) and parent-reported hyperactivity/impulsivity on the Conners (r = −.24, p = 0.04). Associations between inhibitory control as measured by CALM-IT d’ and parent-reported irritability on the ARI (r = −.13, p = 0.27), parent-reported inattentiveness on the Conners (r = −.12, p = 0.30), youth-reported (r = 0.04, p = 0.71) and parent-reported (r = 0.08, p = 0.46) anxiety on the SCARED, and youth-reported (r = 0.21, p = 0.06) and parent-reported (r = 0.16, p = 0.16) depression on the MFQ were nonsignificant. Conclusions: Taken together, findings provide robust support for the feasibility, reliability, validity, and clinical utility of CALM-IT. First, CALM-IT produced largely complete and usable data, and youth showed moderate levels of task engagement. Second, CALM-IT exhibited moderate-to-good test-retest reliability. Third, CALM-IT d’ was positively associated with a latent factor of inhibitory control with a medium effect size. Finally, preliminary support emerged for the clinical relevance of CALM-IT; lower levels of CALM-IT inhibitory control were related to greater youth-reported irritability and parent-reported hyperactivity/impulsivity. Future work will examine associations between youth CALM-IT performance and neural activation during inhibitory control tasks. Keywords: Inhibitory Control, Mobile Application, Developmental Psychopathology Disclosure: Nothing to disclose. P801. Differences in Personality, Cognitive Abilities, Illicit Drug Use, and White Matter Structural Integrity Between Hallucinogen Users and Matched Controls Aviv Aharon-Almagor, Frederick Barrett* Johns Hopkins University School of Medicine, Baltimore, Maryland, United States Background: Classic psychedelic drugs have potential positive enduring effects on personality (MacLean et al., 2011; Erritzoe et al., 2018) and cognition (Bouso et al., 2012; Doss et al., 2021; Rucker et al., 2022) as well as mood (Davis et al., 2021; Carhart-Harris et al., 2016, 2022) and substance use disorders (Johnson et al., 2014, 2017; Bogenschutz et al., 2015). However, enduring effects of psychedelic on human brain structure are unknown, and neural mechanisms supporting the enduring psychedelic effects are not well-understood. Preclinical findings suggest micro-scale structural neuroplastic changes after psychedelic administration (Ly et al., 2018; Shao et al., 2021). The current study investigated the association between psychedelic use, macroscale brain structure, personality, cognitive ability, and illicit drug use in a naturalistic sample. Methods: Data from 53 subjects (age M = 43.1, SD = 15,1; 32 F) reporting ever having used hallucinogens and 53 approximately matched controls (age M = 43.2, SD = 15.8; 31 F) who reported never having used a hallucinogen were drawn from the Nathan Kline Institute-Rockland Sample database (Nooner et al., 2012). All participants completed diffusion tensor imaging (137 directions, 2mm3 voxels, b = 0, 1500 s/mm2), psychological assessments (NEO Five Factor Inventory, Wechsler Abbreviated Scale of Intelligence II or WASI-II, Wechsler Individual Achievement Test second edition abbreviated or WIAT-IIA) and psychiatric assessments (CASI-A and NIDA drug use questionnaires, Structured Clinical Interview for DSM-IV-TR). Controls were matched to hallucinogen users by age, sex, and presence vs absence of a psychiatric diagnosis. Groups were compared on measures of personality, cognitive ability, history of illicit drug use, and the density of white matter tracts determined from probabilistic tractography using linear statistics. Diffusion image preprocessing and analysis was conducted using the FSL toolbox (Jenkinson et al., 2021). Preprocessing included skull stripping, eddy current and motion correction, and local fitting of diffusion tensors using DTIFIT. Data were normalized to MNI template space using FLIRT. Tractography was then performed in native space using BEDPOSTX and the cross-species tractography toolbox (XTRACT; Warrington et al., 2020). XTRACT performs probabilistic, anatomically constrained tractography using a library of validated tractography protocols for 42 white matter tracts. 2 tracts (Left and Right corticospinal tract) were removed due to insufficient number of subjects with full brain data coverage for these areas. White matter tract density for each of the remaining 40 tracts was compared between groups using multi-covariate regression, controlling for sex, age, and the presence or absence of lifetime use of stimulants, cocaine, narcotics, and inhalants. Additional linear models were estimated for each white matter tract that showed a significant difference in density between groups, to evaluate the association between differences in white matter density and differences in questionnaire assessment data. For statistical inference, including correction for multiple comparisons across space, maximal statistic permutation testing with threshold-free cluster enhancement was conducted (Smith et al., 2006). Results: Hallucinogen users reported greater lifetime use of stimulants ((χ)2 = 5.194, p = 0.023), cocaine ((χ)2 = 12.791, p = 0.000), narcotics ((χ)2 = 8.477, p = 0.004), and inhalants ((χ)2 = 6.360, p = 0.012), but not tobacco ((χ)2 = 1.178, p = 0.278), cannabis ((χ)2 = 2.038, p = 0.153), alcohol ((χ)2 = 3.087, p = 0.079), or tranquilizers ((χ)2 = 1.504, p = 0.22). Higher scores were observed in hallucinogen users on the personality trait of openness (t = 2.321, p = 0.014), WASI-II total score (Mann-Whitney U = 1033, p = 0.012), and WIAT-IIA composite standard score (Mann-Whitney U = 8897, p = 0.006). Greater density of white-matter tracts was observed in hallucinogen users in the left superior thalamic radiation (p = 0.031), left arcuate fasciculus (p = 0.026), left perigenual cingulum (p = 0.009), left (p < 0.001) and right (p = 0.035) frontal aslant, right superior longitudinal fasciculus (p = 0.004), and left inferior longitudinal fasciculus (p = 0.017). Less white matter tract density was observed in hallucinogen users in the right inferior longitudinal fasciculus (p < 0.001). Positive associations between white matter tract density and cognitive abilities were observed for WIAT-IIA composite score and the left inferior longitudinal fasciculus (p = 0.0009), and for WASI-II full scale score and the right superior longitudinal fasciculus (p = 0.038). Conclusions: Hallucinogen exposure may result in structural consequence in canonical white matter tracts associated with perception, cognition, and affect. If greater white matter density confers a processing benefit, and lower density confers a deficit, the current findings are consistent with previous findings suggesting improved cognitive function and reduced negative affect among hallucinogen users (Bouso et al., 2012; Barrett et al., 2020; Doss et al., 2021). Given proposed psychoplastogenic effects of hallucinogens (Ly et al., 2018; Shao et al., 2021), it may be that circuits or pathways that are acutely altered during hallucinogen drug action undergo some lasting structural change that can be observed with diffusion imaging methods. These novel findings provide clues to potential neural mechanisms underlying therapeutic effects of hallucinogens. Keywords: Psychedelics, Potential Mechanism, Structural Connectivity, Big Five Personality Factors, Cognitive Performance Disclosure: Wavepaths, Ltd, MindState Design Labs, Inc.: Advisory Board (Self). P802. The Placebo Response in Classic Psychedelics: A Systematic Review of Clinical Trials and Qualitative Analysis Cory Weissman, Nikhita Singhal, Brett D. M. Jones, Richard J. Zeifman University of California - San Diego, San Diego, California, United States Background: The use of classic psychedelics (e.g., psilocybin, ayahuasca, lysergic acid diethylamide [LSD]) as potent mental health treatments is gaining traction, yet significant challenges remain in conducting trials with these substances. Both the role of placebo controls in psychedelic therapy trials and the importance of placebo mechanisms in explaining the efficacy of psychedelic therapy remain understudied. Several factors pose significant challenges to blinding in psychedelic therapy trials. Successfully blinding participants to respective treatment arms is challenging, in part due to strong subjective effects of psychedelic compounds. This issue is important because it may overestimate outcomes in psychedelic therapy trials by increasing expectancy effects in active arm conditions and by diminishing effects in placebo conditions via participant disappointment in not receiving a psychedelic. This is supported by the fact that one study reported a death by suicide in one of their participants 11 days after receiving a very low dose of psilocybin intended as a placebo (Griffiths et al., 2016). The authors note that the subject reported feeling bored, possibly reflecting a nocebo effect. While blinding is clearly a difficult area in this field, surprisingly, it has not been well studied. One previous review suggests that only half of modern psychedelic clinical studies reported on subject blinding and nearly all subjects were able to ascertain whether they were given the psychedelic or placebo. The role of expectancy in placebo response also remains understudied and is a vital component of response in these studies. We review and synthesize the entire existing literature on the placebo response in the context of clinical trials that involve the administration of classic psychedelics to humans in order to guide future clinical trial design and enhance our understanding of this complex area. Methods: We conducted an extensive PROSPERO registered (CRD42020185100) systematic review in accordance with PRISMA guidelines to identify any studies with the use of placebo in the context of a classic psychedelic clinical trial in a psychiatric population. EMBASE, PsycINFO, and PubMed were electronically searched with a combination of medical subject headings and free text keywords from their inception dates to present. The following data was extracted: study authors, publication year, study design, study duration, inclusion/exclusion criteria, participant demographics and baseline characteristics, sample sizes and descriptions of groups, details on interventions/exposures, outcome measures used, outcomes/effects reported, and adverse events. The characteristics and findings of included studies are presented as a systematic narrative synthesis and summarized in tabular format. The narrative synthesis explores findings within and between included studies and includes qualitative outcomes. We assessed the methodological quality of included studies using the Oxford Centre for Evidence Based Medicine (OCEBM) Levels of Evidence. Results: We identified a total of 55 out of 1053 studies in our search that were eligible for inclusion, with publication dates ranging from 1963-2020. Studies were either RCTs or within subject cross-over design. There was also one single-blind non-randomized placebo-controlled within subject trial. In the RCTs, there were 10 on LSD, 17 on psilocybin, 5 on ayahuasca, and 1 on N,N-Dimethyltryptamine (DMT). Among RCTs, 29 studies were on healthy subjects, 3 on life-threatening cancer, 2 on schizophrenia, and 2 on major depressive disorder. For cross-over design studies, 7 were on LSD, 2 on psilocybin, 1 on ayahuasca, and 1 on DMT/6-hydroxy-N-dimethyltryptamine. There were 8 studies on healthy subjects, 2 on schizophrenia, and 1 in alcohol use disorder in these studies. The most common forms of placebo used were empty capsules, niacin, and IV saline. Expectancy was not measured in the vast majority of studies. Clinical outcomes in our review include subjective mental states, physiological measures, creative imagination and mental imagery tests, BDNF and cortisol levels, eyeblink responses, as well as formal measures of clinical depression and anxiety. Conclusions: Our review suggests that the vast majority of placebo-controlled psychedelic therapy studies involve studies in healthy participants with the use of LSD or psilocybin. There is a very limited number of placebo-controlled studies in psychiatric populations, and the quality of placebo controls have been questionable. The use of an adequate placebo control, and assessment and balancing of expectancy, is severely lacking in existing clinical trials. Future psychedelic clinical trials should include adequate assessment of blinding, use of more appropriate controls, and should randomize both treatment arms and treatment expectancy. Active psychopharmacological controls, such as other rapid acting agents, and head-to-head comparison with active treatments should be considered as alternatives. Keywords: Psychedelic Therapy, Placebo, Clinical Trials, Blinding, Expectancy Disclosure: Consultant for GoodCap Pharmaceuticals Inc.: Consultant (Self). P803. Effects of Psychoactive Drugs MDMA, Methamphetamine, and Buprenorphine on Affective Touch Processing in Healthy Humans Anya Bershad, Leah Mayo, Harriet de Wit UCLA, Los Angeles, California, United States Background: MDMA has shown great promise in the treatment of several psychiatric disorders when used in combination with psychotherapy. However, the components of the psychotherapy that accompanies MDMA treatment have not been fully investigated. One novel, yet understudied, component of psychedelic-assisted psychotherapy is therapeutic touch, raising the possibility that drugs like MDMA affect treatment response in part by affecting the somatosensory experience. The effects of psychoactive drugs on the experience of touch are not fully understood. In controlled studies with healthy volunteers, we tested the effects of three different psychoactive drugs on responses to affective touch; 1) the prosocial drug MDMA, 2) the prototypical stimulant methamphetamine, which shares some properties with MDMA without producing reported experiences of social closeness, and 3) the opioid buprenorphine, which has been shown to enhance some dimensions of social reward processing. Methods: Healthy men and women participated in two double-blind placebo-controlled studies using a within-subjects crossover design. In Study 1 (N = 36; two sessions), they received buprenorphine (0.2 mg) or placebo in randomized counterbalanced order. In Study 2 (N = 36; four sessions), they received MDMA (0.75 mg/kg and 1.5 mg/kg), methamphetamine (20 mg), and placebo. In both studies, during expected peak drug effect, they completed an affective touch task to assess pleasantness of either affective touch (slow forearm brushing; 3 cm/s) or nonaffective touch (fast forearm brushing; 30 cm/s). Pleasantness of the touch was assessed with facial electromyography (EMG) and subjective ratings. These measures were examined in relation to ratings of subjective drug effects measured by the Drug Effects Questionnaire (DEQ, e.g. liking the drug and wanting more). Results: As expected, slow touch was rated as more pleasant than fast touch in both studies. Both MDMA (both doses) and buprenorphine (0.2 mg) increased pleasantness ratings of affective touch [MDMA: F(2,70)=7.27, p < 0.05; buprenorphine t(34)=-1.70, p < 0.05], but methamphetamine did not. For MDMA, increases in pleasantness ratings were significantly correlated with “like drug” (r = 0.39, p < 0.05) and “want more” (r = 0.50, p < 0.01). Conclusions: The finding that MDMA, and not methamphetamine, enhanced pleasantness ratings of affective touch is consistent with reports that the drug enhances the experience of touch in recreational settings. This finding suggests that changes in somatosensory processing may also contribute to the drug’s effectiveness in MDMA-assisted psychotherapy. Our finding the opioid buprenorphine also enhances pleasantness of affective touch suggests that this effect is not specific to serotonergic stimulant drugs, but may also occur with other drug classes, and may be dissociable from euphorigenic effects. Future studies with symptomatic participants are needed to extend these findings to a clinical population. Keywords: MDMA, Buprenorphine, Methamphetamine, Affective Touch, Psychophysiology Disclosure: Nothing to disclose. P804. Monosynaptic Circuitry Based CNS Drug Development: A Proven Approach to De-Risk Such Work Sheldon Preskorn* University of Kansas, Wichita, Kansas, United States Background: Most major pharmaceutical companies have abandoned psychiatric drug development. A principal reason is the riskiness of such development. However, there is a proven way to de-risk such development which will be presented in this poster. The reason to why ACNP as an organization and its membership should be interested in this topic is two-fold: (a) psychiatric medication is the major treatment modality for patient suffering from psychiatric disorders and abandonment of such development stagnates the development of new treatment for such illnesses and (b) the research that ACNP members do provides the necessary knowledge to take this circuitry approach to drug development. Methods: The successful development of 7 different single mechanism of action CNS drugs will be present and the features that they have in common. The seven drugs include: aprepitant, lorcaserin, ondansetron, ramelteon, suvorexant, varenicline, and most recently most recent is sublingual dexmedetomidine (SLD). The poster will explain why these 7 drugs were selected as well as why drugs such as ketamine/esketamine and acamprosate. Briefly, the criteria were:A well-defined, monosynaptic circuit mediating the behavior broadly defined such as appetite, agitation, nausea, sleep, and smoking. How a single mechanism of action drug could impact the circuitry to modify the behavioral in a predictable and desirable way, The drug has a single mechanism thus reducing off target effects The behavior is observable rather than subjective and is either dichotomous (e.g., vomiting or not) or highly correlated (e.g., the factors on the Excitatory Component of the Positive and Negative Symptom Scale, PANSS) in contrast to the less correlated symptoms domains in scales used to measure antidepressant (i.e. HDRS or MADRS) or antipsychotic (total PANSS). An objective, surrogate marker may be able such as polysomnography encephalography (PSE) or blood pressure to assess target engagement. Results: In contrast to drugs which do not meet the above criteria such as antidepressants and antipsychotics, the results of the studies with these 7 drug include the following:good agreement between preclinical models and human studies good agreement between phase 2 and phase 3 human studies. few, if any, negative studies in phase 3. Table and graphs will be presented in this poster to illustrate and support the above points. The development of such drugs is not without some development hurdles. One example is that there may be a shifted dose-response curve individuals with the target illness versus normal volunteers. That is generally accepted for dopamine-2 receptors blockers in normal volunteers versus patients with schizophrenia. Another example is blood pressure effects of SLD in normal volunteers versus agitated individuals with bipolar disorder or schizophrenia. However, these differences have been readily addressed in early phase 2 studies to properly determine the dose range for the phase 3 studies. Conclusions: Encouraging the development of mechanistically new drugs to treat psychiatric illness should be a prime concern of ACNP. This poster reviews the development of 7 new psychiatric medication based on well-defined monosynaptic circuitry mediating specific behavioral abnormalities over the last two decades. This approach is also being used for drug development and discovery relative to the model of the Nucleus Accumbens as the central common pathway to drug addiction developed by George Koop. This topic can be discussed during the poster presentation. This knowledge is important for each member of ACNP which will in turn aid the ACNP in advocating for and promoting such drug development in the future. An issue will be how to translate this approach to more complex disease states such as major depression and schizophrenia. That may well be done by defining the circuits underlying the overall syndromic diagnosis. An adage here is that behaviors are the output of brain functions whereas syndromic diagnoses are human made conventions. Keywords: Drug Development, Circuitry-Based Approach, Mechanism of Action Disclosure: Nothing to disclose. P805. Endocannabinoid Contributions to Affective Touch Processing in Humans With or Without Trauma Exposure Histories Connor Haggarty, Madeleine Jones, India Morrison, Markus Heilig, Leah Mayo* University of Calgary, Linköping, Sweden Background: Touch is a vital component of social interactions, contributing to the development and maintenance of social bonds throughout life. The pleasant, affective quality of touch is mediated by C-tactile (CT) afferent nerve fibers that respond most robustly to slow, gentle touch. CT-mediated affective touch is believed to play a role in buffering the effects of psychosocial stress and promoting prosocial behaviors. Thus, our first aim was to explore reactions to affective touch in adults who were exposed to trauma during childhood or adolescence. We then explored how individual variation in the endocannabinoid system, a neuromodulatory system implicated in stress and social behavior, may influence reactions to affective touch in this population. Finally, in a separate study, we assessed how pharmacological manipulation of the endocannabinoid system influenced affective touch processing in healthy adults. Methods: In the first study, adult participants (N = 101) with documented childhood trauma (N = 51) or no trauma history (N = 50) completed tasks assessing perception of affective (e.g., CT-optimal) touch or non-affective (non-CT-optimal) touch. Blood samples were collected to analyze levels of endocannabinoids and cortisol. In the subsequent study, healthy adults (N = 46) were randomized to placebo or a drug that potentiates endocannabinoid signaling and responses to touch were measured. Results: As expected, CT-optimal touch was rated as more pleasant overall (main effect of velocity: F(1,81) = 44.5, p < 0.001, partial η2 = 0.36), though this did not differ between trauma-exposed and non-exposed individuals (touch type x group interaction: p = 0.30). However, this effect was mediated by levels of the endocannabinoid ligand anandamide (AEA; AEA x touch type interaction: F(1,63)=7.13, p = 0.01, partial η2 = 0.10) such that greater AEA levels were associated with reduced preference for CT-touch (r = −0.32; p = 0.01). This was specific to ratings of the affective quality (“pleasantness”) of touch, and not evident in ratings of touch intensity (touch type x group interaction: p = 0.18). In study 2, we further demonstrated the role for AEA in affective touch processing by using PF-04457845 (now known as JZP150) to enhance AEA levels via reduced enzymatic degradation in healthy adults. Here, we again found that individuals with elevated AEA had no preference for affective, CT-optimal touch (group x touch type interaction: F(1,42 = 50.9, p = 0.002, partial η2 = 0.20)). This was again specific to ratings of pleasantness and not intensity (group x touch type interaction: p = 0.68) Conclusions: Overall, we find that increases in the stress-buffering endocannabinoid ligand AEA may play a critical role in mediating the perceived pleasantness of affective touch, as evidence both by individual variation in AEA levels and pharmacological intervention. This data suggests that when AEA is high, the impact of the stress buffering qualities of affective, CT-optimal touch may be minimal. As such, it is possible that individuals with lower AEA, or states that induce decreases in AEA (i.e., following stress exposure), may be particularly responsive to the stress buffering qualities of socially relevant, affective touch, providing insight into populations who may most benefit from social support via affective touch. In addition, it suggests that the endocannabinoid system may be a viable treatment target in individuals whom experience insufficient social contact. Keywords: Affective Touch, Endocannabinoids, Childhood Trauma Disclosure: Nothing to disclose. P806. Lifestyle Factor Influence on TMS-Induced Excitability Joshua Brown, Jamie Kweon, Megan Vigne, Prayushi Sharma, Linda Carpenter Alpert Medical School of Brown University, Providence, Rhode Island, United States Background: Neurophysiology measures, such as motor-evoked potentials (MEPs), have been used with receptor-influencing pharmacology to test purported mechanisms of repetitive transcranial magnetic stimulation (rTMS) in humans. As such studies have been small in number and sample sizes, we sought to retest our previous findings (only ten subjects) with NMDA receptor partial agonist d-cycloserine which showed enhancement of rTMS effects with NMDA agonism. We hypothesized that d-cycloserine would again enhance rTMS excitatory effects. Additionally, MEPs are also known for their high variability, which limits our ability at times to interpret their outcomes. We accordingly gathered demographic and lifestyle information to include in our post-hoc exploratory analyses. Methods: We conducted a double-blind crossover replication study with 10 healthy subjects whereby we gave NMDA receptor partial agonist d-cycloserine (100 mg) or placebo on separate visits. In each visit, drug was given first, followed by baseline excitability probes assessed by motor-evoked potentials (MEPs), then a 300 pulse 10 Hz rTMS “plasticity protocol” at 80% of resting motor threshold, finally followed by repeat MEPs over 1-hour. Post-hoc analysis was performed of covariate data including self-identified musicians and athletes (n = 4), and of self-identified caffeine users (n = 7). Time course data was analyzed with repeated measures ANOVA with and without drug for each covariate condition. Paired-pulse measures were analyzed with t-tests. Significance was set at p < 0.05. Results: The primary outcome measure of the replication study was the 1-hour time course, and was not significant (p = 0.12), but was underpowered to determine a non-effect (this was a replication study which was previously significant with this sample size). However, post-hoc analysis of covariates revealed a significant enhancement of rTMS plasticity effects in musicians and athletes while taking d-cycloserine (p = 0.03). A separate but overlapping analysis of caffeine drinkers revealed blunting of plasticity effects in caffeine drinkers, and a marked enhancement from rTMS only in non-caffeine drinkers in the d-cycloserine condition. (p = 0.01). Paired-pulse measures, which can reveal underlying changes in glutamatergic or GABAergic tone, revealed enhanced intracortical facilitation, reflecting glutamatergic changes (p = 0.02). Conclusions: Our underpowered primary outcome emphasizes the need for better powered motor neurophysiology experiments which are considered foundational for our mechanistic understanding of rTMS. Interestingly, our exploratory post-hoc covariate findings may reflect Hebbian and homeostatic plasticity mechanisms, induced by long-term practice and chronic caffeine intake, respectively. In addition, these extemporaneous factors may account for cortical excitability outcomes, and by extension, clinical outcomes, as they could conceivable affect rTMS effects on target neurons. Keywords: Repetitive Transcranial Magnetic Stimulation (rTMS), Neurophysiology, Pharmacology, NMDA Receptors Disclosure: Nothing to disclose. P807. Mesolimbic Dopamine Release Conveys Causal Associations Huijeong Jeong, Mingkang Zhou, Brenda Wu, Dennis A. Burke, Vijay Mohan K. Namboodiri University of California at San Francisco, San Francisco, California, United States Background: Learning to predict rewards based on environmental cues is essential for survival. It is widely believed that animals learn to predict rewards by updating predictions whenever the outcome deviates from expectations. Such violations of predictions are called reward prediction errors (RPEs) and are thought to facilitate learning. RPEs are the critical teaching signal in the most widely accepted model for cue-reward associative learning—temporal difference reinforcement learning (TDRL). Despite a few exceptions, TDRL RPE has been largely successful at explaining the activity dynamics of dopaminergic cell bodies and release in the nucleus accumbens. Hence, TDRL RPE has become the dominant theory of dopamine’s role as the critical regulator of behavioral learning. An alternative approach to learn cue-reward associations is to infer the cause of meaningful outcomes such as rewards. Since causes must precede outcomes, a viable approach to infer whether a cue causes reward is to learn whether the cue consistently precedes reward. This approach is advantageous as predicting the future is highly demanding in a cue-rich environment but inferring the cause of a rarer meaningful outcome simply requires a memory of previous experience. In other words, if an animal knows that a stimulus it just received is meaningful (e.g., a reward), it can look back in memory to infer its cause. Using this intuition, here we propose a causal inference algorithm that infers whether a cue is a cause of reward. Based on this algorithm, we denote stimuli (cues or rewards) whose cause should be learned by the animal as “meaningful causal targets” and propose that mesolimbic dopamine signals whether a current event is a meaningful causal target using an adjusted net contingency for causal relations (ANCCR) of that event in relation to other meaningful causal targets. We found that ANCCR makes similar predictions as RPE under commonly studied experimental settings. Hence, to distinguish between the two hypotheses (RPE or ANCCR signaling by mesolimbic dopamine release), we performed eight experimental tests by measuring dopamine release in nucleus accumbens core. Methods: Eight adult wild type (C57BL/6; Jackson Laboratory) mice were used for the main experimental tests (six males). Additionally, six adult wild type mice were used to show that mice learn average reward rates in an environment with random delivery of rewards (four males). The animals were individually housed after surgery under a 12 h light/dark cycle. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the UCSF Institutional Animal Care and Use Committee. Behavioral experiments were conducted under water deprivation, in which animals were maintained at ~85–90% of their pre-deprivation weights. Animals underwent surgery for injection of a virus causing expression of a dopamine sensor (AAVDJ-CAG-dLight1.3b) in the nucleus accumbens core. Dopamine measurements were made using photometry of dLight signals using standard frequency modulation approaches. Trace conditioning was done with an auditory tone (3 kHz pulsing tone or 12 kHz constant tone, 75–80 dB) lasting 2 s or 8 s paired with a sucrose reward (15%, ~2.5 µL) 1 s after tone offset. This reward paired tone (CS+) was presented randomly interleaved with another tone (12 kHz constant tone or 3 kHz pulsing tone, 75–80 dB) that did not predict reward (CS−). In another experiment, random unpredictable sucrose drops were delivered with an exponential inter-reward interval at an average of once every 12 s. We first simulated standard temporal difference reinforcement learning algorithms used to model dopamine dynamics. These included the use of a complete serial compound, microstimulus and semi-Markov state space. We compared the expected dynamics of dopamine from these models against those predicted by our ANCCR model. Using this comparison, we developed eight discriminative experimental tests that allowed us to distinguish TDRL RPE from a signaling of ANCCR. The collected data were analyzed across these conditions using standard statistical approaches. Results: We found that mesolimbic dopamine conveys causal associations but not RPE in every case (n = 8 tests in n = 7 animals, p < 0.05 in each test). These included two tests using the delivery of random Poisson trains of rewards, five tests using standard variations assaying a cue-reward association, and one test in a new “trial-less” cue-reward association task. In all cases, we ruled out the standard TDRL RPE signals commonly used to model dopamine dynamics but observed that the results remained consistent with ANCCR, thereby challenging the dominant theory of reward learning in the brain. Conclusions: In summary, we observed that the dynamics of mesolimbic dopamine release in nucleus accumbens core are inconsistent with TDRL RPE across a multitude of experiments but remain consistent with a causal learning algorithm. Our results provide a new conceptual and biological framework for associative learning. Keywords: Dopamine, Associative Learning, Reinforcement Learning Disclosure: Nothing to disclose. P808. Interdigitated Visual and Personal Space-Sensitive Cortical Columns in Human Parietal Cortex: An Ultra-High Resolution fMRI Study Daphne Holt, Zahra Nasiriavanaki, Baktash Babadi, Douglas Greve, Shahin Nasir, Roger Tootell Harvard Medical School, Charlestown, Massachusetts, United States Background: Personal space (PS) is the “safety zone” around the body that people prefer to maintain between themselves and unfamiliar others. Intrusion into one’s PS evokes discomfort and an urge to move away. Numerous studies have described PS abnormalities in neuropsychiatric disorders such as schizophrenia and autism, suggesting that understanding the mechanisms underlying PS regulation may provide insights regarding the pathophysiology of these conditions. Physiological studies in non-human primates have shown that sensory and motor responses to stimuli that intrude into PS rely on the parietal cortex among other brain regions. However, the neural processes involved in calculating and regulating interpersonal distances remain unclear. Thus here we used ultra-high resolution (7Tesla, 1.1 mm isotropic) functional magnetic resonance imaging (fMRI) to investigate the hypothesis that the spatial encoding of interpersonal distance is transformed from purely sensory (retinotopic) to PS-related (body-centric) coordinates within human parietal cortex. Methods: Seven healthy individuals (4 females) participated in multiple MRI scan sessions. In each session, subjects viewed one of three types of stimuli: 1) three-dimensional (3D) face stimuli (8 male and 8 female, with neutral facial expressions) that appeared to move either toward or away from (Approach vs. Withdrawal) the participant (2 sessions); 2) stationary 3D face stimuli that appeared at 9 different virtual distances from the participant, based on the participant’s preferred personal space boundary (one session); 3) geometric random-dot stimuli forming a stereoscopic percept of a regular array of cuboids varying in-depth, either ‘near’ or ‘far’, relative to the fixation plane (one session). The fMRI data were analyzed using FreeSurfer (on the individual cortical surface, 14 hemispheres) without spatial smoothing. A region-of-interest (ROI) in the inferior parietal cortex was defined in each hemisphere using anatomical criteria. To test for a columnar organization of the fMRI responses, correlations were calculated between the selective activity in vertices distributed along each of the three axes in the flattened cortical maps: X and Y within the surface-parallel cortical map, and Z along the perpendicular (radial) axis, at two different cortical depths (superficial and deep). For each of the three axes, correlation values were calculated independently for each subject and compared (following Fisher’s r-to-z transformation) using paired t-tests. To measure the topographic relationships among the four main activity maps (approach-biased, withdrawal-biased, disparity-near biased and disparity-far biased), the degree of overlap of the columnar activity maps across them was calculated (as a percentage of the surface area) when the maps were correctly aligned compared to when the maps were randomly misaligned (i.e., spatially shuffled) 1000 times. Results: In all fourteen hemispheres, activity within the parietal ROI showed a significantly higher correlation in the Approach-vs.-Withdrawal functional contrast when sampled along the radial axis, compared to functional variation along either of the surface-parallel axes (superficial layers: t = 11.38, p < 0.001; deep layers: t = 16.82, p < 0.001). Similarly, comparisons across maps sampled at different cortical depths revealed that disparity-selective Near and Far- biased clusters of activity in parietal cortex were also radially elongated, exhibiting a columnar organization. Lastly, the overlap analyses showed that maps of the PS-sensitive columns were systematically interdigitated (highly non-overlapping across multiple thresholds) with the disparity-sensitive columns in the parietal cortex ROI. Conclusions: Across these experiments, two types of distance encoding were identified in two corresponding columns of activity within human parietal cortex. One category of columns responded selectively to moving and stationary face images presented at virtual distances that were nearer (but not further) than each subject’s behaviorally-defined PS boundary. In the majority of these columns, BOLD response amplitudes increased monotonically and nonlinearly with increasing virtual face proximity (an approach bias). In a complementary subset of these columns, BOLD responses decreased with increasing proximity (a withdrawal bias). A second set of columns responded selectively to disparity-based distances (near or far) in random-dot stimuli, similar to disparity-selective columns described previously in occipital cortex. These results suggest that the transformation of spatial information, from visual to body-centric, may be computed within the parietal cortex via communication across short-range projections between columns. In future studies, similar approaches could be applied to determine whether the PS system, and the laminar organization of association cortices more broadly, are altered in neurodevelopmental disorders. Keywords: Parietal Cortex, High Resolution fMRI, Cortical Columns, Personal Space, Depth Perception Disclosure: Nothing to disclose. P809. Behavioral Economics of Striatal Dopamine Neir Eshel, Gavin Touponse, Allan Wang, Amber Osterman, Amei Shank, Alexa Groome, Lara Taniguchi, Daniel Cardozo Pinto, Jason Tucciarone, Brandon Bentzley, Robert Malenka Stanford University, Stanford, California, United States Background: Although striatal DA release is crucial for reward learning and decision-making, prior studies have disagreed over its role encoding costs, benefits, and motivation. Manipulations of DA function with lesions or pharmacology imply that striatal DA primarily mediates cost calculations; that is, how much effort an animal exerts to obtain rewards. However, these studies often confounded cost and benefit by varying both simultaneously, and used tools that could not examine how DA activity dynamics underlie these behaviors. In contrast, electrophysiological studies of DA activity dynamics suggest that striatal DA more reliably encodes benefit, not cost. These studies, however, were correlational and did not exploit causal tools to explore how DA release influences behavior. Furthermore, DA neuron recording studies have rarely explored changes in motivational state between individuals or across time except in studies of addiction, where there is debate over whether striatal DA release is sensitized or depleted once individuals transition to a highly-motivated, addicted state. Methods: We established a simple operant task that independently varies costs and benefits to generate behavioral economic demand curves in response to sucrose rewards. During a single session, the fixed ratio (FR, or “cost” of reward) varied in 10-minute bins over 50 minutes, while the reward “benefit” remained identical. Between sessions, we varied the concentration and quantity of sucrose reward, such that mice experienced four different “benefits”. By plotting reward consumption across cost for each session, we extracted a quantitative metric of motivation, defined by the willingness to overcome cost, independently from reward dose. At the same time, we measured DA release in both the nucleus accumbens (NAc) and dorsolateral striatum (DLS) using the genetically-encoded sensor GRAB-DA, allowing us to monitor how striatal DA release reflects costs, benefits, and motivational state. In a second cohort of mice, we used the excitatory opsin ChRMINE to stimulate DA inputs to the NAc or DLS while simultaneously recording DA release. These mice performed the identical task, except instead of working for sucrose reward, they worked for optogenetic stimulation of DA release. Finally, in a third cohort of mice, we used the inhibitory opsin NpHR3.0 to inhibit DA inputs to the NAc or DLS while mice worked for sucrose reward. In total we recorded from 84 mice (39 female). Results: Mice successfully learned the task, increasing the number of rewards they earned in each session (Friedman test, P = 0.0002) and decreasing their latency to consume each reward (Friedman test, P = 0.0026). As expected, mouse behavior was sensitive to both reward benefit (Mixed-effects model, fixed effect of quantity, F1,11 = 17.9, P = 0.0014; fixed effect of concentration, F1,11 = 176, P < 0.0001) and cost (Friedman test, P < 0.0001). Examining DA release aligned to the reward-predicting cue, we found that increased sucrose concentration (Wilcoxon signed rank test, P = 0.024) and quantity (Wilcoxon signed rank test, P = 0.003) enhanced DA release in both regions. Surprisingly, increased cost also enhanced DA responses in both regions, despite the cue and reward being held constant (Friedman test, NAc: P = 0.0043; DLS: P < 0.0001). We then generated demand curves for each session, and took advantage of daily variation in subjects’ performance to determine how striatal DA release relates to motivation. Strikingly, we found an inverse relationship between DA release and motivation level. In sessions when mice were more motivated for a given reward, less DA was released in response to fixed rewards in both striatal regions (Mann-Whitney test; NAc: P = 0.001; DLS: P = 0.0049). Furthermore, mice with higher average motivation levels exhibited lower average DA responses in both ventral and dorsal striatum (Spearman correlation; NAc: P = 0.048; DLS: P = 0.028). When we repeated all the above experiments with optogenetic stimulation of DA inputs, we found the same results. As cost increased, optogenetically-evoked DA release in both the NAc (Friedman test, P = 0.0005) and the DLS (Friedman test, P < 0.0001) increased, despite using identical light stimulation parameters. Furthermore, optogenetically-evoked DA release was greater during low motivation sessions in both the NAc (Mann-Whitney test, P = 0.0061) and DLS (Mann-Whitney test, P = 0.0038), and this inverse relationship between motivation and optogenetic striatal DA release held both within and between subjects. Finally, we tested the causal relationship between motivation and striatal DA release in two ways. First, we reduced subjects’ motivation for sucrose by prefeeding them with sucrose before each session. We found that this manipulation increased striatal DA release to the same reward (Wilcoxon signed-rank test; NAc: P = 0.016; DLS: P = 0.016). Second, we used optogenetics to inhibit DA release for sucrose reward, and found that this inhibition increased motivation (Wilcoxon signed-rank test; NAc: P = 0.031; DLS: P = 0.031). In all experiments, we did not observe any behavioral or neural differences between sexes. Conclusions: We reveal that striatal DA release integrates benefit and sunk cost to encode value, and surprisingly, that high motivation dampens these signals. These findings reconcile discrepancies between prior studies on DA and help clarify the role of striatal DA signals in motivated behavior. Keywords: Striatal Dopamine Signaling, Behavioral Economics, Circuit Optogenetics, Intrinsic Motivation, Effort-Cost Benefit Task Disclosures: MapLight, Boehringer Ingelheim: Consultant (Self), Summus: Honoraria (Self).	36456695	PMC9714408	NO-CC CODE	2022-12-06 23:15:03	no	Neuropsychopharmacology. 2022 Dec 1; 47(Suppl 1):371-520	utf-8	Neuropsychopharmacology	2,022	10.1038/s41386-022-01486-z	oa_other

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